Late Blastula Stage Research Articles

Widespread priming of transcriptional regulatory elements by incipient accessibility or RNA polymerase II pause in early embryos of the sea urchin Strongylocentrotus purpuratus.

Transcriptional regulatory elements (TREs) are the primary nodes that control developmental gene regulatory networks. In embryo stages, larvae, and adult differentiated red spherule cells of the sea urchin Strongylocentrotus purpuratus, transcriptionally engaged TREs are detected by Precision Run-On Sequencing (PRO-seq), which maps genome-wide at base pair resolution the location of paused or elongating RNA polymerase II (Pol II). In parallel, TRE accessibility is estimated by the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq). Our analysis identifies surprisingly early and widespread TRE accessibility in 4-cell cleavage embryos that is not necessarily followed by concurrent or subsequent transcription. TRE transcriptional differences identified by PRO-seq provide more contrast among embryonic stages than ATAC-seq accessibility differences, in agreement with the apparent excess of accessible but inactive TREs during embryogenesis. Global TRE accessibility reaches a maximum around the 20-hour late blastula stage, which coincides with the consolidation of major embryo regionalizations and peak histone variant H2A.Z expression. A transcriptional potency model based on labile nucleosome TRE occupancy driven by DNA sequences and the prevalence of histone variants is proposed in order to explain the basal accessibility of transcriptionally inactive TREs during embryogenesis. However, our results would not reconcile well with labile nucleosome models based on simple A/T sequence enrichment. In addition, a large number of distal TREs become transcriptionally disengaged during developmental progression, in support of an early Pol II paused model for developmental gene regulation that eventually resolves in transcriptional activation or silencing. Thus, developmental potency in early embryos may be facilitated by incipient accessibility and transcriptional pause at TREs.

Activation of P53 pathway contributes to Xenopus hybrid inviability

Hybrid incompatibility as a kind of reproductive isolation contributes to speciation. The nucleocytoplasmic incompatibility between Xenopus tropicalis eggs and Xenopus laevis sperm (te×ls) leads to specific loss of paternal chromosomes 3L and 4L. The hybrids die before gastrulation, of which the lethal causes remain largely unclear. Here, we show that the activation of the tumor suppressor protein P53 at late blastula stage contributes to this early lethality. We find that in stage 9 embryos, P53-binding motif is the most enriched one in the up-regulated Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) peaks between te×ls and wild-type X. tropicalis controls, which correlates with an abrupt stabilization of P53 protein in te×ls hybrids at stage 9. Inhibition of P53 activity via either tp53 knockout or overexpression of a dominant-negative P53 mutant or Murine double minute 2 proto-oncogene (Mdm2), a negative regulator of P53, by mRNA injection can rescue the te×ls early lethality. Our results suggest a causal function of P53 on hybrid lethality prior to gastrulation.

A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies.

Cell sorting and germ layer formation in reconstructed starfish embryos.

Germ layer formation is driven by embryonic cell sorting during the early developmental stages. Starfish (Patiria pectinifera) embryos have a connected endoderm and ectoderm, albeit with few contact surfaces between the epithelia. To better understand the association between cell sorting and germ layer formation, we reconstructed P.pectinifera embryos and examined their germ layer formation. Initial observations showed that the presumptive endodermal (pEN) and presumptive ectodermal (pEC) portions of the embryonic body at the late-blastula stage were preserved throughout development. Based on this, cells that were dissociated from each dermal fragment were mixed in a reconstruction experiment. Our results showed that the pEN and pEC cells were located inside and outside the reaggregates, respectively, to form an embryonic body containing two epithelial layers, separated by a blastocoel. During this process, the pEN cells were motile and shifted from smaller clumps to form a large clump. In contrast, in reaggregates formed in separate cultures, the pEN cells showed strong adhesion abilities, whereas the pEC cells underwent epithelialization. Unlike that in pEN cells, the reaggregation of pEC cells preceded cadherin expression. Filamentous actin was similarly observed in both reaggregates. These results suggest that during the reconstruction of starfish embryos, germ layer formation occurs via the sorting of pEN and pEC cells, depending on their adhesiveness, motility, and epithelialization. In vivo, these properties might embody the physiological significance of cell adhesion in the germ layers constituting the epithelial monolayer.

Exposure to ethanol leads to midfacial hypoplasia in a zebrafish model of FASD via indirect interactions with the Shh pathway

BackgroundGene-environment interactions are likely to underlie most human birth defects. The most common known environmental contributor to birth defects is prenatal alcohol exposure. Fetal alcohol spectrum disorders (FASD) describe the full range of defects that result from prenatal alcohol exposure. Gene-ethanol interactions underlie susceptibility to FASD, but we lack a mechanistic understanding of these interactions. Here, we leverage the genetic tractability of zebrafish to address this problem.ResultsWe first show that vangl2, a member of the Wnt/planar cell polarity (Wnt/PCP) pathway that mediates convergent extension movements, strongly interacts with ethanol during late blastula and early gastrula stages. Embryos mutant or heterozygous for vangl2 are sensitized to ethanol-induced midfacial hypoplasia. We performed single-embryo RNA-seq during early embryonic stages to assess individual variation in the transcriptional response to ethanol and determine the mechanism of the vangl2-ethanol interaction. To identify the pathway(s) that are disrupted by ethanol, we used these global changes in gene expression to identify small molecules that mimic the effects of ethanol via the Library of Integrated Network-based Cellular Signatures (LINCS L1000) dataset. Surprisingly, this dataset predicted that the Sonic Hedgehog (Shh) pathway inhibitor, cyclopamine, would mimic the effects of ethanol, despite ethanol not altering the expression levels of direct targets of Shh signaling. Indeed, we found that ethanol and cyclopamine strongly, but indirectly, interact to disrupt midfacial development. Ethanol also interacts with another Wnt/PCP pathway member, gpc4, and a chemical inhibitor of the Wnt/PCP pathway, blebbistatin, phenocopies the effect of ethanol. By characterizing membrane protrusions, we demonstrate that ethanol synergistically interacts with the loss of vangl2 to disrupt cell polarity required for convergent extension movements.ConclusionsOur results show that the midfacial defects in ethanol-exposed vangl2 mutants are likely due to an indirect interaction between ethanol and the Shh pathway. Vangl2 functions as part of a signaling pathway that regulates coordinated cell movements during midfacial development. Ethanol exposure alters the position of a critical source of Shh signaling that separates the developing eye field into bilateral eyes, allowing the expansion of the midface. Collectively, our results shed light on the mechanism by which the most common teratogen can disrupt development.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Chromatin accessibility and histone acetylation in the regulation of competence in early development

As development proceeds, inductive cues are interpreted by competent tissues in a spatially and temporally restricted manner. While key inductive signaling pathways within competent cells are well-described at a molecular level, the mechanisms by which tissues lose responsiveness to inductive signals are not well understood. Localized activation of Wnt signaling before zygotic gene activation in Xenopus laevis leads to dorsal development, but competence to induce dorsal genes in response to Wnts is lost by the late blastula stage. We hypothesize that loss of competence is mediated by changes in histone modifications leading to a loss of chromatin accessibility at the promoters of Wnt target genes. We use ATAC-seq to evaluate genome-wide changes in chromatin accessibility across several developmental stages. Based on overlap with p300 binding, we identify thousands of putative cis-regulatory elements at the gastrula stage, including sites that lose accessibility by the end of gastrulation and are enriched for pluripotency factor binding motifs. Dorsal Wnt target gene promoters are not accessible after the loss of competence in the early gastrula while genes involved in mesoderm and neural crest development maintain accessibility at their promoters. Inhibition of histone deacetylases increases acetylation at the promoters of dorsal Wnt target genes and extends competence for dorsal gene induction by Wnt signaling. Histone deacetylase inhibition, however, is not sufficient to extend competence for mesoderm or neural crest induction. These data suggest that chromatin state regulates the loss of competence to inductive signals in a context-dependent manner.

DNA methylation dynamics during epigenetic reprogramming of medaka embryo

ABSTRACTPost-fertilization epigenome reprogramming erases epigenetic marks transmitted through gametes and establishes new marks during mid-blastula stages. The mouse embryo undergoes dynamic DNA methylation reprogramming after fertilization, while in zebrafish, the paternal DNA methylation pattern is maintained throughout the early embryogenesis and the maternal genome is reprogrammed in a pattern similar to that of sperm during the mid-blastula transition. Here, we show DNA methylation dynamics in medaka embryos, the biomedical model fish, during epigenetic reprogramming of embryonic genome. The sperm genome was hypermethylated and the oocyte genome hypomethylated prior to fertilization. After fertilization, the methylation marks of sperm genome were erased within the first cell cycle and embryonic genome remained hypomethylated from the zygote until 16-cell stage. The DNA methylation level gradually increased from 16-cell stage through the gastrula. The 5-hydroxymethylation (5hmC) levels showed an opposite pattern to DNA methylation (5-mC). The mRNA levels for DNA methyltransferase (DNMT) 1 remained high in oocytes and maintained the same level through late blastula stage and was reduced thereafter. DNMT3BB.1 mRNA levels increased prior to remethylation. The mRNA levels for ten-eleven translocation methylcytosine dioxygenases (TET2 & TET3) were detected in sperm and embryos at cleavage stages, whereas TET1 and TET3 mRNAs decreased during gastrulation. The pattern of genome methylation in medaka was identical to mammalian genome methylation but not to zebrafish. The present study suggests that a medaka embryo resets its DNA methylation pattern by active demethylation and by a gradual remethylation similar to mammals.

Temporal-spatial characteristics of lipea gene expression in Cyprinus carpio and its correlation with fat deposition

PDF HTML阅读 XML下载 导出引用 引用提醒 鲤lipea基因表达的时空特征及与脂肪沉积相关性分析 DOI: 作者: 作者单位: 1. 上海海洋大学, 农业农村部淡水水产种质资源重点实验室, 上海 201306;2. 中国水产科学研究院黑龙江水产研究所, 淡水鱼类育种国家地方联合工程实验室, 黑龙江 哈尔滨 150070 作者简介: 党红阳(1995-),女,硕士研究生,研究方向为水产生物技术.E-mail:danghongyang95@163.com 通讯作者: 中图分类号: S965 基金项目: 国家自然科学基金项目（31302174）；中国水产科学研究院基本科研业务费（2015C04XK01）；国家水产种质资源共享服务平台项目（2018DKA30470）. Temporal-spatial characteristics of lipea gene expression in Cyprinus carpio and its correlation with fat deposition Author: Affiliation: 1. Key Laboratory of Freshwater Fishery Germplasm Resource, Ministry of Agriculture and Rural Affairs;Shanghai Ocean University, Shanghai 201306, China;2. National Local Joint Engineering Laboratory for Freshwater Fish Breeding, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:本文旨在克隆鲤（）基因，探究其时空表达特征及其与脂肪沉积的关系，以期为鲤脂肪沉积分子调控机制研究奠定理论基础。本实验采用RACE技术克隆获得鲤基因，其cDNA全长为3379 bp，包括230 bp的5'非翻译区（5'-UTR）、1067 bp的3'非翻译区（3'-UTR）和编码693个氨基酸的开放阅读框。多序列比对显示，鲤Oncorhynchus mykiss）的氨基酸序列相似性分别为88.17%、64.94%和63.35%，不同物种间序列差异主要集中在C-端的多肽序列上。蛋白质结构分析显示该蛋白含有3个功能域分别具有脂肪酶活性、乙酰基水解酶活性和水解酶活性。应用实时荧光定量PCR技术分析了在各组织中均有表达，腹腔脂肪中相对表达量最高，其次是腹部肌肉，血液中最少。随着胚胎的发育，表达量与背部肌肉脂肪含量呈正相关，但差异不显著（基因对鲤腹腔脂肪沉积可能具有抑制作用。本实验结果可为进一步解析鲤基因的功能和基因调控脂肪沉积分子机制研究提供参考。 Abstract:The objectives of this study were to investigate the expression pattern of Cyprinus carpio), to lay the foundation for studying and understanding the molecular regulation mechanism of fat deposition in common carp. In this study, a full-length cDNA sequence of gene in common carp was cloned by reverse transcription PCR and rapid-amplification of cDNA ends (RACE) technology, and the expression pattern of gene in different tissues of common carp and during its different developmental stages. cDNA of the gene of common carp was 3379 bp, with an open reading frame of 2079 bp, 5' untranslated region was 230 bp, 3' untranslated region was 1067 bp, encoding 693 amino acids. Multiple sequences alignment revealed that the amino acid sequences of Paralichthys olivaceus, and , respectively. It was determined that the sequence differences were mainly concentrated on the C-terminal polypeptide sequence between different species, and the structural analysis of proteins showed that the protein contains three functional domains with lipase activity, acetyl hydrolase activity, and hydrolase activity, respectively. Real-time quantitative PCR showed that the gene was expressed in all tested tissues, and the relative expression level was the highest in visceral fat, followed by abdominal muscles, and lowest in the blood. Its expression declined during the development of embryo; it was the highest in the 8-cell stage, followed by the late blastula stage. It was the lowest in the feeding period. The mRNA expression in different fat tissues negatively correlated with adipose tissue in the abdominal cavity; the highest and the lowest expression levels differed significantly (<0.01). The expression also positively correlated with dorsal muscle but it was not significant ( gene highlights its function and molecular regulation mechanism of fat development in common carp. 参考文献 相似文献 引证文献

The yolk syncytial layer of loach, Misgurnus fossilis (Teleostei) during early development.

The yolk syncytial layer (YSL) of Teleostei is a dynamic multifunctional temporary system. This paper describes the YSL structure of Misgurnus fossilis (Cobitidae) during its early developmental stages, studied using histological methods. YSL formation is prolonged. From the late blastula stage, the basal surface of the YSL is uneven and has protuberances, but becomes smoother during development. There are syncytial 'islands' with 1-2 yolk syncytial nuclei in the yolk mass. During epiboly, gastrulation and early segmentation, loach YSL is of different thickness in different regions along the dorso-ventral and antero-posterior axes of an embryo. The YSL is thickened in the dorsal region of gastrulae compared with the ventral region. Although the development of M. fossilis is similar to the development of zebrafish, there are important differences in YSL formation and organization that await further study and analysis. The study of YSL organization contributes to our knowledge of teleost developmental diversity and to the biology of temporary structures.

Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos.

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus.

Characterization of an amphioxus heat-shock protein gene promoter and its application in vivo.

Amphioxus is a good proxy for studying the evolution and development of mechanisms during the invertebrate to vertebrate transition. However, one of the major limitations of amphioxus as a model organism is the lack of well-developed technical approaches. Promoters of genes encoding heat shock proteins are useful tools for gene function studies. In the present study, we tested heat shock temperatures on 4-cell, 128-cell, late blastula, mid-gastrula and late gastrula amphioxus embryos and optimized the heat shock conditions. We then examined the expression profile of the endogenous Hsp70 gene in embryos heat-shocked under optimum conditions. RT-qPCR revealed that the expression of Hsp70 could be induced in embryos by heat shock after the 128-cell stage, and in situ hybridization showed that the induced Hsp70 expression was predominantly detected in epidermal ectoderm. Further, we constructed two reporter gene expression plasmids carrying the Hsp70 promoter to evaluate its efficiency in vivo. Microinjection experiments showed strong red fluorescence or LacZ staining signals in injected embryos after heat shock treatment. In contrast to endogenous Hsp70, reporter gene expression was found in all three germ layers at equivalent levels. Finally, using the Hsp70 promoter, we conducted a preliminary functional analysis of two amphioxus genes, Vg1 and Cer, which play essential roles in left-right patterning in vertebrates. After heat shock at late blastula, mid-gastrula or early neurula stages, ectopic expression of Vg1 was detected in each group of treated embryos. Phenotype observation at 3-gill-slit larvae stage showed that ectopic Vg1 expression led to two-left-side or left-right reversal phenotypes, and that of Cer caused a two-right-side phenotype. Our study establishes a thermo-inducible gene expression system which will be a useful tool for gene function investigation in amphioxus embryos.

Apolipoprotein C-I mediates Wnt/Ctnnb1 signaling during neural border formation and is required for neural crest development.

In vertebrates, the neural crest and placodes originate in the neural border, which is located between the neural plate and epidermal ectoderm. The neural crest and placodes give rise to a vast array of cell types. Formation of neural crest is a multi-step process, in which Wnt signals are used reiteratively, but it is currently not clear if a Wnt signal is required for neural border formation. Here, we have identified apolipoprotein C-I (apoc1) in a screen for genes regulated by Wnt/Ctnnb1 signaling in late blastula stage Xenopus tropicalis embryos. We show that Xenopus laevis apoc1 encodes a small, secreted protein, and is induced by Wnt/Ctnnb1 signaling. Depletion of Apoc1 protein results in a neural border formation defect and loss of border fates, including neural crest cells. However, unlike another Wnt/Ctnnb1 target, gbx2.2, apoc1 is not required for patterning of the neural border. We further show that gbx2.2 and apoc1 are independently regulated by Wnt signaling. Our results thus suggest that Wnt regulates border formation and patterning by distinct genetic mechanisms.

Nodal signalling inXenopus: the role of Xnr5 in left/right asymmetry and heart development

Nodal class TGF-β signalling molecules play essential roles in establishing the vertebrate body plan. In all vertebrates, nodal family members have specific waves of expression required for tissue specification and axis formation. In Xenopus laevis, six nodal genes are expressed before gastrulation, raising the question of whether they have specific roles or act redundantly with each other. Here, we examine the role of Xnr5. We find it acts at the late blastula stage as a mesoderm inducer and repressor of ectodermal gene expression, a role it shares with Vg1. However, unlike Vg1, Xnr5 depletion reduces the expression of the nodal family member xnr1 at the gastrula stage. It is also required for left/right laterality by controlling the expression of the laterality genes xnr1, antivin (lefty) and pitx2 at the tailbud stage. In Xnr5-depleted embryos, the heart field is established normally, but symmetrical reduction in Xnr5 levels causes a severely stunted midline heart, first evidenced by a reduction in cardiac troponin mRNA levels, while left-sided reduction leads to randomization of the left/right axis. This work identifies Xnr5 as the earliest step in the signalling pathway establishing normal heart laterality in Xenopus.

On the origin of vertebrate somites.

IntroductionSomites, blocks of mesoderm tissue located on either side of the neural tube in the developing vertebrate embryo, are derived from mesenchymal cells in the presomitic mesoderm (PSM) and are a defining characteristic of vertebrates. In vertebrates, the somite segmental boundary is determined by Notch signalling and the antagonistic relationship of the downstream targets of Notch, Lfng, and Delta1 in the anterior PSM. The presence of somites in the basal chordate amphioxus (Branchiostoma floridae) indicates that the last common ancestor of chordates also had somites. However, it remains unclear how the genetic mechanisms underlying somitogenesis in vertebrates evolved from those in ancestral chordates.ResultsWe demonstrate that during the gastrula stages of amphioxus embryos, BfFringe expression in the endoderm of the archenteron is detected ventrally to the ventral limit of BfDelta expression in the presumptive rostral somites along the dorsal/ventral (D/V) body axis. Suppression of Notch signalling by DAPT (a γ-secretase inhibitor that indirectly inhibits Notch) treatment from the late blastula stage reduced late gastrula stage expression of BfFringe in the endodermal archenteron and somite markers BfDelta and BfHairy-b in the mesodermal archenteron. Later in development, somites in the DAPT-treated embryo did not separate completely from the dorsal roof of the archenteron. In addition, clear segmental boundaries between somites were not detected in DAPT-treated amphioxus embryos at the larva stage. Similarly, in vertebrates, DAPT treatment from the late blastula stage in Xenopus (Xenopus laevis) embryos resulted in disruption of somite XlDelta-2 expression at the late gastrula stage. At the tail bud stage, the segmental expression of XlMyoD in myotomes was diminished.ConclusionsWe propose that Notch signalling and the Fringe/Delta cassette for dorso-ventral boundary formation in the archenteron that separates somites from the gut in an amphioxus-like ancestral chordate were co-opted for anteroposterior segmental boundary formation in the vertebrate anterior PSM during evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s40851-015-0033-0) contains supplementary material, which is available to authorized users.

A genome-wide survey of maternal and embryonic transcripts during Xenopus tropicalis development

BackgroundDynamics of polyadenylation vs. deadenylation determine the fate of several developmentally regulated genes. Decay of a subset of maternal mRNAs and new transcription define the maternal-to-zygotic transition, but the full complement of polyadenylated and deadenylated coding and non-coding transcripts has not yet been assessed in Xenopus embryos.ResultsTo analyze the dynamics and diversity of coding and non-coding transcripts during development, both polyadenylated mRNA and ribosomal RNA-depleted total RNA were harvested across six developmental stages and subjected to high throughput sequencing. The maternally loaded transcriptome is highly diverse and consists of both polyadenylated and deadenylated transcripts. Many maternal genes show peak expression in the oocyte and include genes which are known to be the key regulators of events like oocyte maturation and fertilization. Of all the transcripts that increase in abundance between early blastula and larval stages, about 30% of the embryonic genes are induced by fourfold or more by the late blastula stage and another 35% by late gastrulation. Using a gene model validation and discovery pipeline, we identified novel transcripts and putative long non-coding RNAs (lncRNA). These lncRNA transcripts were stringently selected as spliced transcripts generated from independent promoters, with limited coding potential and a codon bias characteristic of noncoding sequences. Many lncRNAs are conserved and expressed in a developmental stage-specific fashion.ConclusionsThese data reveal dynamics of transcriptome polyadenylation and abundance and provides a high-confidence catalogue of novel and long non-coding RNAs.

Transcriptional Regulation of Mesoderm Genes by MEF2D during Early Xenopus Development

In Xenopus, specification of the three germ layers is one of the earliest developmental decisions occurring prior to gastrulation. The maternally-expressed vegetally-localized transcription factor VegT has a central role in cell autonomous specification of endoderm and in the generation of mesoderm-inducing signals. Yet, marginally-expressed transcription factors that cooperate with mesoderm-inducing signals are less investigated. Here we report that the transcription factors MEF2A and MEF2D are expressed in the animal hemisphere before mid-blastula transition. At the initiation of zygotic transcription, expression of MEF2D expands into the marginal region that gives rise to mesoderm. Knockdown of MEF2D delayed gastrulation movements, prevented embryo elongation at the subsequent tailbud stage and caused severe defects in axial tissues. At the molecular level, MEF2D knockdown reduced the expression of genes involved in mesoderm formation and patterning. We also report that MEF2D functions with FGF signaling in a positive feedback loop; each augments the expression of the other in the marginal region and both are necessary for mesodermal gene expression. One target of MEF2D is the Nodal-related 1 gene (Xnr1) that mediates some of MEF2D mesodermal activities. Chromatin immunoprecipitation analysis revealed that MEF2D associates with transcriptional regulatory sequences of the Xnr1 gene. Several MEF2 binding sites within the proximal promoter region of Xnr1 were identified by their in vitro association with MEF2D protein. The same promoter region was necessary but not sufficient to mediate MEF2D activity in a reporter gene assay. In sum, our results indicate that the MEF2D protein is a key transcription factor in the marginal zone acting in a positive feedback loop with FGF signaling that promotes mesoderm specification at late blastula stages.

Pou5f1-Dependent EGF Expression Controls E-Cadherin Endocytosis, Cell Adhesion, and Zebrafish Epiboly Movements

Initiation of motile cell behavior in embryonic development occurs during late blastula stages when gastrulation begins. At this stage, the strong adhesion of blastomeres has to be modulated to enable dynamic behavior, similar to epithelial-to-mesenchymal transitions. We show that, in zebrafish maternal and zygotic (MZ)spg embryos mutant for the stem cell transcription factor Pou5f1/Oct4, which are severely delayed in the epiboly gastrulation movement, all blastomeres are defective in E-cadherin (E-cad) endosomal trafficking, and E-cad accumulates at the plasma membrane. We find that Pou5f1-dependent control of EGF expression regulates endosomal E-cad trafficking. EGF receptor may act via modulation of p120 activity. Loss of E-cad dynamics reduces cohesion of cells in reaggregation assays. Quantitative analysis of cell behavior indicates that dynamic E-cad endosomal trafficking is required for epiboly cell movements. We hypothesize that dynamic control of E-cad trafficking is essential to effectively generate new adhesion sites when cells move relative to each other.

Characterization of differential transcript abundance through time during Nematostella vectensis development

BackgroundNematostella vectensis, a burrowing sea anemone, has become a popular species for the study of cnidarian development. In previous studies, the expression of a variety of genes has been characterized during N. vectensis development with in situ mRNA hybridization. This has provided detailed spatial resolution and a qualitative perspective on changes in expression. However, little is known about broad transcriptome-level patterns of gene expression through time. Here we examine the expression of N. vectensis genes through the course of development with quantitative RNA-seq. We provide an overview of changes in the transcriptome through development, and examine the maternal to zygotic transition, which has been difficult to investigate with other tools.ResultsWe measured transcript abundance in N. vectensis with RNA-seq at six time points in development: zygote (2 hours post fertilization (HPF)), early blastula (7 HPF), mid-blastula (12 HPF), gastrula (24 HPF), planula (5 days post fertilization (DPF)) and young polyp (10 DPF). The major wave of zygotic expression appears between 7–12 HPF, though some changes occur between 2–7 HPF. The most dynamic changes in transcript abundance occur between the late blastula and early gastrula stages. More transcripts are upregulated between the gastrula and planula than downregulated, and a comparatively lower number of transcripts significantly change between planula and polyp. Within the maternal to zygotic transition, we identified a subset of maternal factors that decrease early in development, and likely play a role in suppressing zygotic gene expression. Among the first genes to be expressed zygotically are genes whose proteins may be involved in the degradation of maternal RNA.ConclusionsThe approach presented here is highly complementary to prior studies on spatial patterns of gene expression, as it provides a quantitative perspective on a broad set of genes through time but lacks spatial resolution. In addition to addressing the problems identified above, our work provides an annotated matrix that other investigators can use to examine genes and developmental events that we do not examine in detail here.

A gene network that coordinates preplacodal competence and neural crest specification in zebrafish

Preplacodal ectoderm (PPE) and neural crest (NC) are specified at the interface of neural and nonneural ectoderm and together contribute to the peripheral nervous system in all vertebrates. Bmp activates early steps for both fates during late blastula stage. Low Bmp activates expression of transcription factors Tfap2a and Tfap2c in the lateral neural plate, thereby specifying neural crest fate. Elevated Bmp establishes preplacodal competence throughout the ventral ectoderm by coinducing Tfap2a, Tfap2c, Foxi1 and Gata3. PPE specification occurs later at the end of gastrulation and requires complete attenuation of Bmp, yet expression of PPE competence factors continues well past gastrulation. Here we show that competence factors positively regulate each other's expression during gastrulation, forming a self-sustaining network that operates independently of Bmp. Misexpression of Tfap2a in embryos blocked for Bmp from late blastula stage can restore development of both PPE and NC. However, Tfap2a alone is not sufficient to activate any other competence factors nor does it rescue individual placodes. On the other hand, misexpression of any two competence factors in Bmp-blocked embryos can activate the entire transcription factor network and support the development of NC, PPE and some individual placodes. We also show that while these factors are partially redundant with respect to PPE specification, they later provide non-redundant functions needed for development of specific placodes. Thus, we have identified a gene regulatory network that coordinates development of NC, PPE and individual placodes in zebrafish.