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Calcium: Calcium signalling during embryonic development
Abstract
Consider a hypothetical design specification for an integrated communication-control system within an embryo. It would require short-range (subcellular) and long-range (pan-embryonic) abilities, it would have to be flexible and, at the same time, robust enough to operate in a dynamically changing environment without information being lost or misinterpreted. Although many signalling elements appear, disappear and sometimes reappear during development, it is becoming clear that embryos also depend on a ubiquitous, persistent and highly versatile signalling system that is based around a single messenger, Ca2+.
... ATP is an NT and neuromodulator [20,23,24] associated with the synthesis and release of trophic factors such as fibroblast growth factor 2 (FGF2), nerve growth factor (NGF), and neurotrophin 3 (NT-3) [20,25,26]. Then, the purinergic signaling increases [Ca 2+ ]i modulating the propagation of calcium waves, which play an important role in embryonic development and neural tube formation [27]. The P2X(2-7) and P2Y (1,8) receptors and the ectonucleoside triphosphate diphosphohydrolase-2 (eNTPDase-2) enzyme, modulate ATP signaling in neurulation, early embryonic neurogenesis, and brain development in rats [25,[28][29][30]. ...
... HCs-mediated ATP release could activate P2X3-4, P2Y1,8 receptors [25,[28][29][30], triggering intracellular signaling [20,23]. This may involve intracellular calcium [Ca 2+ ]i, which regulates cell proliferation, migration, differentiation, and cytoskeletal remodeling during embryonic development and neural tube formation [27,94]. Eventually, the alteration of ATP signaling (HCs-Cxs) can lead to NTDs. ...
... The inhibition of the NMDAR [21] and glutaminase 1 (GLS1), (glutaminolysis, GLN → GLU) [22], generates alterations in [Ca 2+ ]i and neural plate cell migration resulting in NTDs. This suggests that Glu and ATP could act as complementary interplay signaling during neurulation and neural tube formation helping the comprehensive understanding of neural tube defects etiology [21,22,27,29,30,94]. ...
Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD+; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis. Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation.
... Calcium signaling is an important feature of embryonic development, and has been implicated in events spanning oocyte maturation, fertilization, gastrulation and cell differentiation (Webb and Miller, 2003). Calcium signals during early development have been characterized in several vertebrate and invertebrate species, leading to the discovery of some highly conserved roles, such as in polyspermy block, and also diversity in spatiotemporal patterns, potentially indicative of species-specific intercellular coordination (Stricker, 1999;Miller, 2003, 2006;Whitaker, 2008). ...
... Having characterized the temporal and spatial dynamics of the Ca2þ transients, we then attempted to determine whether influx of Ca2þ from the extracellular fluid was involved in initiating them. Since our main interest here was to investigate intercellular Ca2þ waves in the context of the extensive cellular rearrangements that occur during gastrulation (Tada and Concha, 2001;Webb and Miller, 2003), we restricted our experimental manipulations to gastrula stages. Ca2þ transients under experimental conditions were compared to those in control embryos (Video S2). ...
... Each of them includes three cysteins, which generate a cystein bridge through sulfur-sulfur bonds between the connexons of two adjacent cells, thereby forming the intercellular gap junction (Ahir and Pratten, 2014). Gap junctions pass Ca2þ ions, and are integral to the spread of Ca2þ waves among neighboring cells in many tissues (Webb and Miller, 2003). ...
We characterized spontaneous Ca2þ signals in Oikopleura dioica embryos from pre-fertilization to gastrula stages
following injection of GCaMP6 mRNA into unfertilized eggs. The unfertilized egg exhibited regular, transient
elevations in intracellular Ca2þ concentration with an average duration of 4–6 s and an average frequency of
about 1 every 2.5 min. Fertilization was accompanied by a longer Ca2þ transient that lasted several minutes.
Thereafter, regular Ca2þ transients were reinstated that spread within seconds among blastomeres and gradually
increased in duration (by about 50%) and decreased in frequency (by about 20%) by gastrulation. Peak amplitudes
also exhibited a dynamic, with a transitory drop occurring at about the 4-cell stage and a subsequent rise.
Each peak was preceded by about 15 s by a smaller and shorter Ca2þ increase (about 5% of the main peak
amplitude, average duration 3 s), which we term the “minipeak”. By gastrulation, Ca2þ transients exhibited a
stereotyped initiation site on either side of the 32-64-cell embryo, likely in the nascent muscle precursor cells, and
spread thereafter symmetrically in a stereotyped spatial pattern that engaged blastomeres giving rise to all the
major tissue lineages. The rapid spread of the transients relative to the intertransient interval created a coordinated
wave that, on a coarse time scale, could be considered an approximate synchronization. Treatment with the
divalent cations Ni2þ or Cd2þ gradually diminished peak amplitudes, had only moderate effects on wave frequency,
but markedly disrupted wave synchronization and normal development. The T-type Ca2þ channel
blocker mibefradil similarly disrupted normal development, and eliminated the minipeaks, but did not affect
wave synchronization. To assess the role of gap junctions in calcium wave spread and coordination, we first
characterized the expression of two Oikopleura connexins, Od-CxA and Od-CxB, both of which are expressed
during pre-gastrulation and gastrula stages, and then co-injected double-stranded inhibitory RNAs together with
CGaMP6 to suppress connexin expression. Connexin mRNA knockdown led to a gradual increase in Ca2þ transient
peak width, a decrease of interpeak interval and a marked disruption of wave synchronization. As seen with
divalent cations and mibefradil, this desynchronization was accompanied by a disruption of normal development.
... Mammalian reproduction and development depend on coordinated programs of cell division, specification, migration and death. These events are organized in space, time and scale by short-and long-range chemical and mechanical signals [1]. As a second messenger, the intracellular calcium signal is capable of decoding and integrating inputs from both the chemical and physical extracellular environment [2]. ...
... The calcium signal is an important participant in the evolution of the developing embryo from a fertilized oocyte, enabling a multitude of biologically distinct and complex processes, such as cell fate determination and organogenesis (reviewed in [1,2]). Such a feat is made possible by the selective expression and engagement of specific components of the calcium signaling toolkit, at precise points in space and time [2]. ...
... Such a feat is made possible by the selective expression and engagement of specific components of the calcium signaling toolkit, at precise points in space and time [2]. Based on studies in other vertebrate model systems-primarily Xenopus, zebrafish and medaka fish-there is strong evidence supporting a role for the calcium signal throughout the embryonic development of vertebrate animals [1]. Early embryonic development in mammals involves complex and dynamic cellular interactions and rearrangements required for the determination of the mammalian body plan. ...
Life begins with calcium. It is the language that a sperm cell uses to respond to instructions from the female reproductive tract to alter its swimming pattern and gain the force required to penetrate the outer layers of the oocyte. The first heartbeat transpires from spontaneous calcium oscillations in embryonic cardiomyocytes. The dynamic balance of calcium between auditory hair cells and the fluid they bathe in enables us to hear our first sound, and our interpretation and response to this sound requires rapid calcium flux through neuronal voltage-sensitive calcium channels. Calcium signaling can decode and integrate informational cues from both the chemical and mechanical cellular microenvironment to drive the form and function of many mammalian organ-systems. Here, we highlight roles for the intracellular calcium signal in the reproductive- and developmental- biology of mammals. A greater appreciation of the signaling pathways that initiate and support life has wide-ranging significance for the fields of reproductive science, neonatology and regenerative medicine. Furthermore, as developmental programs are often reactivated in cancer, an improved understanding of the signaling pathways that underpin mammalian development has important implications for cancer research.
... Although fewer in number, there are several excellent studies that do investigate the role of calcium activity in development using in vivo calcium imaging, but they tend to use completely different imaging and analysis methods, making comparisons between studies and integration of knowledge across studies quite challenging. Given that several comprehensive reviews on the role of calcium activity in development exist (see [18][19][20][21]), the goal of this review is to focus on the methodological aspects of in vivo calcium imaging, specifically the techniques used for analysis by summarizing, comparing, and contrasting the techniques that have been historically used to study calcium. In particular, cleavage stages, neural induction, neuronal subtype formation, and neurotransmitter phenotype specification are explored. ...
... Gray color indicates lowest levels of calcium activity, which is usually in non-neural (nn) tissue. While some studies have reported higher levels of calcium activity near the blastopore areas (a) [18,39], others have shown higher levels in the neural plate (b,e) [40,41] or neural ridge areas (c,e) [41][42][43]. One of the reasons for this difference could be due to application of differential imaging parameters as well as calcium activity analysis methods. ...
The intracellular and intercellular flux of calcium ions represents an ancient and universal mode of signaling that regulates an extensive array of cellular processes. Evidence for the central role of calcium signaling includes various techniques that allow the visualization of calcium activity in living cells. While extensively investigated in mature cells, calcium activity is equally important in developing cells, particularly the embryonic nervous system where it has been implicated in a wide variety array of determinative events. However, unlike in mature cells, where the calcium dynamics display regular, predictable patterns, calcium activity in developing systems is far more sporadic, irregular, and diverse. This renders the ability to assess calcium activity in a consistent manner extremely challenging, challenges reflected in the diversity of methods employed to analyze calcium activity in neural development. Here we review the wide array of calcium detection and analysis methods used across studies, limiting the extent to which they can be comparatively analyzed. The goal is to provide investigators not only with an overview of calcium activity analysis techniques currently available, but also to offer suggestions for future work and standardization to enable informative comparative evaluations of this fundamental and important process in neural development.
... Early embryogenesis depends on a proper control of intracellular homeostasis of ions including Ca 2+ Mg 2+ , protons, and phosphate (Komiya and Runnels, 2015;Lane and Gardner, 2000;Webb and Miller, 2003). Ca 2+ is a versatile and universal messenger that is implicated in the regulation of embryonic development in many species and participates in numerous sequential steps of development including fertilization, development of the blastula and gastrula stage, as well as during organoand somitogenesis (Markova and Lenne, 2012;Seisenberger et al., 2000;Webb and Miller, 2003). ...
... Early embryogenesis depends on a proper control of intracellular homeostasis of ions including Ca 2+ Mg 2+ , protons, and phosphate (Komiya and Runnels, 2015;Lane and Gardner, 2000;Webb and Miller, 2003). Ca 2+ is a versatile and universal messenger that is implicated in the regulation of embryonic development in many species and participates in numerous sequential steps of development including fertilization, development of the blastula and gastrula stage, as well as during organoand somitogenesis (Markova and Lenne, 2012;Seisenberger et al., 2000;Webb and Miller, 2003). Starting from a constant and low Ca 2+ concentration of about 100 nM in the resting state various patterns of changes of the intracellular Ca 2+ concentration are described to occur in conjunction with developmental steps. ...
Early embryogenesis depends on proper control of intracellular homeostasis of ions including Ca2+ and Mg2+. Deletion of the Ca2+ and Mg2+ conducting the TRPM7 channel is embryonically lethal in mice but leaves compaction, blastomere polarization, blastocoel formation, and correct specification of the lineages of the trophectoderm and inner cell mass unaltered despite that free cytoplasmic Ca2+ and Mg2+ is reduced at the two-cell stage. Although Trpm7-/- embryos are able to hatch from the zona pellucida, no expansion of Trpm7-/- trophoblast cells can be observed, and Trpm7-/- embryos are not identifiable in utero at E6.5 or later. Given the proliferation and adhesion defect of Trpm7-/- trophoblast stem cells and the ability of Trpm7-/- ESCs to develop to embryos in tetraploid embryo complementation assays, we postulate a critical role of TRPM7 in trophectoderm cells and their failure during implantation as the most likely explanation of the developmental arrest of Trpm7-deficient mouse embryos.
... Calreticulin is a highly conserved, ubiquitous high-capacity Ca 2ϩ -buffering protein of the endoplasmic reticulum (ER) 3 that is centrally located in a signaling network within the ER lumen (1). Calreticulin is uniquely endowed for this role as it controls ER [Ca 2ϩ ] and the amount of Ca 2ϩ releasable from the ER (2). ...
... Calreticulin is uniquely endowed for this role as it controls ER [Ca 2ϩ ] and the amount of Ca 2ϩ releasable from the ER (2). Calreticulin-mediated ER signaling is poised to regulate transcriptional activity of developmental programs in general, and ESC differentiation in particular (3,4). An important part of the transcriptional circuitry is the Ca 2ϩ /calmodulin-dependent phosphatase, calcineurin (5). ...
Calreticulin is a highly conserved, ubiquitous Ca2+ buffering protein in the endoplasmic reticulum (ER) that controls transcriptional activity of various developmental programs and also of embryonic stem cell (ESC) differentiation. Calreticulin activates calcineurin, which dephosphorylates and induces the nuclear import of the osteogenic transcription regulator- nuclear factor of activated T cells 1 (NFATC1). We investigated whether calreticulin controls a switch between osteogenesis and chondrogenesis in mouse ESCs through NFATC1. We found that in the absence of calreticulin, intranuclear transport of NFATC1 is blocked and that differentiation switches from osteogenic to chondrogenic, a process that could be mimicked by chemical inhibition of NFAT translocation. Glycogen synthase kinase 3ß (GSK3ß)-deactivation, and nuclear-localization of ß-catenin critical to osteogenesis, was abrogated by calreticulin-deficiency or NFAT blockade. Chemically-induced GSK3ß inhibition bypassed the calreticulin/calcineurin axis and increased osteoblast output from both control and calreticulin-deficient ESCs, while suppressing chondrogenesis. Calreticulin-deficient ESCs or cells treated with an NFAT-blocker had enhanced expression of dickkopf WNT-signalling pathway inhibitor 1 ( Dkk1 ), a canonical Wnt pathway antagonist that blocks GSK3ß-deactivation. Addition of recombinant mDKK1 switched osteogenic ESC-differentiation towards chondrogenic differentiation. The results of our study indicate a role for ER calcium signalling via calreticulin in the differentiation of ESCs to closely associated osteoblast or chondrocyte lineages.
... The calcium ion is a versatile and universal second messenger involved in the regulation of embryogenesis (Markova et al., 2015, Slusarski and Pelegri 2007, Webb and Miller 2003. Calcium signals have been shown to play a central role in several developmental processes, such as fertilization and organ formation (Kaneuchi et al., 2015, York-Andersen et al., 2015, Christodoulou and Skourides 2015, Berridge, Bootman and Roderick 2003. ...
... Intercellular calcium waves transmit local information from the initiator cell to a large number of cells in order to coordinate and synchronize their activity during development (Moreno-Juan et al., 2017;Akahoshi , Hotta and Oka 2017, Wallingford et al 2001, Webb and Miller 2003, Özsu and Monteiro 2017. Surprisingly, endogenous calcium waves during embryogenesis of insects have not been documented. ...
Calcium signaling plays a crucial role in the physiology of the organs but also in various aspects of the organogenesis of the embryo. High versatility of calcium signaling is encoded by the dynamic variation of intracellular calcium concentration. While the dynamics of calcium is important, little is known about it throughout the embryogenesis of the largest class of animals, insects. Here, we visualize calcium dynamics throughout embryogenesis of Drosophila using a fluorescent protein-based calcium indicator, GCaMP3, and report calcium transients in epithelium and neuronal tissues. Local calcium transients of varying duration were detected in the outer epithelium, trachea and neural cells. In addition, gap-junction-dependent calcium waves were identified at stage 16 in the outer epithelium and in the trachea at stage 17. Calcium transient waveform analysis revealed different characteristics as a function of the duration, location and frequency. Detailed characterization of calcium transients during embryogenesis of Drosophila will help us better understand the role of calcium signaling in embryogenesis and organogenesis of insects.
... Similarly, mouse embryos treated with bivalent cation chelators such as BAPTA or EGTA, or embryos that lack calreticulin, which is a Ca 2+ -binding chaperone of the endoplasmic reticulum, die in utero due to defective heart development. However, the calcium ionophore ionomycin restored myofibrillogenesis in cardiomyocytes [107,108]. These observations underscore the importance of calcium activity during cardiogenesis and associated developmental heart defects. ...
... Additionally, several calcium-dependent proteins have been linked to pronephros development and function. For example, the proteins polycystin-1 and polycystin-2, encoded by the genes PKD1 and PKD2 respectively, are expressed in renal tissue of both humans and mice during renal development and have been shown to function together to create a calcium-permeable channel [107]. Mutations in PKD1 and PKD2 result in autosomal dominant polycystic kidney disease in humans [113]. ...
Accumulating evidence over the past three decades suggests that altered calcium signaling during development may be a major driving force for adult pathophysiological events. Well over a hundred human genes encode proteins that are specifically dedicated to calcium homeostasis and calcium signaling, and the majority of these are expressed during embryonic development. Recent advances in molecular techniques have identified impaired calcium signaling during development due to either mutations or dysregulation of these proteins. This impaired signaling has been implicated in various human diseases ranging from cardiac malformations to epilepsy. Although the molecular basis of these and other diseases have been well studied in adult systems, the potential developmental origins of such diseases are less well characterized. In this review, we will discuss the recent evidence that examines different patterns of calcium activity during early development, as well as potential medical conditions associated with its dysregulation. Studies performed using various model organisms, including zebrafish, Xenopus, and mouse, have underscored the critical role of calcium activity in infertility, abortive pregnancy, developmental defects, and a range of diseases which manifest later in life. Understanding the underlying mechanisms by which calcium regulates these diverse developmental processes remains a challenge; however, this knowledge will potentially enable calcium signaling to be used as a therapeutic target in regenerative and personalized medicine.
... Well-studied developmental pathways, such as Ras, Wnt, Notch, Jak-STAT, TGF-beta, Hedgehog, PPAR, MAPK, NF-kB, and calcium signaling pathways, were ranked at the top ( Figure 3G; Table S3). [66][67][68][69][70][71][72][73][74] For example, 71% (164/ 232) of Ras pathway genes, 57% (83/147) of Wnt pathway genes, and 56% (28/49) of Notch pathway genes were correlated with eRNAs in at least one developmental stage (Table S3). Thus, eRNAs play critical roles in mediating developmental signaling pathways in early embryos. ...
Enhancer-derived RNAs (eRNAs) play critical roles in diverse biological processes by facilitating their target gene expression. However, the abundance and function of eRNAs in early embryos are not clear. Here, we present a comprehensive eRNA atlas by systematically integrating publicly available datasets of mouse early embryos. We characterize the transcriptional and regulatory network of eRNAs and show that different embryo developmental stages have distinct eRNA expression and regulatory profiles. Paternal eRNAs are activated asymmetrically during zygotic genome activation (ZGA). Moreover, we identify an eRNA, MZGAe1, which plays an important function in regulating mouse ZGA and early embryo development. MZGAe1 knockdown leads to a developmental block from 2-cell embryo to blastocyst. We create an online data portal, M2ED2, to query and visualize eRNA expression and regulation. Our study thus provides a systematic landscape of eRNA and reveals the important role of eRNAs in regulating mouse early embryo development.
... Specifically, during these stages, calcium ions modulate the release of vital secondary messengers, including inositol trisphosphate (IP3) [33], cyclic adenosine monophosphate (cAMP) [34], and diacylglycerol (DAG) [35], which are essential for oocyte activation and its fusion with the spermatozoon [31,32]. In the early stages of embryonic development, a balanced calcium concentration is crucial for proper cellular division and the implantation of the embryo into the uterine lining [31, [200][201][202][203]. Consequently, an imbalance in calcium levels, whether due to a deficiency [204,205] or excess [206], can compromise fertility and reduce the likelihood of a successful pregnancy [207]. ...
The role of minerals in female fertility, particularly in relation to the menstrual cycle, presents a complex area of study that underscores the interplay between nutrition and reproductive health. This narrative review aims to elucidate the impacts of minerals on key aspects of the reproductive system: hormonal regulation, ovarian function and ovulation, endometrial health, and oxidative stress. Despite the attention given to specific micronutrients in relation to reproductive disorders, there is a noticeable absence of a comprehensive review focusing on the impact of minerals throughout the menstrual cycle on female fertility. This narrative review aims to address this gap by examining the influence of minerals on reproductive health. Each mineral’s contribution is explored in detail to provide a clearer picture of its importance in supporting female fertility. This comprehensive analysis not only enhances our knowledge of reproductive health but also offers clinicians valuable insights into potential therapeutic strategies and the recommended intake of minerals to promote female reproductive well-being, considering the menstrual cycle. This review stands as the first to offer such a detailed examination of minerals in the context of the menstrual cycle, aiming to elevate the understanding of their critical role in female fertility and reproductive health.
... During early development, Ca 2+ signals help establish the dorsal-ventral (D-V) axis and contribute to organogenesis [2] . ...
Endoplasmic reticulum (ER) calcium (Ca ²⁺ ) homeostasis is essential for maintaining normal cellular physiological functions. Its disturbance is strongly linked to the onset and progression of human diseases, including cancer, developmental defects, and neurodegenerative disorders. The lack of sensitive ratiometric ER Ca ²⁺ indicators, nevertheless, hinders systematic investigation of ER Ca ²⁺ modulators and the underlying mechanisms. Capitalizing on two ultra-sensitive ER Ca ²⁺ indicators and CRISPR-based genome-wide screening, we identified a set of proteins capable of reducing the ER Ca ²⁺ content. Further comparative analysis and qPCR validation pinpointed adenylate cyclase 9 (AC9), which is upregulated during neuronal differentiation, as a key ER-Ca ²⁺ -reducing regulator. Mechanistically, AC9-mediated production of cAMP is not essential for its ability to reduce ER Ca2+ content. Instead, AC9 inhibits store operated calcium entry (SOCE) by acting on Orai1, ultimately causing attenuation of ER Ca ²⁺ level. More physiologically relevant, upregulation of AC9 in neurons is essential for reducing ER Ca ²⁺ levels during Drosophila brain development. Collectively, this study lays a solid groundwork for further in-depth exploration of the regulatory mechanisms dictating ER Ca ²⁺ homeostasis during neuronal differentiation and brain development.
... Luminal flow rate subjects epithelial cells in the discrete nephron segments to hydrodynamic forces (66). Fluid shear stress has been shown to play an essential role in organ and vascular maturation during embryogenesis and angiogenesis (67)(68)(69), partly mediated by calcium signaling (70)(71)(72). This observation along with findings that vasculature facilitates the formation of the liver bud from stem cellderived multiple lineages (73), led to the investigation of the role of superfusate flow on kidney organoid differentiation and maturation (Fig. 3A). ...
Kidney organoids are three-dimensional structures generated from pluripotent stem cells (PSCs) that are capable of recapitulating the major structures of mammalian kidneys. As this technology is expected to be a promising tool for studying renal biology, drug discovery, and regenerative medicine, the functional capacity of kidney organoids has emerged as a critical question in the field. Kidney organoids produced using several protocols harbor key structures of native kidneys. Here we review the current state, recent advances, and future challenges in the functional characterization of kidney organoids, strategies to accelerate and enhance kidney organoid functions, and access to PSC resources to advance organoid research. The strategies to construct physiologically relevant kidney organoids include the use of organ-on-a-chip technologies that integrate fluid circulation and improve organoid maturation. These approaches result in increased expression of the major tubular transporters and elements of mechanosensory signaling pathways suggestive of improved functionality. Nevertheless, continuous efforts remain crucial to create kidney tissue that more faithfully replicates physiological conditions for future applications in kidney regeneration medicine and their ethical use in patient care.
... Regulating Ca 2+ levels plays a central role in the physiology and survival of cells [16]. In addition, Ca 2+ signaling through calcineurin and NFAT is important for the differentiation of ESCs [17], and it plays an essential role in the early development of vertebrate embryos [18]. Furthermore, there is a developing body of literature suggesting that miRNAs can regulate the expression of genes involved in Ca 2+ transport [19]. ...
MicroRNAs (miRNAs) are important regulators of embryonic stem cell (ESC) biology, and their study has identified key regulatory mechanisms. To find novel pathways regulated by miRNAs in ESCs, we undertook a bioinformatics analysis of gene pathways differently expressed in the absence of miRNAs due to the deletion of Dicer, which encodes an RNase that is essential for the synthesis of miRNAs. One pathway that stood out was Ca2+ signaling. Interestingly, we found that Dicer−/− ESCs had no difference in basal cytoplasmic Ca2+ levels but were hyperresponsive when Ca2+ import into the endoplasmic reticulum (ER) was blocked by thapsigargin. Remarkably, the increased Ca2+ response to thapsigargin in ESCs resulted in almost no increase in apoptosis and no differences in stress response pathways, despite the importance of miRNAs in the stress response of other cell types. The increased Ca2+ response in Dicer−/− ESCs was also observed during purinergic receptor activation, demonstrating a physiological role for the miRNA regulation of Ca2+ signaling pathways. In examining the mechanism of increased Ca2+ responsiveness to thapsigargin, neither store-operated Ca2+ entry nor Ca2+ clearance mechanisms from the cytoplasm appeared to be involved. Rather, it appeared to involve an increase in the expression of one isoform of the IP3 receptors (Itpr2). miRNA regulation of Itpr2 expression primarily appeared to be indirect, with transcriptional regulation playing a major role. Therefore, the miRNA regulation of Itpr2 expression offers a unique mechanism to regulate Ca2+ signaling pathways in the physiology of pluripotent stem cells.
... Calcium ion channel activity is involved in embryo development. 85,86 The Drosophila KCNQ (dKCNQ) is a calmodulin-binding voltage-dependent channel expressed in the nurse cells of ovaries, oocytes, pre-zygotic transition embryos, as well as in the fly brain. 87 Embryos produced by homozygous dKCNQ mutant females showed disorganized mid-cleavage nuclei, and consequently, failed to hatch. ...
Female Aedes aegypti mosquitoes undergo multiple rounds of reproduction, known as gonotrophic cycles. These cycles span the period from blood meal intake to oviposition. Understanding how reproductive success is maintained across gonotrophic cycles allows for the identification of molecular targets to reduce mosquito population growth. Odorant receptor co-receptor (orco) encodes a conserved insect-specific transmembrane ion channel that complexes with tuning odorant receptors (ORs) to form a functional olfactory receptor. orco expression has been identified in the male and female mosquito germline, but its role is unclear. We report an orco-dependent, maternal effect reduction in fertility after the first gonotrophic cycle. This phenotype was removed by CRISPR-Cas9 reversion of the orco mutant locus. Eggs deposited by orco mutant females are fertilized but the embryos reveal developmental defects, reduced hatching, and changes in ion channel signaling gene transcription. We present an unexpected role for an olfactory receptor pathway in mosquito reproduction.
... We detected a down-regulation of STR gene regions in the late prenatal developmental stage and an up-regulation in brains, respectively, highlighting a possible key role of STRs in regulating genes that are important, after birth, during the growth of axons and dendrites with dendritic spines (neuritogenesis) during the first two years of life 34 and, also, important in the maturation of the prefrontal cortex that continues evolving through the adolescence until the early 20s 35,36 . A pathway enrichment analysis using the results in the discovery phase, further, confirmed a major representation of genes across all brain regions that were important in neuron specific biological pathways, such as axon guidance, which is of main importance for neuron connections 37,38 . ...
We performed a genome-wide association study of 143,067 highly polymorphic short tandem repeats (STRs) with MRI brain grey matter volumes (GMVs) on 10,702 UK Biobank (UKB) participants, including 8,751 in the discovery stage and 1,701 in the replication analysis. STRs’ repeat lengths were estimated from the UKB whole-genome sequencing data using Expansion Hunter software.
A total of 262 STRs reached genome-wide significance in the analyses of the autosomal and sex chromosomes’ ( P = 6.9 × 10 ⁻⁸ ) in association with MRI -GMVs. Replication in a second batch extraction in the UKB and linkage disequilibrium (LD) analyses confirmed 12 associations of five STRs with hippocampal, intra-calcarine cortex, and cerebellum volumes with no evidence of single nucleotide polymorphisms (SNP) in LD detected in the surrounding DNA regions. Our study highlights the importance of STR variants involved in the genetic architecture of grey matter volumes.
... Along the V-D axis ( Supplementary Fig. 8B), the calcium signaling pathway was enriched in cluster 6 (muscle) and cluster 19 (pharynx) but not cluster 9, 14 and 17 (anterior region neuron). This pathway plays the roles in regulating muscle contraction and pharynx pumping [67], and contributes to control intercellular communication, germ layer formation and the establishment of body axes during gastrulation [68]. Along the L-R axis (Supplemen-tary Fig. 8C), the MAPK signaling pathway is detected in cluster 1, 7, 8 and 9 (muscle) but not cluster 2 and 19 (anterior region neuron). ...
Caenorhabditis elegans, often referred to as the 'roundworm', provides a powerful model for studying cell autonomous and cell-cell interactions through the direct observation of embryonic development in vivo. By leveraging the precisely mapped cell lineage at single cell resolution, we are able to study at a systems level how early embryonic cells communicate across morphogenetic domains for the coordinated processes of gene expressions and collective cellular behaviors that regulate tissue morphogenesis. In this study, we developed a computational framework for the exploration of the morphogenetic domain cell signaling networks that may regulate C. elegans gastrulation and embryonic organogenesis. We demonstrated its utility by producing the following results, i) established a virtual reference model of developing C. elegans embryos through the spatiotemporal alignment of individual embryo cell nuclear imaging samples; ii) integrated the single cell spatiotemporal gene expression profile with the established virtual embryo model by data pooling; iii) trained a Machine Learning model (Random Forest Regression), which predicts accurately the spatial positions of the cells given their gene expression profiles for a given developmental time (e.g. total cell number of the embryo); iv) enabled virtual 4-dimensional tomographic graphical modeling of single cell data; v) inferred the biology signaling pathways that act in each of morphogenetic domains by meta-data analysis. It is intriguing that the morphogenetic domain cell signaling network seems to involve some crosstalk of multiple biology signaling pathways during the formation of tissue boundary pattern. Lastly, we developed the Software tool 'Embryo aligner version 1.0' and provided it as an Open Source program to the research community for virtual embryo modeling, and phenotype perturbation analyses (https://github.com/csniuben/embryo_aligner/wiki and https://bioinfo89.github.io/C.elegansEmbryonicOrganogenesisweb/).
... Humans have an absolute requirement for some alkali elements (AEs) and alkaline earth elements (AEEs) because of their essential role in a variety of physiological and biochemical processes [7]. Animal studies have shown that AEEs, especially calcium (Ca) and magnesium (Mg), play a critical role in embryonic development [8,9]. However, there is limited research on the role of AEs and AEEs in the development of OFCs. ...
Alkali elements (AEs) and alkaline earth elements (AEEs) play critical roles in numerous physiological and pathological processes. However, the effects of maternal exposure to AEs and AEEs on the risk for fetal orofacial clefts (OFCs) remain unclear. We explored the associations between levels of eight AEs and AEEs in maternal serum during pregnancy and occurrence of fetal OFCs. Concentrations of four AEs and four AEEs in maternal serum of 130 OFC cases and 260 non-malformed controls were assessed. Multilevel mixed-effects logistic regression and bayesian kernel machine regression (BKMR) were performed to evaluate the single and combined effects, respectively, of exposure to AEs and AEEs on OFC risk. When individual elements were analyzed separately as categorical variables, the odds of OFCs increased by 2.08-fold (1.10–3.93) and 2.35-fold (1.24–4.45) for sodium and by 1.98-fold (1.04–3.77) and 1.92-fold (1.21–3.61) for strontium but decreased by 0.54-fold (0.29–0.98) and 0.42-fold (0.22–0.78) for potassium in the second and third tertiles, respectively, with the lowest tertile concentration being used as the referent. When all eight elements were considered as a mixture, potassium and calcium showed protective effects, whereas sodium and strontium increased odds of OFCs in the BKMR model. No joint effect on OFC risk was observed when the eight elements were considered as a mixture. Taken together, higher levels of sodium and strontium in maternal serum were associated with an increased odds of fetal OFCs, whereas higher levels of potassium in maternal serum were associated with a decreased odds of OFCs.
... The most documented explanation on sea urchin embryonic delay as an effect of toxicity is the reduction in the ability to uptake calcium and, in turn, to maintain intracellular homeostasis related to a low extracellular pH; calcium-contaminant trafficking competition also affects the normal gene regulatory network controlling development (Stumpp et al., 2011;Pinsino et al., 2011). Several developmental steps such as fertilization, cleavage, neuronal development, skeletogenesis, cell death, and body modelling are known to be dependent on calcium ion trafficking (Webb and Miller 2003). On the other hand, regulatory studies reveal that the embryos present an early sequence of encoded "fail-safe" regulatory devices (Smith and Davidson 2009). ...
Management of dredged materials disposal is regulated by several environmental normative requirements, and it is often supported by the integration of chemical data with ecotoxicological characterization. The reliability of a bioassay to assess the potential toxicity of dredged sediments requires the selection of quality criteria that should be based on simple analytical methods and easily understandable hazard for politicians and environmental managers. The sea urchin embryo-toxicity bioassay is considered an essential component for evaluating the quality of sediments in harbour areas but its use, when based exclusively on the observation of normal vs. abnormal embryos, may alter the interpretation of the results, overestimating the risk assessment. To improve the reliability of this assay in establishing a causative relationship between quality of sediments and sea urchin embryonic development, here we developed and validated three Integrative Toxicity Indexes (ITI 2.0, ITI 3.0, ITI 4.0), modifying the already-known ITI (here ITI 1.0). Based on this aim, we used Taranto harbour as a model pilot-study to compare results to those obtained from standard criteria. Among the tested indexes, the ITI 4.0, discriminating strictly developmental delay and morphological defects from fertilized egg to gastrula stage, resulted in the most promising.
... Magnesium (Mg), calcium (Ca), strontium (Sr) and barium (Ba) are abundant alkaline earth metals in the Earth's crust, wherein Mg and Ca are essential to human health. Animal experiments have suggested that Mg and Ca play key roles in nervous system development of early embryos (Webb and Miller, 2003;Komiya et al., 2014). The other two metals are not essential and high-level exposure may be detrimental to human health, particularly that of vulnerable fetuses (Kravchenko et al., 2014;Pors Nielsen, 2004). ...
Neural tube defects (NTDs) have a complex etiology. Few studies have assessed alkaline earth metals exposures and occurrence of NTDs. We examined the association between prenatal exposure to magnesium (Mg), calcium (Ca), strontium (Sr), and barium (Ba) and risk for NTDs in a case-control study, and assessed the teratogenic effects of Ba on mice. Placentas were collected from 408 women with NTD-affected pregnancies and 593 women who delivered healthy infants, and concentrations of these metals were determined as prenatal exposure markers. The single effect of individual exposure and joint effect of coexposure to these metals were evaluated with logistic regression and Bayesian kernel machine regression (BKMR), respectively. Barium chloride (BaCl2) was intragastrically administered to pregnant ICR mice and fetal mice were examined for NTDs. Median concentrations of Mg and Ba were higher in NTD cases than in controls (Pall<0.001). In logistic regression, higher levels of Ba were associated with 1.6-fold increased risk for NTDs (95% confidence interval: 1.06–2.43). In BKMR, the joint effect of the four-metal mixture on NTD risk increased steadily with the levels of the mixture. A change in Ba concentration from the 25th to 75th percentile displayed a risk effect when the other three metals were fixed at the 25th, 50th or 75th percentile, while such a change in Ca concentration showed a protective effect when the other metals were held at the 25th or 50th percentile. No interactions among metals were found. In the mouse experiment, dams treated with 200 mg/kg BaCl2 showed 16.8% of NTDs in fetal mice, compared to 2.6% in the untreated control group (P<0.01). Taken together, higher mixture levels of the four alkaline earth metals were associated with increased risk for NTDs, with Ba being the major contributor for the joint effect. Intragastric administration of Ba can induce NTDs in mice.
... At the cellular level, processes regulated by Ca 2+ signalling include cell proliferation 15,16 , differentiation 17 , migration 18,19 and morphogenesis 20 . The importance of Ca 2+ signalling in embryonic development has been recognized for over 20 years 19,[21][22][23][24] , motivating Ca 2+ imaging studies in a range of organisms including C. elegans 25 , Drosophila [26][27][28][29] , the tunicates C. robusta [30][31][32][33] and Oikopleura dioica 34 , zebrafish [35][36][37][38][39] , xenopus 18,[40][41][42][43][44] , butterfly 45 and mouse 46 . However, an important gap in our current knowledge is how Ca 2+ signalling dynamics mediate the morphogenetic processes that give rise to tubular structures such as blood vessels, renal tubules, the neural tube and the notochord. ...
Calcium is a ubiquitous and versatile second messenger that plays a central role in the development and function of a wide range of cell types, tissues and organs. Despite significant recent progress in the understanding of calcium (Ca ²⁺ ) signalling in organs such as the developing and adult brain, we have relatively little knowledge of the contribution of Ca ²⁺ to the development of tubes, structures widely present in multicellular organisms. Here we image Ca ²⁺ dynamics in the developing notochord of Ciona intestinalis . We show that notochord cells exhibit distinct Ca ²⁺ dynamics during specific morphogenetic events such as cell intercalation, cell elongation and tubulogenesis. We used an optogenetically controlled Ca ²⁺ actuator to show that sequestration of Ca ²⁺ results in defective notochord cell intercalation, and pharmacological inhibition to reveal that stretch-activated ion channels (SACs), inositol triphosphate receptor (IP3R) signalling, Store Operated Calcium Entry (SOCE), Sarco/endoplasmic reticulum Ca ²⁺ -ATPase (SERCA) and gap junctions are required for regulating notochord Ca ²⁺ activity during tubulogenesis. Cytoskeletal rearrangements drive the cell shape changes that accompany tubulogenesis. In line with this, we show that Ca ²⁺ signalling modulates reorganization of the cytoskeletal network across the morphogenetic events leading up to and during tubulogenesis of the notochord. We additionally demonstrate that perturbation of the actin cytoskeleton drastically remodels Ca ²⁺ dynamics, suggesting a feedback mechanism between actin dynamics and Ca ²⁺ signalling during notochord development. This work provides a framework to quantitatively define how Ca ²⁺ signalling regulates tubulogenesis using the notochord as model organ, a defining structure of all chordates.
... Ca 2+ signals participate in various aspects of life processes [1,2], including development [3,4] and cardiac function [5]. Dysregulated Ca 2+ signaling correlates with heart diseases [6,7]. ...
... Ca 2+ signals participate in various aspects of life processes [1,2], including development [3,4] and cardiac function [5]. Dysregulated Ca 2+ signaling correlates with heart diseases [6,7]. ...
Type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) regulates the intracellular Ca2+ release from endoplasmic reticulum in human embryonic stem cells (hESCs), cardiovascular progenitor cells (CVPCs), and mammalian cardiomyocytes. However, the role of IP3R2 in human cardiac development is unknown and its function in mammalian cardiomyocytes is controversial. hESC-derived cardiomyocytes have unique merits in disease modeling, cell therapy, and drug screening. Therefore, understanding the role of IP3R2 in the generation and function of human cardiomyocytes would be valuable for the application of hESC-derived cardiomyocytes. In the current study, we investigated the role of IP3R2 in the differentiation of hESCs to cardiomyocytes and in the hESC-derived cardiomyocytes. By using IP3R2 knockout (IP3R2KO) hESCs, we showed that IP3R2KO did not affect the self-renewal of hESCs as well as the differentiation ability of hESCs into CVPCs and cardiomyocytes. Furthermore, we demonstrated the ventricular-like myocyte characteristics of hESC-derived cardiomyocytes. Under the α1-adrenergic stimulation by phenylephrine (10 μmol/L), the amplitude and maximum rate of depolarization of action potential (AP) were slightly affected in the IP3R2KO hESC-derived cardiomyocytes at differentiation day 90, whereas the other parameters of APs and the Ca2+ transients did not show significant changes compared with these in the wide-type ones. These results demonstrate that IP3R2 has minimal contribution to the differentiation and function of human cardiomyocytes derived from hESCs, thus provide the new knowledge to the function of IP3R2 in the generation of human cardiac lineage cells and in the early cardiomyocytes.
... A deficiency or excess of Mg, Ca, Sr and Ba may interfere with fetal organogenesis. Animal studies showed that Ca and Mg are essential for embryonic development (Hurley et al., 1976;Webb and Miller, 2003). Epidemiological studies using questionnaires indicated that increasing intakes of Ca or Mg reduced the risk of CL ± P (Krapels et al., 2004;Sabbagh et al., 2016;Wallenstein et al., 2013). ...
The relationship between alkaline earth elements in utero exposure and the risk of cleft lip with or without cleft palate (CL ± P) remains unclear. We aimed to investigate the associations between the concentration of alkaline earth elements in umbilical cord and risk for CL ± P. A case-control study was carried out in this study, including 78 cases and 142 controls. Association between each metals and the risk of CL ± P were evaluated with conventional logistic regression, bayesian kernel machine regression and weighted quantile sum regression models. Logistic regression model indicated that in utero exposure to higher levels of Barium was associated with increasing risk for CL ± P (odds ratio = 2.79, 95% confidence interval, 1.22–6.38) and for cleft lip with cleft palate (odds ratio = 3.94, 95% confidence interval, 1.45–10.72). Bayesian kernel machine regression model showed the statistical association between the metals mixture and risk difference of CL ± P, and barium was associated with CL ± P risk when all other metals were held fixed at the 25th percentiles (risk difference = 1.07, 95% confidence interval, 1.01–1.14). In weighted quantile sum model, barium accounted for most of the weight index in the combined effect of the metals mixture. The weighted quantile sum index showed that a quartile increase in the index resulted in an increase odds of 1.69 (95% confidence interval, 1.16–2.46) for CL ± P and of 2.11 (95% confidence interval, 1.34–3.35) for CLP. No associations were found in the three statistical models between Calcium, Magnesium and Strontium and the risks of CL ± P. In conclusion, in utero exposure to mixtures of alkaline earth elements was associated with an increased risk for CL ± P, of which barium was likely to be important factors in the development.
... Thus, the device can also be applied for chronic stimulation. Ca 2+ signaling is involved in fertilization, embryogenesis, organogenesis, and other developmental events in a highly coordinated manner [44][45][46][47]. These processes require threedimensional control of the local environment such as gradients of biomolecules, flow conditions, and cell-cell communications. ...
Numerous in vitro studies have been conducted in conventional static cell culture systems. However, most of the results represent an average response from a population of cells regardless of their local microenvironment. A microfluidic probe is a non-contact technology that has been widely used to perform local chemical stimulation within a restricted space, providing elaborated modulation and analysis of cellular responses within the microenvironment. Although microfluidic probes developed earlier have various potential applications, the two-dimensional structure can compromise their functionality and flexibility for practical use. In this study, we developed a three-dimensional microfluidic probe integrated device equipped with vertically oriented microchannels to overcome crucial challenges and tested the potential utility of the device in biological research. We demonstrated that the device tightly regulated spatial diffusion of a fluorescent molecule, and the flow profile predicted by simulation replicated the experimental results. Additionally, the device modulated the physiological Ca2+ response of cells within the restricted area by altering the local and temporal concentrations of biomolecules such as ATP. The novel device developed in this study may provide various applications for biological studies and contribute to further understanding of molecular mechanisms underlying cellular physiology.
... Moreover, zinc has been shown to play an important role in bone development and health. Further, it has demonstrated effects on stem cell behavior as have other ions including iron and calcium [12,35]. Zinc plays an important role in several biological mechanisms, as it is a constituent of more than 200 enzymes involved in transcription, cell signaling and cell growth, differentiation and cancer [23,[36][37][38][39][40][41]. ...
The aim of this project was to evaluate the effects of zinc in osteogenic media on differentiation of adipose-derived stem cells (ASC) into osteoblasts. ASC were plated (50,000 cells/mL) and subjected to treatments of zinc at six different concentrations in osteogenic medium: 8 mM, 4 mM, 0.8 mM, 0.4 mM, 0.08 mM, and 0.04 mM, as well as standard osteogenic medium as a positive control, and ASC culture medium as a negative control. At the end of the 4-week incubation period, cultures were analyzed for osteogenic differentiation, as measured by Alizarin Red S staining, osteocalcin production and Spatial Light Interference Microscopy (SLIM). Here we demonstrate that zinc supplementation to a standard ASC osteogenesis-promoting media formulation has a positive effect on the differentiation of porcine ASC into osteoblasts, and that 0.08 mM is the optimum concentration. Average size and number of osteogenic nodules increased under zinc supplementation, cells changed morphology more rapidly, and the concentration of osteocalcin released during osteogenic differentiation was higher (p=0.0004). Additionally we have demonstrated that a new non-invasive method utilizing SLIM microscopy can provide researchers another tool to probe intracellular and intracultural behavior in vitro, linking changes in cell behavior to differences in culture media formation. This specialized microscopic visualization technique may provide researchers with a good, non-invasive tool to evaluate the effect of molecular additives to media formulations on cell behavior, including potentially screening the effect of small molecule compounds derived from chemical libraries on cellular behavior in vitro.
... Indeed, an important factor in determining the specificity of a signal carried by a ubiquitous second messenger such as Ca 2+ might be the spatial nature of the Ca 2+ signal (Rosenberg and Spitzer, 2011). In some cases, such signals are clearly highly localized, whereas in others they are more global (Spitzer et al., 2000;Webb and Miller, 2003). This might be reflected in the nature of the Ca 2+ store responsible for generating the signal. ...
The role of TPC2-mediated Ca2+ release was recently characterized in zebrafish during the establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, we extend our study to investigate the in vivo role of TPC2 in the regulation of caudal primary motor neuron (CaP) axon extension. We used a combination of: TPC2 knock-down with a translational-blocking morpholino antisense oligonucleotide (MO); TPC2 knock-out via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing; and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H+-ATPase inhibitor) or trans-ned-19 (an NAADP receptor antagonist), and showed that these treatments attenuated CaP Ca2+ signaling and inhibited axon extension. We also characterized the expression of an arc1-like transcript in CaPs grown in primary culture. MO-mediated knock-down of ARC1-like in vivo led to an attenuation of the Ca2+ transients in the CaP growth cones, and an inhibition of axon extension. Together our new data suggest a link between ARC1-like, TPC2 and Ca2+ signaling during axon extension in zebrafish.
... De la misma manera, inhibidores de la N-glicosilación como la tunicamicina o inhibidores del transporte de proteínas del RE al aparato de Golgi provocan estrés por acumulación de las proteínas (23).. El RE es uno de los reguladores más importantes del calcio intracelular (24). Además, las concentraciones de calcio en el RE fluctúan rápida y frecuentemente, ya que el calcio almacenado se moviliza durante la señalización celular (25). La entrada del calcio está regulada a través de la bomba ATPasa sarcoplásmica/ ER (SERCA); y la liberación del mismo, vía el receptor inositol 1,4,5-trifosfato (InsP3R) o por los canales de calcio mediados por rianodina (RyR) (26). ...
La disfunción placentaria ha sido implicada en la fisiopatología
de la preeclampsia. Se considera en
el modelo clásico de las dos oleadas del síndrome que
la conversión deficiente de las arterias espirales lleva
al estrés oxidativo placentario secundario a las alteraciones
de la perfusión, lo cual genera la liberación en
la circulación materna de factores que causan activación
de las células endoteliales. Existe evidencia
científica que indica la presencia de estrés oxidativo
de la placenta y experimentos en explantes de vellosidades
terminales in vitro, han confirmado que el
estrés oxidativo es un estímulo suficiente para liberar
un gran número de citoquinas y factores pro-inflamatorios
desde el trofoblasto. Los experimentos con
explantes han permitido identificar las vías de señalización
intermedias activadas y la importancia de
estas in vivo es confirmada por los mismos cambios
observados luego del parto, cuando se induce el estrés
oxidativo a través de isquemia-reperfusión posterior
a las contracciones uterinas. El estrés oxidativo
puede causar una amplia alteración de la función celular;
sin embargo, esto raramente ocurre en forma
aislada a otras respuestas celulares al estrés. Se han
identificado estrechas relaciones entre el estrés oxidativo
y el estrés del retículo endoplásmico, siendo cada
uno capaz de inducir al otro.
El retículo endoplásmico (RE) es más comúnmente
conocido por su papel en las modificaciones post-plegamiento
de las proteínas, pero más recientemente se
ha sugerido que el orgánelo es también un coordinador
central de diversas vías de señalización que regulan
el metabolismo, proliferación y muerte celular. Su
papel no es sorprendente debido a que la síntesis de
proteínas es fundamental para la integridad-función
celular y es fuertemente dependiente de los procesos
energéticos que requieren un flujo adecuado de
nutrientes y oxígeno. Las alteraciones del RE llevan
a un estado conocido como estrés del retículo endoplásmico
(ERS) y activa una serie de vías de señalización
conocidas en forma colectiva como respuesta del
mal plegamiento proteico (unfolded protein response
o UPR por sus siglas en Ingles). Inicialmente, la UPR
ayuda a restaurar la homeostasis del retículo endoplásmico,
pero si este intento falla se activa la cascada
apoptoica. Estas vías juegan un papel central en
la fisiopatología de las enfermedades crónicas, como
las enfermedades neurodegenerativas y diabetes.
... fertilization, secretion, endocytosis, etc.), morphogenesis and the specialization of bone, heart and neurons, to name a few cell types (e.g. Webb & Miller, 2003;Zhou et al., 2011). CaM [152 amino acids (aas), 17 kDa] is encoded by the calA gene (http://dictybase.org/gene/DDB_G0279407). ...
This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.
... During gastrulation, it is likely that CaMK-IIs encoded by camk2b1 and camk2g1 are activated by ncWnt-mediated Ca 2+ elevations to directly enable cell migration. Oscillations of Ca 2+ are known to occur during gastrulation [7,94], which would activate CaMK-II to enable cell migration [91]. Interestingly, the migration of cells in culture and in embryos is compromised when CaMK-II is either hyperactivated, inhibited or eliminated [91]. ...
The multifunctional Ca²⁺/calmodulin-dependent protein kinase type 2 (CaMK-II) was first discovered in brain tissue and shown to have a central role in long term potentiation, responding to Ca²⁺ elevations through voltage dependent channels. CaMK-II has a unique molecular mechanism that enables it to remain active in proportion to the degree (frequency and amplitude) of Ca²⁺ elevations, long after such elevations have subsided. Ca²⁺ is also a rapid activator of early development and CaMK-II is expressed and activated in early development. Using biochemical, pharmacological and genetic approaches, the functions of CaMK-II overlap remarkably well with those for Ca²⁺ elevations, post-fertilization. Conclusion. Activated CaMK-II plays a central role in decoding Ca²⁺ signals to activate specific events during early development; a majority of the known functions of elevated Ca²⁺ act though CaMK-II.
... Normal development is characterized by diverse morphogenetic tissue cross-link and functionating. 18 Nuclear receptors (NRs), at the subcellular level, are one of the largest groups of association factors in vertebrates, which serve important functions in the regulation of a range of physiological functions and in the maintenance of overall homeostasis. 19 Characteristics of NR genes in vertebrates can be used to screen the toxic potency of chemicals, because most NRs are found to have homologues in humans. ...
... Calcium signaling is vital for both early and late stages of embryonic development [1]. Early in development at the blastomere stage and in the later segmentation phases, calcium signaling is thought to be largely intracellular [2], while in the intervening gastrulation phase, it is largely intercellular signaling [3]. ...
Calcium signaling is essential for embryonic development but the signals upstream of calcium are only partially understood. Here, we investigate the role of the intracellular glutathione redox potential in calcium signaling using the Chac1 protein of zebrafish. A member of the γ-glutamylcyclotransferase family of enzymes, the zebrafish Chac1 is a glutathione-degrading enzyme that acts only on reduced glutathione. The zebrafish chac1 expression was seen early in development, and in the latter stages, in the developing muscles, brain and heart. The chac1 knockdown was embryonic lethal, and the developmental defects were seen primarily in the myotome, brain and heart where chac1 was maximally expressed. The phenotypes could be rescued by the WT Chac1 but not by the catalytically inactive Chac1 that was incapable of degrading glutathione. The ability of chac1 to alter the intracellular glutathione redox potential in the live animals was examined using Grx1-roGFP2. The chac1 morphants lacked the increased degree of cellular oxidation seen in the WT zebrafish. As calcium is also known to be critical for the developing myotomes, brain and heart, we further investigated if the chac1 knockdown phenotypes were a consequence of the lack of calcium signals. We observed using GCaMP6s, that calcium transients normally seen in the developing embryos were strongly attenuated in these knockdowns. The study thus identifies Chac1 and the consequent change in intracellular glutathione redox potential as important upstream activators of calcium signaling during development.
... Calcium ions (Ca 2+ ) are vital intracellular second messengers involved in multiple functions of cells, including proliferation, differentiation, fertilization, development, muscle contraction, cell death, learning, and memory (33)(34)(35). The voltage-gated calcium channel is a multi-subunit protein complex consisting of a channel-forming α1 subunit and three regulatory subunits, α2δ, β, and γ (36-38). ...
Resistance to platinum-based combination chemotherapy is the main cause of poor prognosis in patients with advanced esophageal squamous cell carcinoma (ESCC). Previously, we showed that CACNA2D3 (voltage-dependent subunit alpha 2 delta 3 of a calcium channel complex) was significantly downregulated and functioned as a tumor suppressor in ESCC, but its role in the chemosensitivity of ESCC to cisplatin remained unknown. Here, we found that the expression of CACNA2D3 was significantly associated with poor platinum response in ESCC patients from the Gene Expression Omnibus database. Overexpression of CACNA2D3 increased sensitivity to cisplatin in ESCC in vitro, whereas knockdown of CACNA2D3 increased cisplatin resistance. CACNA2D3 promoted cisplatin-induced apoptosis by modulating intracellular Ca²⁺ stores. In vivo experiments further showed that overexpression of CACNA2D3 enhanced cisplatin anti-tumor effects in a xenograft mouse model. CACNA2D3 overexpression also resulted in the attenuation of PI3K and Akt phosphorylation. Treatment with the PI3K/Akt inhibitor LY294002 restored the chemosensitivity of CACAN2D3-knockdown cells to cisplatin. In conclusion, the results of the current study indicate that CACAN2D3 enhances the chemosensitivity of ESCC to cisplatin via inducing Ca²⁺-mediated apoptosis and suppressing PI3K/Akt pathways. Therefore, regulating the expression of CACNA2D3 is a potential new strategy to increase the efficacy of cisplatin in ESCC patients.
... The role of calcium signaling for embryogenesis and organogenesis of early vertebrates is well documented. For example, spontaneous calcium activity has been observed after egg fertilization (17)(18)(19), during early development of zebra fish (20) and frog (21,22), in stem cells (23,24), and in developing neurons (25,26). ...
The central role of calcium signaling during development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used immunofluorescence and time‐lapse calcium imaging of cultured explanted embryonic rat kidneys to study the role of calcium signaling for branching morphogenesis. In mesenchymal cells, we recorded spontaneous calcium activity that was characterized by irregular calcium transients. The calcium signals were dependent on release of calcium from intracellular stores in the endoplasmic reticulum. Down‐regulation of the calcium activity, both by blocking the sarco‐endoplasmic reticulum Ca²⁺‐ATPase and by chelating cytosolic calcium, resulted in retardation of branching morphogenesis and a reduced formation of primitive nephrons but had no effect on cell proliferation. We propose that spontaneous calcium activity contributes with a stochastic factor to the self‐organizing process that controls branching morphogenesis, a major determinant of the ultimate number of nephrons in the kidney.—Fontana, J. M., Khodus, G. R., Unnersjö‐Jess, D., Blom, H., Aperia, A., Brismar, H. Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney. FASEB J. 33, 4089–4096 (2019). www.fasebj.org
... A change in its composition can be indicative of disease, such as cancer [5][6][7], but is also associated with major physiological events, such as embryo fertilisation and development [8][9][10]. Of particular significance is the impact of extracellular metal ions and other analytes on embryonic health during early development [11,12]. Selective detection of these species has thus attracted considerable attention as a means to monitor embryo health in clinical applications, such as in vitro fertilisation (IVF) [13][14][15]. ...
Cell-permeable fluorescent chemosensors (calcein, monochlorobimane, and a recently reported spiropyran-based sensor SP2) have been incorporated into yeast total lipid extract-based liposomes to suppress inherent cell permeability to allow the detection of extracellular Ca2+, GSH, and Zn2+, respectively. The repurposed sensors have enhanced aqueous solubility and the ability to quantitatively measure biologically relevant concentrations of Ca2+ (0.25 mM–1 mM), Zn2+ (6.25 µM–50 µM), and GSH (0.25 mM–1 mM) by fluorescence in aqueous media. In addition, the liposomal sensors are nontoxic to HEK293 cells and have the ability to detect exogenously added Zn2+ (1 mM), Ca2+ (1 mM), or GSH (1 mM) near cells without internalisation. This new sensing platform provides a means to repurpose a range of intracellular fluorescent sensors to specifically detect extracellular analytes, while also improving biocompatibility for overall enhanced use in a wide range of biomedical applications.
... Thus, the intracellular machinery regulating such transport determines whether such signals will be able to correctly modulate these processes. Calcium signaling is one mechanism that is able to modify organelle transport, enzymatic activity, downstream growth factor signaling pathways such as the FGF pathway, growth cone behavior, and cytoskeletal dynamics in many cell types, and it is a key feature of neural development (25)(26)(27). Indeed, a diverse landscape of patterned Ca 2+ activities within RGCs and newborn neurons has become evident, offering clues to the physiology of RGCs and their long fibers (28,29). ...
Significance
Metabolic abnormalities during gestation have been previously linked to increased likelihood of dyslexia, autism, and attention deficit hyperactivity disorder, but the factors involved and how they may act during cortical development is unknown. Here, we found in rodents that obesity and hyperglycemia affect the primary stem cells of the cortex (radial glial cells), reducing their ability to perform neurogenesis and form the radial scaffold needed for neuronal migration. At the subcellular level, excess glucose degraded calcium activity and the transport of mitochondria in the radial scaffold. Consequent delayed neuronal production and migration were observed. These results reveal aspects of neurogenesis as well as the sensitivity of the cortical development process to certain gestational metabolic influences.
Key message
Plants exhibit a unique pattern of cytosolic Ca²⁺ dynamics to correlate with microtubules to regulate cytokinesis, which significantly differs from those observed in animal and yeast cells.
Abstract
Calcium (Ca²⁺) transients mediated signaling is known to be essential in cytokinesis across eukaryotic cells. However, the detailed spatiotemporal dynamics of Ca²⁺ during plant cytokinesis remain largely unexplored. In this study, we employed GCaMP5, a genetically encoded Ca²⁺ sensor, to investigate cytokinetic Ca²⁺ transients during cytokinesis in Nicotiana tabacum Bright Yellow-2 (BY-2) cells. We validated the effectiveness of GCaMP5 to capture fluctuations in intracellular free Ca²⁺ in transgenic BY-2 cells. Our results reveal that Ca²⁺ dynamics during BY-2 cell cytokinesis are distinctly different from those observed in embryonic and yeast cells. It is characterized by an initial significant Ca²⁺ spike within the phragmoplast region. This spike is followed by a decrease in Ca²⁺ concentration at the onset of cytokinesis in phragmoplast, which then remains elevated in comparison to the cytosolic Ca²⁺ until the completion of cell plate formation. At the end of cytokinesis, Ca²⁺ becomes uniformly distributed in the cytosol. This pattern contrasts with the typical dual waves of Ca²⁺ spikes observed during cytokinesis in animal embryonic cells and fission yeasts. Furthermore, applications of pharmaceutical inhibitors for either Ca²⁺ or microtubules revealed a close correlation between Ca²⁺ transients and microtubule organization in the regulation of cytokinesis. Collectively, our findings highlight the unique dynamics and crucial role of Ca²⁺ transients during plant cell cytokinesis, and provides new insights into plant cell division mechanisms.
Orofacial clefts (OFCs) have been linked to various toxic and essential trace elements (TETEs) worldwide. However, review estimation is absent. Therefore, addressing the hypothesis that TETEs are associated with OFCs is the main area of this review. A systematic literature search was conducted using electronic databases through PubMed, Web of Science, Scopus, Science Direct, and Google Scholar between 2004 and August 2022. The “AND” and “OR” operators were used to make our search results inclusive and restrictive as follows: (“Toxic element*” OR “Heavy metal*”) AND (“Toxic element*” OR “Lead OR Arsenic OR Mercury*”)) AND (“Essential trace element*” OR “Zinc OR Selenium OR Copper*”)) AND (“Orofacial cleft*” OR “Cleft lip*” OR “Cleft palate*”) AND (“Infant*” OR “Newborn*” OR “Neonate*”)). The presence of toxic elements was linked to the development of OFCs. The results showed that higher levels of toxic elements in various biological sample types were related to increased risks for OFCs. Increased concentrations of essential trace elements (ETEs) lowered the risk of OFCs. Maternal consumption of diets rich in ETEs, including zinc (Zn), selenium (Se), copper (Cu), cobalt (Co), and molybdenum (Mo), was linked to a more pronounced reduction in the risk of OFCs. Based on the findings, it is acceptable to infer that maternal exposure to toxic elements, whether through environmental contaminants or dietary sources, was associated with an elevated risk of OFCs. Furthermore, the study revealed that ETEs exhibited a potential protective role in reducing the incidence of OFCs. This observation highlights the importance of reducing exposure to toxic elements during pregnancy and suggests that optimizing maternal intake of ETEs could be an effective preventive strategy.
Background
Noncanonical Wnts are morphogens that can elevate intracellular Ca²⁺, activate the Ca²⁺/calmodulin‐dependent protein kinase, CaMKII, and promote cell movements during vertebrate gastrulation.
Results
Zebrafish express seven CaMKII genes during embryogenesis; two of these, camk2b1 and camk2g1, are necessary for convergent extension (CE) cell movements. CaMKII morphant phenotypes were observed as early as epiboly. At the 1–3 somite stage, neuroectoderm and paraxial cells remained unconverged in both morphants. Later, somites lacked their stereotypical shape and were wider, more closely spaced, and body gap angles increased. At 24hpf, somite compression and notochord undulation coincided with a shorter and broader body axis. A camk2b1 crispant was generated which phenocopied the camk2b1 morphant. The levels of cell proliferation, apoptosis and paraxial and neuroectodermal markers were unchanged in morphants. Hyperactivation of CaMKII during gastrulation by transient pharmacological intervention (thapsigargin) also caused CE defects. Mosaically expressed dominant‐negative CaMKII recapitulated these phenotypes and showed significant midline bifurcation. Finally, the introduction of CaMKII partially rescued Wnt11 morphant phenotypes.
Conclusions
Overall, these data support a model whereby cyclically activated CaMKII encoded from two genes enables cell migration during the process of CE.
Metallic elements play a pivotal role in maternal and fetal health. Metals can cross the placental barrier and be absorbed by fetuses, where they may affect closure of the neural tube during embryonic development. Neural tube defects (NTDs), which result from aberrant closure of the neural tube three to four weeks post-conception, have a multifactorial and complex etiology that combines genetic variants and environmental exposure. Recent advances in population-level association studies have investigated the link between maternal environmental exposure and NTDs, particularly the influence of metals on the incidence of NTDs. Herein, we present a broad and qualitative review of current literature on the association between maternal and prenatal metal exposure via the maternal peripheral blood, amniotic fluid, placenta, umbilical cord, and maternal hair, and the risk of developing NTDs. Specifically, we identify the various aggravating or attenuating effects of metallic exposure on the risk of NTD formation. This review provides novel insights into the association between environmental metals and NTDs and has important applications for NTD prevention and mitigating environmental exposure to metals.
Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared to static controls (Homan et al., 2019; Takasato et al., 2015). However, their physiological function has yet to be systematically investigated. Here, we measured mechanoinduced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 d, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0±22.1 nM (n=9; p≤0.001) was observed when microperfused tubules from organoids >40 d in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 d in culture. Nonperfused tubules (41 d) subjected to a 10-fold increase in bath flow rate also exhibited a 3-fold increase in [Ca2+]i from baseline (p<0.001). Mechanosensitive Piezo1 channels contribute to the flow-induced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan et al., EB 2019). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 d in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (p≤0.001) in segments from organoids cultured for >30 d, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture.
Stiffness and forces are two fundamental quantities essential to living cells and tissues. However, it has been a challenge to quantify both 3D traction forces and stiffness (or modulus) using the same probe in vivo. Here, we describe an approach that overcomes this challenge by creating a magnetic microrobot probe with controllable functionality. Biocompatible ferromagnetic cobalt-platinum microcrosses were fabricated, and each microcross (about 30 micrometers) was trapped inside an arginine-glycine-apartic acid-conjugated stiff poly(ethylene glycol) (PEG) round microgel (about 50 micrometers) using a microfluidic device. The stiff magnetic microrobot was seeded inside a cell colony and acted as a stiffness probe by rigidly rotating in response to an oscillatory magnetic field. Then, brief episodes of ultraviolet light exposure were applied to dynamically photodegrade and soften the fluorescent nanoparticle-embedded PEG microgel, whose deformation and 3D traction forces were quantified. Using the microrobot probe, we show that malignant tumor-repopulating cell colonies altered their modulus but not traction forces in response to different 3D substrate elasticities. Stiffness and 3D traction forces were measured, and both normal and shear traction force oscillations were observed in zebrafish embryos from blastula to gastrula. Mouse embryos generated larger tensile and compressive traction force oscillations than shear traction force oscillations during blastocyst. The microrobot probe with controllable functionality via magnetic fields could potentially be useful for studying the mechanoregulation of cells, tissues, and embryos.
We recently described calcium signaling in the appendicularian tunicate Oikopleura dioica during pre-gastrulation stages, and showed that regularly occurring calcium waves progress throughout the embryo in a characteristic spatiotemporal pattern from an initiation site in muscle lineage blastomeres (Mikhaleva et al., 2019). Here, we have extended our observations to the period spanning from gastrulation to post-hatching stages. We find that repetitive Ca2+ waves persist throughout this developmental window, albeit with a gradual increase in frequency. The initiation site of the waves shifts from muscle cells at gastrulation and early tailbud stages, to the central nervous system at late tailbud and post-hatching stages, indicating a transition from muscle-driven to neurally driven events as tail movements emerge. At these later stages, both the voltage gated Na + channel blocker tetrodotoxin (TTX) and the T-type Ca2+ channel blocker and nAChR antagonist mecamylamine eliminate tail movements. At late post-hatching stages, mecamylamine blocks Ca2+ signals in the muscles but not the central nervous system. Post-gastrulation Ca2+ signals also arise in epithelial cells, first in a haphazard pattern in scattered cells during tailbud stages, evolving after hatching into repetitive rostrocaudal waves with a different frequency than the nervous system-to-muscle waves, and insensitive to mecamylamine. The desynchronization of Ca2+ waves arising in different parts of the body indicates a shift from whole-body to tissue/organ-specific Ca2+ signaling dynamics as organogenesis occurs, with neurally driven Ca2+ signaling dominating at the later stages when behavior emerges.
Background
Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown.
Methods
We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids.
Results
Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C- γ 1, but not nephrin or phospholipase C- ε 1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis.
Conclusions
These findings establish podocyte cell–autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.
Transient receptor potential (TRP) channels play prominent roles in ion homeostasis by their ability to control cation influx. Mouse placentation is governed by the processes of trophoblast proliferation, invasion, differentiation, and fusion, all of which require calcium signaling. Although certain TRP channels have been shown to contribute to maternal–fetal transport of magnesium and calcium, a role for TRP channels in specific trophoblast functions has been disregarded. Using qRT-PCR and in situ hybridisation, the spatio-temporal expression pattern of TRP channels in the mouse placenta across gestation (E10.5–E18.5) was assessed. Prominent expression was observed for Trpv2, Trpm6, and Trpm7. Calcium microfluorimetry in primary trophoblast cells isolated at E14.5 of gestation further revealed the functional activity of TRPV2 and TRPM7. Finally, comparing TRP channels expression in mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESC) confirmed the specific expression of TRPV2 during placental development. Moreover, TRP channel expression was similar in mTSCs compared to primary trophoblasts and validate mTSC as a model to study TRP channels in placental development. Collectivity, our results identify a specific spatio-temporal TRP channel expression pattern in trophoblasts, suggesting a possible involvement in regulating the process of placentation.
Nucleokinesis, literally the movement of the nucleus, may or may not be coupled with cellular motility. In this chapter, we present our point of view on the moving nucleus. We depict the structure of the nucleus, discuss how the chromatin is organized within the nucleus, highlight nuclear substructure, and describe the molecules that anchor it to the cytoskeleton. We further discuss diseases that arise when these molecules malfunction. How the nucleus connects with the cytoskeleton is key in understanding the relationships between these two different domains. The structure and the role of the bridging complex, the LINC complex, are presented as well. As an example for nuclear motility, we focus on dynamic nucleokinesis during interkinetic nuclear movement. Collectively, we aim to introduce the concept that the migrating nucleus is more than just a passive cargo.
The roles of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in embryonic development remain unclear. We performed a comprehensive analysis of lncRNA and circRNA profiles in rabbit embryos at different stages by whole transcriptome sequencing. We identified 719 lncRNAs and 744 circRNAs that were differentially expressed between stages S1, S2 and S3. A total of 241 differentially expressed lncRNAs and 166 differentially expressed circRNAs were significantly involved in embryonic morphogenesis and development. An RNA network was established and of the embryonic development-associated RNAs, the lncRNAs TCONS_00009253 and TCONS_00010436 were persistently downregulated, while circRNA_07129, circRNA_15209, and circRNA_12526 were persistently upregulated, and their co-expressed mRNAs TBX1, WNT3 and FGFR2 were persistently downregulated during embryonic development. These candidate RNAs were mainly involved in the Wnt, PI3K-Akt, and calcium signaling pathways. This study reports candidate lncRNAs and circRNAs that may be indispensable for the morphogenesis and development of rabbit embryos.
Coilia nasus (C. nasus) is an important anadromous fish species that resides in the Yangtze River in China. However, wild C. nasus have suffered serious damage as a result of overfishing and environmental pollution. We performed comparative liver and brain transcriptome analyses of C. nasus from the Jingjiang (JJ) and Dangtu (DT) sections of the Yangtze River. The results indicate that, during migration, most signal pathways in C. nasus livers were downregulated, indicating that the liver has a function in energy conservation. The brain assumes more of a regulatory role, and the signal transduction pathways and relevant genes were upregulated. This study provides genetic information for screening the key regulatory genes of gonad development of C. nasus, which can be applied in the artificial breeding of C. nasus, providing high-quality fish fry for proliferation and release and may also contribute to efforts towards the restoration of wild C. nasus.
We have visualized many of the Ca²⁺ signaling events that occur during the early stages of zebrafish development using complementary luminescent and fluorescent imaging techniques. We initially microinject embryos with the luminescent Ca²⁺ reporter, f-holo-aequorin, and using a custom-designed luminescent imaging system, we can obtain pan-embryonic visual information continually for up to the first ~24 h postfertilization (hpf). Once we know approximately when and where to look for these Ca²⁺ signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca²⁺ reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images. These protocols allow us to identify the specific cell types and even the particular subcellular domain (e.g., nucleus or cytoplasm) generating the Ca²⁺ signal. Here, we outline the techniques we use to precisely microinject f-holo-aequorin or calcium green-1 dextran into embryos without affecting their viability or development. We also describe how to inject specific regions of early embryos in order to load localized embryonic domains with a particular Ca²⁺ reporter. These same techniques can also be used to introduce other membrane-impermeable reagents into embryos, including Ca²⁺ channel antagonists, Ca²⁺ chelators, fluorescent dyes, RNA, and DNA.
Ionizable strategies are routinely used to enhance the solubility and dissolution rates of various pharmaceuticals. These chemicals may directly affect aquatic environment once discharged from factories, hospitals or livestock farms. Here, we assessed the potential side effect of tetracyclines (TCs) on the development of zebrafish embryos. Tetracycline hydrochloride decreased water pH from 6.4 to 4.4 at 30 mg/L. Acidified water exceeded the tolerance of zebrafish embryos in pure water during the early ten hours post fertilization (hpf). Interestingly, we found that Ca²⁺ in the embryo medium could increase the tolerance of embryos to acidified water. Furthermore, we found that the protection of Ca²⁺ was not due to the formation of TCs-Ca²⁺ complexes under acidic condition, based on spectral analysis. Meanwhile we showed that exogenous addition of Ca²⁺ could inhibit the accumulation of Ca²⁺ from the cytoplasm to the surface of embryos. These results may shed light on the strategies for protecting aquatic animals from acidic environments.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, “quin2”, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
Early morphogenesis of the teleost embryo is characterized by three orchestrated cell movements. Epiboly leads to spreading of the blastoderm over an uncleaved yolk cell while involution around the blastoderm margin and convergence movements towards the dorsal side generate the mesendodermal inner cell sheet and the axis rudiment, respectively. Irradiation of zebrafish zygotes with ultraviolet light selectively impairs epiboly resulting in embryos with open blastopores but well-formed anterior axes. Gastrulation movements are only marginally affected by ultraviolet irradiation. Involution of marginal cells in epiboly-retarded embryos takes place prior to 50% epiboly and thus appears independent of epiboly. Expression of dorsal and anterior marker genes is unaffected by ultraviolet irradiation.
The ultraviolet light effect is not restricted to the zygote stage as irradiation of later embryonic stages also impairs epiboly. The ultraviolet-sensitive targets may thus be maternally encoded components of the machinery driving epiboly. These targets appear to be microtubules: firstly, irradiated embryos show disorganized and less microtubules in the cytoplasmic layer of the yolk sphere; secondly, the ultraviolet light effect can be mimicked by the microtubule-depolymerising agent nocodazole. We suggest that epiboly is driven, at least partially, by motors that use microtubules radiating from the yolk syncytial layer into the yolk cytoplasmic layer. Together with an observed constrictive behaviour of the blastoderm margin, we propose a two-force model of epiboly: epiboly is initiated and driven by a pulling force dependent on microtubules in the yolk cytoplasmic layer; contraction at the margin operates in addition to aid closure of the blastopore.
Wnt ligands working through Frizzled receptors have a differential ability to stimulate release of intracellular calcium (Ca2+) and activation of protein kinase C (PKC). Since targets of this Ca2+ release could play a role in Wnt signaling, we first tested the hypothesis that Ca2+/calmodulin-dependent protein kinase II (CamKII) is activated by some Wnt and Frizzled homologs. We report that Wnt and Frizzled
homologs that activate Ca2+ release and PKC also activate CamKII activity in Xenopus embryos, while Wnt and Frizzled homologs that activate β-catenin function do not. This activation occurs within 10 min after
receptor activation in a pertussis toxin-sensitive manner, concomitant with autophosphorylation of endogenous CamKII. Based
on data that Wnt-5A and Wnt-11 are present maternally in Xenopus eggs, and activate CamKII, we then tested the hypothesis that CamKII participates in axis formation in the early embryo.
Measurements of endogenous CamKII activity from dorsal and ventral regions of embryos revealed elevated activity on the prospective
ventral side, which was suppressed by a dominant negative Xwnt-11. If this spatial bias in CamKII activity were involved in
promoting ventral cell fate one might predict that elevating CamKII activity on the dorsal side would inhibit dorsal cell
fates, while reducing CamKII activity on the ventral side would promote dorsal cell fates. Results obtained by expression
of CamKII mutants were consistent with this prediction, revealing that CamKII contributes to a ventral cell fate.
Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell–cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell–cell adhesion, convergent extension movements, and cell differentiation. Here we show that endogenous Wnt/β-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. Our data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/β-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/β-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/β-catenin pathway upstream of β-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/β-catenin signaling cascade downstream of β-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus.
In most deuterostome eggs, fertilization is marked by an abrupt and transient increase in intracellular calcium concentration. The transient takes the form of a propagating wave and is the signal for the onset of development. For those interested in cell signalling, the two obvious questions to ask are how the wave is initiated and how it propagates through the egg cytoplasm. Answers have come largely from experiments in frog, hamster, mouse and sea urchin eggs. One explanation of signal transduction at fertilization makes an analogy with transmembrane signalling in somatic cells, where a family of G-protein-linked receptors pass activating signals across the plasma membrane. Another, older idea is that it is the fusion of sperm and egg that is responsible for detonating the calcium explosion at fertilization. We discuss the relative merits of the two ideas. Both are plausible; the creative tension between them has led to experiments that broaden our view of signal transduction at fertilization.
Activation of the inositol lipid signaling system results in cytosolic Ca2+ oscillations and intra- and intercellular Ca2+ waves in many isolated cell preparations. However, this form of temporal and spatial organization of signaling has not been
demonstrated in intact tissues. Digital imaging fluorescence microscopy was used to monitor Ca2+ at the cellular and subcellular level in intact perfused rat liver loaded with fluorescent Ca2+ indicators. Perfusion with low doses of vasopressin induced oscillations of hepatocyte Ca2+ that were coordinated across entire lobules of the liver by propagation of Ca2+ waves along the hepatic plates. At the subcellular level these periodic Ca2+ waves initiated from the sinusoidal domain of cells within the periportal region and propagated radially across cell-cell
contacts into the pericentral region, or until terminated by annihilation collision with other Ca2+ wave fronts. With increasing agonist dose, the frequency but not the amplitude of the Ca2+ waves increased. Intracellular Ca2+ wave rates were constant, but transcellular signal propagation was determined by agonist dose, giving rise to a dose-dependent
increase in the rate at which Ca2+ waves spread through the liver. At high vasopressin doses, a single Ca2+ wave was observed and the direction of Ca2+ wave propagation was reversed, initiating in the pericentral region and spreading to the periportal region. It is concluded
that intercellular Ca2+ waves may provide a mechanism to coordinate responses across the functional units of the liver.
Wntgenes encode secreted proteins which are implicated in receptor-mediated cell–cell signaling events important in embryogenesis, but the second messenger systems modulated byWntshave not been identified. We report that ectopic expression ofXwnt-5A in zebrafish embryos enhances the frequency of intracellular Ca2+transients in the enveloping layer of the blastodisc, whereasXwnt-8 does not. These transients are independent of extracellular Ca2+. Consistent with the observed Ca2+transients playing a role in responses of embryos toXwnt-5A, we report that the ligand-activated serotonin type 1C receptor, which stimulates PI cycle activity and Ca2+signaling independent ofWnts,phenocopies embryonic responses toXwnt-5A. These results suggest that intercellular signaling by a subset of vertebrate Wnts involves modulation of a intracellular Ca2+signaling pathway, which may arise from phosphatidylinositol cycle activity.
The ionophorous properties of a new antibiotic, ionomycin, have been studied. It was found that the antibiotic is capable of extracting calcium ion from the bulk of an aqueous phase into an organic phase. The antibiotic also acts as a mobile ion carrier to transport the cation across a solvent barrier. The divalent cation selectivity order for ionomycin as determined by ion competition experiments was found to be: Ca greater than Mg greater than Sr = Ba, where the binding of strontium and barium by the antibiotic is insignificant. The antibiotic also binds La3+ to some extent, but its complexation with monovalent alkali metal ions is negligible. Measurement of the binding of ionomycin with Ca2+ indicates that ionomycin complexes and transports calcium ion in a one to one stoichiometry.
We investigated the effects of calcium removal and calcium ionophores on the behavior and ultrastructure of cultured chick dorsal root ganglia (DRG) neurons to identify possible mechanisms by which calcium might regulate neurite outgrowth. Both calcium removal and the addition of calcium ionophores A23187 or ionomycin blocked outgrowth in previously elongating neurites, although in the case of calcium ionophores, changes in growth cone shape and retraction of neurites were also observed. Treatment with calcium ionophores significantly increased growth cone calcium. The ability of the microtubule stabilizing agent taxol to block A23187-induced neurite retraction and the ability of the actin stabilizing agent phalloidin to reverse both A23187-induced growth cone collapse and neurite retraction suggested that calcium acted on the cytoskeleton. Whole mount electron micrographs revealed an apparent disruption of actin filaments in the periphery (but not filopodia) of growth cones that were exposed to calcium ionophores in medium with normal calcium concentrations. This effect was not seen in cells treated with calcium ionophores in calcium-free medium or cells treated with the monovalent cation ionophore monensin, indicating that these effects were calcium specific. Ultrastructure of Triton X-100 extracted whole mounts further indicated that both microtubules and microfilaments may be more stable or extraction resistant after treatments which lower intracellular calcium. Taken together, the data suggest that calcium may control neurite elongation at least in part by regulating actin filament stability, and support a model for neurite outgrowth involving a balance between assembly and disassembly of the cytoskeleton.
We have determined or redetermined the calcium dissociation constants of seven BAPTA-type buffers with KD's in the range from 0.4 microM to about 20 mM in 300 mM KCl. These include four newly synthesized ones: 5-nitro BAPTA; 5,5'-dinitro BAPTA; 5-methyl-5'-nitro BAPTA; and 5-methyl-5'-formyl BAPTA. Moreover, we tabulate dissociation constants or KD's for BAPTA and eleven BAPTA-type buffers, compare most of them with an empirical curve based upon so-called Hammett values, and predict KD's for several still unsynthesized but potentially valuable buffers.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
Although the regulation of events in the cell division cycle by calcium or other cations has been the subject of much interest and speculation, experimental studies have been hampered by the difficulty of measuring submicromolar intracellular free calcium concentrations ([Ca2+]i) over an entire cell cycle. We now describe experiments using a new fluorescent calcium chelator, fura-2 (see Fig. 1c for structure), for continuous measurement of [Ca2+]i from fertilization through the first cleavage of individual eggs of the sea urchin Lytechinus pictus. We also show for comparison the results of parthenogenetic activation by ammonia. In addition to the known transient rise of [Ca2+]i at fertilization, further peaks are now revealed during pronuclear migration, nuclear envelope breakdown, the metaphase/anaphase transition and cleavage. Parthenogenetic activation by ammonia also elicits a sustained rise starting at nuclear envelope breakdown.
A23187 is a carboxylic acid antibiotic which simultaneously uncouples oxidative phosphorylation and inhibits ATPase of rat liver mitochondria incubated in a magnesium-free medium. The antibiotic acts as a freely mobile carrier to transport calcium and magnesium, but not potassium, from an aqueous medium buffered at pH 7.4 into a bulk organic phase. A23187 causes a progressive release of endogenous magnesium and inorganic phosphate from mitochondria incubated with substrate, but little change in calcium or potassium content. If EDTA is added, A23187 severely depletes intramitochondrial calcium and magnesium in <30 s and produces loss of intramitochondrial potassium.
Release of State 4 succinate oxidation by A23187 is inhibited by lanthanum chloride, ruthenium red, ((ethylene bis)oxyethylene nitrilo)tetraacetic acid (EGTA), EDTA, MgCl2, and ATP. Inhibition by EGTA (or EDTA) of A23187′s uncoupling is reversed readily by calcium but not magnesium. In the presence of A23187 plus EGTA, mitochondria retain only 2 to 3 nmoles of Mg²⁺ per mg of protein but remain coupled and phosphorylate ADP at 80 to 85% of control rates with β-hydroxybutyrate or succinate as substrate. Under these conditions, coupled phosphorylation supported by glutamate, and ATP hydrolysis are severely depressed, but increasing concentrations of magnesium in the incubating medium prevent these inhibitions. It is proposed that a cyclic, energy-dissipating flux of mitochondrial calcium accounts for uncoupling by A23187, whereas inhibition of ATPase and glutamate oxidation and increased potassium permeability of mitochondria result from antibiotic-mediated magnesium efflux.
Results are presented for the purity of three commercial brands of the Ca chelator EGTA [ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid] as determined with pH-metric and oxalate precipitation methods and other techniques. The effect that the observed degree of hydration (2-4%) has on calculated values for [Ca2+] in Ca-EGTA solutions is described with reference to a theoretical dose-response relationship. In addition to the corrections for purity, the adjustments necessary to allow for ionic strength and the correction of pH readings to yield true [H+] activity or concentration values are described. These several corrections result in both a shift and a shape change for the dose-response curve compared with the curve calculated without these corrective procedures. Some authors have already taken some of these aspects into account, but this has not generally been made clear in the literature heretofore. The points raised may help to account for the wide variation of reported [Ca2+] ranges for Ca-sensitive cellular processes.
In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500-1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer.
At the onset of epiboly, the external yolk syncytial layer narrows, the syncytial nuclei become tightly packed and the network of intercrossing microtubules surrounding them becomes denser. Soon after, there is a vegetal expansion of the blastoderm and of the yolk syncytial layer with its network of intercrossing microtubules. Concomitantly, the yolk cytoplasmic layer diminishes and its set of animal-vegetal microtubules becomes shorter.
We investigated the involvement of microtubules in epiboly using the microtubule depolymerizing agent nocodazole and a stabilizing agent taxol. In embryos treated with nocodazole, microtubules were absent and epibolic movements of the yolk syncytial nuclei were blocked. In contrast, the vegetal expansion of the enveloping layer and deep cells was only partially inhibited. The process of endo-cytosis, proposed to play a major role in epiboly of the yolk syncytial layer (Betchaku, T. and Trinkaus, J. P. (1986) Am. Zool. 26, 193-199), was still observed in nocodazole-treated embryos. Treatment of embryos with taxol led to a delay in all epibolic movements.
We propose that the yolk cell microtubules contribute either directly or indirectly to all epibolic movements. However, the epibolic movements of the yolk syncytial layer nuclei and of the blastoderm are not coupled, and only movements of the yolk syncytial nuclei are absolutely dependent on microtubules. We hypothesize that the microtubule network of the syncytial layer and the animal-vegetal set of the yolk cytoplasmic layer contribute differently to various aspects of epiboly. Models that address the mechanisms by which the two microtubule arrays might function during epiboly are discussed.
Depolarization of leech neurons growing on extracellular matrix extract (ECM) leads to cessation of neurite outgrowth, rounding up of the peripheral regions of the growth cone, loss of filopodia, and neurite retraction. These responses depend on the influx of calcium (Neely, 1993). The aim of the present experiments was to analyze how the cytoskeleton becomes reorganized as growth cones change their morphology. Immunocytochemistry revealed a loss of microfilaments in the tips of neurites growing on ECM after depolarization. Leech neurons cultured on a different substrate, the plant lectin concanavalin A (ConA), continue to grow during and after depolarization (Grumbacher-Reinert and Nicholls, 1992; Neely, 1993). As expected, we did not observe any change in the distribution of microfilaments after depolarization on ConA. Since there is evidence that this lack of response is due to a reduced calcium influx during depolarization of neurons on ConA (Ross et al., 1988), the effect of the calcium ionophore A23187 on the outgrowth of these cells was analyzed. In the absence of depolarization, this ionophore caused cessation of growth cone motility and a loss of microfilaments, while microtubules were not affected. Cytochalasin D, a microfilament-disrupting agent, induced changes in growth cone morphology and neurite retraction similar to those observed after depolarization and calcium influx. Application of phalloidin, a drug that stabilizes microfilaments, inhibited depolarization-induced retraction of neurites on ECM. By contrast, stabilization of microtubules with taxol did not prevent depolarization from inducing changes in growth cone morphology and neurite growth. These experiments show that changes in growth cone morphology and motility of leech neurons induced by depolarization and calcium influx are accompanied by a dramatic change in the organization of microfilaments, but not microtubules.
Spontaneous local increases in the concentration of intracellular calcium, called "calcium sparks," were detected in quiescent
rat heart cells with a laser scanning confocal microscope and the fluorescent calcium indicator fluo-3. Estimates of calcium
flux associated with the sparks suggest that calcium sparks result from spontaneous openings of single sarcoplasmic reticulum
(SR) calcium-release channels, a finding supported by ryanodine-dependent changes of spark kinetics. At resting intracellular
calcium concentrations, these SR calcium-release channels had a low rate of opening (approximately 0.0001 per second). An
increase in the calcium content of the SR, however, was associated with a fourfold increase in opening rate and resulted in
some sparks triggering propagating waves of increased intracellular calcium concentration. The calcium spark is the consequence
of elementary events underlying excitation-contraction coupling and provides an explanation for both spontaneous and triggered
changes in the intracellular calcium concentration in the mammalian heart.
Calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes. This role in signalling has to be conducted against the rigid homeostatic mechanisms that ensure that the resting level of Ca2+ is kept low (i.e. between 20 and 100 nmol l-1) in order to avoid the cytotoxic effects of a prolonged elevation of [Ca2+]. Cells have evolved a sophisticated signalling system based on the generation of brief pulses of Ca2+ which enables this ion to be used as a messenger, thus avoiding its toxic effects. Such Ca2+ spikes usually result from the coordinated release of Ca2+ from internal stores using either inositol 1,4,5-trisphosphate or ryanodine receptors. Using Ca2+ imaging techniques, the opening of individual channels has now been visualized and models have been proposed to explain how these elementary events are coordinated to generate the global Ca2+ signals that regulate cellular activity.
Regulation of intracellular Ca2+ (Ca-i) plays a central role in cell survival, proliferation, and differentiation. We previously reported that immature oligodendroglia (OLs) are less susceptible than mature OLs to cell death following increases in Cai (Benjamins and Nedelkoska [1995] Neurochem. Res. 21:471-479). The N20.1 murine OL cell line provides a model of an intermediate stage of OL maturation in which to study responses to Cai increases with regard to viability, as well as the expression of mRNAs for myelin basic protein (MBP), proteolipid protein (PLP), DM-20, SCIP, and the immediate early genes ZIF268, c-fos, and c-jun. Cells were treated with the calcium ionophore A23187 or thapsigargin for 1, 3, and 18 hr. A23187 at 1.0 mu M had no significant effect on cell detachment or death, whereas thapsigargin at 1.0 mu M slightly increased both. With both agents, SCIP, MBP, and PLP mRNA levels were unaffected by 3 hr, but markedly reduced after 18 hours. DM-20 mRNA levels remained unchanged at both time points. With both agents, ZIF268, c-fos, and c-jun mRNA levels were unaffected after 1 hr; c-jun mRNA levels showed a significant increase after 3 hr of thapsigargin treatment. Thus, in N20.1 cells, increased calcium affects the IEG c-jun first, SCIP is coordinately decreased with MBP and PLP mRNAs at a later time point, and DM-20 message is under different regulation than PLP. (C) 1999 Wiley-Liss, Inc.
Intercellular Ca2+ ivaves may be propagated through multicellular systems by the diffusion, through gap junctions, of the second messenger inositol 1,4,5-trisphosphate (IP3). This intercellular IP3 signaling provides a mechanism by which cooperative cell activity may be coordinated.
We have injected medaka fish zygotes with recombinant aequorin and visualized the resulting patterns of luminescence to reveal patterns of free calcium during early development. We have co-injected fluorescein-labeled aequorin to correct for nonuniformities in aequorin (as opposed to calcium) distributions by visualizing the resulting patterns of fluorescence as opposed to luminescence. We have also coinjected a calcium buffer to facilitate calcium diffusion, dissipate apparent calcium gradients, and thus confirm their reality. An exploratory study shows zones of elevated free calcium at the vegetal as well as the animal pole during the first day of development and thus up to the beginning of gastrulation. A closer study during the first 6 h, and thus through ooplasmic segregation and early cleavage, shows a steady zone of high calcium at the vegetal pole and a slowly oscillating one at the animal pole. The latter is particularly strong during ooplasmic segregation and cytokinesis. This report contains the first unambiguous evidence of relatively steady zones of high cytosolic calcium during the development of an animal egg.
We used indirect immunofluorescence to study microtubule arrays in the medaka egg between fer- tilization (normalized time, T,, , = 0) and the first cleavage (T, = 1 .O). Eggs were fixed at various times after fertil- ization and examined with conventional fluorescence mi- croscopy, laser scanning confocal microscopy, and three- dimensional fluorescence microscopy. Soon after the eggs were fertilized (T, = 0.02) we saw microtubules oriented perpendicular to the plane of the plasma membrane but none parallel to the plasma membrane. Later (T, = 0.08), we saw an array of microtubules oriented more or less parallel to the plasma membrane but having no apparent preferred orientation with respect to the animal-vegetal axis of the egg. In the interpolar regions of the egg, this network increased in density by T, = 0.24 and remained a constant feature of the ooplasm until the first cleavage. From T,, = 0.30 to 0.76 the polar regions of the egg con- tained dense arrays of organized microtubules. At the animal pole, microtubules radiated from a site near the pronuclei; while at the vegetal pole, an array of parallel microtubules was present. Injection of the weak (Ku = 1.5 PM) calcium buffer &Sdibromo-BAPTA disrupted the radial pattern of microtubules near the animal pole but had no apparent effect on the parallel array of micro- tubules near the vegetal pole. Because this buffer has pre- viously been shown to suppress a zone of elevated cytosolic calcium at the animal pole and to disrupt ooplasmic seg- regation in this egg, the results of the present study (1) are consistent with a model in which microtubules are required for ooplasmic segregation in the medaka egg, and (2) suggest that the normal function of a microtubule- organizing center at the animal pole of the egg requires a zone of elevated calcium.
Injection of the weak (K, = 1.5 PM) calcium buffer 5,5'-dibromo-BAPTA into fertilized medaka eggs inhibited the formation of the blastodisc at the animal pole, the movement of oil droplets toward the vegetal pole, and cytokinesis. These inhibitory actions were de- pendent upon the concentration of the buffer but were independent of free (Ca*') in the injectate. Because this buffer has previously been shown to substantially suppress zones of elevated calcium at the animal and vegetal poles of the medaka egg, the results of the present study suggest that these zones are necessary for normal segregation of the ooplasm and its inclusions in the medaka egg.
Members of the vertebrate Wnt family have been subdivided into two functional classes according to their biological activities. Some Wnts signal through the canonical Wnt-1/wingless pathway by stabilizing cytoplasmic β-catenin. By contrast other Wnts stimulate intracellular Ca2+ release and activate two kinases, CamKII and PKC, in a G-protein-dependent manner. Moreover, putative Wnt receptors belonging to the Frizzled gene family have been identified that preferentially couple to the two prospective pathways in the absence of ectopic Wnt ligand and that might account for the signaling specificity of the Wnt pathways. As Ca2+ release was the first described feature of the noncanonical pathway, and as Ca2+ probably plays a key role in the activation of CamKII and PKC, we have named this Wnt pathway the Wnt/Ca2+ pathway.
The release of Ca2+ from intracellular stores is an important trigger for secretion in many cell types. Depending on the spatial relationship between the intracellular Ca2+ stores and the site of exocytosis, the Ca2+ signal can be very local or spread throughout the entire cell. Here, we review how the release of Ca2+ from inositol trisphospate (IP3)-sensitive stores contributes differently to the stimulus-secretion coupling in three types of secretory cells (acinar cells of the pancreas, gonadotrophs, and corticotrophs of the anterior pituitary gland). We propose that in both pancreatic acinar cells and pituitary gonadotrophs the IP3-sensitive stores may be in close proximity to the sites of exocytosis such that the concentration of Ca2+ at these sites are transiently much higher than the average cytosolic Ca2+ concentration. In contrast, the local Ca2+ gradient is less prominent in pituitary corticotrophs. Finally, some recent technical developments that may contribute significantly to future investigations of local Ca2+ signals are discussed. BioEssays 21:861–865, 1999. © 1999 John Wiley & Sons, Inc.
Summary both the anesthetized and awake brain, is strong scat- tering of light by neural tissue. This problem is reduced Two-photon microscopy has enabled anatomical and in two-photon microscopy because of excitation with functional fluorescence imaging in the intact brain of near-infrared light. In addition, two-photon excitation is rats. Here, we extend two-photon imaging from anes- confined to the focal plane, permitting efficient fluores- thetized, head-stabilized to awake, freely moving ani- cence detection (Denk et al., 1995). Due to these proper- mals by using a miniaturized head-mounted micro- ties, two-photon microscopy has been the method of scope. Excitation light is conducted to the microscope choice for obtaining optical sections relatively deep in in a single-mode optical fiber, and images are scanned neural tissue; imaging depths of 0.5 mm have been using vibrations of the fiber tip. Microscope perfor- achieved in neocortex (Svoboda et al., 1997; Kleinfeld mance was first characterized in the neocortex of an- et al., 1998). As a second problem, there are brain pulsa- esthetized rats. We readily obtained images of vascu- tions and movements, which cause motion artifacts and lature filled with fluorescently labeled blood and of prevent stable imaging. This problem is manageable layer 2/3 pyramidal neurons filled with a calcium indi- in anesthetized animals but is exacerbated in awake cator. Capillary blood flow and dendritic calcium tran- animals (Fee, 2000). Movement artifacts are expected sients were measured with high time resolution using to be especially severe in freely moving animals, with line scans. In awake, freely moving rats, stable imaging the question arising whether in unrestrained animals was possible except during sudden head movements. stable imaging on the cellular scale is possible at all. Imaging of dendritic activity has been possible in awake,
The human kidney is composed of roughly 1.2-million renal tubules that must maintain their tubular structure to function properly. In autosomal dominant polycystic kidney disease (ADPKD) cysts develop from renal tubules and enlarge independently, in a process that ultimately causes renal failure in 50% of affected individuals1,2. Mutations in either PKCD1 or PKD2 are associated with ADPKD but the function of these genes is unknown. PKD1 is thought to encode a membrane protein, polycystin-1, involved in cell-cell or cell-matrix interactions3-5, whereas the PKD2 gene product, polycystin-2, is thought to be a channel protein6. Here we show that polycystin-1 and -2 interact to produce new calcium-permeable non-selective cation currents. Neither polycystin- 1 nor-2 alone is capable of producing currents. Moreover, disease-associated mutant forms of either potycystin protein that are incapable of heterodimerization do not result in new channel activity. We also show that polycystin-2 is localized in the cell in the absence of polycystin-1, but is translocated to the plasma membrane in its presence. Thus, polycystin-1 and -2 co-assemble at the plasma membrane to produce a new channel and to regulate renal tubular morphology and function.
A series of Ca2+-selective chelators incorporating a photosensitive o-nitrobenzhydryl ether, alcohol, or ester (λmax 350-360 nm, ε ≈5500 M-1 cm-1) were synthesized. The key step of the syntheses required the novel and mild trimethylsilyl triflate catalyzed Friedel-Crafts alkylation of a N,N-dialkylaniline by a nitrobenzaldehyde or its acetal. Before photolysis, the chelators show dissociation constants for Ca2+ of about 10-7 M, roughly matching the typical free [Ca2+] inside unstimulated cells. Considerable adjustment of the affinities is possible by subtle variations in the stereochemistry of the linkage between the two halves of the binding site. Irradiation around 365 nm smoothly converts the chelators into o-nitrosobenzophenones whose Ca2+ affinity is 10-30-fold weaker than the unphotolyzed compounds. The photolyses have quantum efficiencies of 0.01-0.04 and release Ca2+ with rate constants of 5-3000 s-1 after a flash, with free benzhydrols remarkably faster than their ethers. Therefore, these chelators can be used to generate controlled fast jumps in intracellular free [Ca2+] to mimic and analyze a host of important cellular responses, especially in nerve and muscle.
Two approaches were explored toward the goal of synthesizing physiologically useful Ca2+-selective chelators whose Ca2+ affinities increase markedly upon photolysis. In the first approach, the known Ca2+-selective chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was masked with a variety of photoremovable protecting groups on one of its four carboxyl groups, reducing its affinity for Ca2+ to ∼ 105 M-1. Upon irradiation around 365 nm, free chelator with an affinity constant ∼ 107 M-1 was regenerated but with very low quantum efficiencies (≤0.007). A more successful approach was to manipulate one of the coordinating nitrogens by remote inductive effects. Addition of an electron-withdrawing diazoacetyl substituent to one ring of BAPTA, para to the amino group, resulted in "diazo-2", a chelator with a Ca2+ affinity of 4.5 × 105 M-1. Photochemical rearrangement of the diazoacetyl group converted it into an electron-donating carboxymethyl group, causing the Ca2+ affinity to increase 30-fold to 1.4 × 107 M-1. The photolysis of Ca2+-free diazo-2 had a quantum efficiency with 365-nm light (λmax 370 nm, ε ∼ 22000 M-1 cm-1) of ∼0.03 and generated the high-affinity chelator with rate constants of 2300 s-1 after a flash. Ca2+ was then bound with association and dissociation rate constants of 8.0 × 108 M-1 s-1 and 58 s-1, respectively. Diazo-2 was incorporated into rat fibroblasts either by microinjection or by incubation as the membrane-permeable, enzymatically labile tetrakis(acetoxymethyl) ester and, when flash-photolyzed, caused a drop in intracellular free [Ca2+] to or below resting values of ∼ 10-7 M. An even larger increase in affinity (1600-fold) was obtained by substituting both phenyl rings of BAPTA with diazoacetyl substituents. Therefore, these chelators can be used to generate controlled fast decrements in intracellular free [Ca2+] to mimic or ablate a host of important cellular responses, especially in nerve and muscle.
The induction of intracellular Ca2+ release in pre-implantation mouse embryos accelerates their subsequent rate of development in vitro through a calmodulin-dependent mechanism [Stachecki J.J., Armant D.R. Transient release of calcium from inositol 1,4,5-trisphosphate-specific stores regulates mouse pre-implantation development. Development 1996; 122: 2485-2496]. To examine the hypothesis that intracellular Ca2+ signaling alters embryonic gene expression, individual transcript levels were compared by mRNA differential display before and 1 h after intracellular Ca2+ mobilization with ethanol in mouse blastocysts. Ten up-regulated and four down-regulated genes were observed, representing 3.5% of approximately 400 transcripts that were resolved. After sequencing, most of the DNA fragments appeared to be novel; however, two amplicons that increased after Ca2+ mobilization were identified as arginase and ubiquitin conjugating enzyme (E2). The up-regulation of arginase mRNA (3.5-fold after 2 h) was confirmed by reverse transcription and the polymerase chain reaction using specific oligonucleotide primers derived from the deduced mouse embryo sequence. A corresponding 2.5-fold increase in arginase enzymatic activity peaked 9 h after ethanol exposure. Increased expression of arginase and other genes may mediate the onset of rapid cell proliferation and differentiation that is induced by Ca2+ signaling during pre-implantation development.
Thesis (Ph.D. in Zoology)--Graduate School of Arts and Sciences, University of Pennsylvania, 1937. "Reprinted from the Journal of cellular and comparative pysiology, vol. 10, no. 3, October 1937". Bibliography: p. 303-304.
Surface contour, contact relations, and cortical structure of marginal cells of the enveloping layer (EVL) and of the yolk syncytial layer (YSL) of Fundulus heteroclitus were studied before and during epiboly with transmission and scanning electron microscopy. The contacts of the marginal cells of the EVL with the underlying YSL involve only the most marginal part of each cell and consist of a mixture of tight and close junctions apically and wider appositions more proximally. This junctional complex is very extensive (2.1–2.4 μm) prior to the onset of epiboly, very restricted (0.5–0.8 μm) during early epiboly up to a mid‐gastrula, more extensive again (0.8–1.0 μm) at late midgastrula, and still more extensive (2.0–2.3 μm) at late gastrula toward the end of epiboly. At this time, the margin of each marginal cell is embedded in the YSL. Several lines of evidence suggest that these marginal contacts are stable and therefore that epiboly of the EVL occurs passively, in response to pull exerted by the independently expanding YSL.
As the external YSL (E‐YSL) narrows during the earliest phase of epiboly, its surface becomes more and more convoluted. Since the network of 4–6 nm microfilaments in the cortical cytoplasm of the E‐YSL thickens with increasing convolution of its surface, it seems possible that a contractile force resides in the E‐YSL cortex which simultaneously throws the surface of the E‐YSL into folds, narrows the E‐YSL, and exerts tension on the attached margin of the EVL. Networks of thin microfilaments are also found in the cortex of EVL cells, especially in the leading edge of the marginal cells (as in fibroblasts and epithelial cells in vitro) and in the cortex of the yolk cytoplasmic layer (YCL), where they are presumably responsible for the contractile tension of this layer; 10‐nm microfilaments are also present but have a different distribution. They are arranged in bundles in both the marginal cytoplasm of each marginal cell and in the YSL beneath the marginal contact, running parallel to the contact and circumferentially relative to the whole egg. This arrangement in this location coincides with constriction of the egg in this marginal region and suggests that these thick filaments might provide the contractile force for the constriction, along with the thin filaments with which they are associated. The morphological relationship between the E‐YSL and the YCL during epiboly is also described.
SEM reveals that the surface of the internal YSL (I‐YSL) is covered with long microvilli at the beginning of gastrulation and that they disappear and are replaced by shorter microvilli as epiboly progresses. Estimation of the amount of surface in the long microvilli at the beginning of epiboly indicates that there is enough membrane on the surface of the I‐YSL at this time to account for epibolic expansion up to a late gastrula. Although it is not known how the surface membrane in these microvilli might be redistributed, the presence within them of abundant microfilaments that appear to insert in the plasma membrane suggests that this process might be accomplished by these presumed contractile elements.
Finally, measurements of cell surface expansion and calculations of cell number show that the number of cells in the EVL remains approximately constant during epiboly. Clearly, cell division is not a factor in the epibolic expansion of the enveloping layer. Instead, there is a marked thinning of individual cells.
Rat basophilic leukaemia cells, like mast cells from which they are derived, have surface Fc epsilon receptors that trigger secretion of inflammatory mediators when crosslinked. Both GTP-binding proteins and a rise in cytosolic calcium concentration ([Ca2+]i) are implicated in the secretory mechanism. Here we use a video-imaging technique to report that transient rises in [Ca2+]i initiated in an individual cell can spread from cell to cell in a wave-like pattern by means of a secreted intermediate, in the absence of gap-junctional communication. We find that the leukaemia cells, peritoneal mast cells and mucosal mast cells have cell-surface P2-type purinergic receptors that can trigger similar [Ca2+]i transients. We provide evidence that ATP is rapidly released, and that it can amplify [Ca2+]i signals and initial secretory responses during antigen-stimulation of rat basophilic leukaemia cells.
Two types of calcium (Ca2+) signaling-propagating intercellular Ca2+ waves of increasing intracellular Ca2+ concentration ([Ca2+]i) and nonpropagating oscillations in [Ca2+]i-co-exist in a variety of cell types. To investigate this difference in Ca2+ signaling, airway epithelial cells were loaded with heparin, an inositol 1,4,5-triphosphate (IP3) receptor antagonist, by pulsed, high-frequency electroporation. Heparin inhibited propagation of intercellular Ca2+ waves but not oscillations of [Ca2+]i. In heparin-free cells, Ca2+ waves propagated through cells displaying [Ca2+]i oscillations. Depletion of intracellular Ca2+ pools with the Ca2+-pump inhibitor thapsigargin also inhibited the propagation of Ca2+ waves. These studies demonstrate that the release of Ca2+ by IP3 is necessary for the propagation of intercellular Ca2+ waves and suggest that IP3 moves through gap junctions to communicate intercellular Ca2+ waves.
Zygotic expression of the Drosophila TGF beta family member decapentaplegic (dpp) is required for the development of the dorsal embryonic structures. By injecting dpp transcripts into young embryos, we find that 2- to 4-fold increases in the concentration of injected RNA elicit progressively more dorsal cell fates: only low levels of dpp permit development of ventral ectoderm, intermediate dpp levels drive dorsal epidermal development, and high dpp levels drive cells to differentiate as the most dorsal pattern element, the amnioserosa. Localized dpp RNA injections into embryos that lack all known maternal and zygotic dorsal-ventral polarity indicate that dpp can both define embryonic polarity and organize detailed patterning within the ectoderm. We infer that dpp acts as an extracellular morphogen and that the graded activity of dpp specifies the pattern of ectodermal cell fates in the Drosophila embryo.
Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser
scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a
stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and
other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence
and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This
technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly
photolytic release of caged effector molecules.
1. The blastoderm of the Oryzias latipes (medaka, Teleostei) embryo begins to contract rhythmically, about once per min at 25 degrees C, during epiboly. When the blastoderm was mechanically detached from the rest of the egg, it contracted into a pear-shaped ball and also continued to contract rhythmically. 2. The optimal [Ca2+] for the rhythmic contractions was approximately 1 mM. 3. The contractions stopped in media containing La3+, Ni2+, Mn2+, Co2+ or Ba2+. 4. A number of organic calcium antagonists--cinnarizine, D600, diltiazem, nifedipine, TMB-8 and verapamil--had no apparent effect on the contractions. However, the contractions were inhibited by papaverine, caffeine, and a mixture of TMB-8 and verapamil. 5. The contractions stopped in a medium containing 25 mM K+ or cytochalasin D. 6. We conclude that microfilaments cause the contractions, that each rhythmic contraction is preceded or accompanied by an increase in cytoplasmic free [Ca2+], and that Ca2+ enters the cytoplasm from both an extracellular and an intracellular pool.
The decapentaplegic gene complex (DPP-C) has been implicated in several events in pattern formation during Drosophila development. During embryogenesis, the DPP-C participates in the establishment of dorsal-ventral specification. Later, it is required for the correct morphogenesis of the imaginal disks, which will form much of the adult epidermis. We have undertaken a molecular analysis of the DPP-C to determine what role it plays in positional information. It appears to be a large genetic unit (greater than 40 kilobases (kb] consisting mostly of cis-regulatory information controlling the expression of a set of overlapping transcripts that differ at their 5' ends, but share the bulk of their transcribed sequences. Here, we describe the sequence analysis of two complementary DNAs comprising 4.0 kb of a 4.5-kb transcript. The C-terminus of the protein thereby deduced exhibits strong sequence homology (25-38% amino-acid identity) to the C-termini of a class of mammalian proteins that includes transforming growth factor-beta (TGF-beta), inhibin and Müllerian inhibiting substance (MIS). These proteins act on target cells to produce a variety of responses, such as stimulation or inhibition of cell division or differentiation. The homology suggests that the DPP-C protein contributes to correct morphogenesis as a secreted factor involved in the differential regulation of cell growth. This is the first report of a member of the TGF-beta gene family in a non-mammalian organism, and indicates that one or more members of this gene family existed before arthropod and vertebrate lineages diverged.
Chlorpromazine and trifluoperazine HCl, antipsychotic drugs known to bind to calmodulin, reversibly inhibited elevation of neural folds by interfering with the contractile activity of apical microfilament bundles in developing chick neuroepithelial cells.
A new family of high-affinity buffers and optical indicators for Ca2+ is rationally designed and synthesized. The parent compound is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relative of the well-known chelator EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in which methylene links between oxygen and nitrogen are replaced by benzene rings. BAPTA and its derivatives share the high (greater than 10(5)) selectivity for Ca2+ over Mg2+ of EGTA but are very much less affected by pH changes and are faster at taking up and releasing Ca2+. The affinity of the parent compound for Ca2+ (dissociation constant 1.1 x 10(-7) M in 0.1 M KCl) may be strengthened or weakened by electron-releasing or -withdrawing substituents on the aromatic rings. The Ca2+ and Mg2+ affinities may further be altered by replacing the ether oxygens by heterocyclic nitrogen atoms. The compounds described are fluorescent Ca2+ indicators absorbing in the ultraviolet region; the very large spectral shifts observed on binding Ca2+ fit the prediction that complexation should hinder the conjugation of the nitrogen lone-pair electrons with the aromatic rings. Derivatives with quinoline nuclei are notable for their high sensitivity of fluorescent quantum yield to the binding of Ca2+ but not of Mg2+. Preliminary biological tests have so far revealed little or no binding to membranes or toxic effects following intracellular microinjection.
Invagination of the optic vesicle to form the optic cup is an important event in the formation of the eye in the early embryo. To obtain support for earlier conclusions that a contractile process is involved, calcium dependency of optic cup formation was tested. Heads were excised from chicken embryos at the optic vesicle stage of development (stage 13) and incubated in nutrient medium containing antagonists or agonists of calcium transport. Invagination was reversibly inhibited by the Ca2+ antagonists verapamil and papaverine. It was initiated in a precocious fashion by the Ca2+ ionophore A23187 but only in the presence of external Ca2+. Neither caffeine, theophylline, nor A23187 (in the absence of external Ca2+) were able to initiate precocious optic cup formation. Trifluoperazine and chlorpromazine reversibly inhibited optic cup formation while chlorpromazine sulfoxide had no effect at the concentrations used. The binding of [3H]trifluoperazine to isolated stage 13 heads revealed a class of Ca2+-dependent binding sites having a Kd similar to that of calmodulin. These results indicate a Ca2+-dependence for optic cup formation and that the source of the Ca2+ may be extracellular. This Ca2+ dependence probably is conferred to the system by calmodulin.
Activation of the phosphoinositide (PI) pathway has been shown to be involved in the compaction of blastomeres in mouse embryos and in embryonic axis formation in Xenopus and in zebrafish embryos. Here we investigate Ca2+ signals in individual blastomeres of zebrafish embryos with the goal to better understand the role of PI and Ca2+ signaling for early vertebrate embryogenesis. Initial studies showed that the inositol 1,4,5-trisphosphate (IP3) concentration increases after the 32-cell stage of development, suggesting that IP3-mediated Ca2+ signals may be present during the blastula stage. Ca2+ signals were measured by identifying individual cells using confocal imaging of a nuclear localized Ca2+ indicator. Using this in situ indicator, changes in Ca2+ concentration were measured over several hours in each cell of a series of sections through the developing embryo. Transient increases in Ca2+ concentration that lasted 20-50 sec (Ca2+ spikes) were first triggered during the 32- to 128-cell stage in cells of the outer embryonic cell layer. These cells develop epithelial characteristics and specialize into the enveloping layer (EVL). No Ca2+ activity was observed during the earlier cleavage cycles or in deep blastomeres. Ca2+ spikes remained restricted to the EVL until the end of the blastula stage. Ca2+ spikes in neighboring EVL cells often occurred in the same short time interval, indicating that small groups of EVL cells can synchronize their activity. When averaged over several cell cycles, Ca2+ activity showed an even distribution in the EVL and did not indicate future polarities.
1. Ca2+ liberation induced in Xenopus oocytes by a poorly metabolized derivative of inositol 1,4,5-trisphosphate (3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate; 3-F-InsP3) was visualized using a video-rate confocal microscope to image fluorescence signals reported by the indicator dye calcium green-1. 2. Low (10-30 nM) intracellular concentrations of 3-F-InsP3 evoked Ca2+ release as localized transient 'puffs'. Progressively higher concentrations (30-60 nM) gave rise to abortive Ca2+ waves triggered by puffs, and then (> 60 nM) to a sustained elevation of Ca2+ followed by the appearance of propagating Ca2+ waves. At concentrations up to that giving waves, the frequency of puffs increased as about the third power of [InsP3], whereas their amplitudes increased only slightly. 3. The rise of cytosolic Ca2+ during a puff began abruptly, and peaked within about 50 ms. The peak free Ca2+ level was about 180 nM, and the total amount of Ca2+ liberated was several attomoles (10(-18) mol), too much to be accounted for by opening of a single InsP3-gated channel. The subsequent decline of Ca2+ occurred over a few hundred milliseconds, determined largely by diffusion of Ca2+ away from the release site, rather than by resequestration. Lateral spread of Ca2+ was restricted to a few micrometres, consistent with an effective diffusion coefficient for Ca2+ ions of about 27 microns2 s-1. 4. The peak amplitudes of puffs recorded at a given site were distributed in a roughly Gaussian manner, and a small proportion of sites consistently gave puffs much larger than the main population. Intervals between successive puffs at a single site were exponentially distributed, except for a progressive fall-off in puffs seen at intervals shorter than about 10 s. Thus, triggering of puffs appeared to be stochastically determined after recovery from a refractory period. 5. There was little correlation between the occurrence of puffs at sites more than a few micrometres apart, indicating that puff sites can function autonomously, but closely (ca 2 microns) adjacent sites showed highly correlated behaviour. 6. Puffs arose from sites-present at a density of about 1 per 30 microns2 in the animal hemisphere, located within a narrow band about 5-7 microns below the plasma membrane. 7. We conclude that Ca2+ puffs represent a 'quantal' unit of InsP3-evoked Ca2+ liberation, which may arise because local regenerative feedback by cytosolic Ca2+ ions causes the concerted opening of several closely clustered InsP3 receptor channels.
Calcium ions (Ca2+) appear to participate in the regulation of several aspects of cell division. Evidence is accumulating that transients or local gradients in the [Ca2+] contribute to different events including nuclear envelope breakdown and reformation, cleavage furrow formation and growth, and cell plate formation. At present there is little direct evidence that Ca2+ transients trigger the onset of anaphase. However, studies with exogenously applied Ca2+ indicate that spindle fibers and the movement of chromosomes at anaphase are exquisitely sensitive to the ion at physiological levels. Although Ca2+ is involved with many processes there are many gaps in our understanding, particularly pertaining to exactly when and where the ion concentration changes are expressed, which events and macromolecules are targeted, and what the processes are that control Ca2+.
N-cadherin cDNA was cloned from a zebrafish embryonic cDNA library. Analysis of the deduced amino acid sequence of this molecule (ZN-cadherin) revealed a high degree of homology to N-cadherins of other species, except that its pre-sequence is considerably shorter. Nevertheless, following transfection into chinese hamster ovary (CHO) cells, the expressed protein was functionally active, namely participated in calcium-dependent intercellular interactions. Moreover, ectopic over-expression of ZN-cadherin, following mRNA microinjection into 2-4 cell embryos, caused microaggregation and uneven segregation of deep cells, resulting in distorted embryos. Developmental Northern and Western blot analyses indicated that both the mRNA and the protein first appear at gastrulation. In-situ hybridization showed that ZN-cadherin mRNA was initially present in all deep cells, and later became restricted to various epithelial and neural tissues. Whole-mount immunostaining indicated that while ZN-cadherin was already present at 50% epiboly, it became associated with cell junctions only 4-5 h later. In developing somites ZN-cadherin expression was prominent but transient. High levels of the protein were detected in epithelial somites and its expression was apparently down regulated concomitantly with the onset of myogenesis.
The best known calcium waves move at about 5-30 microns/s (at 20 degrees C) and will be called fast waves to distinguish them from slow (contractile) ones which move at 0.1-1 microns/s as well as electrically propagated, ultrafast ones. Fast waves move deep within cells and seem to underlie most calcium signals. Their velocity and hence mechanism has been remarkably conserved among all or almost all eukaryotic cells. In fully active (but not overstimulated) cells of all sorts, their mean speeds lie between about 15-30 microns/s at 20 degrees C. Their amplitudes usually lie between 3-30 microM and their frequencies from one per 10-300 s. They are propagated by a reaction diffusion mechanism governed by the Luther equation in which Ca2+ ions are the only diffusing propagators, and calcium induced calcium release, or CICR, the only reaction; although this reaction traverses various channels which are generally modulated by IP3 or cADPR. However, they may be generally initiated by a second, lumenal mode of CICR which occurs within the ER. Moreover, they are propagated between cells by a variety of mechanisms. Slow intracellular waves, on the other hand, may be mechanically propagated via stretch sensitive calcium channels.
The synthesis and properties of a caged calcium are described. The compound is an ortho-nitrophenyl derivative of EGTA. It is synthesized in 10 steps and with 24% overall yield. The photosensitive chelator, nitrophenyl-EGTA, has a Kd value for Ca2+ of 80 nM and for Mg2+ of 9 mM. Upon exposure to UV radiation (approximately 350 nm), the chelator is cleaved, yielding iminodiacetic acid photoproducts with low Ca affinity (Kd = 1 mM). The quantum yield of photolysis of nitrophenyl-EGTA in the presence of Ca2+ is 0.23 and in the absence of Ca2+ is 0.20. In experiments with chemically skinned skeletal muscle fibers, a fully relaxed fiber equilibrated with nitrophenyl-EGTA-Ca2+ complex, in the presence of 1 mM free Mg2+, maximally contracted after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension was achieved in 18 ms at 15 degrees C. Nitrophenyl-EGTA provides a tool for the investigation of the mechanism of Ca(2+)-dependent physiological processes, since under conditions of normal intracellular Ca2+ and Mg2+ concentrations, only Ca2+ is bound by the photolabile chelator and on illumination released rapidly and in high photochemical yield.
Cadherins compose a family of calcium-dependent cell adhesion molecules that are involved in the segregation of differentiating tissues during development. Each cadherin has a unique spatial and temporal pattern of expression. As has been observed for other cadherins, B-cadherin, when expressed in mouse L929 fibroblasts, confers upon them a calcium-dependent cell aggregation activity. A monoclonal antibody to B-cadherin was isolated and used to determine the pattern of expression of B-cadherin in the developing chick embryo. Antibody staining and in situ hybridization reveal that B-cadherin protein and mRNA are found in diverse epithelia derived from each of the three primary germ layers, where their expression is strikingly regulated during differentiation. In some instances, the regional distribution of B-cadherin within a tissue reflects the distinct functional regions of the tissue. Patterns of staining are similar to, but distinct from, those seen with anti-LCAM antibodies. Although there are many examples where B-cadherin and LCAM are coexpressed, there are also distinctive regional differences.