Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps.

Spemann's organizer has potent neural inducing and mesoderm dorsalizing activities in the Xenopus gastrula. A third activity, the organizer's ability to induce a secondary gut, has been difficult to analyze experimentally due to the lack of early gene markers. Here we introduce endodermin, a pan-endodermal gene marker, and use it to demonstrate that chordin (Chd), a protein secreted by the organizer region, is able to induce endodermal differentiation in Xenopus. The ability of chd, as well as that of noggin, to induce endoderm in animal cap explants is repressed by the ventralizing factor BMP-4. When FGF signaling is blocked by a dominant-negative FGF receptor in chd-injected animal caps, neural induction is inhibited and most of the explant is induced to become endoderm. The results suggest that proteins secreted by the organizer, acting together with known peptide growth factors, regulate differentiation of the endodermal germ layer.     

BMP-2 modulates the proliferation and differentiation of normal and cancerous gastric cells

Fibroblast growth factor (FGF) is established as an initiator of signaling events critical for neurogenesis and mesoderm formation during early Xenopus embryogenesis. However, less is known about the role FGF signaling plays in endoderm specification. Here, we show for the first time that endoderm-specific genes are induced when FGF signaling is blocked in animal cap explants. This block of FGF signaling is also responsible for a significant enhancement of endodermal gene expression in animal cap explants that are injected with a dominant-negative BMP-4 receptor (DNBR) RNA or treated with activin, however, neural and mesoderm gene expression is diminished. Consistent with these results, the injection of dominant-negative FGF receptor (DNFR) RNA expands endodermal cell fate boundaries while FGF treatment dramatically reduces endoderm in whole embryos. Taken together, these results indicate that inhibition of FGF signaling promotes endoderm formation, whereas the presence of active FGF signaling is necessary for neurogenesis/mesoderm formation.     

Induction of tooth and eye by transplantation of activin A-treated, undifferentiated presumptive ectodermal Xenopus cells into the abdomen

Activin A can induce the Xenopus presumptive ectoderm (animal cap) to form different types of mesoderm and endoderm at different concentrations and the animal cap treated with activin can function as an organizer during early development. The dissociated Xenopus animal cap cells treated with activin form an aggregate and it develops into various tissues in vitro. In this study, to induce jaw cartilage from undifferentiated cells effectively, we developed a culture method to manipulate body patterning in vitro, using activin A and dissociated animal cap cells. An aggregate consisting only of activin A-treated dissociated cells developed into endodermal tissues. However, when activin A-treated cells were mixed with untreated cells at a ratio of 1:5, the aggregate developed cartilage with the maxillofacial regional marker genes, goosecoid, Xenopus Distal-less 4 and X-Hoxa2. When this aggregate was transplanted into the abdominal region of host embryos, maxillofacial structures containing cartilage and eye developed. We raised these embryos to adulthood and found that tooth germ had developed in the transplanted tissue. Here, we show the induction of jaw cartilage, tooth germ and eye structures from animal caps using activin A in the aggregation culture method. This differentiation system will help to promote a better understanding of the regulating mechanisms of body patterning and tooth induction in vertebrates.     

GATA factors as key regulatory molecules in the development of Drosophila endoderm

Essential roles for GATA factors in the development of endoderm have been reported in various animals. A Drosophila GATA factor gene, serpent ( srp , dGATAb , ABF ), is expressed in the prospective endoderm, and loss of srp activity causes transformation of the prospective endoderm into ectodermal foregut and hindgut, indicating that srp acts as a selector gene to specify the developmental fate of the endoderm. While srp is expressed in the endoderm only during early stages, it activates a subsequent GATA factor gene, dGATAe , and the latter continues to be expressed specifically in the endoderm throughout life. dGATAe activates various functional genes in the differentiated endodermal midgut. An analogous mode of regulation has been reported in Caenorhabditis elegans , in which a pair of GATA genes, end-1/3 , specifies endodermal fate, and a downstream pair of GATA genes, elt-2/7 , activates genes in the differentiated endoderm. Functional homology of GATA genes in nature is apparently extendable to vertebrates, because endodermal GATA genes of C. elegans and Drosophila induce endoderm development in Xenopus ectoderm. These findings strongly imply evolutionary conservation of the roles of GATA factors in the endoderm across the protostomes and the deuterostomes.     

Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate

During embryonic development, organs arise along the gut tube as a series of buds in a stereotyped anterior-posterior (A-P) pattern. Using chick-quail chimeras and in vitro tissue recombination, we studied the interactions governing the induction and maintenance of endodermal organ identify focusing on the pancreas. Though several permissive signals in pancreatic development have been previously identified, here we provide evidence that lateral plate mesoderm sends instructive signals to the endoderm, signals that induce expression of the pancreatic genes Pdx1, p48, Nkx6.1, glucagon, and insulin. Moreover, this instructive signal directs cells to form ectopic insulin-positive islet-like clusters in endoderm that would otherwise form more rostral organs. Once generated, endocrine cells no longer require interaction with mesoderm, but nonendocrine cells continue to require permissive signals from the mesoderm. Stimulation of activin, BMP, or retinoic acid signaling is sufficient to induce Pdx1 expression in endoderm anterior to the pancreas. Lateral plate mesoderm appears to pattern the endoderm in a posterior-dominant fashion as first noted in the patterning of the neural tube at the same embryonic stage. These findings argue for a central role of the mesoderm in coordinating the A-P pattern of all three primary germ layers.     

An endoderm-specific GATA factor gene, dGATAe, is required for the terminal differentiation of the Drosophila endoderm

GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent (srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. We have identified another endoderm-specific Drosophila GATA factor gene, dGATAe. Expression of dGATAe is first detected at stage 8 in the endoderm, and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of dGATAe, and misexpression of srp resulted in ectopic dGATAe expression. Embryos that either lacked dGATAe or were injected with double-stranded RNA (dsRNA) corresponding to dGATAe failed to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of dGATAe induced ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the dGATAe cDNA also induced endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein (hkb), and finally, two GATA factor genes, srp and dGATAe.     

GATA4, 5 and 6 mediate TGFbeta maintenance of endodermal gene expression in Xenopus embryos

The individual contributions of the three vertebrate GATA factors to endoderm formation have been unclear. Here we detail the early expression of GATA4, 5 and 6 in presumptive endoderm in Xenopus embryos and their induction of endodermal markers in presumptive ectoderm. Induction of HNF3beta by all three GATA factors was abolished when protein synthesis was inhibited, showing that these inductions are indirect. In contrast, whereas induction of Sox17alpha and HNF1beta by GATA4 and 5 was substantially reduced when protein synthesis was inhibited, induction by GATA6 was minimally affected, suggesting that GATA6 is a direct activator of these early endodermal genes. GATA4 induced GATA6 expression in the same assay and antisense morpholino oligonucleotides (MOs), designed to knock down translation of GATA6, blocked induction of Sox17alpha and HNF1beta by GATA4, suggesting that GATA4 induces these genes via GATA6 in this assay. All three GATA factors were induced by activin, although GATA4 and 6 required lower concentrations. GATA MOs inhibited Sox17alpha and HNF1beta induction by activin at low and high concentrations in the order: GATA6>GATA4>GATA5. Together with the timing of their expression and the effects of GATA MOs in vivo, these observations identify GATA6 as the predominant GATA factor in the maintenance of endodermal gene expression by TGFbeta signaling in gastrulating embryos. In addition, examination of gene expression and morphology in later embryos, revealed GATA5 and 6 as the most critical for the development of the gut and the liver.     

Two essential processes in the formation of a dorsal axis during gastrulation ofCynops embryo

The isolated upper marginal zone from the initial stage ofCynops gastrulation is not yet determined to form the dorsal axis mesoderm: notochord and muscle. In this experiment, we will indicate where the dorsal mesoderm-inducing activity is localized in the very early gastrula, and what is an important event for specification of the dorsal axis mesoderm during gastrulation. Recombination experiments showed that dorsal mesoderm-inducing activity was localized definitively in the endodermal epithelium (EE) of the lower marginal zone, with a dorso-ventral gradient; and the EE itself differentiated into endodermal tissues, mainly pharyngeal endoderm. Nevertheless, when dorsal EE alone was transplanted into the ventral region, a secondary axis with dorsal mesoderm was barely formed. However, when dorsal EE was transplanted with the bottle cells which by themselves were incapable of mesoderm induction, a second axis with well-developed dorsal mesoderm was observed. When the animal half with the lower marginal zone was rotated 180° and recombined with the vegetal half, most of the rotated embryos formed only one dorsal axis at the primary blastopore side. The present results suggest that there are at least two essential processes in dorsal axis formation: mesoderm induction of the upper marginal zone by endodermal epithelium of the lower marginal zone, and dorsalization of the upper dorsal marginal zone evoked during involution.     

Vegfc is required for vascular development and endoderm morphogenesis in zebrafish

During embryogenesis, complex morphogenetic events lead endodermal cells to coalesce at the midline and form the primitive gut tube and associated organs. While several genes have recently been implicated in endoderm differentiation, we know little about the genes that regulate endodermal morphogenesis. Here, we show that vascular endothelial growth factor C (Vegfc), an angiogenic as well as a lymphangiogenic factor, is unexpectedly involved in this process in zebrafish. Reducing Vegfc levels using morpholino antisense oligonucleotides, or through overexpression of a soluble form of the VEGFC receptor, VEGFR-3, affects the coalescence of endodermal cells in the anterior midline, leading to the formation of a forked gut tube and the duplication of the liver and pancreatic buds. Further analyses indicate that Vegfc is additionally required for the initial formation of the dorsal endoderm. We also demonstrate that Vegfc is required for vasculogenesis as well as angiogenesis in the zebrafish embryo. These data argue for a requirement of Vegfc in the developing vasculature and, more surprisingly, implicate Vegfc signalling in two distinct steps during endoderm development, first during the initial differentiation of the dorsal endoderm, and second in the coalescence of the anterior endoderm to the midline.