Dorsal Mesoderm Research Articles

An RNA interference approach for functional studies in the sea urchin and its use in analysis of Nodal signaling gradients.

Dicer substrate interfering RNAs (DsiRNAs) destroy targeted transcripts using the RNA-Induced Silencing Complex (RISC) through a process called RNA interference (RNAi). This process is ubiquitous among eukaryotes. Here we report the utility of DsiRNA in embryos of the sea urchin Lytechinus variagatus (Lv). Specific knockdowns phenocopy known morpholino and inhibitor knockdowns, and DsiRNA offers a useful alternative to morpholinos. Methods for designing and obtaining specific DsiRNAs that lead to destruction of targeted mRNA are described. DsiRNAs directed against pks1, an enzyme necessary for pigment production, show how successful DsiRNA perturbations are monitored by RNA in situ analysis and by qPCR to determine relative destruction of targeted mRNA. DsiRNA-based knockdowns phenocopy morpholino- and drug-based inhibition of nodal and lefty. Other knockdowns demonstrate that the RISC operates early in development as well as on genes that are first transcribed hours after gastrulation is completed. Thus, DsiRNAs effectively mediate destruction of targeted mRNA in the sea urchin embryo. The approach offers significant advantages over other widely used methods in the urchin in terms of cost, and ease of procurement, and offers sizeable experimental advantages in terms of ease of handling, injection, and knockdown validation.

Two Homeobox Transcription Factors, Goosecoid and Ventx1.1, Oppositely Regulate Chordin Transcription in Xenopus Gastrula Embryos.

The reciprocal inhibition between two signaling centers, the Spemann organizer (dorsal mesoderm) and ventral region (mesoderm and ectoderm), collectively regulate the overall development of vertebrate embryos. Each center expresses key homeobox transcription factors (TFs) that directly control target gene transcription. Goosecoid (Gsc) is an organizer (dorsal mesoderm)-specific TF known to induce dorsal fate and inhibit ventral/ectodermal specification. Ventx1.1 (downstream of Bmp signaling) induces the epidermal lineage and inhibits dorsal organizer-specific genes from the ventral region. Chordin (Chrd) is an organizer-specific secreted Bmp antagonist whose expression is primarily activated by Gsc. Alternatively, chrd expression is repressed by Bmp/Ventx1.1 in the ventral/epidermal region. However, the regulatory mechanisms underlying the transcription mediated by Gsc and Ventx1.1 remain elusive. Here, we found that the chrd promoter contained two cis-acting response elements that responded negatively to Ventx1.1 and positively to Gsc. In the ventral/ectodermal region, Ventx1.1 was directly bound to the Ventx1.1 response element (VRE) and inhibited chrd transcription. In the organizer region, Gsc was bound to the Gsc response elements (GRE) to activate chrd transcription. The Gsc-mediated positive response on the chrd promoter completely depended on another adjacent Wnt response cis-acting element (WRE), which was the TCF7 (also known as Tcf1) binding element. Site-directed mutagenesis of VRE, GRE, or WRE completely abolished the repressive or activator activity of Ventx1.1 and Gsc, respectively. The ChIP-PCR results confirmed the direct binding of Ventx1.1 and Gsc/Tcf7 to VRE and GRE/WRE, respectively. These results demonstrated that chrd expression is oppositely modulated by homeobox TFs, Ventx1.1, and Gsc/Tcf7 during the embryonic patterning of Xenopus gastrula.

Microsurgical Methods to Make the Keller Sandwich Explant and the Dorsal Isolate.

This protocol summarizes preparation of the dorsal marginal zone sandwich explant (a.k.a. the "Keller sandwich") and the dorsal isolate from Xenopus embryos. The Keller sandwich is assembled from two early gastrula stage dorsal marginal zone (DMZ) explants. DMZ explants isolated before involution maintain planar patterning processes and block radial signals that might be exchanged between pre- and postinvolution tissues. DMZ explants isolated later in gastrulation, but subsequently opened and flattened may have both planar and radial patterning. The epithelial margins of DMZ explants in Keller sandwiches heal and basal contacts form between the deep layers of the two DMZ explants. The dorsal isolate is dissected from mid- to late-gastrula stage embryos after involution and archenteron formation. Germ-layer contacts between dorsal endoderm, mesoderm, and ectoderm generated by gastrulation movements are maintained in the dorsal isolate. These two explants can be used to study tissue, cell, and subcellular processes relevant to convergent extension, from patterning to cell behaviors, and their collective biomechanics. Skills needed to dissect the Keller sandwich are greater than those needed to dissect animal cap ectoderm and can be mastered in a few weeks; skills needed to dissect the dorsal isolate are similar to those needed to dissect animal caps and can be learned in a week.

FGF-mediated establishment of left-right asymmetry requires Rab7 function in the dorsal mesoderm in Xenopus.

Gastrulation denotes a very important developmental process, which includes significant structural tissue rearrangements and patterning events that shape the emerging vertebrate organism. At the end of gastrulation, the three body axes are spatially defined while the left-right axis still lacks any molecular or morphological polarity. In most vertebrates, this is established during neurulation by a symmetry breaking LR organizer. However, this mesoderm-derived structure depends on proper induction and specification of the mesoderm, which in turn requires involvement of several signaling pathways. Endocytosis and the endosomal machinery offer manifold platforms for intracellular pathway regulation, especially late endosomes claim increasing attention. The late endosomal regulator Rab7 has been linked to mesoderm specification during gastrulation. Distinct axial defects due to compromised dorsal mesoderm development in rab7-deficient Xenopus embryos suggested a requirement of Rab7 for FGF-dependent mesoderm patterning and LR asymmetry. Here we specifically addressed such a role of Rab7, demonstrating a functional requirement for LR organizer development and symmetry breakage. Using different FGF/MAPK pathway components we show that Rab7 participates in dorsal mesoderm patterning. We suggest a hierarchical classification of Rab7 upstream of MAPK-dependent mesoderm specification, most probably at the level of the small GTPase Ras. Thus, this study affords an insight on how the Rab7-regulated endosomal machinery could participate in signal transduction to enable correct mesoderm specification and left-right asymmetry.

Bmp Signal Gradient Modulates Convergent Cell Movement via Xarhgef3.2 during Gastrulation of Xenopus Embryos.

Gastrulation is a critical step in the establishment of a basic body plan during development. Convergence and extension (CE) cell movements organize germ layers during gastrulation. Noncanonical Wnt signaling has been known as major signaling that regulates CE cell movement by activating Rho and Rac. In addition, Bmp molecules are expressed in the ventral side of a developing embryo, and the ventral mesoderm region undergoes minimal CE cell movement while the dorsal mesoderm undergoes dynamic cell movements. This suggests that Bmp signal gradient may affect CE cell movement. To investigate whether Bmp signaling negatively regulates CE cell movements, we performed microarray-based screening and found that the transcription of Xenopus Arhgef3.2 (Rho guanine nucleotide exchange factor) was negatively regulated by Bmp signaling. We also showed that overexpression or knockdown of Xarhgef3.2 caused gastrulation defects. Interestingly, Xarhgef3.2 controlled gastrulation cell movements through interacting with Disheveled (Dsh2) and Dsh2-associated activator of morphogenesis 1 (Daam1). Our results suggest that Bmp gradient affects gastrulation cell movement (CE) via negative regulation of Xarhgef3.2 expression.

Foxd4l1.1 Negatively Regulates Chordin Transcription in Neuroectoderm of Xenopus Gastrula.

Inhibition of the bone morphogenetic proteins (BMPs) is the primary step toward neuroectoderm formation in vertebrates. In this process, the Spemann organizer of the dorsal mesoderm plays a decisive role by secreting several extracellular BMP inhibitors such as Chordin (Chrd). Chrd physically interacts with BMP proteins and inhibits BMP signaling, which triggers the expression of neural-specific transcription factors (TFs), including Foxd4l1.1. Thus, Chrd induces in a BMP-inhibited manner and promotes neuroectoderm formation. However, the regulatory feedback mechanism of Foxd4l1.1 on mesodermal genes expression during germ-layer specification has not been fully elucidated. In this study, we investigated the regulatory mechanism of Foxd4l1.1 on chrd (a mesodermal gene). We demonstrate that Foxd4l1.1 inhibits chrd expression during neuroectoderm formation in two ways: First, Foxd4l1.1 directly binds to FRE (Foxd4l1.1 response elements) within the chrd promoter region to inhibit transcription. Second, Foxd4l1.1 physically interacts with Smad2 and Smad3, and this interaction blocks Smad2 and Smad3 binding to activin response elements (AREs) within the chrd promoter. Site-directed mutagenesis of FRE within the chrd(-2250) promoter completely abolished repressor activity of the Foxd4l1.1. RT-PCR and reporter gene assay results indicate that Foxd4l1.1 strongly inhibits mesoderm- and ectoderm-specific marker genes to maintain neural fate. Altogether, these results suggest that Foxd4l1.1 negatively regulates chrd transcription by dual mechanism. Thus, our study demonstrates the existence of precise reciprocal regulation of chrd transcription during neuroectoderm and mesoderm germ-layer specification in Xenopus embryos.

Goosecoid Controls Neuroectoderm Specification via Dual Circuits of Direct Repression and Indirect Stimulation in Xenopus Embryos.

Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.

Temporal transcriptomic profiling reveals dynamic changes in gene expression of Xenopus animal cap upon activin treatment

Activin, a member of the transforming growth factor-β (TGF-β) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann’s organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.

Furry is required for cell movements during gastrulation and functionally interacts with NDR1

Gastrulation is a key event in animal embryogenesis during which germ layer precursors are rearranged and the embryonic axes are established. Cell polarization is essential during gastrulation, driving asymmetric cell division, cell movements, and cell shape changes. The furry (fry) gene encodes an evolutionarily conserved protein with a wide variety of cellular functions, including cell polarization and morphogenesis in invertebrates. However, little is known about its function in vertebrate development. Here, we show that in Xenopus, Fry plays a role in morphogenetic processes during gastrulation, in addition to its previously described function in the regulation of dorsal mesoderm gene expression. Using morpholino knock-down, we demonstrate a distinct role for Fry in blastopore closure and dorsal axis elongation. Loss of Fry function drastically affects the movement and morphological polarization of cells during gastrulation and disrupts dorsal mesoderm convergent extension, responsible for head-to-tail elongation. Finally, we evaluate a functional interaction between Fry and NDR1 kinase, providing evidence of an evolutionarily conserved complex required for morphogenesis.

The saga of neural induction: almost a century of research

Neural induction is a developmental process that allows cells from the ectoderm (the target tissue) to acquire a neural fate in response to signals coming from a specific adjacent embryonic region, the dorsal mesoderm (the inducing tissue). This process described in 1924 in amphibian embryos has not received a molecular explanation until the mid-1990s. Most of the work on neural induction has been carried out in amphibians. At these times, although the role played by the membrane of the target tissue had been suggested, no definitive work had been performed on the transduction of the neuralizing signal. Between 1990 and 2019 our aim was to decipher this transduction. We have underlined the necessary and sufficient role played by calcium signaling to induce ectoderm cells towards a neural fate from the activation of calcium channels to the direct transcription of early neural genes by calcium.

Fgf-driven Tbx protein activities directly induce myf5 and myod to initiate zebrafish myogenesis.

ABSTRACTSkeletal muscle derives from dorsal mesoderm formed during vertebrate gastrulation. Fibroblast growth factor (Fgf) signalling cooperates with Tbx transcription factors to promote dorsal mesoderm formation, but their role in myogenesis has been unclear. Using zebrafish, we show that dorsally derived Fgf signals act through Tbx16 and Tbxta to induce slow and fast trunk muscle precursors at distinct dorsoventral positions. Tbx16 binds to and directly activates the myf5 and myod genes, which are required for commitment to myogenesis. Tbx16 activity depends on Fgf signalling from the organiser. In contrast, Tbxta is not required for myf5 expression, but binds a specific site upstream of myod that is not bound by Tbx16 and drives (dependent on Fgf signals) myod expression in adaxial slow precursors, thereby initiating trunk myogenesis. After gastrulation, when similar muscle cell populations in the post-anal tail are generated from tailbud, declining Fgf signalling is less effective at initiating adaxial myogenesis, which is instead initiated by Hedgehog signalling from the notochord. Our findings suggest a hypothesis for ancestral vertebrate trunk myogenic patterning and how it was co-opted during tail evolution to generate similar muscle by new mechanisms.This article has an associated ‘The people behind the papers’ interview.

Application of a human mesoderm tissue elongation system in vitro derived from human induced pluripotent stem cells to risk assessment for teratogenic chemicals

Toxic compounds from the mother’s diet and medication in addition to genetic factors and infection during pregnancy remain risks for various congenital disorders and misbirth. To ensure the safety of food and drugs for pregnant women, establishment of an in vitro system that morphologically resembles human tissues has been long desired. In this study, we focused on dorsal mesoderm elongation, one of the critical early development events for trunk formation, and we established in vitro autonomous elongating tissues from human induced pluripotent stem cells (hiPSCs). This artificial tissue elongation is regulated by MYOSIN II and FGF signaling, and is diminished by methylmercury or retinoic acid (RA), similar to in vivo human developmental disabilities. Moreover, our method for differentiation of hiPSCs requires only a short culture period, and the elongation is cell number-independent. Therefore, our in vitro human tissue elongation system is a potential tool for risk assessment assays for identification of teratogenic chemicals via human tissue morphogenesis.

The regulatory proteins DSCR6 and Ezh2 oppositely regulate Stat3 transcriptional activity in mesoderm patterning during Xenopus development

Embryonic cell fate specification and axis patterning requires integration of several signaling pathways that orchestrate region-specific gene expression. The transcription factor signal transducer and activator of transcription 3 (Stat3) plays important roles during early development, but it is unclear how Stat3 is activated. Here, using Xenopus as a model, we analyzed the post-translational regulation and functional consequences of Stat3 activation in dorsoventral axis patterning. We show that Stat3 phosphorylation, lysine methylation, and transcriptional activity increase before gastrulation and induce ventral mesoderm formation. Down syndrome critical region gene 6 (DSCR6), a RIPPLY family member that induces dorsal mesoderm by releasing repressive polycomb group proteins from chromatin, bound to the Stat3 C-terminal region and antagonized its transcriptional and ventralizing activities by interfering with its lysine methylation. Enhancer of zeste 2 polycomb-repressive complex 2 subunit (Ezh2) also bound to this region; however, its methyltransferase activity was required for Stat3 methylation and activation. Loss of Ezh2 resulted in dorsalization of ventral mesoderm and formation of a secondary axis. Furthermore, interference with Ezh2 phosphorylation also prevented Stat3 lysine methylation and transcriptional activity. Thus, inhibition of either Ezh2 phosphorylation or Stat3 lysine methylation compensated for the absence of DSCR6 function. These results reveal that DSCR6 and Ezh2 critically and post-translationally regulate Stat3 transcriptional activity. Ezh2 promotes Stat3 activation in ventral mesoderm formation independently of epigenetic regulation, whereas DSCR6 specifies dorsal fate by counteracting this ventralizing activity. This antagonism helps pattern the mesoderm along the dorsoventral axis, representing a critical facet of cell identity regulation during development.

Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis.

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up-regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan-Trk inhibitor K252a or the TrkA inhibitor GW-441756, but not by the TrkB inhibitor ANA-12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.

The roles and controls of GATA factors in blood and cardiac development

GATA factors play central roles in the programming of blood and cardiac cells during embryonic development. Using the experimentally accessible Xenopus and zebrafish models, we report observations regarding the roles of GATA‐2 in the development of blood stem cells and GATA‐4, ‐5, and ‐6 in cardiac development. We show that blood stem cells develop from the dorsal lateral plate mesoderm and GATA‐2 is required at multiple stages. Firstly, GATA‐2 is required to make the cells responsive to VEGF‐A signalling by driving the synthesis of its receptor, FLK‐1/KDR. This leads to differentiation into the endothelial cells that form the dorsal aorta. GATA‐2 is again required for the endothelial‐to‐haematopoietic transition that takes place later in the floor of the dorsal aorta. GATA‐2 expression is dependent on BMP signalling for each of these inputs into blood stem cell programming. GATA‐4, ‐5, and ‐6 work together to ensure the specification of cardiac cells during development. We have demonstrated redundancy within the family and also some evolution of the functions of the different family members. Interestingly, one of the features that varies in evolution is the timing of expression relative to other key regulators such as Nkx2.5 and BMP. We show that the GATA factors, Nkx2.5 and BMP regulate each other and it would appear that what is critical is the mutually supportive network of expression rather than the order of expression of each of the component genes. In Xenopus and zebrafish, the cardiac mesoderm is adjacent to an anterior population of cells giving rise to blood and endothelium. This population is not present in mammals and we have shown that, like the cardiac population, the blood and endothelial precursors require GATA‐4, ‐5, and ‐6 for their development. Later, blood‐specific or cardiac‐specific regulators determine the ultimate fate of the cells, and we show that these regulators act cross‐antagonistically. Fibroblast growth factor (FGF) signalling drives the cardiac fate, and we propose that the anterior extension of the FGF signalling field during evolution led to the recruitment of the blood and endothelial precursors into the heart field ultimately resulting in a larger four chambered heart. Zebrafish are able to successfully regenerate their hearts after injury. To understand the pathways involved, with a view to determining why humans cannot do this, we profiled gene expression in the cardiomyocytes before and after injury, and compared those proximal to the injury with those more distal. We were able to identify an enhancement of the expression of regulators of the canonical Wnt pathway proximal to the injury, suggesting that changes in Wnt signalling are responsible for the repair response to injury.

Mesoderm and endoderm internalization in the Xenopus gastrula.

Mesoderm and endoderm internalization in the Xenopus embryo are based on a number of region-specific morphogenetic processes that co-act in the vegetal half of the gastrula. In the multilayered wall surrounding the blastocoel, the apical layer engages in bottle cell formation and associated invagination and involution movements, and in cell intercalation in the plane of the layer. Of these epithelial-type processes, only bottle cell formation has been analyzed mechanistically. In the deep layers of the blastocoel wall, cell-on-cell migration drives the internalization of mesoderm by various forms of involution and of the endodermal cell mass by vegetal rotation. In the mesoderm, cells migrate in a mesenchymal mode with the aid of locomotory protrusions, whereas cells of the vegetal cell mass resemble free bottle cells that engage in ingression-type amoeboid migration. Cells rearrange by differential migration leading to parallel or orthogonal forms of intercalation and respective types of convergent extension. The interaction of the various apical and deep layer processes gives rise to dorsal multilayer invagination, ventrolateral internal involution, peak involution and orthogonal convergent extension of the dorsal posterior mesoderm, vegetal rotation, and blastopore constriction. It is speculated how these multilayer gastrulation movements could be derived from mechanisms in invertebrate single-epithelium gastrulae.

Evolution of cis-regulatory modules for the head organizer gene goosecoid in chordates: comparisons between Branchiostoma and Xenopus

BackgroundIn cephalochordates (amphioxus), the notochord runs along the dorsal to the anterior tip of the body. In contrast, the vertebrate head is formed anterior to the notochord, as a result of head organizer formation in anterior mesoderm during early development. A key gene for the vertebrate head organizer, goosecoid (gsc), is broadly expressed in the dorsal mesoderm of amphioxus gastrula. Amphioxus gsc expression subsequently becomes restricted to the posterior notochord from the early neurula. This has prompted the hypothesis that a change in expression patterns of gsc led to development of the vertebrate head during chordate evolution. However, molecular mechanisms of head organizer evolution involving gsc have never been elucidated.ResultsTo address this question, we compared cis-regulatory modules of vertebrate organizer genes between amphioxus, Branchiostoma japonicum, and frogs, Xenopus laevis and Xenopus tropicalis. Here we show conservation and diversification of gene regulatory mechanisms through cis-regulatory modules for gsc, lim1/lhx1, and chordin in Branchiostoma and Xenopus. Reporter analysis using Xenopus embryos demonstrates that activation of gsc by Nodal/FoxH1 signal through the 5′ upstream region, that of lim1 by Nodal/FoxH1 signal through the first intron, and that of chordin by Lim1 through the second intron, are conserved between amphioxus and Xenopus. However, activation of gsc by Lim1 and Otx through the 5′ upstream region in Xenopus are not conserved in amphioxus. Furthermore, the 5′ region of amphioxus gsc recapitulated the amphioxus-like posterior mesoderm expression of the reporter gene in transgenic Xenopus embryos.ConclusionsOn the basis of this study, we propose a model, in which the gsc gene acquired the cis-regulatory module bound with Lim1 and Otx at its 5′ upstream region to be activated persistently in anterior mesoderm, in the vertebrate lineage. Because Gsc globally represses trunk (notochord) genes in the vertebrate head organizer, this cooption of gsc in vertebrates appears to have resulted in inhibition of trunk genes and acquisition of the head organizer and its derivative prechordal plate.

Ventx1.1 competes with a transcriptional activator Xcad2 to regulate negatively its own expression

Dorsoventral patterning of body axis in vertebrate embryo is tightly controlled by a complex regulatory network of transcription factors. Ventx1.1 is known as a transcriptional repressor to inhibit dorsal mesoderm formation and neural differentiation in Xenopus. In an attempt to identify, using chromatin immunoprecipitation (ChIP)-Seq, genome-wide binding pattern of Ventx1.1 in Xenopus gastrulae, we observed that Ventx1.1 associates with its own 5′-flanking sequence. In this study, we present evidence that Ventx1.1 binds a cis-acting Ventx1.1 response element (VRE) in its own promoter, leading to repression of its own transcription. Site-directed mutagenesis of the VRE in the Ventx1.1 promoter significantly abrogated this inhibitory autoregulation of Ventx1.1 transcription. Notably, Ventx1.1 and Xcad2, an activator of Ventx1.1 transcription, competitively co-occupied the VRE in the Ventx1.1 promoter. In support of this, mutation of the VRE down-regulated basal and Xcad2-induced levels of Ventx1.1 promoter activity. In addition, overexpression of Ventx1.1 prevented Xcad2 from binding to the Ventx1.1 promoter, and vice versa. Taken together, these results suggest that Ventx1.1 negatively regulates its own transcription in competition with Xcad2, thereby fine-tuning its own expression levels during dorsoventral patterning of Xenopus early embryo.

Ventx1.1 as a Direct Repressor of Early Neural Gene zic3 in Xenopus laevis.

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.

Roles for Xenopus aquaporin-3b (aqp3.L) during gastrulation: Fibrillar fibronectin and tissue boundary establishment in the dorsal margin

Aquaporins and aquaglyceroporins are a large family of membrane channel proteins that allow rapid movement of water and small, uncharged solutes into and out of cells along concentration gradients. Recently, aquaporins have been gaining recognition for more complex biological roles than the regulation of cellular osmotic homeostasis. We have identified a specific expression pattern for Xenopus aqp3b (also called aqp3.L) during gastrulation, where it is localized to the sensorial (deep) layer of the blastocoel roof and dorsal margin. Interference with aqp3b expression resulted in loss of fibrillar fibronectin matrix in Brachet's cleft at the dorsal marginal zone, but not on the free surface of the blastocoel. Detailed observation showed that the absence of fibronectin matrix correlated with compromised border integrities between involuted mesendoderm and noninvoluted ectoderm in the marginal zone. Knockdown of aqp3b also led to delayed closure of the blastopore, suggesting defects in gastrulation movements. Radial intercalation was not affected in aqp3b morphants, while the data presented are consistent with impeded convergent extension movements of the dorsal mesoderm in response to loss of aqp3b. Our emerging model suggests that aqp3b is part of a mechanism that promotes proper interaction between cells and the extracellular matrix, thereby playing a critical role in gastrulation.