Gastrulation Defects Research Articles

A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

BackgroundENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome.ResultsWe isolated 11 lethal lines that map to the region of chromosome 4 between D4Mit117 and D4Mit281. These lines form 10 complementation groups. The majority of lines die during embryonic development between E5.5 and E12.5 and display defects in gastrulation, cardiac development, and craniofacial development. One line displayed postnatal lethality and neurological defects, including ataxia and seizures.ConclusionThese eleven mutants allow us to query gene function within the distal region of mouse chromosome 4 and demonstrate that new mouse models of mammalian developmental defects can easily and quickly be generated and mapped with the use of ENU-mutagenesis in combination with balancer chromosomes. The low number of mutations isolated in this screen compared with other balancer chromosome screens indicates that the functions of genes in different regions of the genome vary widely.

Characterization of the functional properties of the neuroectoderm in mouse Cripto-/-embryos showing severe gastrulation defects

During development of the mammalian embryo, there is a complex relation between formation of the mesoderm and the neuroectoderm. In mouse, for example, the role of the node and its mesendoderm derivatives in anterior neural specification is still debated. Mouse Cripto(-/-) embryos could potentially help settle this debate because they lack almost all embryonic endoderm and mesoderm, including the node and its derivatives. In the present paper, we show that Cripto(-/-) embryos can still form functional neural stem cells that are able to differentiate and maintain a neural phenotype both in vivo and in vitro. These data suggest that signals emanating from the mesoderm and endoderm might not be essential for the formation and differentiation of neural stem cells. However, we use grafting experiments to show that the Cripto(-/-) isthmus (the secondary organizer located at the midbrain-hindbrain boundary) loses its inductive ability. We further show that the Cripto(-/-)isthmus expresses lower amounts of the isthmic signalling molecule, Fgf8. Since nearby tissues remain competent to respond to exogenously added Fgf8, this reduction in Fgf8 levels in the Cripto(-/-) isthmus is the potential cause of the loss of patterning ability in graft experiments. Overall, we interpret our data to suggest that the mammalian node and primitive streak are essential for the development of the regional identities that control the specification and formation of the secondary organizers within the developing brain.

Maternal Interferon Regulatory Factor 6 is required for the differentiation of primary superficial epithelia in Danio and Xenopus embryos

Early in the development of animal embryos, superficial cells of the blastula form a distinct lineage and adopt an epithelial morphology. In different animals, the fate of these primary superficial epithelial (PSE) cells varies, and it is unclear whether pathways governing segregation of blastomeres into the PSE lineage are conserved. Mutations in the gene encoding Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic forms of cleft lip and palate, consistent with a role for Irf6 in development of oral epithelia, and mouse Irf6 targeted null mutant embryos display abnormal differentiation of oral epithelia and skin. In Danio rerio (zebrafish) and Xenopus laevis (African clawed frog) embryos, zygotic irf6 transcripts are present in many epithelial tissues including the presumptive PSE cells and maternal irf6 transcripts are present throughout all cells at the blastula stage. Injection of antisense oligonucleotides with ability to disrupt translation of irf6 transcripts caused little or no effect on development. By contrast, injection of RNA encoding a putative dominant negative Irf6 caused epiboly arrest, loss of gene expression characteristic of the EVL, and rupture of the embryo at late gastrula stage. The dominant negative Irf6 disrupted EVL gene expression in a cell autonomous fashion. These results suggest that Irf6 translated in the oocyte or unfertilized egg suffices for early development. Supporting the importance of maternal Irf6, we show that depletion of maternal irf6 transcripts in X. laevis embryos leads to gastrulation defects and rupture of the superficial epithelium. These experiments reveal a conserved role for maternally-encoded Irf6 in differentiation of a simple epithelium in X. laevis and D. rerio. This epithelium constitutes a novel model tissue in which to explore the Irf6 regulatory pathway.

Comparative Phosphoproteomics of Zebrafish Fyn/Yes Morpholino Knockdown Embryos

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

The C. elegans Zonula Occludens Ortholog Cooperates with the Cadherin Complex to Recruit Actin during Morphogenesis

The dramatic cell-shape changes necessary to form a multicellular organism require cell-cell junctions to be both pliable and strong. The zonula occludens (ZO) subfamily of membrane-associated guanylate kinases (MAGUKs) are scaffolding molecules thought to regulate cell-cell adhesion [1-3], but there is little known about their roles in vivo. To elucidate the functional role of ZO proteins in a living embryo, we have characterized the sole C. elegans ZO family member, ZOO-1. ZOO-1 localizes with the cadherin-catenin complex during development, and its junctional recruitment requires the transmembrane proteins HMR-1/E-cadherin and VAB-9/claudin, but surprisingly, not HMP-1/alpha-catenin or HMP-2/beta-catenin. zoo-1 knockdown results in lethality during elongation, resulting in the rupture of epidermal cell-cell junctions under stress and failure of epidermal sheet sealing at the ventral midline. Consistent with a role in recruiting actin to the junction in parallel to the cadherin-catenin complex, zoo-1 loss of function reduces the dynamic recruitment of actin to junctions and enhances the severity of actin filament defects in hypomorphic alleles of hmp-1 and hmp-2. These results show that ZOO-1 cooperates with the cadherin-catenin complex to dynamically regulate strong junctional anchorage to the actin cytoskeleton during morphogenesis.

Mesp1 Acts as a Master Regulator of Multipotent Cardiovascular Progenitor Specification

During embryonic development, multipotent cardiovascular progenitor cells are specified from early mesoderm. Using mouse ESCs in which gene expression can be temporally regulated, we have found that transient expression of Mesp1 dramatically accelerates and enhances multipotent cardiovascular progenitor specification through an intrinsic and cell autonomous mechanism. Genome-wide transcriptional analysis indicates that Mesp1 rapidly activates and represses a discrete set of genes, and chromatin immunoprecipitation shows that Mesp1 directly binds to regulatory DNA sequences located in the promoter of many key genes in the core cardiac transcriptional machinery, resulting in their rapid upregulation. Mesp1 also directly represses the expression of key genes regulating other early mesoderm and endoderm cell fates. Our results demonstrate that Mesp1 acts as a key regulatory switch during cardiovascular specification, residing at the top of the hierarchy of the gene network responsible for cardiovascular cell-fate determination.

Pnas4 is a novel regulator for convergence and extension during vertebrate gastrulation

Recent studies show that human Pnas4 might be tumor associated, while its function remains unknown. Here, we investigate the developmental function of Pnas4 using zebrafish as a model system. Knocking down Pnas4 causes gastrulation defects with a shorter and broader axis, as well as a posteriorly mis-positioned prechordal plate, due to the defective convergence and extension movement. Conversely, over-expression of Pnas4 mRNA leads to an elongated body axis. We further demonstrate that Pnas4 is required cell-autonomously for dorsal convergence but not for anterior migration. In addition, genetic interaction assays indicate that Pnas4 might act in parallel with non-canonical Wnt signal in the regulation of cell movement. Our data suggest that Pnas4 is a key regulator of cell movement during gastrulation.

Identification of presomitic mesoderm (PSM)-specific Mesp1 enhancer and generation of a PSM-specific Mesp1/Mesp2-null mouse using BAC-based rescue technology

Bacterial artificial chromosome (BAC) modification technology is a powerful method for the identification of enhancer sequences and genetic modifications. Using this method, we have analyzed the Mesp1 and/or Mesp2 enhancers and identified P1-PSME, a PSM-specific enhancer of Mesp1, which contains a T-box binding site similar to the previously identified P2-PSME. Hence, Mesp1 and Mesp2 use different enhancers for their PSM-specific expression. In addition, we find that these two genes also use distinct enhancers for their early mesodermal expression. Based on these results, we generated a PSM-specific Mesp1/Mesp2-null mouse by introducing a BAC clone, from which only early mesodermal Mesp1 expression is possible, into the Mesp1/Mesp2 double knockout (dKO) genetic background. This successfully rescued gastrulation defects due to the lack of the early mesoderm in the dKO mouse and we thereby obtained a PSM-specific Mesp1/Mesp2-null mouse showing a lack of segmented somites.

Inhibiting Akt

The Akt family of protein kinases is involved in mediating cell survival signals, and cancers are often associated with mutations in genes encoding negative regulators of Akt activity, such as the lipid phosphatase PTEN. Activation of Akt involves recruitment to the plasma membrane, followed by phosphorylation by activating kinases. Cinar et al . report that the kinases Mst1 and Mst2 inhibit Akt activity in a mechanism that is independent of the kinase activity of Mst. Fractionation of lipid rafts (Triton X-100-insoluble membranes) showed that ~10% of Akt1 was present in this compartment in the prostate cancer cell line LNCaP (a PTEN-deficient cell line in which Akt1 is constitutively active), and coimmunoprecipitation followed by mass spectrometry analysis revealed that Mst1 (also known as STK4) was in a complex with Akt1 in this compartment. The interaction appeared to be direct in that purified proteins interacted. Mst1 is cleaved by caspase into a C-terminal domain containing the regulatory region (Mst1-C) and a N-terminal domain containing the kinase domain (Mst1-N). Full-length Mst1, Mst1-C, and Mst1-N interacted with a tagged form of Akt1 when overexpressed in HEK 293T cells. A kinase-inactive form of the Mst1-N also interacted with Akt1, suggesting that kinase activity was not required for the interaction. All three forms of Mst1 interacted with the C-terminal hydrophobic domain of Akt1 based on experiments with Akt1 mutants. Proteins expressed in transfected cells [Mst1, Mst1-N, and Mst1-C and GFP-Myr-Akt1 (myristoylated and tagged with green fluorescent protein)] and in LNCaP cells (endogenous phosphorylated Akt1 and transfected Mst1-N and Mst1-C) showed that although the full-length Mst1 and Mst1-C colocalized with Akt1 at the plasma membrane and cytoplasm, Mst1-N did not colocalize with Akt1 at the membrane; instead, in the LNCaP cells it colocalized with Akt1 in the nucleus. RNA interference experiments with LNCaP cells showed that depletion of either Mst1 or the related protein Mst2 enhanced Akt1 activity, whereas overexpression of the Mst proteins inhibited Akt1 activity. Expression of Mst1, Mst1-C, Mst1-N, or the kinase-inactive Mst1-N reverted a gastrulation defect induced by expression of constitutively active Akt (Myr-Akt1) in zebrafish embryos, further confirming the negatively regulatory role of Mst. Finally, immunohistochemical analysis of a human prostate cancer tissue array showed that tissues with the highest levels of activated Akt had the lowest levels of Mst1, suggesting that loss of this inhibitory input may be clinically relevant during cancer progression. B. Cinar, P.-K. Fang, M. Lutchman, D. Di Vizio, R. M. Adam, N. Pavlova, M. A. Rubin, P. C. Yelick, M. R. Freeman, The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1. EMBO J . 26 , 4523-4534 (2007). [PubMed]

Alterations of the cytoskeleton in all three embryonic lineages contribute to the epiboly defect of Pou5f1/Oct4 deficient MZ spg zebrafish embryos

Pou5f1/Oct4 is a transcription factor required for pluripotency of embryonic stem cells in mammals. Zebrafish pou5f1 deficient maternal and zygotic spiel ohne grenzen (MZ spg) mutant embryos develop severe gastrulation defects, are dorsalized, and defective in endoderm formation. Here we analyze in detail gastrulation defects, which are manifested by a severe delay in epiboly progression. All three embryonic lineages in MZ spg embryos behave abnormally during epiboly: the yolk cell forms an altered array of cortical microtubules and F-Actin, with large patches of microtubule free areas; the enveloping layer (EVL) is delayed in the coordinated cell shape changes of marginal cells, that may be mediated by F-Actin; the deep layer cells (DEL), forming the embryo proper, are non-autonomously affected in their motility and do not enter the space opening by epiboly of the EVL. Analysis of adhesiveness as well as high resolution in vivo time lapse image analysis of DEL cells suggests changed adhesive properties and inability to migrate properly on EVL and yolk syncytial layer (YSL) surfaces. Our data further reveal that during epiboly the EVL may actively probe the YSL by filopodia formation, rather than just being passively pulled vegetalwards. Our findings on the effect of Pou5f1 on cell behavior may be relevant to understand stem cell behavior and tumorigenesis involving Pou5f1.

PGAP1 Knock-out Mice Show Otocephaly and Male Infertility

A palmitate linked to the inositol in glycosylphosphatidylinositol (GPI) is removed in the endoplasmic reticulum immediately after the conjugation of GPI with proteins in most cells. Previously, we identified PGAP1 (post GPI attachment to proteins 1) as a GPI inositoldeacylase that removes the palmitate from inositol. A defect in PGAP1 caused a delay in the transport of GPI-anchored proteins (GPI-APs) from the endoplasmic reticulum to the cell surface in Chinese hamster ovary cells, although the cell-surface expression of GPI-APs in the steady state was normal. Nevertheless, in most cells, GPI-APs undergo deacylation. To elucidate the biological significance of PGAP1 in vivo, we established PGAP1 knock-out mice. Most PGAP1 knock-out mice showed otocephaly, a developmental defect, and died right after birth. However, some survived with growth retardation. Male knock-out mice showed severely reduced fertility despite the capability of ejaculation. Their spermatozoa were normal in number, motility, and ability to ascend the uterus, but were unable to go into the oviduct. In vitro, PGAP1-deficient spermatozoa showed weak attachment to the zona pellucida and a severely diminished rate of fertilization. Therefore, an extra acyl chain in GPI anchors caused severe deleterious effects to development and sperm function.

XRab40 Participates in a Ubiquitin Ligase Complex

Noting that some Rab GTPases (guanosine triphosphatases) have been implicated in embryogenesis, Lee et al . injected mRNA encoding Rab proteins into developing Xenopus embryos and found that the protein encoded by one of them inhibited gastrulation. This protein, XRab40, contained not only a GTPase domain but also a SOCS (suppressors of cytokine signaling) box domain. SOCS-box proteins associate with Elongin B and Elongin C as part of Cullin-based ubiquitin ligase complexes, and a combination of mass spectrometry analysis, yeast two-hybrid assays, and pull-down assays revealed that XRab40 interacted with the Xenopus homologs of Elongins B and C and Cullin5, and with the XRap2 GTPase. The SOCs box was required for XRab40 binding to XElonginB, XElonginC, and XCul5, whereas the GTPase domain was required for interaction with XRap2. XRab40 localized to the Golgi, and this localization depended on the SOCs box. Although wild-type XRab40 rescued gastrulation defects produced by antisense morpholino oligonucleotides directed against XRab40 (XRab40 Mo), mutants that did not localize to the Golgi failed to do so. Moreover, morpholinos directed against XCul5 (XCul5 Mo), which inhibited localization of XRab40 to the Golgi, elicited a phenotype similar to that of XRab40 Mo, as did a Cul5 dominant-negative mutant. XRab40 knockdown or mislocalization led to XRap2 mislocalization and a decrease in XRap2 ubiquitination (but not its stability). XRap2 bound to XMINK (Misshapen/Nck-interacting kinase) and morpholinos directed against XMINK also elicited gastrulation defects. Analyses of the ability to rescue morpholino-induced phenotypes indicated that XMINK acted downstream of XRab40 and XRap2. XMINK bound to Dishevelled (Dsh), translocation of which to the plasma membrane has been implicated in noncanonical Wnt signaling. Morpholinos directed against XRab40, XCul5, and XMINK inhibited Dsh localization to the plasma membrane when the Wnt pathway was activated. Thus, the authors conclude that XRab40 participates in a Cullin-based ubiquitin ligase complex, which regulates noncanonical Wnt signaling by way of XRap2 and XMINK and plays a critical role in Xenopus gastrulation. R. H. K. Lee, H. Iioka, M. Ohashi, S.-i. Iemura, T. Natsume, N. Kinoshita, XRab40 and XCullin5 form a ubiquitin ligase complex essential for the noncanonical Wnt pathway. EMBO J. 26 , 3592-3606 (2007). [PubMed]

PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation

ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis.

Foxa2 IS ESSENTIAL FOR MURINE ENDOMETRIAL GLAND DEVELOPMENT AND FERTILITY

Foxa2 is a member of the winged helix/forkhead box (Fox) transcription factor family is expressed in the glandular epithelium of the murine uterus. During embryonic development Foxa2 is required for the formation of the node and notochord and ablation of this gene results in defects in gastrulation, neural tube patterning and gut morphogenesis. The function of Foxa2 in the adult mouse uterus was investigated by the conditional ablation of Foxa2. This was accomplished by crossing mice with floxed Foxa2 alleles, Foxa2loxP/loxP, with the PRCre mouse model. The PRcre mouse ablates loxP-flanked genes in the pituitary, corpus luteum of the ovary, uterus and mammary gland. Foxa2loxP/loxP; PRCre (or Foxa2d/d for short) mice showed significantly reduced fertility and sterility with increasing age. Morphological examination of the uteri of these mice showed a severe reduction in the number of endometrial glands. Functional analysis of the uterus was investigating by the artificial induction of the uterine decidual response. This was accomplished by treating ovariectomized mice with progesterone and estrogen and initiating the decidual reaction with a scratch trauma. Decidualization was assayed by measuring an increase in uterine weight and expression of alkaline phosphatase in the endometrial stroma cells. The Foxa2d/d mice showed a severe inhibition of the decidual response when compared to Foxa2loxP/loxP control mice. This lack of decidual response could be rescued by intrauterine injection of 10 μl of Leukemia Inhibitory factor (100ng/μl), LIF, prior to the initiation of the decidual response. This analysis demonstrates that Foxa2 regulates endometrial gland development. Mice with loss of endometrial glands cannot support implantation most likely due to the loss of LIF which is a requisite for fertility in the mouse. This work was supported by NICHD/NIH as part of the Cooperative Program on Trophoblast-Maternal Tissue Interactions (U01 HD042311) and the Specialized Cooperative Center for Reproductive Research (U54 DK59820). (platform)

Generation of a conditional Ppap2b/Lpp3 null allele

Lpp3, formerly known as Pap2b, is a lipid phosphohydrolase enzyme. Some of its substrates and products are lipids with potent biological and signaling activities such as phosphatidic acid, lysophosphatidic acid, sphingosine-1-phosphate, diacylglycerol, sphingosine, and ceramide. Lpp3 is dynamically expressed during development and is widely distributed in adult tissues. Targeted inactivation of Lpp3 gene (Ppap2b) in the mouse results in embryonic lethality because of defects in extraembryonic vascular development and gastrulation. To study the participation of Lpp3 later in development and in specific cell lineages we generated a conditional allele of Ppap2b. This was accomplished by flanking critical exons, responsible for its catalytic activity with loxP sites. A generalized Cre-mediated recombination of this allele yielded a phenotype fundamentally identical to our previously reported Ppap2b null allele.

Retinoic acid-inducible G protein-coupled receptors bind to frizzled receptors and may activate non-canonical Wnt signaling

Frizzled (Fz) seven-pass transmembrane receptors are Wnt receptors and function in a variety of developmental pathways. Here we identify retinoic acid-inducible gene-1, 2, 3, and 4 (RAIG1, 2, 3, and 4) as potential Fz binding proteins. RAIG proteins are seven-pass transmembrane receptors, and Xenopus RAIG2, 3, and 4 are expressed in early gastrula. XRAIG2 can activate small GTPases, such as RhoA, Rac, and Cdc42, and c-jun N-terminal kinase, thus exhibit activities that overlap with non-canonical Wnt/Fz signaling. Injection of XRAIG2 mRNA into Xenopus embryo causes a severe shortened and bent body axis due to defective gastrulation movements, reminiscent of abnormal non-canonical Wnt signaling. XRAIG2 affects convergent extension in activin-treated animal caps, which can be partially rescued by co-injection of a dominant-negative form of Cdc42. In zebrafish embryo, XRAIG2 also causes Ca 2+ flux, one of the consequences of non-canonical Wnt signaling. These results suggest a possible crosstalk/integration between Wnt/Frizzled and RAIG signal transduction pathways.

SDF-1α regulates mesendodermal cell migration during frog gastrulation

During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1α expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1α caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-1α-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1α, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation.

Soluble membrane-type 3 matrix metalloprioteinase causes changes in gene expression and increased gelatinase activity during Xenopus laevis development

Matrix metalloproteinases (MMPs) are a family of endopeptidases that cleave and remodel the extracellular matrix (ECM). Membrane-type 3 MMP (MT3-MMP) is a membrane-anchored MMP, which has recently been shown to 'shed' from the cell surface in a soluble form upon proteolytic cleavage. Shed MT-MMPs can activate gelatinase-A in vitro and have been directly linked to the metastatic potential of many cancers. Here we examined the effect of ectopic expression of full-length tethered and shed (soluble) forms of MT3-MMP during Xenopus laevis development. Injection of mRNA coding for full-length tethered MT3-MMP resulted in the delayed onset of gastrulation and subsequent defects. Phenotype severity and the frequency of embryo death were dose-dependent. Dose-dependent defects were also observed with the injection of mRNA of the soluble form, but the phenotypes and frequencies of death were greater. Histological analysis of injected embryos demonstrated defects in the organization of axial structures, such as the neural tube and somites. Embryos injected with full-length MT3-MMP mRNA showed no significant changes in expression levels of the tissue specific genes endodermin, chordin and muscle actin when examined by semi-quantitative RT-PCR. In contrast, embryos injected with the soluble form of MT3-MMP exhibited decreased expression of these same marker genes. In addition, while full-length tethered MT3-MMP failed to alter gelatinase activity, a 50% increase was measured in response to injection of the soluble form, suggesting that the two forms of this protein could play distinct roles during embryogenesis.

BMP type I receptor ALK2 is essential for proper patterning at late gastrulation during mouse embryogenesis

Bone morphogenetic proteins (BMPs) have multiple functions during vertebrate development. Previously, it was shown that BMP type I receptor ALK2 (also known as ACVRI, ActRI, or ActRIA) was important for normal mouse gastrulation by deleting exon 4 or exon 5 of Alk2. Recently, flanking exon 7 by loxP sites generated a conditional allele for Alk2. To assess whether the deletion of exon 7 causes functional null of ALK2, and does not produce a dominant negative form or a partially functional form of ALK2, we performed a comparative analysis between Alk2 homozygous mutant embryos with an exon 5 deletion (Alk2(Delta5/Delta5)) and embryos with an exon 7 deletion (Alk2(Delta7/Delta7)). Both Alk2(Delta5/Delta5) and Alk2(Delta7/Delta7) mutants showed identical morphological gastrulation defects. Histological examinations and molecular marker analyses revealed identical abnormal gastrulation phenotypes in Alk2(Delta5/Delta5) and Alk2(Delta7/Delta7) mutants. Although Fgf8 was expressed in the primitive streak of Alk2(Delta5/Delta5) and Alk2(Delta7/Delta7) mutants, Brachyury, Wnt3a, and Tbx6 were dramatically downregulated in Alk2(Delta5/Delta5) and Alk2(Delta7/Delta7) mutants. These results indicate that deletion of exon 7 for Alk2 leads to a functionally null mutation in vivo, and Alk2 is crucial for sustaining the proper gastrulation events in early mouse embryogenesis.

A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation

Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-β family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression.