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Pattern formation in the lateral line of zebrafish
Abstract
The lateral line of fish and amphibians is a sensory system that comprises a number of individual sense organs, the neuromasts, arranged in a defined pattern on the surface of the body. A conspicuous part of the system is a line of organs that extends along each flank (and which gave the system its name). At the end of zebrafish embryogenesis, this line comprises 7–8 neuromasts regularly spaced between the ear and the tip of the tail. The neuromasts are deposited by a migrating primordium that originates from the otic region. Here, we follow the development of this pattern and show that heterogeneities within the migrating primordium prefigure neuromast formation.
... The lateral line (LL) is a mechanosensory system that is found in amphibians and fish [14]. Although it has been lost in the tetrapod superclass, the internal structure known as the inner ear has been conserved [15]. ...
... The neuromasts are distributed along the body surface in species-specific patterns [15]. The LL sensory system can be subdivided into two major components: (i) The anterior lateral line (aLL), which includes neuromasts on the head and an aLL ganglion (aLLg) located anterior to the ear, while (ii) the posterior lateral line (pLL) includes neuromasts from the trunk to the caudal fin and a pLL ganglion (pLLg) located posterior to the ear [14,15]. Neuromast hair cells are innervated by peripheral bipolar sensory neurons which extend their central axons towards the hindbrain in a somatotopic manner (also referred to as neurotopic), meaning that neurons that innervate anterior neuromasts project their axons towards ventrolateral areas of the hindbrain, while neurons that innervate posterior neuromasts project their axons towards more dorsal areas [14,18,19]. ...
... The LL sensory system can be subdivided into two major components: (i) The anterior lateral line (aLL), which includes neuromasts on the head and an aLL ganglion (aLLg) located anterior to the ear, while (ii) the posterior lateral line (pLL) includes neuromasts from the trunk to the caudal fin and a pLL ganglion (pLLg) located posterior to the ear [14,15]. Neuromast hair cells are innervated by peripheral bipolar sensory neurons which extend their central axons towards the hindbrain in a somatotopic manner (also referred to as neurotopic), meaning that neurons that innervate anterior neuromasts project their axons towards ventrolateral areas of the hindbrain, while neurons that innervate posterior neuromasts project their axons towards more dorsal areas [14,18,19]. ...
Following an injury, axons of both the central nervous system (CNS) and peripheral nervous system (PNS) degenerate through a coordinated and genetically conserved mechanism known as Wallerian degeneration (WD). Unlike central axons, severed peripheral axons have a higher capacity to regenerate and reinnervate their original targets, mainly because of the favorable environment that they inhabit and the presence of different cell types. Even though many aspects of regeneration in peripheral nerves have been studied, there is still a lack of understanding regarding the dynamics of axonal degeneration and regeneration, mostly due to the inherent limitations of most animal models. In this scenario, the use of zebrafish (Danio rerio) larvae combined with time-lapse microscopy currently offers a unique experimental opportunity to monitor the dynamics of the regenerative process in the PNS in vivo. This review summarizes the current knowledge and advances made in understanding the dynamics of the regenerative process of PNS axons. By using different tools available in zebrafish such as electroablation of the posterior lateral line nerve (pLLn), and laser-mediated transection of motor and sensory axons followed by time-lapse microscopy, researchers are beginning to unravel the complexity of the spatiotemporal interactions among different cell types during the regenerative process. Thus, understanding the cellular and molecular mechanisms underlying the degeneration and regeneration of peripheral nerves will open new avenues in the treatment of acute nerve trauma or chronic conditions such as neurodegenerative diseases.
... In this section, we first summarize the main mechanisms underlying the processes of interest, that are inferred by a scan of the experimental literature, referring in particular to [1,2,5,[7][8][9][10][11][12][13][14][15][16][17][18][19], and that will constitute the simplified assumptions at the basis of the proposed mathematical model. Then, we will outline the modelling literature specifically focusing on the early development of zebrafish PLL. ...
... For the estimate of the remaining parameters, i.e. v rep (or v adh and V adh ), x fgf , x sdf and a sdf 4 , we have run preliminary simulations of the normal PLL development and fitted the resulting outcomes with the corresponding empirical evidence, both qualitatively and quantitatively (i.e. in terms of morphology and effective mean placode velocity). Specifically, we look for the set of parameter values that results in both (i) an elongation of the placode before the beginning of its migration (said L) within the range [140, 175] µm, according to [15]; and (ii) an effective mean primordium speed (said v PLL ) in the range [0.018, 0.02] μm s −1 , accounting for measurements in [5]. With this strategy, we have fixed reasonable values for adhesion/repulsion coefficients (i.e. ...
... At the end of the observation period, i.e. at t F ¼ 46 hpf, see figure 4l, the embryonic PLL comprises five deposited proto-neuromasts and three terminal rosettes which form the remaining main body of the primordium. Interestingly, the proposed reference simulation (which is characterized by L ≈ 166 µm and v PLL % 0:02 mm s À1 ) is in good agreement with biological evidence also in terms of distance between pairs of deposited proto-neuromasts which ranges between (240, 280) µm, close to the experimental counterparts [3,15]. ...
The morphogenesis of zebrafish posterior lateral line (PLL) is a good predictive model largely used in biology to study cell coordinated reorganization and collective migration regulating pathologies and human embryonic processes. PLL development involves the formation of a placode formed by epithelial cells with mesenchymal characteristics which migrates within the animal myoseptum while cyclically assembling and depositing rosette-like clusters (progenitors of neuromast structures). The overall process mainly relies on the activity of specific diffusive chemicals, which trigger collective directional migration and patterning. Cell proliferation and cascade of phenotypic transitions play a fundamental role as well. The investigation on the mechanisms regulating such a complex morphogenesis has become a research topic, in the last decades, also for the mathematical community. In this respect, we present a multiscale hybrid model integrating a discrete approach for the cellular level and a continuous description for the molecular scale. The resulting numerical simulations are then able to reproduce both the evolution of wild-type (i.e. normal) embryos and the pathological behaviour resulting form experimental manipulations involving laser ablation. A qualitative analysis of the dependence of these model outcomes from cell-cell mutual interactions, cell chemical sensitivity and internalization rates is included. The aim is first to validate the model, as well as the estimated parameter values, and then to predict what happens in situations not tested yet experimentally.
This article is part of the theme issue ‘Multi-scale analysis and modelling of collective migration in biological systems'.
... Follower cells undergo apical constriction to form discrete rosette-shaped clusters of about 20 cells (so-called proneuromasts) [Lecaudey et al., 2008], which synchronously slow their migration and decouple from the rest of the migrating primordium [Gompel et al., 2001;Haas & Gilmour, 2006]. In this way, the primordium intermittently deposits five proneuromasts along the flank of the fish before reaching the tail at about 40hpf, at which point it fragments to form 2-3 terminal proneuromasts in relatively quick succession [Gompel et al., 2001]. ...
... Follower cells undergo apical constriction to form discrete rosette-shaped clusters of about 20 cells (so-called proneuromasts) [Lecaudey et al., 2008], which synchronously slow their migration and decouple from the rest of the migrating primordium [Gompel et al., 2001;Haas & Gilmour, 2006]. In this way, the primordium intermittently deposits five proneuromasts along the flank of the fish before reaching the tail at about 40hpf, at which point it fragments to form 2-3 terminal proneuromasts in relatively quick succession [Gompel et al., 2001]. ...
... In between proneuromasts, the pLLP continuously deposits a chain of more mesenchymal-like progenitors called interneuromast cells which later go on to form additional neuromasts (Fig 1.5a) [Gompel et al., 2001;Grant et al., 2005;. Furthermore, the primordium is closely pursued by outgrowing axons projected from the lateral line ganglion, which innervate proneuromasts upon deposition [Metcalfe, 1985;Gilmour et al., 2004]. ...
During animal development, embryonic cells assemble into intricately structured organs by working together in organized groups capable of implementing tightly coordinated collective behaviors, including patterning, morphogenesis and migration. Although many of the molecular components and basic mechanisms underlying such collective phenomena are known, the complexity emerging from their interplay still represents a major challenge for developmental biology. Here, we first clarify the nature of this challenge and outline three key strategies for addressing it: precision perturbation, synthetic developmental biology, and data-driven inference. We then present the results of our effort to develop a set of tools rooted in two of these strategies and to apply them to uncover new mechanisms and principles underlying the coordination of collective cell behaviors during organogenesis, using the zebrafish posterior lateral line primordium as a model system. To enable precision perturbation of migration and morphogenesis, we sought to adapt optogenetic tools to control chemokine and actin signaling. This endeavor proved far from trivial and we were ultimately unable to derive functional optogenetic constructs. However, our work toward this goal led to a useful new way of perturbing cortical contractility, which in turn revealed a potential role for cell surface tension in lateral line organogenesis. Independently, we hypothesized that the lateral line primordium might employ plithotaxis to coordinate organ formation with collective migration. We tested this hypothesis using a novel optical tool that allows targeted arrest of cell migration, finding that contrary to previous assumptions plithotaxis does not substantially contribute to primordium guidance. Finally, we developed a computational framework for automated single-cell segmentation, latent feature extraction and quantitative analysis of cellular architecture. We identified the key factors defining shape heterogeneity across primordium cells and went on to use this shape space as a reference for mapping the results of multiple experiments into a quantitative atlas of primordium cell architecture. We also propose a number of data-driven approaches to help bridge the gap from big data to mechanistic models. Overall, this study presents several conceptual and methodological advances toward an integrated understanding of complex multi-cellular systems.
... The lateral line is a sense organ, present in fish and amphibians, that is formed by a set of mechanosensory hair cells distributed in a species-specific pattern over the surface of the animal's body. These multicellular structures, named neuromasts, have the function to detect displacements and vibrations of the surrounding water: the lateral line is indeed involved in several aspects of the individual life as, for instance, prey detection, predator avoidance, and sexual courtship ( Ghysen and Dambly-Chaudière, 2004;Gompel et al., 2001 ). In zebrafish, the biological system of our interest, the lateral line extends from the head to the caudal fin and divides in two major components: the so-called anterior lateral line (aLL), which comprises the neuromasts present on the head, and the posterior lateral line (pLL), which conversely includes the neuromasts on the trunk and the tail along each side of the animal ( Ghysen and Dambly-Chaudière, 2004;Gompel et al., 2001 ). ...
... These multicellular structures, named neuromasts, have the function to detect displacements and vibrations of the surrounding water: the lateral line is indeed involved in several aspects of the individual life as, for instance, prey detection, predator avoidance, and sexual courtship ( Ghysen and Dambly-Chaudière, 2004;Gompel et al., 2001 ). In zebrafish, the biological system of our interest, the lateral line extends from the head to the caudal fin and divides in two major components: the so-called anterior lateral line (aLL), which comprises the neuromasts present on the head, and the posterior lateral line (pLL), which conversely includes the neuromasts on the trunk and the tail along each side of the animal ( Ghysen and Dambly-Chaudière, 2004;Gompel et al., 2001 ). During the embryonic stage of life of the zebrafish, the pLL consists of a primordium , i.e., a proto-organ which is first recognized around 18 hpf (hours-post-fertilization), located just posterior of the otic vesicle and formed by nearly 100 epithelial cells ( Gompel et al., 2001;Haas and Gilmour, 2006 ). ...
... In zebrafish, the biological system of our interest, the lateral line extends from the head to the caudal fin and divides in two major components: the so-called anterior lateral line (aLL), which comprises the neuromasts present on the head, and the posterior lateral line (pLL), which conversely includes the neuromasts on the trunk and the tail along each side of the animal ( Ghysen and Dambly-Chaudière, 2004;Gompel et al., 2001 ). During the embryonic stage of life of the zebrafish, the pLL consists of a primordium , i.e., a proto-organ which is first recognized around 18 hpf (hours-post-fertilization), located just posterior of the otic vesicle and formed by nearly 100 epithelial cells ( Gompel et al., 2001;Haas and Gilmour, 2006 ). In particular, these component cells present several mesenchymal determinants, i.e., loss in apicobasal polarity, reduced expression of adhesive proteins and increased numbers of activated dynamic filopodia ( Lecaudey et al., 2008 ). ...
The description of the cell spatial pattern and characteristic distances is fundamental in a wide range of physio-pathological biological phenomena, from morphogenesis to cancer growth. Discrete particle models are widely used in this field, since they are focused on the cell-level of abstraction and are able to preserve the identity of single individuals reproducing their behavior. In particular, a fundamental role in determining the usefulness and the realism of a particle mathematical approach is played by the choice of the intercellular pairwise interaction kernel and by the estimate of its parameters. The aim of the paper is to demonstrate how the concept of H-stability, deriving from statistical mechanics, can have important implications in this respect. For any given interaction kernel, it in fact allows to a priori predict the regions of the free parameter space that result in stable configurations of the system characterized by a finite and strictly positive minimal interparticle distance, which is fundamental when dealing with biological phenomena. The proposed analytical arguments are indeed able to restrict the range of possible variations of selected model coefficients, whose exact estimate however requires further investigations (e.g., fitting with empirical data), as illustrated in this paper by series of representative simulations dealing with cell colony reorganization, sorting phenomena and zebrafish embryonic development.
... The pLL is generated by the deposition of neuromasts by a migrating primordium (pLLP) during development. The migratory pLLP consists of around 140 cells, which emerge behind the otic placode at around 20-22 hpf and begin to migrate towards the tip of the tail, which is completed by 42 hpf (Gompel et al., 2001;Dalle Nogare and Chitnis, 2017). During this migration, they deposit cells as proto-neuromasts at regular intervals throughout the horizontal myoseptum to generate 5 neuromasts (L1-L5) in the trunk region and 2-3 terminal neuromasts at the tip of the tail (Gompel et al., 2001). ...
... The migratory pLLP consists of around 140 cells, which emerge behind the otic placode at around 20-22 hpf and begin to migrate towards the tip of the tail, which is completed by 42 hpf (Gompel et al., 2001;Dalle Nogare and Chitnis, 2017). During this migration, they deposit cells as proto-neuromasts at regular intervals throughout the horizontal myoseptum to generate 5 neuromasts (L1-L5) in the trunk region and 2-3 terminal neuromasts at the tip of the tail (Gompel et al., 2001). These neuromasts undergo a process of morphogenesis, become epithelized and mature into rosettes with sensory hairs at their center (Lecaudey et al., 2008;Hava et al., 2009). ...
The lateral line system is a mechanosensory organ of fish and amphibians that detects changes in water flow and is formed by the coordinated action of many signalling pathways. These signalling pathways can easily be targeted in zebrafish using pharmacological inhibitors to decipher their role in lateral line system development at cellular and molecular level. We have identified two uncharacterized proteins, whose mRNA are expressed in the lateral line system of zebrafish. One of these proteins, uncharacterized protein LOC564095 precursor is conserved across vertebrates and its mRNA is expressed in posterior lateral line primordium (pLLP). The other uncharacterized protein LOC100536887 is present only in the teleost fishes and its mRNA is expressed in neuromasts. We show that inhibition of retinoic acid (RA) signalling reduces the expression of both of these uncharacterized genes. It is reported that inhibition of RA signalling during gastrulation starting at 7 hours post fertilization (hpf) abrogates pLLP formation and inhibition of RA signalling at 10 hpf delays the initiation of pLLP migration. Here, we show that inhibition of RA signalling before and during segmentation (9-16 hpf) results in delayed initiation and reduced speed of pLLP migration as well as inhibition of posterior neuromasts formation.
... The zebrafish posterior lateral line system (pLLS) is a unique model for studying collective cell migration as well as axonal outgrowth and pathfinding during development of a sensory organ (Metcalfe et al., 1985;Kimmel et al., 1995;Gilmour et al., 2002;Dambly-Chaudière et al., 2007;Aman and Piotrowski, 2008;Lush and Piotrowski, 2014;Romero-Carvajal et al., 2015). Its development starts with the formation of the posterior primary primordium (PrimI), which arises from the disintegration of the posterior primary sensory placode (Gompel et al., 2001;Nikaido et al., 2017). PrimI migrates along the horizontal myoseptum and deposits up to 6 proneuromasts before reaching the tip of the tail at 48 hpf (Gompel et al., 2001;Lecaudey et al., 2008). ...
... Its development starts with the formation of the posterior primary primordium (PrimI), which arises from the disintegration of the posterior primary sensory placode (Gompel et al., 2001;Nikaido et al., 2017). PrimI migrates along the horizontal myoseptum and deposits up to 6 proneuromasts before reaching the tip of the tail at 48 hpf (Gompel et al., 2001;Lecaudey et al., 2008). Deposited proneuromasts differentiate into neuromasts, the sensory organs of the pLLS. ...
The posterior lateral line system (pLLS) of aquatic animals comprises small clustered mechanosensory organs along the side of the animal. They develop from proneuromasts, which are deposited from a migratory primordium on its way to the tip of the tail. We here show, that the Neural Cell Adhesion Molecule Ncam1b is an integral part of the pathways initiating and regulating the development of the pLLS in zebrafish. We find that morpholino-knockdowns of ncam1b (i) reduce cell proliferation within the primordium, (ii) reduce the expression of Fgf target gene erm, (iii) severely affect proneuromast formation, and (iv) affect primordium migration. Ncam1b directly interacts with Fgf receptor Fgfr1a, and a knockdown of fgfr1a causes similar phenotypic changes as observed in ncam1b-morphants. We conclude that Ncam1b is involved in activating proliferation by triggering the expression of erm. In addition, we demonstrate that Ncam1b is required for the expression of chemokine receptor Cxcr7b, which is crucial for directed primordial migration. Finally, we show that the knockdown of ncam1b destabilizes proneuromasts, suggesting a further function of Ncam1b in strengthening the cohesion of proneuromast cells.
... The posterior lateral lines, which are sensory organs that sense water flow, have been used as a model system for studying collective cell migration, as well as organ morphogenesis (Chitnis, Nogare, & Matsuda, 2012;Nogare & Chitnis, 2017;Piotrowski & Baker, 2014). During development of the lateral line in zebrafish, the PLLP, which is composed of approximately 120 cells, appears at a region adjacent to the otic vesicle at 20 hr postfertilization (hpf) and migrates toward the posterior end along with the horizontal midline of the embryonic body until it reaches the tip of the tail at around 48 hpf (Gompel et al., 2001;Nogare & Chitnis, 2017). During their collective migration, small groups of ~20 cells are segregated from the trailing (i.e., anterior) side of the PLLP and form functional sensory organs called neuromasts by 5 days postfertilization (dpf; Gompel et al., 2001;Nogare & Chitnis, 2017). ...
... During development of the lateral line in zebrafish, the PLLP, which is composed of approximately 120 cells, appears at a region adjacent to the otic vesicle at 20 hr postfertilization (hpf) and migrates toward the posterior end along with the horizontal midline of the embryonic body until it reaches the tip of the tail at around 48 hpf (Gompel et al., 2001;Nogare & Chitnis, 2017). During their collective migration, small groups of ~20 cells are segregated from the trailing (i.e., anterior) side of the PLLP and form functional sensory organs called neuromasts by 5 days postfertilization (dpf; Gompel et al., 2001;Nogare & Chitnis, 2017). ...
Collective cell migration, in which cells assemble and move together, is an essential process in embryonic development, wound healing, and cancer metastasis. Chemokine signaling guides cell assemblies to their destinations. In zebrafish posterior lateral line primordium (PLLP), a model system for collective cell migration, it has been proposed that the chemokine ligand Cxcl12a secreted from muscle pioneer cells (MPs) and muscle fast fibers (MFFs), which are distributed along with the horizontal midline, binds to the receptor Cxcr4b in PLLP, and that Cxcl12a–Cxcr4b signaling guides the anterior‐to‐posterior migration of PLLP along the horizontal midline. However, how the surrounding tissues affect PLLP migration remains to be elucidated. Here we investigated the relationship between the PLLP and the surrounding tissues and found that a furrow between the dorsal and ventral myotomes is generated by Sonic hedgehog (Shh) signaling–dependent MP and MFF differentiation, and that the PLLP migrates in this furrow. When transient inhibition of Shh signaling impaired both the furrow formation and differentiation of cxcl12a ‐expressing MPs/MFFs, directional PLLP migration was severely perturbed. Furthermore, when differentiated MPs and MFFs were ablated by femtosecond laser irradiations, the furrow remained and PLLP migration was relatively unaffected. These results suggest that the furrow formation between the dorsal and ventral myotomes is associated with the migratory behavior of PLLP.
... The morphogenesis of the Drosophila eye is also facilitated by rosettes [38]. In zebrafish lateral-line development, the lateral line is composed of mechanosensory organs called neuromasts, which are formed from rosettes composed of 20 or more cells [39][40][41]. In other vertebrates, rosettes have been observed in the neural plate of chick [42] and mouse [43] embryos, in the development of the mouse visceral endoderm [44], the kidney tubule [45], and the pancreas [46]. ...
... This mechanism can be manifested in several ways. For example, in Drosophila body-axis elongation, rosettes are due to planar polarized constriction [28,29,31,33,34,36], while in gastrulation and neural tube closure, actomyosin structures in the apical cell surface constrict to form rosettes [39][40][41]. Also, when cells delaminate or extrude from epithelia [51][52][53][54], a vertex with more than four edges can be left behind that becomes a center of a rosette. ...
Models for confluent biological tissues often describe the network formed by cells as a triple-junction network, similar to foams. However, higher-order vertices or multicellular rosettes are prevalent in developmental and in vitro processes and have been recognized as crucial in many important aspects of morphogenesis, disease, and physiology. In this work, we study the influence of rosettes on the mechanics of a confluent tissue. We find that the existence of rosettes in a tissue can greatly influence its rigidity. Using a generalized vertex model and the effective medium theory, we find a fluid-to-solid transition driven by rosette density and intracellular tensions. This transition exhibits several hallmarks of a second-order phase transition such as a growing correlation length and a universal critical scaling in the vicinity of a critical point. Furthermore, we elucidate the nature of rigidity transitions in dense biological tissues and other cellular structures using a generalized Maxwell constraint counting approach, which answers a long-standing puzzle of the origin of solidity in these systems.
... The PLLP consists of more than 100 cells that migrate together, along the length of the zebrafish embryo from head to tail (approximately 22-48 hours post-fertilization). The PLLP completes its migration over the course of a day, migrating around 2.3 mm [1]. During migration, the cells in the trailing region re-organize to deposit clusters (neuromasts) in their wake, a process that we consider in a future study. ...
... The PLLP consists of more than 100 cells (%4-5 cells wide) when migration is initiated, which migrate about 2.3 mm down the horizontal myoseptum during the time interval 22-48 hours post-fertilization [1]. Cells in the trailing region re-organize into rosettes, are periodically deposited and later develop into neuromasts, to form sensory organs of the posterior lateral line. ...
Collective cell migration plays an important role in development. Here, we study the posterior lateral line primordium (PLLP) a group of about 100 cells, destined to form sensory structures, that migrates from head to tail in the zebrafish embryo. We model mutually inhibitory FGF-Wnt signalling network in the PLLP and link tissue subdivision (Wnt receptor and FGF receptor activity domains) to receptor-ligand parameters. We then use a 3D cell-based simulation with realistic cell-cell adhesion, interaction forces, and chemotaxis. Our model is able to reproduce experimentally observed motility with leading cells migrating up a gradient of CXCL12a, and trailing (FGF receptor active) cells moving actively by chemotaxis towards FGF ligand secreted by the leading cells. The 3D simulation framework, combined with experiments, allows an investigation of the role of cell division, chemotaxis, adhesion, and other parameters on the shape and speed of the PLLP. The 3D model demonstrates reasonable behaviour of control as well as mutant phenotypes.
... A total of 5-6 primary neuromasts are deposited along the trunk in this manner, and then as the PLLp approaches the tip of the tail it fragments to form an additional 2-3 terminal neuromasts. The migration and dispersion of neuromasts concludes at about 42 hpf, with the last of the primary neuromasts fully differentiated by about 54 hpf (Gompel et al. 2001;López-Schier et al. 2004). In addition to sdf1a and cxcr4b/cxcr7b, other signaling pathways that have been implicated in this process include Wnt/E-catenin, FGF, and Notch/Delta (Ma and Raible 2009). ...
... The ALL includes the neuromasts present on the head, jaw and opercle, and their sensory neurons that lie rostral to the ear. The PLL includes the neuromasts on the trunk and tail, and its sensory neurons form the PLL ganglion, caudal to the ear(Gompel et al. 2001;Ghysen and Dambly-Chaudière 2004). In zebrafish embryos, the PLL is a line of seven to eight neuromasts that are formed from a primordium of precursor cells that originates from the otic region and begins to migrate caudally(Figure 1.4A). ...
By utilizing the zebrafish as a research model, the function of specific cell signalling pathway components in development was investigated. This revealed new roles for the so-called Jak2/Stat5/Cis pathway in blood and sensory organ development, and a conserved prolactin pathway despite divergent functions between species.
... Myosin-driven contraction constricts the shared interfaces of the aligned cells, pulling them into a rosette structure with a centrally shared interface. These multicellular rosettes are a common feature during the morphogenesis of various organs (Gompel et al., 2001;Lienkamp et al., 2012;Villasenor et al., 2010). ...
Here, we report the generation of a transgenic Lifeact–EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviors and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we visualize cells undergoing apical constriction and large-scale actin structures such as multicellular rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact–EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton.
... Moreover, the Smpx protein was also localized at the level of the head neuromasts (Supp. Fig. S1B-E) that form the anterior lateral line (ALL), a structure that develops independently from the PLL 25 . In all 120 hpf neuromasts, Smpx localized quite exclusively in the cytoplasm of the cells (Supp. ...
The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable—and easily accessible—tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.
... Most multicellular rosettes are transient epithelial structures that contain five or more cells that interface at a central point, where apical membranes of these cells constrict. Multicellular rosettes are observed in many developmental contexts, including convergent extension during Drosophila embryogenesis, posterior Lateral Line (pLL) formation in zebrafish, vertebrate kidney tubule elongation, as well as numerous others (Blankenship et al., 2006;Gompel et al., 2001;Lienkamp et al., 2012). ...
Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of ∼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.
... Myosin-driven contracaon of supracellular acan cables constricts the lateral edges of the cells along the cable, pulling them into a rosepe structure with a centrally shared interface. These mulacellular rosepes are a common feature during morphogenesis of various organs (Gompel et al., 2001, Lienkamp et al., 2012, Villasenor et al., 2010. ...
Here we report the generation of a transgenic LifeactEGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high resolution live imaging approaches, we present the dynamic behaviours and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we reveal the dynamics of cells undergoing apical constriction and the emergence of large scale actin structures such as supracellular cables and rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the LifeactEGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton.
... In zebrafish, the posterior lateral line system genesis (PLL) is started at the end of 24 hpf by the migration of cells cluster (100 cells) called the primordium PLL (PLLp) under the skin near the ear to the end of the tail [14]. During this step, the PLLp periodically deposits neuromasts (L1, L2 … L7) along the body and will finish its migration, by the establishment of 2-3 terminal neuromasts (TN) at the level of the tail. ...
Endocrine-disrupting chemicals (EDCs), including polychlorinated biphenyls (PCBs), bisphenol A (BPA), pharmaceutical drugs, and pesticides, affect a variety of hormone-regulated physiological pathways in humans and wildlife. The occurrence of these EDCs in the aquatic environment is linked with vertebrates’ health alteration. EDCs exhibit lipophilic characteristics and bind to hydrophobic areas of steroid receptors, such as the estrogen receptor, which are involved in vertebrate developmental regulation. Mainly, EDCs modify the transcription of several genes involved in individual homeostasis. Zebrafish conserve many developmental pathways found in humans, which makes it an appreciated model system for EDCs research studies, especially on early organ development. In the current chapter, we emphasize on latest published papers of EDCs effects on lateral line regeneration in zebrafish larvae. Similarly, we describe other special impacts of EDCs exposure. In conclusion, we make the case that the zebrafish lateral line exposed to EDCs can provide important insights into human health.
... In zebrafish, hair cells are found in the lateral line (LL) system located in the apical part of the mechanosensory organs known as neuromasts. During embryonic and larval development, a population of approximately 140 cells, named primordium, located in the head region caudally migrates to the tip of the tail (Gompel et al., 2001;Nogare et al., 2017) and deposits 5 groups of ~20 cells along the posterior LL (pLL), completing the formation of pioneer neuromasts in about 40 h post fertilization (hpf) (Dambly-Chaudière et al., 2007). In zebrafish larvae, each neuromast in the pLL is named by its position (from L1 to L5, in a rostral to caudal direction) and is composed of a central cluster of hair cells surrounded by non-sensory supporting and mantle cells forming together an epithelial rosette (Montalbano et al., 2018). ...
Zebrafish possess hair cells on the body surface similar to that of mammals inner hear, in particular in the neuromasts, and due to its ability in regenerating damaged hair cells, is regularly used as a powerful animal model to study in vivo cytotoxicity.
Among the factors leading to hair cell disruption, metal ions are of particular concern since they are important environmental pollutants. To date, several studies on zebrafish hair cell regeneration after metal exposure exist, while no data on regeneration during continuous metal exposure are available. In the present study, neuromast hair cell disruption and regeneration were assessed in zebrafish larvae for the first time during zinc (Zn) and cadmium (Cd) continuous exposure and a visual and molecular approach was adopted.
Fluorescent vital dye DASPEI was used to assess hair cell regeneration and the gene expression of claudin b (cldnb) and phoenix (pho), was analyzed. Metallotionein-2 (mt2) gene expression was used as standard molecular marker of metal toxicity and confirmed the higher toxicity of Cd compared to Zn. In addition, Cd caused a delay in hair cell regeneration compared to Zn. Molecular analysis showed cldnb gene expression increased in relation to the metal concentrations used, confirming the involvement of this gene in hair cell regeneration. On the contrary, a dramatic decrease of pho gene expression was observed in Cd exposed groups, suggesting a negative impact of Cd on pho expression, thus negatively interfering with hair cell regeneration in zebrafish larvae exposed to this metal.
... The lateral line system consists of numerous sensory organs, neuromasts, which are typically arranged in superficial lines covering the head and body. The neuromasts are innervated by sensory neurons situated in two ganglia, giving rise to two separated networks: the anterior lateral line (ALL) projecting around the head and the posterior lateral line (PLL) projecting along the body (Gompel et al., 2001; Figure 1A). Neuromasts consist of hair cells that have cilia protruding from the skin, enabling the detection of water flow and play a crucial role in behaviors such as rheotaxis, predator avoidance, and schooling (Coombs et al., 2014;Olszewski et al., 2012). ...
The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprized of clusters of superficial hair cells called neuromasts. Modulation occurs via excitatory and inhibitory efferent neurons located in the brain. Using mosaic transgenic labeling we provide an anatomical overview of the lateral line projections made by individual inhibitory efferent neurons in 5-day old zebrafish larvae. For each hemisphere we estimate there to be six inhibitory efferent neurons located in two different nuclei. Three distinct cell types were classified based on their projections; to the anterior lateral line around the head, to the posterior lateral line along the body, or to both. Our analyses corroborate previous studies employing back-fills, but our transgenic labeling allowed a more thorough characterization of their morphology. We found that individual inhibitory efferent cells connect to multiple neuromasts and that a single neuromast is connected by multiple inhibitory efferent cells. The efferent axons project to the sensory ganglia and follow the sensory axon tract along the lateral line. Time-lapse imaging revealed that inhibitory efferent axons do not migrate with the primordium as the primary sensory afferent does, but follow with an 8–14 h lag. These data bring new insights into the formation of a sensory circuit and support the hypothesis that different classes of inhibitory efferent cells have different functions. Our findings provide a foundation for future studies focussed toward unraveling how and when sensory perception is modulated by different efferent cells.
... Zebrafish has emerged as a good model for investigating the ototoxic side-effects of drugs (Buck et al., 2012;d'Alençon et al., 2010;Gompel et al., 2001) and for screening otoprotectants (Kruger et al., 2016;Rocha-Sanchez et al., 2018). Zebrafish has sensory organs called neuromast that run along the length of lateral line, where mechanosensory hair cells are located (Namdaran et al., 2012;Pichler and Lagnado, 2019). ...
Generation of reactive oxygen species, a critical factor in cisplatin-induced ototoxicity, leads to the formation of peroxynitrite, which in turn results in the nitration of susceptible proteins. Previous studies indicated that LMO4, a transcriptional regulator, is the most abundantly nitrated cochlear protein after cisplatin treatment and that LMO4 nitration facilitates ototoxicity in rodents. However, the role of this mechanism in regulating cisplatin-induced hair cell loss in non-mammalian models is unknown. As the mechanosensory hair cells in the neuromasts of zebrafish share many features with mammalian inner ear and is a good model for studying ototoxicity, we hypothesized that cisplatin treatment induces protein nitration and Lmo4 degradation in zebrafish hair cells, thereby facilitating hair cell loss. Immunostaining with anti-parvalbumin revealed a significant decrease in the number of hair cells in the neuromast of cisplatin treated larvae. In addition, cisplatin treatment induced a significant decrease in the expression of Lmo4 protein and a significant increase in nitrotyrosine levels, in the hair cells. The cisplatin-induced changes in Lmo4 and nitrotyrosine levels strongly correlated with hair cell loss, implying a potential link. Furthermore, a significant increase in the expression of activated Caspase-3 in zebrafish hair cells, post cisplatin treatment, suggested that cisplatin-induced decrease in Lmo4 levels is accompanied by apoptosis. These findings suggest that nitrative stress and Lmo4 degradation are important factors in cisplatin-induced hair cell loss in zebrafish neuromasts and that zebrafish could be used as a model to screen the otoprotective efficacy of compounds that inhibit protein nitration.
... In recent years, the Zebrafish Posterior Lateral Line primordium (PLLp) has emerged as a powerful model for studying a wide range of cellular and developmental processes, including cell-cell signaling, tissue patterning, and collective migration (Chitnis et al., 2012;Friedl and Gilmour, 2009;Ghysen and Dambly-Chaudière, 2007). This group of 100-150 cells is initially specified adjacent to the otic vesicle and migrates caudally down the length of the embryo over the course of~24 hr (Gompel et al., 2001;Nogare et al., 2017). As this primordium migrates, cells in the trailing domain are progressively reorganized into apically constricted epithelial rosettes, each cradling a central sensory hair cell progenitor (Nechiporuk and Raible, 2008). ...
The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium.
... In zebrafish, the migration of the primordium of the pLL to the tail begins at 20-22 hpf from the pLL placode, situated at the hindbrain, just posterior to the otic placode (Dambly-Chaudiere et al., 2003). Then, the primordium migrates caudally, followed by the formation of proneuromasts that differentiate in the following few hours (Gompel et al., 2001). Two reciprocally antagonistic signaling centers primarily control the progress: the Wnt signaling center in the prominent domain of migrating primordia and the FGF signaling pathway in the trailing domain (Aman and Piotrowski, 2008;Ma and Raible, 2009). ...
Histone demethylase PHF8 is crucial for multiple developmental processes, and hence, the awareness of its function in developing auditory organs needs to be increased. Using in situ hybridization (ISH) labeling, the mRNA expression of PHF8 in the zebrafish lateral line system and otic vesicle was monitored. The knockdown of PHF8 by morpholino significantly disrupted the development of the posterior lateral line system, which impacted cell migration and decreased the number of lateral line neuromasts. The knockdown of PHF8 also resulted in severe malformation of the semicircular canal and otoliths in terms of size, quantity, and position during the inner ear development. The loss of function of PHF8 also induced a defective differentiation in sensory hair cells in both lateral line neuromasts and the inner ear. ISH analysis of embryos that lacked PHF8 showed alterations in the expression of many target genes of several signaling pathways concerning cell migration and deposition, including the Wnt and FGF pathways. In summary, the current findings established PHF8 as a novel epigenetic element in developing auditory organs, rendering it a potential candidate for hearing loss therapy.
... Rosettes are multicellular structures that interface at a central point. Rosette formation has been observed in many contexts including Drosophila eye morphogenesis, zebrafish lateral line development, mouse and Xenopus kidney tubule formation, and pancreatic branching in mice [2][3][4][5] . Our studies here utilize the left-right organizer, Kupffer's vesicle (KV), in the vertebrate model Danio rerio to characterize a mechanism of rosette and subsequent lumen formation. ...
Multicellular rosettes are transient epithelial structures that serve as intermediates during diverse organ formation. We have identified a unique contributor to rosette formation in zebrafish Kupffer’s vesicle (KV) that requires cell division, specifically the final stage of mitosis termed abscission. KV utilizes a rosette as a prerequisite before forming a lumen surrounded by ciliated epithelial cells. Our studies identify that KV-destined cells remain interconnected by cytokinetic bridges that position at the rosette’s center. These bridges act as a landmark for directed Rab11 vesicle motility to deliver an essential cargo for lumen formation, CFTR (cystic fibrosis transmembrane conductance regulator). Here we report that premature bridge cleavage through laser ablation or inhibiting abscission using optogenetic clustering of Rab11 result in disrupted lumen formation. We present a model in which KV mitotic cells strategically place their cytokinetic bridges at the rosette center, where Rab11-associated vesicles transport CFTR to aid in lumen establishment. De novo lumen formation during vertebrate left–right organizer development is required for body axis establishment. Here the authors utilize zebrafish to demonstrate that the position and cleavage of the cytokinetic bridge in dividing left-right organizer cells dictates tissue morphogenesis.
... The sensory organs are derived from several neurogenic, cephalic placodes/ primordia that either migrate into the trunk or into the head 30 . As they migrate, primordia periodically deposit clusters of cells that differentiate into sensory organs [31][32][33] . PrimordiumI (primI) and primordiumII (primII) both migrate into the trunk but arise from different placodes (primary placode and D0 placode, respectively). ...
Planar cell polarity (PCP) plays crucial roles in developmental processes such as gastrulation, neural tube closure and hearing. Wnt pathway mutants are often classified as PCP mutants due to similarities between their phenotypes. Here, we show that in the zebrafish lateral line, disruptions of the PCP and Wnt pathways have differential effects on hair cell orientations. While mutations in the PCP genes vangl2 and scrib cause random orientations of hair cells, mutations in wnt11f1, gpc4 and fzd7a/b induce hair cells to adopt a concentric pattern. This concentric pattern is not caused by defects in PCP but is due to misaligned support cells. The molecular basis of the support cell defect is unknown but we demonstrate that the PCP and Wnt pathways work in parallel to establish proper hair cell orientation. Consequently, hair cell orientation defects are not solely explained by defects in PCP signaling, and some hair cell phenotypes warrant re-evaluation.
... The sensory organs are derived from several neurogenic, cephalic placodes/ primordia that either migrate into the trunk or into the head 30 . As they migrate, primordia periodically deposit clusters of cells that differentiate into sensory organs [31][32][33] . PrimordiumI (primI) and primordiumII (primII) both migrate into the trunk but arise from different placodes (primary placode and D0 placode, respectively). ...
Planar cell polarity (PCP) plays crucial roles in developmental processes such as gastrulation, neural tube closure and hearing. Wnt pathway mutants are often classified as PCP mutants due to similarities between their phenotypes. Here, we show that in the zebrafish lateral line, disruptions of the PCP and Wnt pathways have differential effects on hair cell orientations. While mutations in the PCP genes vangl2 and scrib cause random orientations of hair cells, mutations in wnt11f1, gpc4 and fzd7a/b induce hair cells to adopt a concentric pattern. This concentric pattern is not caused by defects in PCP but is due to misaligned support cells. The molecular basis of the support cell defect is unknown but we demonstrate that the PCP and Wnt pathways work in parallel to establish proper hair cell orientation. Consequently, hair cell orientation defects are not solely explained by defects in PCP signaling, and some hair cell phenotypes warrant re-evaluation. Planar cell polarity (PCP) regulates hair cell orientation in the zebrafish lateral line. Here, the authors show that mutating Wnt pathway genes (wnt11f1, fzd7a/b, and gpc4) causes concentric hair cell patterns not regulated by PCP, thus showing PCP/Wnt pathway genes have different consequences on hair cell orientation.
... Let us remark that agent-based models of this type with a finite number of cells are also interesting in detailed models where differential adhesion is important, see Carrillo et al. (2018a) and the references therein. They include rosette formation in the early migration of the zebrafish lateral line primordium ( Gompel et al., 2001;Ghysen and Dambly-Chaudière, 2001 ), whose dynamics are fundamental for the correct embryonic development of the animal, and zebrafish stripe skin patterning ( Volkening and Sandstede, 2015 ). ...
We discuss several continuum cell-cell adhesion models based on the underlying microscopic assumptions. We propose an improvement on these models leading to sharp fronts and intermingling invasion fronts between different cell type populations. The model is based on basic principles of localized repulsion and nonlocal attraction due to adhesion forces at the microscopic level. The new model is able to capture both qualitatively and quantitatively experiments by Katsunuma et al. (2016). We also review some of the applications of these models in other areas of tissue growth in developmental biology. We finally explore the resulting qualitative behavior due to cell-cell repulsion.
... Let us remark that agentbased models of this type with a finite number of cells are also interesting in detailed models where differential adhesion is important, see [11] and the references therein. They include rosette formation in the early migration of the zebrafish lateral line primordium [17,18], whose dynamics are fundamental for the correct embryonic development of the animal, and zebrafish stripe skin patterning [48]. We now put further assumptions on the scaling of these potentials reflecting the attraction for distances less than some cut-off radius R together with the volume size restriction modelled by localized repulsion [9]. ...
We discuss several continuum cell-cell adhesion models based on the underlying microscopic assumptions. We propose an improvement on these models leading to sharp fronts and intermingling invasion fronts between different cell type populations. The model is based on basic principles of localized repulsion and nonlocal attraction due to adhesion forces at the microscopic level. The new model is able to capture both qualitatively and quantitatively experiments by Katsunuma et al. (2016) [J. Cell Biol. 212(5), pp. 561--575]. We also review some of the applications of these models in other areas of tissue growth in developmental biology. We finally explore the resulting qualitative behavior due to cell-cell repulsion.
... Zebrafish lateral line, comprised of anterior lateral line (aLL) and posterior lateral line (pLL), is a mechanosensory system to sense surrounding environments. From about 20 hpf on, the pLL primordium (pLLP) migrates along the horizontal myoseptum and deposits a cluster of cells at regular intervals to form proneuromasts, which ultimately generate five to six primary neuromasts in the trunk region and two to three terminal neuromasts in the tail region (Metcalfe, 1985;Gompel et al., 2001). Recently, zebrafish pLLP has emerged as a favorable model to study collective cell migration, which requires Fgf, Wnt/b-catenin, TGFb and Cxcl12a/ Cxcr4b/Cxcr7b chemokine signals (Aman and Piotrowski, 2008;Lecaudey et al., 2008;Valentin et al., 2007;Xing et al., 2015). ...
Prpf4 (pre-mRNA processing factor 4), a key component of spliceosome, plays critical roles in pre-mRNA splicing and its mutations result in retinitis pigmentosa due to photoreceptor defects. In this study, we characterized a zebrafish prpf4t243 mutant harboring a Tol2 transposon-based gene trap cassette in the third intron of the prpf4 gene. Cells in the brain and spinal cord gradually undergo p53-dependent apoptosis after 28 hpf in prpf4t243 mutants, suggesting that a widespread function of prpf4 in neural cell survival. In addition, prpf4 is essential for survival of posterior lateral line primordial (pLLP) cells. prpf4 deficiency perturbs Fgf, Wnt/β-catenin and chemokine signaling pathways and impairs pLLP migration. RNA-Seq analysis suggests that prpf4 deficiency may impair spliceosome assembly, leading to compensatory upregulation of core spliceosomal genes and alteration of pre-mRNA splicing. Taken together, our studies uncover an essential role of prpf4 in pre-mRNA splicing, cell survival and pLLP migration.
... The morphogenesis of the Drosophila eye is also facilitated by rosettes [30]. In zebrafish lateral line development, the lateral line is composed of mechanosensory organs called neuromasts which are formed from rosettes composed of 20 or more cells [31]. In other vertebrates, rosettes have been observed in the development of the mouse visceral endoderm [32], the kidney tubule [33] and the pancreas [34]. ...
Models for confluent biological tissues often describe the network formed by cells as a triple-junction network, similar to foams. However, higher order vertices or multicellular rosettes exist prevalently in developmental and {\it in vitro} processes and have been recognized as crucial in many important aspects of development, disease, and physiology. In this work, we study the influence of rosettes on the mechanics of a confluent tissue. We find that the existence of rosettes in a tissue can greatly influence its rigidity. Using a generalized vertex model and effective medium theory we find a fluid-to-solid transition driven by rosette density and intracellular tensions. This phase transition exhibits several hallmarks of a second-order phase transition such as a growing correlation length and a universal critical scaling in the vicinity a critical point. Further, we elucidate the nature of rigidity transitions in dense tissues using a generalized Maxwell constraint counting to answer a long-standing puzzle of the origin of solidity in the vertex model.
... Cranial neuromasts are called the anterior lateral line and are present on the head. Trunk neuromasts are called the posterior lateral line and include the neuromasts on the trunk and tail [10,25]. Specifically, we focused on trunk neuromast observation using 0.1% YO-PRO. ...
Backgroud
Diabetic sensorineural damage is a complication of the sensory neural system, resulting from long-term hyperglycemia. Red ginseng (RG) has shown efficacy for treatment of various diseases, including diabetes mellitus; however, there is little research about its benefit for treating sensorineural damage. Therefore, we aim to evaluate RG efficacy in alloxan-induced diabetic neuromast (AIDN) zebrafish.
Methods
In this study, we developed and validated an AIDN zebrafish model. To assess RG effectiveness, we observed morphological changes in live neuromast zebrafish. Also, zebrafish has been observed to have an ultrastructure of hair-cell cilia under SEM. Thus, we recorded these physiological traits to assess hair cell function. Finally, we confirmed that RG promoted neuromast recovery via NGF signaling pathway markers.
Results
First, we established an AIDN zebrafish model. Using this model, we showed via live neuromast imaging that RG fostered recovery of sensorineural damage. Damaged hair cell cilia were recovered in AIDN zebrafish. Furthermore, RG rescued damaged hair cell function through cell membrane ion balance.
Conclusion
Our data suggest that RG potentially facilitates recovery in AIDN zebrafish, and its mechanism seems to be promotion of the Nerve growth factor (NGF) pathway through increased expression of trkA, TRPV1 and p-MAPK.
... Rosette organization is one such process occurring in various tissues and organs in a variety of organisms, which might reflect its functional significance or selective advantage. Rosette structures have been observed in Drosophila epithelial morphogenesis (Blankenship et al., 2006), zebrafish posterior lateral line primordium (Gompel et al., 2001) and Kupffer's vesicles (Oteiza et al., 2010), Xenopus kidney development (Lienkamp et al., 2012), the chick pre-streak blastoderm (Yanagawa et al., 2011), mouse pancreatic branching morphogenesis (Villasenor et al., 2010) and visceral endoderm development (Trichas et al., 2012), and vertebrate neural tube formation (Afonso and Henrique, 2006;Eom et al., 2011;Haigo et al., 2003;Nishimura and Takeichi, 2008;Harding et al., 2014). ...
Neural rosette formation is a critical morphogenetic process during neural development, whereby neural stem cells are enclosed in rosette niches to equipoise proliferation and differentiation. How neural rosettes form and provide a regulatory micro-environment remains to be elucidated. We employed the human embryonic stem cell-based neural rosette system to investigate the structural development and function of neural rosettes. Our study shows that neural rosette formation consists of 5 types of cell movements: intercalation, constriction, polarization, elongation, and lumen formation. Ca2+ signaling plays a pivotal role in the five steps by regulating the actions of the cytoskeletal complexes, ACTIN, MYOSIN II, and TUBULIN during intercalation, constriction, and elongation. These in turn control the polarizing elements, ZO-1, PARD3, and β-CATENIN during polarization and lumen formation in neural rosette formation. We further demonstrated that the dismantlement of neural rosettes, mediated by the destruction of cytoskeletal elements, promoted neurogenesis and astrogenesis prematurely, indicating that an intact rosette structure is essential for orderly neural development.
... In addition, a cluster of cells can move as a connected group. This collective movement, which is also used by border cells in the Drosophila ovary and primordial cells during development of the zebrafish lateral line organ (Gompel et al., 2001;Montell et al., 1992), is characterized by maintenance of cell-cell junctions throughout the migration process (reviewed in Friedl and Gilmour (2009)). ...
Homeostasis of tissues is tightly regulated at the cellular, tissue and organismal level. Interestingly, tumor cells have found ways to hijack many of these physiological processes at all the different levels. Here we review how intravital microscopy techniques have provided new insights into our understanding of tissue homeostasis and cancer progression. In addition, we highlight the different strategies that tumor cells have adopted to use these physiological processes for their own benefit. We describe how visualization of these dynamic processes in living mice has broadened to our view on cancer initiation and progression.
... The lateral line system is a series of HC bundles known as neuromasts that are located around the head and along the horizontal axis of zebrafish [7]. A neuromast is composed of a core of HCs surrounded by supporting cells (SCs) [7][8][9][10]. HCs in the lateral line neuromasts of the zebrafish are reported to be morphologically and functionally similar to inner ear HCs [11,12]. ...
Loss of hair cells occurs after radiotherapy, which is a major treatment modality for head and neck cancers. In the lateral line neuromasts of zebrafish, hair cells regenerate rapidly from supporting cells after damage from ototoxins. To investigate hair cell regeneration after radiation damage, zebrafish larvae were exposed to radiation, and hair cells were counted and cell proliferation was detected in neuromasts. After irradiation exposure, cell proliferation was inhibited in neuromasts and the number of supporting cells remained stable. There was a gradual loss of hair cells in lateral line neuromasts, which was not followed by regeneration. An activator of Wnt signaling (1-azakenpaullone) promoted robust regeneration of hair cells in irradiated neuromasts. By the quantitative real-time PCR and immunofluorescence, dkk2, an inhibitory Wnt ligand, was identified upregulated in irradiated neuromasts. Accelerating the death process of irradiated hair cells by treatment with neomycin also restored the regenerative capacity of neuromasts. However, a proportion of the new hair cells died within several days after forced regeneration and baseline activity of proliferation in supporting cells remained unimproved. In conclusion, these findings suggested that radiation suppressed hair cell regeneration in zebrafish lateral line neuromasts through inhibition of Wnt signaling in supporting cells possibly by secreting anti-proliferation factors like dkk2. Maintaining a healthy supporting cell pool is vital for regeneration of hair cells.
... However, as discussed previously, any cells possessing a reduced amount of epithelial features (such as fewer or more dynamic junctions and a less structured apico-basal surface) are often able to migrate actively. In addition, although epithelial cells can exchange positions and migrate autonomously (Ewald et al., 2012;Gompel et al., 2001), the physical connection between cells means that the movement of just one cell will influence the behaviour of neighbouring cells. Thus, migratory active epithelial cells are only capable of collective cell migration, and the more cell-cell adhesion and junctions there are between cells, the more 'collective' the migratory process will be (Fig. 1B). ...
During development, cells often switch between static and migratory behaviours. Such transitions are fundamental events in development and are linked to harmful consequences in pathology. It has long been considered that epithelial cells either migrate collectively as epithelial cells, or undergo an epithelial-to-mesenchymal transition and migrate as individual mesenchymal cells. Here, we assess what is currently known about in vivo cell migratory phenomena and hypothesise that such migratory behaviours do not fit into alternative and mutually exclusive categories. Rather, we propose that these categories can be viewed as the most extreme cases of a general continuum of morphological variety, with cells harbouring different degrees or combinations of epithelial and mesenchymal features and displaying an array of migratory behaviours.
... The lateral line sensory system allows animals to orient themselves in water, sense water flow and the presence of predators or prey. The formation of the posterior lateral line starts when an ectoderm-derived placode, called the primordium, delaminates at the level of the otic vesicle and starts migrating along the trunk toward the tail tip Dambly-Chaudiere, 2004, 2007;Gompel et al., 2001;Sarrazin et al., 2010). During migration, the primordium will drop small rosette-like cell clusters called neuromasts, which are the sensory organs of this system . ...
Collective cell migration is an essential process during embryonic development and diseases such as cancer, and still much remains to be learned about how cell intrinsic and environmental cues are coordinated to guide cells to their targets. The migration-dependent development of the zebrafish sensory lateral line proves to be an excellent model to study how proteoglycans control collective cell migration in a vertebrate. Proteoglycans are extracellular matrix glycoproteins essential for the control of several signaling pathways including Wnt/β-catenin, Fgf, BMP and Hh. In the lateral line primordium the modified sugar chains on proteoglycans are important regulators of cell polarity, ligand distribution and Fgf signaling. At least five proteoglycans show distinct expression patterns in the primordium; however, their individual functions have not been studied. Here, we describe the function of glypican4 during zebrafish lateral line development. glypican4 is expressed in neuromasts, interneuromast cells and muscle cells underlying the lateral line. knypek(fr6)/glypican4 mutants show severe primordium migration defects and the primordium often U-turns and migrates back toward the head. Our analysis shows that Glypican4 regulates the feedback loop between Wnt/β-catenin/Fgf signaling in the primordium redundantly with other Heparan Sulfate Proteoglycans. In addition, the primordium migration defect is caused non-cell autonomously by the loss of cxcl12a-expressing muscle precursors along the myoseptum via downregulation of Hh. Our results show that glypican4 has distinct functions in primordium cells and cells in the environment and that both of these functions are essential for collective cell migration.
... Those deposited throughout the skin on the head, jaw, operculum and the sensory neurons from rostral to the ear are called the anterior lateral line (ALL). The primordium extends from the ear to the tail by only approximately 100 cells to form the PLL (Gompel et al., 2001;Ghysen and Dambly-Chaudiere, 2004). Appropriate spatial expression patterns of the chemokine receptors Cxcr4b and Cxcr7b, which are regulated by Wnt and Fgf signaling, direct the collective migration of the PLL primordium (Haas and Gilmour, 2006;Dambly-Chaudiere et al., 2007). ...
Subtle morphogenesis begins during trunk development after the segmentation period in zebrafish. Sprouting angiogenesis forms the trunk vascular system after the extension of the dorsal aorta and cardinal veins. The posterior lateral line primordium migrates from head to tail, forming the sensory organs on the skin. It is still unclear whether this morphogenesis requires several master regulators to control the ongoing process. Hypoxia-inducible factor 2-alpha (Hif2-alpha) plays an essential role in the maturation of both central neural system and digestive system during embryogenesis. We hypothesized that Hif2-alpha also regulates trunk development. Through loss-of-function experiments, we found that Hif2-alpha, but not Hif1-alpha, is necessary for angiogenesis and the formation of neuromasts in the trunk. Hif2-alpha regulates the expression of spleen tyrosine kinase (Syk), maintaining sprouting in the trunk. Furthermore, Hif2-alpha also regulates lymphoid enhancer factor 1 (Lef1), moderating the deposition of neuromasts. The regulation of Syk and Lef1 expression by Hif2-alpha occurs through the direct binding of Hif2-alpha to the hypoxia-responsive elements (HRE) of syk and lef1. This study provides the first evidence that Hif2-alpha plays a pivotal role in the vertebrate embryonic development of intersegmental vessels and the mechanosensory system in the zebrafish trunk.
... Primary neuromasts are named by their position, from L1 (the most anterior one) to L8. Each neuromast is composed of a central core of hair cells surrounded by mantle cells, supporting cells, and progenitor cells, and is innervated by the peripheral projections of afferent neurons located in the PLL ganglion [17][18][19][20]. Together, these peripheral projections form the PLL nerve (PLLn). ...
Background:
Regenerating damaged tissue is a complex process, requiring progenitor cells that must be stimulated to undergo proliferation, differentiation and, often, migratory behaviors and morphological changes. Multiple cell types, both resident within the damaged tissue and recruited to the lesion site, have been shown to participate. However, the cellular and molecular mechanisms involved in the activation of progenitor cell proliferation and differentiation after injury, and their regulation by different cells types, are not fully understood. The zebrafish lateral line is a suitable system to study regeneration because most of its components are fully restored after damage. The posterior lateral line (PLL) is a mechanosensory system that develops embryonically and is initially composed of seven to eight neuromasts distributed along the trunk and tail, connected by a continuous stripe of interneuromastic cells (INCs). The INCs remain in a quiescent state owing to the presence of underlying Schwann cells. They become activated during development to form intercalary neuromasts. However, no studies have described if INCs can participate in a regenerative event, for example, after the total loss of a neuromast.
Results:
We used electroablation in transgenic larvae expressing fluorescent proteins in PLL components to completely ablate single neuromasts in larvae and adult fish. This injury results in discontinuity of the INCs, Schwann cells, and the PLL nerve. In vivo imaging showed that the INCs fill the gap left after the injury and can regenerate a new neuromast in the injury zone. Further, a single INC is able to divide and form all cell types in a regenerated neuromast and, during this process, it transiently expresses the sox2 gene, a neural progenitor cell marker. We demonstrate a critical role for Schwann cells as negative regulators of INC proliferation and neuromast regeneration, and that this inhibitory property is completely dependent on active ErbB signaling.
Conclusions:
The potential to regenerate a neuromast after damage requires that progenitor cells (INCs) be temporarily released from an inhibitory signal produced by nearby Schwann cells. This simple yet highly effective two-component niche offers the animal robust mechanisms for organ growth and regeneration, which can be sustained throughout life.
... (C) A recording with the electrode placed at the center of the neuromast, the 2f microphonic has equal amplitude peaks compared to those in B. The amplitudes noted for the microphonic potentials in A, B, and C were quantified from the absolute area of the waveform between stimulus onset (position 1 on the 20 Hz stimulus trace) and 10 ms after stimulus offset (position 2) divided by the duration of the position 1e2 interval. straightforward to perform electrophysiological recordings on hair cells within primary neuromasts of the posterior lateral line (L1eL4) originating from the first primordium (primI; (Gompel et al., 2001;Pujol-Martí & López-Schier, 2013)). As mentioned above, these primary neuromasts show planar polarity along the anterioreposterior axis and contain hair cells that respond to stimuli of opposing polarity (López-Schier et al., 2004;Nicolson et al., 1998). ...
During sensory transduction, external physical stimuli are translated into an internal biological signal. In vertebrates, hair cells are specialized mechanosensory receptors that transduce sound, gravitational forces, and head movements into electrical signals that are transmitted with remarkable precision and efficiency to afferent neurons. Hair cells have a conserved structure between species and are also found in the lateral line system of fish, including zebrafish, which serve as an ideal animal model to study sensory transmission in vivo. In this chapter, we describe the methods required to investigate the biophysical properties underlying mechanosensation in the lateral line of the zebrafish in vivo from microphonic potentials and single hair cell patch-clamp recordings to single afferent neuron recordings. These techniques provide real-time measurements of hair-cell transduction and transmission following delivery of controlled and defined stimuli and their combined use on the intact zebrafish provides a powerful platform to investigate sensory encoding in vivo.
... Lateral line placodes develop anterior and posterior to the otic placode, generating, respectively, the anterior and posterior lateral line systems (ALL and PLL; Metcalfe et al., 1985; Andermann et al., 2002). The PLL placode gives rise to two cell populations: a stationary population which forms the PLL ganglion, and a migratory component which will deposit regularly spaced clusters of cells, the proneuromasts, along its way from the otic region to the tip of the tail (Metcalfe et al., 1985; Gompel et al., 2001). Deposited cells will differentiate as hair cells and accessory cells of two types: inner accessory (support) cells that surround the hair cells and a rim of outer accessory (mantle) cells. ...
Age-related hearing loss (ARHL) is a debilitating disorder for millions worldwide. While there are multiple underlying causes of ARHL, one common factor is loss of sensory hair cells. In mammals, new hair cells are not produced postnatally and do not regenerate after damage, leading to permanent hearing impairment. By contrast, fish produce hair cells throughout life and robustly regenerate these cells after toxic insult. Despite these regenerative abilities, zebrafish show features of ARHL. Here, we show that aged zebrafish of both sexes exhibited significant hair cell loss and decreased cell proliferation in all inner ear epithelia (saccule, lagena, utricle). Ears from aged zebrafish had increased expression of pro-inflammatory genes and significantly more macrophages than ears from young adult animals. Aged zebrafish also had fewer lateral line hair cells and less cell proliferation than young animals, although lateral line hair cells still robustly regenerated following damage. Unlike zebrafish, African turquoise killifish (an emerging aging model) only showed hair cell loss in the saccule of aged males, but both sexes exhibit age-related changes in the lateral line. Our work demonstrates that zebrafish exhibit key features of auditory aging, including hair cell loss and increased inflammation. Further, our finding that aged zebrafish have fewer lateral line hair cells yet retain regenerative capacity, suggests a decoupling of homeostatic hair cell addition from regeneration following acute trauma. Finally, zebrafish and killifish show species-specific strategies for lateral line homeostasis that may inform further comparative research on aging in mechanosensory systems.
Developmental studies have shown that the evolutionarily conserved Wnt planar cell polarity (PCP) pathway is essential for the development of a diverse range of tissues and organs including the brain, spinal cord, heart and sensory organs as well as establishment of the left–right body axis. Germline mutations in the highly conserved PCP gene VANGL2 in humans have only been associated with central nervous system malformations and functional testing to understand variant impact has not been performed. Here we report three new families with missense variants in VANGL2 associated with heterotaxy and congenital heart disease p.(Arg169His), non-syndromic hearing loss p.(Glu465Ala), and congenital heart disease with brain defects p.(Arg135Trp). To test the in vivo impact of these and previously described variants, we have established clinically-relevant assays using mRNA rescue of the vangl2 mutant zebrafish. We show that all variants disrupt Vangl2 function, although to different extents and depending on the developmental process. We also begin to identify that different VANGL2 missense variants may be haploinsufficient and discuss evidence in support of pathogenicity. Together, this study demonstrates that zebrafish present a suitable pipeline to investigate variants of unknown significance and suggests new avenues for investigation of the different developmental contexts of VANGL2 function that are clinically meaningful.
Aberrant activation of the Hh pathway promotes cell proliferation and multi-drug resistance (MDR) in several cancers, including Acute Myeloid Leukemia (AML). Notably, only one Hh inhibitor, glasdegib, has been approved for AML treatment, and most patients eventually relapse, highlighing the urgent need ti discover new therapeutic targets. Hh signal is transduced through the membrane of the primary cilium, a structure expressed by non-proliferating mammalian cells, whose stabilization depends on the activity of HDAC6. Here we describe a positive correlation between Hh, HDAC6, and MDR genes in a cohort of adult AML patients, human leukemic cell lines, and a zebrafish model of Hh overexpression. The hyper-activation of Hh or HDAC6 in zebrafish drove the increased proliferation of hematopoietic stem and progenitor cells (HSPCs). Interestingly, this phenotype was rescued by inhibition of HDAC6 but not of Hh. Also, in human leukemic cell lines, a reduction in vitality was obtained through HDAC6, but not Hh inhibition. Our data showed the presence of a cross-talk between Hh and HDAC6 mediated by stabilization of the primary cilium, which we detect for the first time in zebrafish HSPCs. Inhibition of HDAC6 activity alone or in combination therapy with the chemotherapeutic agent cytarabine, efficiently rescued the hematopoietic phenotype. Our results open the possibility to introduce HDAC6 as therapeutic target to reduce proliferation of leukemic blasts in AML patients.
Se estudió la morfología de las escamas situadas en ambas líneas laterales de D. eleginoides y fueron comparadas con las escamas corporales obtenidas de 12 sectores diferentes a lo largo de los ejes antero-posterior y dorso-ventral del tronco. Las escamas de ambas líneas son del tipo cicloídeas y ovaladas; están constituidas por una placa de la escama, un tubo central con una abertura anterior (ausente en algunas escamas del sector medio del tronco) y otra posterior. El análisis morfológico no mostró diferencias entre las escamas de ambas líneas; sin embargo, las escamas corporales se diferencian de las escamas de la línea lateral por la ausencia del tubo central. La mayoría de las escamas del sector anterior del cuerpo son cicloídeas y sólo algunas son del tipo ctenoídeas; mientras que en la zona media y posterior es característica la presencia de ambos tipos de escamas en el lado derecho e izquierdo del cuerpo. Los resultados obtenidos entregan nuevos antecedentes morfológicos básicos tanto para las escamas presentes en ambas líneas laterales como las corporales. Es recomendable en el caso de las escamas de las líneas laterales continuar con estudios histológicos para determinar la conexión nerviosa entre ambas y obtener así una visión más completa del sistema mecanosensorial de D. eleginoides.
The aim of this work is to provide a mathematical model to describe the early stages of the embryonic development of zebrafish posterior lateral line (PLL). In particular, we focus on evolution of PLL proto-organ (said primordium), from its formation to the beginning of the cyclical behavior that amounts in the assembly of immature proto-neuromasts towards its caudal edge accompanied by the deposition of mature proto-neuromasts at its rostral region. Our approach has an hybrid integro-differential nature, since it integrates a microscopic/discrete particle-based description for cell dynamics and a continuous description for the evolution of the spatial distribution of chemical substances (i.e., the stromal-derived factor SDF1a and the fibroblast growth factor FGF10). Boolean variables instead implement the expression of molecular receptors (i.e., Cxcr4/Cxcr7 and fgfr1). Cell phenotypic transitions and proliferation are included as well. The resulting numerical simulations show that the model is able to qualitatively and quantitatively capture the evolution of the wild-type (i.e., normal) embryos as well as the effect of known experimental manipulations. In particular, it is shown that cell proliferation, intercellular adhesion, FGF10-driven dynamics, and a polarized expression of SDF1a receptors are all fundamental for the correct development of the zebrafish posterior lateral line.
Synopsis
The posterior Lateral Line primordium migrates under the skin forming and depositing neuromasts. Wnt, Fgf and Notch interactions determine specification of a sensory hair cell progenitor at the center of each periodically formed protoneuromast. It recruits surrounding cells to form epithelial rosettes as each protoneuromast matures. The primordium migrates along a path defined by Cxcl12a expression. Ackr3b in trailing cells degrades Cxcl12a, while Cxcr4b determines directed migration in response to a self-generated Cxcl12a gradient. Finally, Emx2, Notch and the Planar Polarity pathways determine the mirror-symmetric organization of sibling sensory hair cells produced by division of each sensory hair cell progenitor.
Vertebrate organs are arranged in a stereotypic, species-specific position along the animal body plan. Substantial morphological variation exists between related species, especially so in the vastly diversified teleost clade. It is still unclear how tissues, organs and systems can accommodate such diverse scaffolds. Here, we use the sequential formation of neuromasts in the posterior lateral line (pLL) system of medaka fish to address the tissue-interactions defining a pattern. We show that the pLL pattern is established independently of its neuronal wiring, and demonstrate that the neuromast precursors that constitute the pLL behave as autonomous units during pattern construction. We uncover the necessity of epithelial integrity for correct pLL patterning by disrupting keratin 15 (krt15) and creating epithelial lesions that lead to novel neuromast positioning. By using krt15/wt chimeras, we determined that the new pLL pattern depends exclusively on the mutant epithelium, which instructs wt neuromasts to locate ectopically. Inducing epithelial lesions by 2-photon laser ablation during pLL morphogenesis phenocopies krt15 genetic mutants and reveals that epithelial integrity defines the final position of the embryonic pLL neuromasts. Our results show that a fine-balance between primordium intrinsic properties and instructive interactions with the surrounding tissues is necessary to achieve proper organ morphogenesis and patterning. We speculate that this logic likely facilitates the accommodation of sensory modules to changing and diverse body plans.
Interactions between primordium cells and their environment determines the self-organization of the zebrafish posterior Lateral Line primordium as it migrates under the skin from the ear to the tip of the tail forming and depositing neuromasts to spearhead formation of the posterior Lateral Line sensory system. In this review we describe how the NetLogo agent-based programming environment has been used in our lab to visualize and explore how self-generated chemokine gradients determine collective migration, how the dynamics of Wnt signaling can be used to predict patterns of neuromast deposition, and how previously defined interactions between Wnt and Fgf signaling systems have the potential to determine the periodic formation of center-biased Fgf signaling centers in the wake of a shrinking Wnt system. We also describe how NetLogo was used as a database for storing and visualizing the results of in toto lineage analysis of all cells in the migrating primordium. Together, the models illustrate how this programming environment can be used in diverse ways to integrate what has been learnt from biological experiments about the nature of interactions between cells and their environment, and explore how these interactions could potentially determine emergent patterns of cell fate specification, morphogenesis and collective migration of the zebrafish posterior Lateral Line primordium.
Normally folded prion protein (PrPC) and its functions in healthy brains remain underappreciated compared to the intense study of its misfolded forms (“prions”, PrPSc) during the pathobiology of prion diseases. This impedes development of therapeutic strategies in Alzheimer and Prion diseases. Disrupting the zebrafish homologs of PrPC has provided novel insights, however mutagenesis of the zebrafish paralog prp2 did not recapitulate previous dramatic developmental phenotypes, suggesting redundancy with the prp1 paralog. Here we generated zebrafish prp1 loss-of-function mutant alleles, and compound prp1-/- ;prp2-/- mutants. Zebrafish prp1-/- and compound prp1-/- ;prp2-/- mutants resemble mammalian Prnp knockouts insofar as they lack overt phenotypes, which surprisingly contrasts reports of severe developmental phenotypes when either prp1 or prp2 are knocked down acutely. Previous studies suggest that PrPC participates in neural cell development/adhesion, including in zebrafish where loss of prp2 affects adhesion and deposition pattern of lateral line neuromasts. Contrasting the expectation that functions of prp1 would be redundant to prp2, they appear to have opposing functions in lateral line neurodevelopment. Similarly, loss of prp1 blunted the seizure susceptibility phenotypes observed in prp2 mutants, contrasting the expected exacerbation of phenotypes if these prion gene paralogs were serving redundant roles. In sum, prion mutant fish lack the overt phenotypes previously predicted, and instead have subtle phenotypes similar to mammals. No evidence was found for functional redundancy in the zebrafish prion gene paralogs, and the phenotypes observed when each gene is disrupted individually are consistent with ancient functions of prion proteins in neurodevelopment and modulation of neural activity.
A description of zebrafish posterior Lateral Line (pLL) primordium development at single cell resolution together with the dynamics of Wnt, FGF, Notch and chemokine signaling in this system has allowed us to develop a framework to understand the self-organization of cell fate, morphogenesis and migration during its early development. The pLL primordium migrates under the skin, from near the ear to the tip of the tail, periodically depositing neuromasts. Nascent neuromasts, or protoneuromasts, form sequentially within the migrating primordium, mature, and are deposited from its trailing end. Initially broad Wnt signaling inhibits protoneuromast formation. However, protoneuromasts form sequentially in response to FGF signaling, starting from the trailing end, in the wake of a progressively shrinking Wnt system. While proliferation adds to the number of cells, the migrating primordium progressively shrinks as its trailing cells stop moving and are deposited. As it shrinks, the length of the migrating primordium correlates with the length of the leading Wnt system. Based on these observations we show how measuring the rate at which the Wnt system shrinks, the proliferation rate, the initial size of the primordium, its speed, and a few additional parameters allows us to predict the pattern of neuromast formation and deposition by the migrating primordium in both wild-type and mutant contexts. While the mechanism that links the length of the leading Wnt system to that of the primordium remains unclear, we discuss how it might be determined by access to factors produced in the leading Wnt active zone that are required for collective migration of trailing cells. We conclude by reviewing how FGFs, produced in response to Wnt signaling in leading cells, help determine collective migration of trailing cells, while a polarized response to a self-generated chemokine gradient serves as an efficient mechanism to steer primordium migration along its relatively long journey.
The lateral line system of teleost fishes presents large variations of patterns and forms, usually thought of as adaptive. This raises the question of how divergent adult patterns are achieved, and how selective pressures have contributed to this divergence. Our understanding of the development of this sensory system has much improved over the past 10 years, mostly through work on the zebrafish. Because this progress is restricted to a single species, we cannot yet answer questions about the determinism of lateral line evolution, but we can at least propose plausible and testable hypotheses. Here we review the mechanisms that mediate the transition from embryonic to adult pattern in the zebrafish posterior lateral line system (PLL), and we show that the adult pattern is largely determined by developmental events that take place during early larval life. We also show that simple variations in the use of the same mechanisms account for the very different patterns observed in juvenile zebrafish and blue-fin tuna, and could potentially account for many or all of the patterns observed in other adult teleosts. We conclude that, in the case of the lateral line at least, large variations in pattern depend on minor changes in the deployment of conserved developmental programs, with uncertain adaptive value. We propose that organisms neurally adapt to whatever tools they are provided with by their own development, and use them as best as they can, thereby giving the impression that such tools were actually selected for.
The expression of the surfactant-associated proteins in bronchiolar cells remains to be defined. We used in situ hybridization to identify sites of message expression of the surfactant-associated proteins A, B, and C (SP-A, SP-B, and SP-C) in adult and fetal rat lung. The expression of these messages by in situ hybridization was also compared with the localization of SP-A by immunocytochemistry. The localization of SP-A was used to identify type II cells and nonciliated bronchiolar epithelial (Clara) cells in these sections. The cRNA antisense probes for SP-A, SP-B, and SP-C appeared to hybridize over type II cells. Sense probes showed no localization or apparent specific hybridization. Messages for both SP-A and SP-B were also found in nonciliated bronchiolar epithelial (Clara) cells. However, no message for SP-C was observed in these cells. Clara cells from terminal to large bronchioles lacked detectable mRNA for SP-C. Expression of surfactant protein mRNAs was not detectable in type I cells, alveolar macrophages, interstitial cells, or vascular cells. Similarly, in fetal rat lung the messages for SP-A and SP-B but not SP-C were detected in bronchiolar cells. We conclude that rat Clara cells do not express SP-C mRNA, and thus SP-C can be regarded as a specific marker for rat type II cells.
Neuromasts, the mechanoreceptors of the lateral line system of fishes and aquatic amphibians, have previously been thought
to develop exclusively from embryonic epidermal placodes. Use of fate mapping techniques shows that neuromasts of the head
and body of zebrafish, Siamese fighting fish, and Xenopus are also derived from neural crest. Neural crest migrates away from
the neural tube in developing vertebrates to form much of the peripheral nervous system, pigment cells, and skeletal elements
of the head. The data presented here demonstrate that neuromasts are derived from both neural crest and epidermal placodes.
The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain.
The semaphorin/collapsin gene family encodes secreted and transmembrane proteins several of which can repulse growth cones. Although the in vitro activity of Semaphorin III/D/Collapsin 1 is clear, recent analyses of two different strains of semaphorin III/D/collapsin 1 knockout mice have generated conflicting findings. In order to clarify the in vivo action of this molecule, we analyzed sema Z1a, a zebrafish homolog of semaphorin III/D/collapsin 1. The expression pattern of sema Z1a suggested that it delimited the pathway of the growth cones of a specific set of sensory neurons, the posterior ganglion of the lateral line, in zebrafish. To examine the in vivo action of this molecule, we analyzed (1) the pathways followed by lateral line growth cones in mutants in which the expression of sema Z1a is altered in an interesting way, (2) response of lateral line growth cones to exogenous Sema Z1a in living embryos, and (3) the pathway followed by lateral line growth cones when Sema Z1a is misexpressed by cells along their normal route. The results suggest that a repulsive action of Sema Z1a helps guide the growth cones of the lateral line along their normal pathway.
Fgf-10-deficient mice (Fgf-10(-/-)) were generated to determine the role(s) of Fgf-10 in vertebrate development. Limb bud initiation was abolished in Fgf-10(-/-) mice. Strikingly, Fgf-10(-/-) fetuses continued to develop until birth, despite the complete absence of both fore- and hindlimbs. Fgf-10 is necessary for apical ectodermal ridge (AER) formation and acts epistatically upstream of Fgf-8, the earliest known AER marker in mice. Fgf-10(-/-) mice exhibited perinatal lethality associated with complete absence of lungs. Although tracheal development was normal, main-stem bronchial formation, as well as all subsequent pulmonary branching morphogenesis, was completely disrupted. The pulmonary phenotype of Fgf-10(-/-) mice is strikingly similar to that of the Drosophila mutant branchless, an Fgf homolog.
We examined the topography of the lateral line primary projection in zebrafish larvae by double labeling. The projections of two identified neuromasts of the posterior lateral line are seen as two separate sets of fibers that show reproducible spatial relationships: the projection of the anterior neuromast is always ventrolateral to that of a more posteriorly located neuromast. The same rule applies to the projection of anterior lateral line neuromasts. The position of the neuromasts along the antero posterior axis of the fish therefore is represented in the central projection of the sensory neurons. This somatotopy is similar to, and may be at the origin of, the tonotopic projection of the cochlear hair cells in mammals.
In the mature mouse lung, the proximal-distal (P-D) axis is delineated by two distinct epithelial subpopulations: the proximal bronchiolar epithelium and the distal respiratory epithelium. Little is known about the signaling molecules that pattern the lung along the P-D axis. One candidate is Bone Morphogenetic Protein 4 (Bmp4), which is expressed in a dynamic pattern in the epithelial cells in the tips of growing lung buds. Previous studies in which Bmp4 was overexpressed in the lung endoderm (Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. M. (1996) Development 122, 1693-1702) suggested that this factor plays an important role in lung morphogenesis. To further investigate this question, two complementary approaches were utilized to inhibit Bmp signaling in vivo. The Bmp antagonist Xnoggin and, independently, a dominant negative Bmp receptor (dnAlk6), were overexpressed using the surfactant protein C (Sp-C) promoter/enhancer. Inhibiting Bmp signaling results in a severe reduction in distal epithelial cell types and a concurrent increase in proximal cell types, as indicated by morphology and expression of marker genes, including the proximally expressed hepatocyte nuclear factor/forkhead homologue 4 (Hfh4) and Clara cell marker CC10, and the distal marker Sp-C. In addition, electron microscopy demonstrates the presence of ciliated cells, a proximal cell type, in the most peripheral regions of the transgenic lungs. We propose a model in which Bmp4 is a component of an apical signaling center controlling P-D patterning. Endodermal cells at the periphery of the lung, which are exposed to high levels of Bmp4, maintain or adopt a distal character, while cells receiving little or no Bmp4 signal initiate a proximal differentiation program.
Morphogenesis of the mouse lung involves reciprocal interactions between the epithelial endoderm and the surrounding mesenchyme, leading to an invariant early pattern of branching that forms the basis of the respiratory tree. There is evidence that Fibroblast growth factor 10 (Fgf10) and Bone Morphogenetic Protein 4 (Bmp4), expressed in the distal mesenchyme and endoderm, respectively, play important roles in branching morphogenesis. To examine these roles in more detail, we have exploited an in vitro culture system in which isolated endoderm is incubated in Matrigel(TM) substratum with Fgf-loaded beads. In addition, we have used a Bmp4(lacZ) line of mice in which lacZ faithfully reports Bmp4 expression. Analysis of lung endoderm in vivo shows a dynamic pattern of Bmp4(lacZ) expression during bud outgrowth, extension and branching. In vitro, Fgf10 induces both proliferation and chemotaxis of isolated endoderm, whether it is derived from the distal or proximal lung. Moreover, after 48 hours, Bmp4(lacZ) expression is upregulated in the endoderm closest to the bead. Addition of 30-50 ng/ml of exogenous purified Bmp4 to the culture medium inhibits Fgf-induced budding or chemotaxis, and inhibits overall proliferation. By contrast, the Bmp-binding protein Noggin enhances Fgf-induced morphogenesis. Based on these and other results, we propose a model for the combinatorial roles of Fgf10 and Bmp4 in branching morphogenesis of the lung.
The central projection of the fish lateral line displays somatotopic ordering. In order to know when and how this ordering is established, we have labelled single sensory neurones and followed the growth of their neurites. We show that the neuromast cells and the corresponding neurones are not related by a fixed lineage, and also that somatotopic differences between anterior and posterior line neurones, and among neurones of the posterior line, are present before innervation of the sense organs. We propose that the position of the central projection defines the peripheral position that the neurone will innervate.
Recent loss-of-function studies in mice show that the transcription factor GATA6 is important for visceral endoderm differentiation. It is also expressed in early bronchial epithelium and the observation that this tissue does not receive any contribution from Gata6 double mutant embryonic stem (ES) cells in chimeric mice suggests that GATA6 may play a crucial role in lung development. The aim of this study was to determine the role of GATA6 in fetal pulmonary development. We show that Gata6 mRNA is expressed predominantly in the developing pulmonary endoderm and epithelium, but at E15.5 also in the pulmonary mesenchyme. Blocking or depleting GATA6 function results in diminished branching morphogenesis both in vitro and in vivo. TTF1 expression is unaltered in chimeric lungs whereas SPC and CC10 expression are attenuated in abnormally branched areas of chimeric lungs. Chimeras generated in a ROSA26 background show that endodermal cells in these abnormally branched areas are derived from Gata6 mutant ES cells, implicating that the defect is intrinsic to the endoderm. Taken together, these data demonstrate that GATA6 is not essential for endoderm specification, but is required for normal branching morphogenesis and late epithelial cell differentiation.
Early studies of the developing lateral lines of amphibians demonstrated that a variety of well-orchestrated cellular behaviors give rise to a rather simple morphological pattern in the embryo. In 1904, Harrison first demonstrated that neuromasts of the posterior lateral lines of frogs (Rana palustris, R. Sylvatica) are derived from cells that migrate from the head region, and that the posterior lateral line nerve connects the posterior lateral line ganglion with the migrating primordium. Later, in studies of the salamander (Ambystoma punctatum) Stone (1922) demonstrated that the posterior lateral line ganglion cells and the primordial cells that form neuromasts both arise from a common postotic placode. Cells in the rostra1 part of the placode become the sensory neurons, and cells in the caudal part form the migratory primordium. As the primordium migrates caudally along the midbody line, clusters of cells are deposited at intervals from the trailing end of the primordium. These cell clusters subsequently differentiate to become the first, or “primary”, neuromasts of the midbody line. Thus, the development of the posterior lateral line involves a variety of fundamentally important but poorly understood cellular activities, such as the formation and differentiation of a placode, initiation and guidance of cell migration, and axon outgrowth and guidance. The posterior lateral line system may prove to be a good model system in which to study these phenomena because of its relative simplicity (being a simple linear array of receptors in the embryo) and accessibility (the receptors are located superficially within the epidermis) and because of the considerable background of information that is already available regarding the development of this system.
Hepatocyte nuclear factor-3β (HNF-3β), a nuclear protein of the winged helix family of transcription factors, is known to play a critical role in the formation of the embryonic node, notochord, and foregut endoderm. HNF-3β influences the expression of a number of target genes in the respiratory epithelium, activating transcription of thyroid transcription factor-1, surfactant protein-B and clara cell secretory protein. In order to discern the role of HNF-3β in differentiation and gene expression in the lung, HNF-3β was expressed in developing respiratory epithelial cells of transgenic mice, under the control of the human surfactant protein C gene promoter. Pulmonary abnormalities were observed in the lungs of fetal mice bearing the HNF-3β transgene. Differentiation of distal respiratory epithelial cells was arrested in the early pseudoglandular stage. Branching morphogenesis and vasculogenesis were markedly disrupted in association with decreased E-cadherin and vascular endothelial growth factor expression. HNF-3β limits cellular diversity of developing respiratory epithelium and alters lung morphogenesis in vivo, suggesting that precise temporal–spatial regulation of HNF-3β expression is critical for respiratory epithelial cell differentiation and lung morphogenesis. Dev. Dyn. 1997;210: 305–314. © 1997 Wiley-Liss, Inc.
We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley-Liss, Inc.
The zinc-finger transcription factor GATA-1 (previously known as GF-1, NF-E1 or Eryf 1 binds to GATA consensus elements in regulatory regions of the alpha- and beta-globin gene clusters and other erythroid cell-specific genes. Analysis of the effects of mutations in GATA-binding sites in cell culture and in binding assays in vitro, as well as transactivation studies with GATA-1 expression vectors in heterologous cells, have provided indirect evidence that this factor is involved in the activation of globin and other genes during erythroid cell maturation. GATA-1 is also expressed in megakaryocytes and mast cells, but not in other blood cell lineages or in non-haemopoietic cells. To investigate the role of this factor in haematopoiesis in vivo, we disrupted the X-linked GATA-1 gene by homologous recombination in a male (XY) murine embryonic stem cell line and tested the GATA-1-deficient cells for their ability to contribute to different tissues in chimaeric mice. The mutant embryonic stem cells contributed to all non-haemopoietic tissues tested and to a white blood cell fraction, but failed to give rise to mature red blood cells. This demonstrates that GATA-1 is required for the normal differentiation of erythroid cells, and that other GATA-binding proteins cannot compensate for its absence.
This study reports that zn-1, a monoclonal antibody, labels hair cells but not supporting cells in the inner ear and the lateral line of the axolotl salamander, Ambystoma mexicanum. Zn-1 immunocytochemically labels the cytoplasm and stereocilia of mature hair cells in the sacculus, in the utriculus, and in the mechanoreceptive neuromast organs of the lateral line. Lower levels of labeling mark newly formed hair cells in the periphery of the sacculus and in regenerating neuromasts. Zn-1 also selectively labels neuronal processes and perikarya in the lateral line nerves and ganglia and the VIIIth cranial nerve and ganglion. Processes and perikarya are labeled by zn-1 in the dorsolateral medulla oblongata, at sites of termination of the afferent octaval and lateral line neurons. Western blot analysis revealed that zn-1 labels one or more proteins with molecular weights of 80 and 160 kDa. The identity of these protein bands remains to be determined. The presence of a specific epitope expressed in both hair cells and neurons, but not in supporting cells, in the vestibular and auditory epithelia of the ear and in the mechanoreceptive neuromasts of the lateral line suggests shared cytogenetic heritages. These findings are consistent with a close evolutionary relationship between otic and lateral line senses, such as that inherent to the theoretical evolutionary scheme outlined in van Bergeijk's "acousticolateralis hypothesis." The protein recognized by zn-1 is as yet unidentified, but its conservative evolution suggests that it may serve an important function in the statoacoustic and lateral line systems.
As we reported earlier, type II alveolar epithelial cells make their appearance in the early embryonic mouse lung around day 14.2, and show distinctive ultrastructural features. The present study focuses on the ultrastructural characteristics of the inclusion bodies by investigating embryos aged 17-19 days (birth on day 19), using transmission electron microscopy. Late embryonic type II cells appear also as low-columnar or cuboid cells having large, approximately round nuclei and cytoplasm displaying typical features of a differentiated cell. The inclusion bodies show a widespread distribution and are extremely variable in appearance. Schematically we discern five main types, namely cytoplasmic, granular/flocculent, multivesicular, dense, and (multi)lamellar, which occur with intermediate and composite forms. All these inclusion bodies frequently contain glycogen particles, and show a structural relation to profiles of endoplasmic reticulum which are wrapped around them. Other distinctive properties are the osmiophily of multivesicular inclusion bodies, and the presence of vesicles in many dense inclusion bodies. The possible interrelationship, and the differences in various aspects of electron density, suggest that the five main types of inclusion bodies may represent different stages in the formation of mature multilamellar bodies.
Growing retinal axons home to their distant target, the tectum, even when they are displaced from their normal pathway. This argues for long-range guidance mechanisms in the embryonic brain. Growth cones may orientate to diffusible attractants released from the target, as proposed in other systems, or they may use a stable distribution of positional information in the neuroepithelium. To distinguish between these possibilities, small pieces of the presumptive optic tract, through which retinal axons will normally grow, were rotated by approximately 90 degrees either clockwise or counterclockwise. When the retinal axons later encountered the rotated neuroepithelium, they also turned clockwise or counterclockwise, in correspondence with the direction of rotation. This demonstrates that long-range navigation of retinal axons in the vertebrate brain is based partly on stable, local positional factors, rather than on remote diffusible factors.
The phylogenetic and ontogenetic changes in the octavolateralis system of sarcopterygian fish and tetrapods, presumed to be important for the formation of an amphibian auditory system, are reviewed. The lateral line system shows rudimentation of lines and loss of ampullary electroreceptors in many amphibians; in some amphibians it never develops. The metamorphic changes of the lateral-line system show different patterns in the different amphibian lineages with metamorphic retention in most urodeles and metamorphic loss in most anurans. The multitude of both ontogenetic and phylogenetic changes of the lateral line system among amphibians do exclude any prediction as to how this system might have changed in ancestral amniotes. The most important auditory epithelium of the tetrapod inner ear, the basilar papilla, seems to be primitively present in all tetrapods and Latimeria. In two amphibian lineages there is a trend towards rudimentation and loss of the basilar papilla. Only in the third order, the anurans, a tympanic ear develops and the inner ear shows a progressive evolution of the auditory epithelia. Together with the known differences in the periotic labyrinth of amphibians and amniotes, this scenario suggests a parallel evolution of the amniotic and anuran auditory periphery. All mechanoreceptive hair cells of the lateral line system and the inner ear appear to receive a common and bilateral efferent innervation. Among amphibians this pattern is represented only in some urodeles, whereas anurans show a derived pattern with loss of a bilateral component and presumably also of a common neuromast/inner ear component. Changes in the rhombencephalic nuclei which receive octavo-lateralis afferent fibers show a trend towards development of auditory nuclei only in the anuran lineage. The phylogenetic appearance of an auditory nucleus in this lineage coincides with the complete absence of formation of ampullary electroreceptors. In contrast, the earlier claim of a correlation between a metamorphic loss of the lateral line system and the formation of an auditory nucleus is not supported by more recent data: an auditory nucleus develops in anurans already prior to metamorphosis and is present in all anurans even when they retain the neuromast system. In anurans with a metamorphic loss of the neuromasts, the second order neurons degenerate as well. This independence of the auditory and the second order lateral line nuclei is further substantiated by their separate projection to other brain areas, like the torus semicircularis of the midbrain, and their functional properties.(ABSTRACT TRUNCATED AT 400 WORDS)
We studied the anatomy of neuromasts, afferent sensory neurons, and efferent neurons of the midbody branch of the posterior lateral line in larvae of the zebrafish (Brachydanio rerio), 5 days after fertilization. This simple sensory system consists often or 11 neuromasts, 15–20 sensory neurons, and about nine efferent neurons. The neuromasts are typical free neuromasts and both afferent and efferent synapses are present on hair cells within them. The sensory neurons project into a single longitudinal column of neuropil in the hindbrain. The sensory terminals appear by light microscopy to contact the dorsolateral dendrite of the ipsilateral Mauthner cell. Three types of efferent neurons are present; two types in the hindbrain and one type in the diencephalon. We provide several lines of evidence that demonstrate that these central neurons are efferent to the lateral line.
We conclude from this morphology that the larval system includes all of the components of the adult system and is probably functional at this early stage. We also found that larvae have all of the efferent neurons found in adult zebrafish, while the number of neuromasts and sensory neurons will increase during subsequent development.
The development of neuromasts and sensory neurons of the posterior lateral line was studied in zebrafish (Brachydanio rerio) in order to determine the relationship between growing axons of sensory neurons and the migratory cellular primordium of midbody line neuromasts. Scanning electron microscopy revealed that a primary system of six neuromasts develops during the second day after fertilization and evidence is presented that these arise from cells of a migratory primordium. The primordium is first detected in the postauditory region immediately adjacent to the developing sensory ganglion. Growth cones of posterior lateral line sensory neurons are found within the premigratory primordium when it is adjacent to the ganglion. At later times growth cones of these sensory neurons are found within the primordium as it migrates caudally along the midbody line.
These results demonstrate that although the growth cones of the sensory neurons grow over a considerable distance to their final destination, they are never very far from their target cells (or target cell precursors), which migrate with them and may even lead them.
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Transgenic animals bearing a chimeric gene containing 5'-flanking regions of the human surfactant protein C (SP-C) gene ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene were analyzed by in situ hybridization histochemistry to determine the temporal and spatial distribution of transgene expression during organogenesis of the murine lung. Ontogenic expression of the SP-C-CAT gene was compared to that of the endogenous SP-C gene and to the Clara cell CC10 gene. High levels of SP-C-CAT expression were observed as early as Day 10 of gestation in epithelial cells of the primordial lung buds. Low levels of endogenous SP-C mRNA were detected a day later, but only in the more distal epithelial cells of the newly formed, primitive, lobar bronchi. On Gestational Days 13 through 16, transcripts for both the endogenous and chimeric gene were restricted to distal epithelial elements of the branching bronchial tubules and were no longer detected in the more proximal regions of the bronchial tree. Although high levels of SP-C-CAT expression were maintained throughout organogenesis, endogenous SP-C expression increased dramatically on Gestational Day 15, coincident with acinar tubule differentiation at the lung periphery. Low levels of endogenous CC10 expression were detected by Gestational Day 16 in both lobar and segmental bronchi. By the time of birth, CC10 transcripts were expressed at high levels in the trachea and at all levels of the bronchial tree; endogenous SP-C mRNA was restricted to epithelial cells of the terminal alveolar saccules; and SP-C-CAT expression was now detected in both alveolar and bronchiolar epithelial cells. These results indicate that (1) cis-acting regulatory elements of the human SP-C gene can direct high levels of foreign gene expression to epithelial cells of the embryonic mouse lung; (2) expression of the human SP-C-CAT chimeric gene is developmentally regulated, exhibiting a morphogenic expression pattern similar, but not identical, to that of the endogenous murine SP-C gene; (3) the embryonic expression of endogenous SP-C and chimeric SP-C-CAT transcripts identifies progenitor cells of the distal respiratory epithelium; and (4) differentiation of bronchial epithelium is coincident with loss of SP-C expression and subsequent acquisition of CC10 expression in proximal regions of the developing bronchial tubules.
Transforming growth factor-beta 1 (TGF-beta 1) influences the morphogenesis of many organs, regulating cell growth, differentiation, gene expression, extracellular matrix deposition, and angiogenesis. In order to assess the effects of TGF-beta 1 on lung development in vivo, transgenic mice were generated bearing a chimeric gene composed of human surfactant protein C (SP-C) gene promoter and the porcine TGF-beta 1 cDNA mutated to ensure constitutive activation of the TGF-beta 1 peptide. Because of the perinatal loss related to the SP-C-TGF-beta 1 transgene, embryos bearing the transgene were obtained on Days 16 and 18.5 of gestation. TGF-beta 1 was selectively expressed in respiratory epithelial cells of the transgenic embryos. Body weight, length, and lung size were not altered in the transgenic embryos; however, lung morphogenesis of Day 18.5 transgenic mice was arrested in a late pseudoglandular stage of development, while that of their nontransgenic littermates was typical of the saccular stage. Lungs of transgenic mice on Day 16 contained fewer acinar buds than those of nontransgenic littermates. At both ages, epithelial cell differentiation, assessed by the expression of Clara cell secretory protein2 and pro-SP-C, was inhibited. While collagen III deposition was not affected by the transgene, collagen I expression was persistent in terminal airways of fd 18.5 transgenic lungs. The distribution of alpha-smooth muscle actin was markedly altered, being detected in the mesenchyme surrounding the distal leading edges of epithelial tubules in the SP-C-TGF-beta 1 transgenic mice. Expression of TGF-beta 1 in the developing respiratory epithelium of transgenic mice arrested lung sacculation and epithelial cell differentiation in vivo, supporting the role of TGF-beta family members in lung morphogenesis and differentiation.
The zinc finger transcription factor GATA4 has been implicated in heart development based on its early expression in precardiogenic splanchnic mesoderm and its ability to activate the expression of a number of cardiac-specific genes. To determine the role of GATA4 in embryogenesis, we generated mice homozygous for a GATA4 null allele. Homozygous GATA4 null mice arrested in development between E7.0 and E9.5 because of severe developmental abnormalities. Mutant embryos most notably lacked a primitive heart tube and foregut and developed partially outside the yolk sac. In the mutants, the two bilaterally symmetric promyocardial primordia failed to migrate ventrally but instead remained lateral and generated two independent heart tubes that contained differentiated cardiomyocytes. We show that these deformities resulted from a general loss in lateral to ventral folding throughout the embryo. GATA4 is most highly expressed within the precardiogenic splanchnic mesoderm at the posterior lip of the anterior intestinal portal, corresponding to the region of the embryo that undergoes ventral fusion. We propose that GATA4 is required for the migration or folding morphogenesis of the precardiogenic splanchnic mesodermal cells at the level of the AIP.
We have identified and characterized c-hairy1, an avian homolog of the Drosophila segmentation gene, hairy. c-hairy1 is strongly expressed in the presomitic mesoderm, where its mRNA exhibits cyclic waves of expression whose temporal periodicity corresponds to the formation time of one somite (90 min). The apparent movement of these waves is due to coordinated pulses of c-hairy1 expression, not to cell displacement along the anteroposterior axis, nor to propagation of an activating signal. Rather, the rhythmic c-hairy mRNA expression is an autonomous property of the paraxial mesoderm. These results provide molecular evidence for a developmental clock linked to segmentation and somitogenesis of the paraxial mesoderm, and support the possibility that segmentation mechanisms used by invertebrates and vertebrates have been conserved.
NKX2.1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. We demonstrate that septation of the anterior foregut along the dorsoventral axis, into distinct tracheal and esophageal structures, is blocked in mouse embryos carrying a homozygous targeted disruption of the Nkx2.1 locus. This is consistent with the loss of Nkx2.1 expression, which defines the dorsoventral boundary within the anterior foregut in wild-type E9 embryos. Failure in septation between the trachea and the esophagus in Nkx2.1(-/-) mice leads to the formation of a common lumen that connects the pharynx to the stomach, serving both as trachea and as esophagus, similar in phenotype to a human pathologic condition termed tracheoesophageal fistula. The main-stem bronchi bifurcate from this common structure and connect to profoundly hypoplastic lungs. The mutant lungs fail to undergo normal branching embryogenesis, consist of highly dilated sacs that are not capable of sustaining normal gas exchange functions, and lead to immediate postnatal death. In situ hybridization suggests reduced Bmp-4 expression in the mutant lung epithelium, providing a possible mechanistic clue for impaired branching. Functional deletion of Nkx2. 1 blocks pulmonary-specific epithelial cell differentiation marked by the absence of pulmonary surfactant protein gene expression. Altered expression of temporally regulated genes such as Vegf demonstrates that the lung in Nkx2.1(-/-) mutant embryos is arrested at early pseudoglandular (E11-E15) stage. These results demonstrate a critical role for Nkx2.1 in morphogenesis of the anterior foregut and the lung as well as in differentiation of pulmonary epithelial cells.
The gene coding for the murine transcription factor GATA6 was inactivated by insertion of a beta-galactosidase marker gene. The analysis of heterozygote GATA6/lacZ mice shows two inductions of GATA6 expression early in development. It is first expressed at the blastocyst stage in part of the inner mass and in the trophectoderm. The second wave of expression is in parietal endoderm (Reichert's membrane) and the mesoderm and endoderm that form the heart and gut. Inactivation leads to a lethality shortly after implantation (5.5 days postcoitum). Chimeric experiments show this to be caused by an indirect effect on the epiblast due to a defect in an extraembryonic tissue.
GATA-3 is a zinc-finger transcription factor that is essential for both early T cell development and Th2 cell differentiation. To quantify GATA-3 expression during T cell development in vivo in the mouse, the GATA-3 gene was targeted by insertion of a lacZ reporter by homologous recombination in embryonic stem (ES) cells. Although we could detect GATA-3+ cells throughout T cell development in the thymus, the proportions of GATA-3+ cells varied considerably between the distinct differentiation stages. The two periods of TCR alpha and beta gene recombination, which occur in quiescent or slowly dividing cells, were associated with low proportions of GATA-3+ cells. Conversely, the stage of rapidly proliferating cells, which insulates these two waves of TCR rearrangement, was characterized by a large proportion of GATA-3+ cells. In addition, we generated chimeric mice by injection of GATA-3-deficient, lacZ-expressing ES cells into wild-type blastocysts. In this in vivo competition analysis, no contribution of GATA-3-deficient cells to the T cell lineage was detected, not even in the earliest CD44+CD25- double-negative (CD4-CD8-) cell stage in the thymus. These results parallel data implicating other GATA family members as key regulators of proliferation and survival of early hematopoietic cells. We therefore propose that GATA-3 is required for the expansion of T cell progenitors, and for the control of subsequent proliferation steps, which alternate periods of TCR recombination in the thymus.
Semaphorins/collapsins are a large family of secreted and cell surface molecules that are thought to guide growth cones to their targets. Although some members are clearly repulsive to specific growth cones in vitro, the in vivo role of many of these molecules in vertebrate embryos is still unclear. As a first step towards clarifying the in vivo role of semaphorins/collapsins, we analyzed semaZ1a in the simple and well-characterized zebrafish embryo. SemaZ1a is a secreted molecule that is highly homologous to Sema III/D/collapsin-1, and it can collapse chick dorsal root ganglion growth cones in vitro. It is expressed in highly specific patterns within the developing embryo, which suggests that it influences outgrowth by a variety of growth cones including those of the posterior lateral line ganglion. Consistent with this hypothesis, the peripherally extending growth cones of posterior lateral line neurons retract and partially collapse during normal outgrowth.
To form a diffusible interface large enough to conduct respiratory gas exchange with the circulation, the lung endoderm undergoes extensive branching morphogenesis and alveolization, coupled with angiogenesis and vasculogenesis. It is becoming clear that many of the key factors determining the process of branching morphogenesis, particularly of the respiratory organs, are highly conserved through evolution. Synthesis of information from null mutations in Drosophila and mouse indicates that members of the sonic hedgehog/patched/smoothened/Gli/FGF/FGFR/sprouty pathway are functionally conserved and extremely important in determining respiratory organogenesis through mesenchymal-epithelial inductive signaling, which induces epithelial proliferation, chemotaxis and organ-specific gene expression. Transcriptional factors including Nkx2.1, HNF family forkhead homologues, GATA family zinc finger factors, pou and hox, helix-loop-helix (HLH) factors, Id factors, glucocorticoid and retinoic acid receptors mediate and integrate the developmental genetic instruction of lung morphogenesis and cell lineage determination. Signaling by the IGF, EGF and TGF-beta/BMP pathways, extracellular matrix components and integrin signaling pathways also directs lung morphogenesis as well as proximo-distal lung epithelial cell lineage differentiation. Soluble factors secreted by lung mesenchyme comprise a 'compleat' inducer of lung morphogenesis. In general, peptide growth factors signaling through cognate receptors with tyrosine kinase intracellular signaling domains such as FGFR, EGFR, IGFR, PDGFR and c-met stimulate lung morphogenesis. On the other hand, cognate receptors with serine/threonine kinase intracellular signaling domains, such as the TGF-beta receptor family are inhibitory, although BMP4 and BMPR also play key inductive roles. Pulmonary neuroendocrine cells differentiate earliest in gestation from among multipotential lung epithelial cells. MASH1 null mutant mice do not develop PNE cells. Proximal and distal airway epithelial phenotypes differentiate under distinct transcriptional control mechanisms. It is becoming clear that angiogenesis and vasculogenesis of the pulmonary circulation and capillary network are closely linked with and may be necessary for lung epithelial morphogenesis. Like epithelial morphogenesis, pulmonary vascularization is subject to a fine balance between positive and negative factors. Angiogenic and vasculogenic factors include VEGF, which signals through cognate receptors flk and flt, while novel anti-angiogenic factors include EMAP II.
Pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) remains a major therapeutic problem. Moreover, the pathogenesis of pulmonary hypoplasia in case of CDH is controversial. In particular, little is known about early lung development in this anomaly. To investigate lung development separate from diaphragm development we used an in vitro modification of the 2, 4-dichlorophenyl-p-nitrophenylether (Nitrofen) animal model for CDH. This enabled us to investigate the direct effects of Nitrofen on early lung development and branching morphogenesis in an organotypic explant system without the influence of impaired diaphragm development. Epithelial cell differentiation of the lung explants was assessed using surfactant protein-C and Clara cell secretory protein-10 mRNA expression as markers. Furthermore, cell proliferation and apoptosis were investigated. Our results indicate that Nitrofen negatively influences branching morphogenesis of the lung. Initial lung anlage formation is not affected. In addition, epithelial cell differentiation and cell proliferation are attenuated in lungs exposed to Nitrofen. These data indicate that Nitrofen interferes with early lung development before and separate from (aberrant) diaphragm development. Therefore, we postulate the dual-hit hypothesis, which explains pulmonary hypoplasia in CDH by two insults, one affecting both lungs before diaphragm development and one affecting the ipsilateral lung after defective diaphragm development.
The numbers and positions of cells undergoing cell death and proliferation in the neuromasts of 10 day old zebrafish larvae were assessed to investigate the ability of supporting cells to differentiate into hair cells. Evaluations of cell death and proliferation showed that a subpopulation of cells located in the centre of the neuromast undergo cell death, and a different subpopulation located at the periphery proliferate. This suggests that cell death of hair cells and proliferation of mantle supporting cells occurs as part of normal development, creating constant turnover of hair cells. We show that the caspase inhibitor zVADfmk reduces cell death while the aminoglycoside neomycin specifically induces an increased amount of cell death in the central population of cells. Both of these treatments affect the rate of proliferation of the peripheral subpopulation of cells in the neuromast suggesting that a feedback mechanism occurs regulating cell death and proliferation. We propose that the dying population of cells are hair cells and the proliferating cells are 'mantle' supporting cells, which is in agreement with previous observations suggesting that supporting cells can give rise to hair cells following hair cell death.
Vertebrate segmentation initiates with the subdivision of the paraxial mesoderm into a regular array of somites. Recent evidence suggests that the segmentation clock - a biochemical oscillator acting in the unsegmented paraxial mesoderm cells in most vertebrates - controls cyclic Notch signalling, resulting in periodic formation of somite boundaries.
Expression of a mouse atonal homologue, math1, defines cells with the potential to become sensory hair cells in the mouse inner ear (Science 284 (1999) 1837) and Notch signaling limits the number of cells that are permitted to adopt this fate (Nat. Genet. 21 (1999) 289; J. Neurocytol. 28 (1999) 809). Failure of lateral inhibition mediated by Notch signaling is associated with an overproduction of ear hair cells in the zebrafish mind bomb (mib) and deltaA mutants (Development 125 (1998a) 4637; Development 126 (1999) 5669), suggesting a similar role for these genes in limiting the number of hair cells in the zebrafish ear. This study extends the analysis of proneural and neurogenic gene expression to the lateral line system, which detects movement via clusters of related sensory hair cells in specialized structures called neuromasts. We have compared the expression of a zebrafish atonal homologue, zath1, and neurogenic genes, deltaA, deltaB and notch3, in neuromasts and the posterior lateral line primordium (PLLP) of wild-type and mib mutant embryos. We describe progressive restriction of proneural and neurogenic gene expression in the migrating PLLP that appears to correlate with selection of hair cell fate in maturing neuromasts. In mib mutants there is a failure to restrict expression of zath1 and Delta homologues in the neuromasts revealing similarities with the phenotype previously described in the ear.
Organization and development of the zebrafish posterior lateral line
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The level of the tissue-speci®c factor GATA-1 affects the cell-cycle machin-ery The transcription factor GATA-3 is neces-sary and suf®cient for Th2 cytokine gene expression in CD4 T cells
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Over-expression of GATA-6 in Xenopus embryos blocks differentiation of heart precursors (published erratum appears in EMBO J A stable lead by modi®cation of Sato's method
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Manipulating the Mouse Embryo. A Laboratory Manual, Cold Spring Harbor Labora-tory Press, Cold Spring Harbor Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium
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Whit-sett and Tichelaar, 1999) MASH1 and HES1 regulate the differentiation of pulmonary neuroendocrine cells TTF1 was shown to positively in¯uence All rights reserved
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HES1, TTF1, HFH4 and GATA6 have been implicated in epithelial cell differentiation and surfactant protein produc-tion (Perl and Whitsett, 1999; Warburton et al., 2000; Whit-sett and Tichelaar, 1999). MASH1 and HES1 regulate the differentiation of pulmonary neuroendocrine cells (Ito et al., 2000). TTF1 was shown to positively in¯uence BMP4 Mechanisms of Development 105 (2001) 105±114 0925-4773/01/$ -see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-4773(01)00386-0 www.elsevier.com/locate/modo * Corresponding author. Tel.: 131-10-408-7593; fax: 131-10-408-9468.
Über die Sinnesorgane der Seitenlinie bei Fische und Amphibian
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Experimentelle Untersuchung über die Entwicklung der Sinnesorgane der Seitenlinie bei den Amphibien
- Harrisson