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Precocious Neural Differentiation in mir-302-Knockout Embryos (A) Tuj1 immunohistochemistry to visualize b-III-tubulin+ post-mitotic neural cells. Quantification of Tuj1+ cells was calculated as the percentage of Tuj1+ cells out of total neuroepithelial cells (DAPI+) at E9.5. Error bars represent SD (n = 3 embryos, six sections/embryo). *p < 0.05, **p < 0.005. The scale bar represents 200 mm. (B) Immunohistochemistry against Ki67 was used to identify cycling cells, and Tuj1 was used to identify neurons. Quantification of Ki67+ cells was calculated as the percentage of Ki67+ cells out of total neuroepithelial cells at E9.5. Error bars represent SD (n = 3 embryos, six sections per embryo). *p < 0.05, **p < 0.005. Increased Ki67+ staining and increased Tuj1+ staining in the knockout is due to a greater number of Ki67+ cells that are also Tuj1+. The scale bar represents 100 mm. (C) In situ hybridization using probe against Btg2 to identify neurogenic dividing cells. (D) In situ hybridization using probe against Hes5 to identify neural progenitors. Transverse sections of embryonic hindbrain counterstained with Fast Red. (E) Individual neuroepithelial cells were plated at clonal density, incubated for 24 hr, fixed, then stained for Tuj1. Pairs of cells that were generated from a single precursor were scored for three possible division types. Quantification represents the average of >150 cells per embryo and genotype (n = 3 embryos). *p < 0.05, **p < 0.005.
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The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepith...
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... for Tuj1 in the hindbrain at E8.5 showed no staining in either wild-type or knockout neural tubes. However, small numbers of Tuj1+ cells were seen in knockout hindbrains as early as E9.0 but not in wild-type ( Figure S4-1A). By E9.5, there was a striking increase in the number of Tuj1+ cells in both the midbrain and hindbrain of the knockout embryos compared with wild-type (Figures 4A, S4-1B, and S4-1C). ...
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... indicate area of dorsolateral bending. as Tuj1+/Ki67+ cells in knockout embryos compared with wild- type, which is consistent with the simultaneous expansion of progenitors and precocious differentiation ( Figure 4B). ...
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... is expressed in and required for neurogenic pro- genitor divisions starting around E9.5 ( Iacopetti et al., 1999;Farioli-Vecchioli et al., 2009). mir-302-knockout embryos showed increased Btg2 expression at E9.5 and E10.5 ( Figures 4C and S4-1F). At E9.5, increased expression was seen in the mid and hindbrain region. ...
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... addition to Btg2, other markers of neuroepithelial progenitors, such as Hes1 and Hes5, were analyzed ( Ishibashi et al., 1995;Hatakeyama et al., 2004). Hes5 showed increased staining in the midbrain and hindbrain at both E8.5 and E9.5 ( Figures 4D and S4-1G). Similarly, Hes1 also showed ectopic expression in the hindbrain neuroepithe- lium at E9.5 that extended into the midbrain and forebrain ( Figure S4-1H). ...
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... showed increased staining in the midbrain and hindbrain at both E8.5 and E9.5 ( Figures 4D and S4-1G). Similarly, Hes1 also showed ectopic expression in the hindbrain neuroepithe- lium at E9.5 that extended into the midbrain and forebrain ( Figure S4-1H). ...
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... more directly measure neurogenic divisions, we performed analysis of daughter pairs arising from individual dividing cells derived from dissociated anterior neural tubes. Individual dividing cells had three possible outcomes: symmetric division into two Tuj1À progenitors (P/P), symmetric division into two Tuj1+ neurons (N/N), or asymmetric division into one Tuj1À pro- genitor and one Tuj1+ neuron (P/N) ( Figure 4E). Quantification of these divisions showed a preponderance of the P/P divisions in both wild-type and knockout embryos ($90%). ...
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... of these divisions showed a preponderance of the P/P divisions in both wild-type and knockout embryos ($90%). However, there was a small yet highly significant increase in the number of N/N divisions in knockout embryos ( Figure 4E). There was also a trend toward increased P/N divisions (p = 0.35). ...
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... contrast to the changes in proliferation, apoptosis, and dif- ferentiation, there did not appear to be any changes in embry- onic axis specification associated with mir-302 loss. Knockout mice showed normal anterior-posterior patterning, as indicated by FoxG1 (forebrain), En-2, Gbx2 (midbrain), and Krox20 (hind- brain) expression ( Figure S4-2A and S4-2B). Additionally, there were no obvious differences in dorsal or ventral patterning of the spinal cord at E9.5, as measured by Msx1/2, Pax3, Pax6, and Nkx2.2 immunostaining ( Figure S4-2C). ...
Citations
... Furthermore, the elevated levels of miR-302b have been associated with caspase-3-induced apoptosis in SH-SY5Y cells (Yadav et al., 2011). Investigations using a miR-302-deficient mouse model revealed that the absence of miR-302 disrupted processes such as cell proliferation, apoptosis, differentiation, and neural tube closure during early neurodevelopment (Parchem et al., 2015). This underlines the pivotal role of miR-302 in neuronal differentiation and embryonic viability. ...
Chemical-induced neurotoxicity is increasingly recognized to accelerate the development of neurodegenerative disorders (NDs), which pose an increasing health burden to society. Attempts are being made to develop drugs that can cross the blood–brain barrier and have minimal or no side effects. Nobiletin (NOB), a polymethoxylated flavonoid with anti-oxidative and anti-inflammatory effects, has been demonstrated to be a promising compound to treat a variety of NDs. Here, we investigated the potential role of NOB in sodium arsenate (NA)-induced deregulated miRNAs and target proteins in human neural progenitor cells (hNPCs). The proteomics and microRNA (miRNA) profiling was done for different groups, namely, unexposed control, NA-exposed, NA + NOB, and NOB groups. Following the correlation analysis between deregulated miRNAs and target proteins, RT-PCR analysis was used to validate the selected genes. The proteomic analysis showed that significantly deregulated proteins were associated with neurodegeneration pathways, response to oxidative stress, RNA processing, DNA repair, and apoptotic process following exposure to NA. The OpenArray analysis confirmed that NA exposure significantly altered miRNAs that regulate P53 signaling, Wnt signaling, cell death, and cell cycle pathways. The RT-PCR validation studies concur with proteomic data as marker genes associated with autophagy and apoptosis (HO-1, SQSTM1, LC-3, Cas3, Apaf1, HSP70, and SNCA1) were altered following NA exposure. It was observed that the treatment of NOB significantly restored the deregulated miRNAs and proteins to their basal levels. Hence, it may be considered one of its neuroprotective mechanisms. Together, the findings are promising to demonstrate the potential applicability of NOB as a neuroprotectant against chemical-induced neurotoxicity.
... According to mounting evidences, the Hippo pathway regulates the proliferation, migration, apoptosis, and differentiation of nerve cells as well as the formation of synapses, the corpus callosum, and the cortex, all of which are important for the development of the nervous system (Li et al., 2021;Sahu and Mondal, 2021). In general, gga-miR-302 participates in neurulation, which is a distinguishing characteristic of vertebrate development and is necessary for healthy CNS development and embryo survival (Parchem et al., 2015). ...
In the early stages of embryonic development, a precise and strictly controlled hierarchy of gene expression is essential to ensure proper development of all cell types and organs. To better understand this gene control process, we constructed a small RNA library from 1- to 5-day-old chick embryos, and identified 2,459 miRNAs including 827 existing, 695 known, and 937 novel miRNAs with bioinformatic analysis. There was absolute high expression of a number of miRNAs in each stage, including gga-miR-363-3p (Em1d), gga-miR-26a-5p (Em2d and Em3d), gga-miR-10a-5p (Em4d), and gga-miR-199-5p (Em5d). We evaluated enriched miRNA profiles, identifying VEGF, Insulin, ErbB, MAPK, Hedgehog, TLR and Hippo signaling pathways as primary regulatory mechanisms enabling complex morphogenetic transformations within tight temporal constraints. Pathway analysis revealed miRNAs as pivotal nodes of interaction, coordinating cascades of gene expression critical for cell fate determination, proliferation, migration, and differentiation across germ layers and developing organ systems. Weighted Gene Co-Expression Network Analysis (WGCNA) generated hub miRNAs whose modular connections spanned regulatory networks, including: gga-miR-181a-3p (blue module), coordinating immunegenesis and myogenesis; gga-miR-126-3p (brown module), regulating vasculogenesis and angiogenesis; gga-miR-302c-5p (turquoise module), enabling pluripotency and self-renew; and gga-miR-429-3p (yellow module), modulating neurogenesis and osteogenesis. The findings of this study extend the knowledge of miRNA expression in early embryonic development of chickens, providing insights into the intricate gene control process that helps ensure proper development.
... These miRs included miR302a-3p, miR302b-3p, miR302c-3p, miR302d-3p, miR302a-5p, miR-142a-3p, miR-335-3p, miR-335-5p, and miR-211-5p. Previous studies have shown that the miR302 cluster is important in mechanosensitivity, neural differentiation, and reprogramming [59][60][61][62][63][64] . Among the upregulated DE miRs, the Meg3-Mirg locus was highly represented. ...
Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes.
... 169 MicroRNA-302 has a vital function in embryonic survival and development. 176 Additionally, acute ethanol exposure at GD12 caused social motivation behavior impairments in PD42 offspring. 167 These behavioral impairments were associated with altered changes (both up and down) in many miRNAs in the ventral striatum and amygdala tissues. ...
During pregnancy, alcohol abuse and its detrimental effects on developing offspring are major public health, economic and social challenges. The prominent characteristic attributes of alcohol (ethanol) abuse during pregnancy in humans are neurobehavioral impairments in offspring due to damage to the central nervous system (CNS), causing structural and behavioral impairments that are together named fetal alcohol spectrum disorder (FASD). Development-specific alcohol exposure paradigms were established to recapitulate the human FASD phenotypes and establish the underlying mechanisms. These animal studies have offered some critical molecular and cellular underpinnings likely to account for the neurobehavioral impairments associated with prenatal ethanol exposure. Although the pathogenesis of FASD remains unclear, emerging literature proposes that the various genomic and epigenetic components that cause the imbalance in gene expression can significantly contribute to the development of this disease. These studies acknowledged numerous immediate and enduring epigenetic modifications, such as methylation of DNA, post-translational modifications (PTMs) of histone proteins, and regulatory networks related to RNA, using many molecular approaches. Methylated DNA profiles, PTMs of histone proteins, and RNA-regulated expression of genes are essential for synaptic and cognitive behavior. Thus, offering a solution to many neuronal and behavioral impairments reported in FASD. In the current chapter, we review the recent advances in different epigenetic modifications that cause the pathogenesis of FASD. The information discussed can help better explain the pathogenesis of FASD and thereby might provide a basis for finding novel therapeutic targets and innovative treatment strategies.
... Moreover, studies have shown that the defective proliferation of neural stem/progenitor cells leads to microcephaly in mice [11,12]. Increased proliferation of the neuroepithelium during neural tube closure results in exencephaly [13,14]. Similar to the studies in animal models, there has been much convincing evidence about Huntington's disease (HD) in the human brain exhibiting greater neural progenitor cell (NPC) proliferation, proportional to the severity of the gene defect [15]. ...
The maintenance of neural stem cells (NSCs) plays a critical role in neurodevelopment and has been implicated in neurodevelopmental disorders (NDDs). However, the underlying mechanisms linking defective human neural stem cell self-renewal to NDDs remain undetermined. Our previous study found that Supt16 haploinsufficiency causes cognitive and social behavior deficits by disrupting the stemness maintenance of NSCs in mice. However, its effects and underlying mechanisms have not been elucidated in human neural stem cells (hNSCs). Here, we generated Supt16+/− induced pluripotent stem cells (iPSCs) and induced them into hNSCs. The results revealed that Supt16 heterozygous hNSCs exhibit impaired proliferation, cell cycle arrest, and increased apoptosis. As the RNA-seq analysis showed, Supt16 haploinsufficiency inhibited the PI3K/AKT/mTOR pathway, leading to rising autophagy, and further resulted in the dysregulated expression of multiple proteins related to cell proliferation and apoptotic process. Furthermore, the suppression of Supt16 heterozygous hNSC self-renewal caused by autophagy activation could be rescued by MHY1485 treatment or reproduced in rapamycin-treated hNSCs. Thus, our results showed that Supt16 was essential for hNSC self-renewal and its haploinsufficiency led to cell cycle arrest, impaired cell proliferation, and increased apoptosis of hNSCs by regulating the PI3K/AKT/mTOR/autophagy pathway. These provided a new insight to understand the causality between the Supt16 heterozygous NSCs and NDDs in humans.
... While miRNAs associated with primed pluripotency, such as miR-302, were among the most highly expressed miRNAs at E7.5 and E8.5, miRNAs associated with naïve pluripotency, such as miR-293 (part of the naïve-specific miR-290 ~ 295 cluster), were expressed exclusively at E7.5 (SI Appendix, Fig. S4D). To validate the expression of miR-302, we used an established mir-302-eGFP reporter mouse (42) and analyzed expression at E7.25 to E9.5 (SI Appendix, Fig. S4E). MiR-302-eGFP was enriched within the cranial region compared with the trunk (Fig. 4B and SI Appendix, Fig. S4E). ...
... Bulk mRNA sequencing of neural crest cells revealed no significant change in expression of the mir-302 host gene, Larp7 (SI Appendix, Fig. S5B). Consistent with previous findings, we observed a cranial neural tube closure defect upon mir-302 deletion (SI Appendix, Fig. S5E) (42). Since miR-302 is expressed in both anterior ectoderm and neural crest cells (Fig. 4), maintains embryonic stem cell self-renewal (22)(23)(24), and regulates developmental timing of differentiation within the neural tube (42), we first asked whether miR-302 prevents precocious specification of cranial neural crest. ...
... Our observation of two distinct populations of premigratory cells that were differentially affected by loss of miR-302 provides an additional mechanistic explanation as to why some defects occur in cranial neural crest cartilage versus neuronal lineages in humans. Since the function of miR-302 is conserved in mice and humans (42) and Sox9 deletion results in defects in cranial chondrocyte differentiation (44), we hypothesize that disruption of the miR-302 regulatory axis may underlie some human craniofacial and neurological disorders. Consistent with our findings, Dicer was reported to be up-regulated in chicken neural crest cells (68). ...
Developmental potential is progressively restricted after germ layer specification during gastrulation. However, cranial neural crest cells challenge this paradigm, as they develop from anterior ectoderm, yet give rise to both ectodermal derivatives of the peripheral nervous system and ectomesenchymal bone and cartilage. How cranial neural crest cells differentiate into multiple lineages is poorly understood. Here, we demonstrate that cranial neural crest cells possess a transient state of increased chromatin accessibility. We profile the spatiotemporal emergence of premigratory neural crest and find evidence of lineage bias toward either a neuronal or ectomesenchymal fate, with each expressing distinct factors from earlier stages of development. We identify the miR-302 miRNA family to be highly expressed in cranial neural crest cells and genetic deletion leads to precocious specification of the ectomesenchymal lineage. Loss of mir-302 results in reduced chromatin accessibility in the neuronal progenitor lineage of neural crest and a reduction in peripheral neuron differentiation. Mechanistically, we find that mir-302 directly targets Sox9 to slow the timing of ectomesenchymal neural crest specification and represses multiple genes involved in chromatin condensation to promote accessibility required for neuronal differentiation. Our findings reveal a posttranscriptional mechanism governed by miRNAs to expand developmental potential of cranial neural crest.
... miR-302 and miR-290 are highly expressed in pluripotent cells and are required for neurulation. Loss of miR-302 causes precocious neuronal differentiation [86]. Moreover, miR-302 stimulates BMP signaling by repressing BMP inhibitors TOB2, DAZAP2, and SLAIN1 in hESCs [87]. ...
The evolutionarily conserved RNA helicase DDX6 is a central player in post-transcriptional regulation, but its role during embryogenesis remains elusive. We here show that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 knockout (KO) mouse embryos and employing an in vitro epiblast-like cell (EpiLC) induction system. Our study unveils that DDX6 is an important BMP signaling regulator. Deletion of Ddx6 causes the aberrant upregulation of the negative regulators of BMP signaling, which is accompanied by enhanced expression of Nodal and related genes. Ddx6 KO pluripotent cells acquire higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal and Eomes expression in the primitive streak likely promotes endoderm cell fate specification while inhibiting mesoderm differentiation. We also genetically dissected major DDX6 pathways by generating Dgcr8 , Dcp2 , and Eif4enif1 KO models in addition to Ddx6 KO. We found that the miRNA pathway mutant Dgcr8 KO phenocopies Ddx6 KO, indicating that DDX6 mostly works along with the miRNA pathway during early development, whereas its P-body-related functions are dispensable. Therefore, we conclude that DDX6 prevents aberrant upregulation of BMP signaling inhibitors by participating in miRNA-mediated gene silencing processes. Overall, this study delineates how DDX6 affects the development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function.
... The interaction between the miRNA and mRNA target is primarily mediated through nucleotides 2-8 at the 5′ end of the miRNA (Brennecke et al., 2005;Lewis et al., 2003). This region, the seed sequence, defines a miRNA family: a group of miRNAs that act largely redundantly on an overlapping set of target genes due to their identical seed sequence (Alvarez-Saavedra and Horvitz, 2010;Parchem et al., 2015). Supplemental base pairing between the 3′ end of the miRNA and the target RNA occurs in some cases, conferring differences in target repertoire of miRNA family members (Brancati and Großhans, 2018;Broughton et al., 2016;Helwak et al., 2013;Isana Veksler-Lublinsky, 2022). ...
MicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. Here, we show that the mir-35 miRNA family undergoes selective decay at the transition from embryonic to larval development in C. elegans. The seed sequence of the miRNA is necessary and largely sufficient for this regulation. Sequences outside the seed (3′ end) regulate mir-35 abundance in the embryo but are not necessary for sharp decay at the transition to larval development. Enzymatic modifications of the miRNA 3′ end are neither prevalent nor correlated with changes in decay, suggesting that miRNA 3′ end display is not a core feature of this mechanism and further supporting a seed-driven decay model. Our findings demonstrate that seed-sequence-specific decay can selectively and coherently regulate all redundant members of a miRNA seed family, a class of mechanism that has great biological and therapeutic potential for dynamic regulation of a miRNA family’s target repertoire.
... Many scientists demonstrated that these tiny inhibitory RNAs can also act as a RNA based inducer and fulfill the regulatory role in various cellular processes, including cell development (Chakraborty et al., 2017;Parchem et al., 2015), differentiation, proliferation and apoptosis (Fu et al., 2019) which sparked the interest of the majority of the scientific community towards miRNA research globally Zhou et al., 2019). Evidence from an array of studies suggested miRNAs association in controlling cellular transitions during differentiation (Hussen et al., 2021;Wijesinghe et al., 2022) as well as stem cell regulation (Lanzillotti et al., 2021) because of their broad range of potential therapeutic targets. ...
MicroRNAs are small non-protein coding RNAs of 19–24 nucleotides that operate as regulators of gene expression in the eukaryotic system at both the transcriptional and post-transcriptional levels. Promising evidences suggest that most of the miRNAs and the related components of the RNA interference pathway are decisively significant in the regulation of nearly all cellular processes of both animals and plants. MicroRNA has crucial role in different metabolic processes like cell division, growth, proliferation, differentiation as well as apoptosis of the cell. Scientists are given more insight into this novel idea of cross kingdom regulation system by miRNA after discovery of exogenous plant MIR168a influencing the transport system of cholesterol in mice by down regulating the receptor adopter protein 1 expression of law density lipoprotein. These types of influential exploration about cross kingdom regulation of miRNAs have taken more intention towards its role in animal as well as microbial system. In human health, the animal sources, plant, microbes derived miRNAs and diet based miRNA plays significant role. Dysregulation of miRNAs is correlated with development of various pathological events such as neurological disorders, cardiovascular diseases, cancer, AMR, which are the most threatened field for human society. Here we detailed about the role of miRNA in human health with diverse source of miRNAs and its role in therapeutic and diagnosis of disease like cancer in human.
... Therefore, we considered the possibility that the G1/S restriction point may regulate phasic expression. MicroRNAs of the miR-302 family suppress the G1/S restriction point 20,21 and are expressed in the pluripotent epiblast at E5.5 before decreasing with varied timing across all germ layers, including ectoderm, by E9.5 22,23 . Small RNA sequencing showed that miR-302 family miRNAs are the most abundant family at E7.5 (18.8%), before declining to 1.0% at E9.5 ( Supplementary Fig. 4A-C). ...
... We made the comparison at E8.5, an intermediate stage in the onset of periodic transcription. We sorted cells with active mir-302 loci using a green fluorescent protein (GFP) reporter that had been knocked into the mutated allele ( Supplementary Fig. 4D) 22 . As expected, mir-302 target genes were upregulated in knockouts (Supplementary Fig. 4E). ...
... Early phasic expression in mir-302−/− precedes differentiation defects. Next we asked how miR-302 mediated suppression of phasic expression relates to its established role in developmental timing of cellular differentiation 22,29 . In particular, given that mir-302 removal leads to premature differentiation in multiple cell types 22,30 , we asked if the precocious phasic expression seen at E7.5 was upstream or downstream of the differentiation phenotype. ...
Pluripotent embryonic stem cells have a unique cell cycle structure with a suppressed G1/S restriction point and little differential expression across the cell cycle phases. Here, we evaluate the link between G1/S restriction point activation, phasic gene expression, and cellular differentiation. Expression analysis reveals a gain in phasic gene expression across lineages between embryonic days E7.5 and E9.5. Genetic manipulation of the G1/S restriction point regulators miR-302 and P27 respectively accelerates or delays the onset of phasic gene expression in mouse embryos. Loss of miR-302-mediated p21 or p27 suppression expedites embryonic stem cell differentiation, while a constitutive Cyclin E mutant blocks it. Together, these findings uncover a causal relationship between emergence of the G1/S restriction point with a gain in phasic gene expression and cellular differentiation.
