Embryonic Axis Specification Research Articles

Canonical and Non-Canonical Wnt Signaling Generates Molecular and Cellular Asymmetries to Establish Embryonic Axes.

The formation of embryonic axes is a critical step during animal development, which contributes to establishing the basic body plan in each particular organism. Wnt signaling pathways play pivotal roles in this fundamental process. Canonical Wnt signaling that is dependent on β-catenin regulates the patterning of dorsoventral, anteroposterior, and left-right axes. Non-canonical Wnt signaling that is independent of β-catenin modulates cytoskeletal organization to coordinate cell polarity changes and asymmetric cell movements. It is now well documented that components of these Wnt pathways biochemically and functionally interact to mediate cell-cell communications and instruct cellular polarization in breaking the embryonic symmetry. The dysfunction of Wnt signaling disrupts embryonic axis specification and proper tissue morphogenesis, and mutations of Wnt pathway genes are associated with birth defects in humans. This review discusses the regulatory roles of Wnt pathway components in embryonic axis formation by focusing on vertebrate models. It highlights current progress in decoding conserved mechanisms underlying the establishment of asymmetry along the three primary body axes. By providing an in-depth analysis of canonical and non-canonical pathways in regulating cell fates and cellular behaviors, this work offers insights into the intricate processes that contribute to setting up the basic body plan in vertebrate embryos.

Analysis of maternal effect genes from maternal mRNA in eggs of Sogatella furcifera

To understand how many kinds of mRNAs in female adults can be transferred into the eggs and the molecular basis of embryonic axis specification in Sogatella furcifera, we performed de novo transcriptome sequencing of six cDNA libraries of female adults and unfertilized eggs. Total 60,306 unigenes were obtained, with an average length of 1114.51 bp and N50 length of 2112 bp. Total 2900 differentially expressed genes with 496 upregulated and 2404 downregulated were found in unfertilized egg compared to female adult. Most of mRNAs in female adult could be passed into its eggs. Total 65 maternal effect genes were identified, including many homologous genes involved in embryonic axis specialization of D. melanogaster. This study provide transcriptome resources to elucidate the functions of maternal effect genes and the molecular basis of embryonic axis specification in S. furcifera in the future.

Preprint Highlight: Polarized Disheveled dissolution and condensation drives embryonic axis specification in oocytes.

Body axes establishment during animal embryogenesis often relies on the intrinsic asymmetry generated early on in the oocyte by the Wnt/β-catenin pathway. However, it's still unclear how the initial asymmetry arises. Here imaging experiments show that in starfish embryos, Disheveled (Dvl), an essential cytoplasmic transducer of Wnt signaling, becomes polarized at the vegetal pole via a dissolution-condensation mechanism coupled to meiotic progression. The authors further show that Dvl reformation is dependent on a maternally expressed Wnt signaling receptor Frizzled (Fzd1). This study identifies a novel link between the cell cycle and the body plan establishment and thus is relevant to a broad audience of developmental and evolutionary cell biologists.

Transcriptome Analysis of mRNAs and Long Non-Coding RNAs During Subsequent Embryo Development of Porcine Cloned Zygotes After Vitrification.

Cryopreservation of porcine cloned zygotes has important implications for biotechnology and biomedicine research; however, lower embryo developmental potential remains an urgent problem to be resolved. For exploring the sublethal cryodamages during embryo development, this study was designed to acquire the mRNA and long non-coding RNA (lncRNA) profiles of 2-cells, 4-cells and blastocysts derived from vitrified porcine cloned zygotes using transcriptome sequencing. We identified 167 differentially expressed (DE) mRNAs and 516 DE lncRNAs in 2-cell stage, 469 DE mRNAs and 565 lncRNAs in 4-cell stage, and 389 DE mRNAs and 816 DE lncRNAs in blastocyst stage. Functional enrichment analysis revealed that the DE mRNAs during embryo development were involved in many regulatory mechanisms related to cell cycle, cell proliferation, apoptosis, metabolism and others. Moreover, the target genes of DE lncRNAs in the three embryonic stages were also enriched in many key GO terms or pathways such as “defense response”, “linoleic acid metabolic process”, “embryonic axis specification”, “negative regulation of protein neddylation”, etc., In conclusion, the present study provided comprehensive transcriptomic data about mRNAs and lncRNAs for the vitrified porcine cloned zygotes during different developmental stages, which contributed to further understand the potential cryodamage mechanisms responsible for impaired embryo development.

Polarized Dishevelled dissolution and reassembly drives embryonic axis specification in sea star oocytes

The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes.1 However, the mechanisms that generate cellular asymmetries within the oocyte and then transduce that polarity to organismal scale body axes are poorly understood outside of select model organisms. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled (Dvl) is a cytoplasmic Wnt pathway effector required for axis development in diverse species,2-4 but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dvl localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-reassembly mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially regulated Dvl dynamics.

Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles

Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. Here, we demonstrate that Hecw, a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons.

The first steps in the life of a worm: Themes and variations in asymmetric division in C. elegans and other nematodes.

Starting with Boveri in the 1870s, microscopic investigation of early embryogenesis in a broad swath of nematode species revealed the central role of asymmetric cell division in embryonic axis specification, blastomere positioning, and cell fate specification. Notably, across the class Chromadorea, a conserved theme emerges-asymmetry is first established in the zygote and specifies its asymmetric division, giving rise to an anterior somatic daughter cell and a posterior germline daughter cell. Beginning in the 1980s, the emergence of Caenorhabditis elegans as a model organism saw the advent of genetic tools that enabled rapid progress in our understanding of the molecular mechanisms underlying asymmetric division, in many cases defining key paradigms that turn out to regulate asymmetric division in a wide range of systems. Yet, the consequence of this focus on C. elegans came at the expense of exploring the extant diversity of developmental variation exhibited across nematode species. Given the resurgent interest in evolutionary studies facilitated in part by new tools, here we revisit the diversity in this asymmetric first division, juxtaposing molecular insight into mechanisms of symmetry-breaking, spindle positioning and fate specification, with a consideration of plasticity and variability within and between species. In the process, we hope to highlight questions of evolutionary forces and molecular variation that may have shaped the extant diversity of developmental mechanisms observed across Nematoda.

Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation ismediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that inDrosophila the small ubiquitin-like protein SUMOand the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.

Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis.

The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. We study the biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, we show that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, we demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, we suggest that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.

Relation of Nodal expression to the specification of the dorsal-ventral axis and tissue patterning in the starfish Patiria pectinifera.

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid-blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left-right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF-beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.

In Situ Hybridization: Fruit Fly Embryos and Tissues

AbstractIt is well known that transcript localization controls important biological processes, including cell fate determination, cell polarity, cell migration, morphogenesis, neuronal function, and embryonic axis specification. Thus, the sub‐cellular visualization of transcripts in ‘their original place’ (in situ) is an important tool to infer and understand their trafficking, stability, translation, and biological functions. This has been made possible through the use of labeled ‘anti‐sense’ probes that can be readily detected after hybridization to their ‘sense’ counterparts. The following is a series of protocols for conducting in situ hybridization in Drosophila (fruit fly) embryos or tissues. Probe‐detection methods include a relatively simple alkaline phosphatase reaction, as well as higher‐resolution and higher‐throughput versions using fluorescence‐conjugated tyramide labeling. New modifications that enhance probe penetration and detection in various tissues are also provided. © 2017 by John Wiley & Sons, Inc.

High-throughput mathematical analysis identifies Turing networks for patterning with equally diffusing signals.

The Turing reaction-diffusion model explains how identical cells can self-organize to form spatial patterns. It has been suggested that extracellular signaling molecules with different diffusion coefficients underlie this model, but the contribution of cell-autonomous signaling components is largely unknown. We developed an automated mathematical analysis to derive a catalog of realistic Turing networks. This analysis reveals that in the presence of cell-autonomous factors, networks can form a pattern with equally diffusing signals and even for any combination of diffusion coefficients. We provide a software (available at http://www.RDNets.com) to explore these networks and to constrain topologies with qualitative and quantitative experimental data. We use the software to examine the self-organizing networks that control embryonic axis specification and digit patterning. Finally, we demonstrate how existing synthetic circuits can be extended with additional feedbacks to form Turing reaction-diffusion systems. Our study offers a new theoretical framework to understand multicellular pattern formation and enables the wide-spread use of mathematical biology to engineer synthetic patterning systems.

POS-1 Promotes Endo-mesoderm Development by Inhibiting the Cytoplasmic Polyadenylation of neg-1 mRNA

The regulation of mRNA translation is of fundamental importance in biological mechanisms ranging from embryonic axis specification to the formation of long-term memory. POS-1 is one of several CCCH zinc-finger RNA-binding proteins that regulate cell fate specification during C. elegans embryogenesis. Paradoxically, pos-1 mutants exhibit striking defects in endo-mesoderm development but have wild-type distributions of SKN-1, a key determinant of endo-mesoderm fates. RNAi screens for pos-1 suppressors identified genes encoding the cytoplasmic poly(A)-polymerase homolog GLD-2, the Bicaudal-C homolog GLD-3, and the protein NEG-1. We show that NEG-1 localizes in anterior nuclei, where it negatively regulates endo-mesoderm fates. In posterior cells, POS-1 binds the neg-1 3' UTR to oppose GLD-2 and GLD-3 activities that promote NEG-1 expression and cytoplasmic lengthening of the neg-1 mRNA poly(A) tail. Our findings uncover an intricate series of post-transcriptional regulatory interactions that, together, achieve precise spatial expression of endo-mesoderm fates in C. elegans embryos.

Contribution of Maternal NODAL to Term Pregnancy

Contribution of Maternal NODAL to Term Pregnancy

In Situ Hybridization: Fruit Fly Embryos and Tissues

AbstractIt is well known that transcript localization controls important biological processes, including cell fate determination, cell polarity, cell migration, morphogenesis, neuronal function, and embryonic axis specification. Thus, the sub‐cellular visualization of transcripts in ‘their original place’ (in situ) is an important tool to infer and understand their trafficking, stability, translation, and biological functions. This has been made possible through the use of labeled ‘anti‐sense’ probes that can be readily detected after hybridization to their ‘sense’ counterparts. The following is a series of protocols for conducting in situ hybridization in Drosophila embryos or tissues. These methods include standard alkaline phosphatase methods, as well as higher resolution and throughput variations using fluorescence‐based probe detection. New modifications that enhance probe penetration and detection in various tissues are also provided. Curr. Protoc. Essential Lab. Tech. 4:9.3.1‐9.3.24. © 2010 by John Wiley & Sons, Inc.

Regulation of TCF3 by Wnt-Dependent Phosphorylation during Vertebrate Axis Specification

A commonly accepted model of Wnt/β-catenin signaling involves target gene activation bya complex of β-catenin with a T-cell factor (TCF) family member. TCF3 is a transcriptional repressor that has been implicated in Wnt signaling and plays key roles in embryonic axis specification and stem cell differentiation. Here we demonstrate that Wnt proteins stimulate TCF3 phosphorylation in gastrulating Xenopus embryos and mammalian cells. This phosphorylation event involves β-catenin-mediated recruitment of homeodomain-interacting protein kinase 2 (HIPK2) to TCF3 and culminates in the dissociation of TCF3 from a target gene promoter. Mutated TCF3 proteins resistant to Wnt-dependent phosphorylation function as constitutive inhibitors of Wnt-mediated activation of Vent2 and Cdx4 during anteroposterior axis specification. These findings reveal an alternative invivo mechanism of Wnt signaling that involves TCF3 phosphorylation and subsequent derepression of target genes and link this molecular event to a specific developmental process.

Assisted fertilization and embryonic axis formation in higher primates

In naturally fertilized embryos of various organisms, the spermatozoon provides a localized cue to initiate early embryonic patterning. In mice, the sperm entry point (SEP) may reorient the first cleavage division, which separates the zygote into two halves that follow distinct fates. However, it is unknown whether the mechanical injection of spermatozoa into an oocyte by intracytoplasmic sperm injection (ICSI), a technique commonly used in human assisted reproduction, possesses such a role. Rhesus macaque embryos fertilized by ICSI were examined in order to determine the consequences of placing the spermatozoon at specific positions in the ooplasm and whether this can provide new information about patterning in mammalian eggs. The SEP specified by the injected spermatozoa was most often localized near the first cleavage plane and was mainly distributed along the boundary zone that separates the embryonic and abembryonic parts of the monkey blastocyst. Moreover, the ICSI data, when compared with naturally fertilized mouse embryos, showed a similar outcome in terms of cleavage axes and first embryonic axis specification. As there are no studies to date regarding sperm entry in human oocytes and its influence on embryonic development, this investigation using the rhesus macaque as a clinical model is noteworthy.

Evidence for a composite anterior determinant in the hover flyEpisyrphus balteatus(Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage

Most insect embryos develop from a monolayer of cells around the yolk, but only part of this blastoderm forms the embryonic rudiment. Another part forms extra-embryonic serosa. Size and position of the serosa anlage vary between species, and previous work raises the issue of whether such differences co-evolve with the mechanisms that establish anteroposterior (AP) polarity of the embryo. AP polarity of the Drosophila embryo depends on bicoid, which is necessary and sufficient to determine the anterior body plan. Orthologs of bicoid have been identified in various cyclorrhaphan flies and their occurrence seems to correlate with a mid-dorsal serosa or amnioserosa anlage. Here, we introduce with Episyrphus balteatus (Syrphidae) a cyclorrhaphan model for embryonic AP axis specification that features an anterodorsal serosa anlage. Current phylogenies place Episyrphus within the clade that uses bicoid mRNA as anterior determinant, but no bicoid-like sequence could be identified in this species. Using RNA interference (RNAi) and ectopic mRNA injection, we obtained evidence that pattern formation along the entire AP axis of the Episyrphus embryo relies heavily on the precise regulation of caudal, and that anterior pattern formation in particular depends on two localized factors rather than one. Early zygotic activation of orthodenticle is separated from anterior repression of caudal, two distinct functions which in Drosophila are performed jointly by bicoid, whereas hunchback appears to be regulated by both factors. Furthermore, we found that overexpression of orthodenticle is sufficient to confine the serosa anlage of Episyrphus to dorsal blastoderm. We discuss our findings in a phylogenetic context and propose that Episyrphus employs a primitive cyclorrhaphan mechanism of AP axis specification.

Development of polarized female germline cysts in the polychaete, Ophryotrocha labronica

Polarized oogenic cysts are clonal syncytia in which cellular compartments (cystocytes) differentiate as either oocytes or nurse cells-supportive germ cells that transport material to the oocyte through cytoplasmic bridges. Although polarized cysts have evolved independently in several animal phyla, most research on this phenomenon has focused on insects, particularly Drosophila melanogaster. To provide a foundation for comparative studies of cyst polarization, we have examined the coelomic development of polarized cysts in an annelid worm, Ophryotrocha labronica, using a variety of microscopic techniques. This species has elegantly simple cysts, each consisting of two interconnected cells (one oocyte and one nurse cell) free of somatic follicular tissue. These cysts arise from segmental ovaries, but are released to the coelom early in oogenesis, where they carry out most of their development. Although the oocyte remains transcriptionally active through oogenesis, nurse cells in this species are robust biosynthetic factories, with highly polyploid nuclei, enormous nucleoli, numerous mitochondria, and abundant rough endoplasmic reticulum. Ultimately, the nurse cell dumps its cytoplasm into oocyte, detaches, and undergoes programmed cell death. Although in insects, and possibly some polychaetes, the overall polarity of the cyst is linked to the specification of future embryonic axes in the oocyte, in Ophryotrocha this appears not to be the case. Our findings provide a detailed analysis of cyst development, addressing earlier misconceptions in the literature and adding new insights. This foundation shows the potential of Ophryotrocha labronica as a model system for further experimental analysis of nurse cell-oocyte differentiation.

Establishing Asymmetry

Fertilization of the Xenopus egg leads to the asymmetrical distribution of components of the Wnt/β-catenin pathway to what will become the dorsal side of the embryo. This leads to asymmetric signaling by Nodal, a transforming growth factor–β ligand critical to embryonic axis specification and to mesoderm induction. Martello et al . found that microRNAs (miRNAs) from gastrula-stage Xenopus embryos inhibited embryonic Nodal signaling. Computational analysis revealed binding sites for two related miRNAs, miR-15 and miR-16, in the 3′ untranslated region (3′ UTR) of mRNA encoding the type II Nodal receptor Acvr2a. Activity of a reporter containing a wild-type miR-15–binding site was reduced in Xenopus embryos and human HepG2 cells compared with that of a reporter containing a mutated binding site. In addition, activity of the former was further decreased by overexpression of miR-15 or its primary precursor (pri-miR-15/16). Injection of miR-15 inhibited endogenous Nodal signaling and attenuated downstream sequelae such as mesoderm induction and development of Spemann’s organizer, phenotypic defects that were rescued by coinjection of Acvr2a mRNA lacking the 3′ UTR. In contrast, inhibitory oligonucleotides directed against miR-15 and miR-16 increased Acvr2a abundance and promoted expansion of Spemann’s organizer. The abundance and activity of miR-15 and miR-16 were greatest ventrally; Acvr2a abundance and Smad2 phosphorylation (p-Smad2, a marker for Nodal signaling) were greatest dorsally. Injection of pri-miR-15/16 led to a decrease in dorsal p-Smad2, whereas inhibition of miR-15 and miR-16 led to an increase in ventral p-Smad2. Overexpression of Xwnt-8 or β-catenin mRNA decreased the abundance and activity of miR-15 and miR-16, whereas β-catenin knockdown increased them. Thus, miR-15 and miR-16 appear to constitute a link between asymmetry in Wnt/β-catenin and asymmetry in Nodal signaling and, thereby, to play a crucial role in the regulation of embryonic patterning. G. Martello, L. Zacchigna, M. Inui, M. Montagner, M. Adorno, A. Mamidi, L. Morsut, S. Soligo, U. Tran, S. Dupont, M. Cordenonsi, O. Wessely, S. Piccolo, MicroRNA control of Nodal signalling. Nature 449 , 183-188 (2007). [PubMed]