Anterior endoderm is sufficient to rescue foregut apoptosis and heart tube morphogenesis in an embryo lacking retinoic acid.
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The vitamin A deficient (VAD) quail embryo lacks active retinoids, fails to express normally GATA-4, and develops a nonlooping heart tube morphogenetic defect that is a model for congenital cardiomyopathy. VAD quail embryos, or chick embryos depleted specifically for GATA factors, show in addition abnormal foregut development, characterized by apoptosis of the endoderm cells associated with presumptive myocardium during the process of heart tube formation. Exogenous retinoic acid or transplantation of normal chick embryo anterior endoderm is sufficient to rescue apoptosis as well as GATA-4 expression and results in normal development and heart tube morphogenesis. Normal posterior endoderm also contains retinoids but is unable to rescue the VAD defect. Our results indicate that a retinoid-dependent transcriptional program, mediated at least in part by GATA factors, is critical in presumptive foregut endoderm for normal heart tube morphogenesis. (+info)
Endodermal Nodal-related signals and mesoderm induction in Xenopus.
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In Xenopus, mesoderm induction by endoderm at the blastula stage is well documented, but the molecular nature of the endogenous inductive signals remains unknown. The carboxy-terminal fragment of Cerberus, designated Cer-S, provides a specific secreted antagonist of mesoderm-inducing Xenopus Nodal-Related (Xnr) factors. Cer-S does not inhibit signalling by other mesoderm inducers such as Activin, Derriere, Vg1 and BMP4, nor by the neural inducer Xnr3. In the present study we show that Cer-S blocks the induction of both dorsal and ventral mesoderm in animal-vegetal Nieuwkoop-type recombinants. During blastula stages Xnr1, Xnr2 and Xnr4 are expressed in a dorsal to ventral gradient in endodermal cells. Dose-response experiments using cer-S mRNA injections support the existence of an endogenous activity gradient of Xnrs. Xnr expression at blastula can be activated by the vegetal determinants VegT and Vg1 acting in synergy with dorsal (beta)-catenin. The data support a modified model for mesoderm induction in Xenopus, in which mesoderm induction is mediated by a gradient of multiple Nodal-related signals released by endoderm at the blastula stage. (+info)
Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha.
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HNF-4alpha is a transcription factor of the nuclear hormone receptor family that is expressed in the hepatic diverticulum at the onset of liver development. Mouse embryos lacking HNF-4alpha fail to complete gastrulation due to dysfunction of the visceral endoderm. This early embryonic lethality has so far prevented any analyses of the contribution of HNF-4alpha toward liver development and hepatocyte differentiation. However, we have shown that complementation of HNF-4alpha(-/-) embryos with a tetraploid embryo-derived wild-type visceral endoderm rescues this early developmental arrest and allows HNF-4alpha(-/-) embryos to proceed normally through midgestation stages of development. Examination of these rescued embryos revealed that HNF-4alpha was dispensable for specification and early development of the liver. However, HNF-4alpha(-/-) fetal livers failed to express a large array of genes whose expression in differentiated hepatocytes is essential for a functional hepatic parenchyma, including genes encoding several apolipoproteins, metabolic proteins, and serum factors. In addition, we have demonstrated that HNF-4alpha is essential for expression of the transcription factors HNF-1alpha and PXR within the fetal liver. We therefore conclude that HNF-4alpha is both essential for hepatocyte differentiation during mammalian liver development and also crucial for metabolic regulation and liver function. (+info)
Xenopus Xenf: an early endodermal nuclear factor that is regulated in a pathway distinct from Sox17 and Mix-related gene pathways.
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We report a novel zygotic gene encoding a Xenopus endodermal nuclear factor, Xenf. Expression of Xenf starts at the late blastula stages and is decreased after gastrulation. Xenf shows no structural homology to any known proteins. When GFP-tagged Xenf is overexpressed in Xenopus cells, Xenf protein is localized to the nucleus, associating closely with the chromosomes. In animal cap assays, Xenf expression is strongly activated by mRNA injection of Vg1 and VegT, maternal vegetal genes that can induce endodermal differentiation. In contrast, Xenf is not induced by endoderm-inducing zygotic transcription factors such as Sox17 and Mix-related genes. In turn, Xenf does not activate expression of Sox17, Mixer or Milk. Thus, Xenf is regulated by maternal vegetal positional information in a parallel manner to Sox17 and Mix-related gene pathways. (+info)
BMP2 is required for early heart development during a distinct time period.
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BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart. (+info)
Liver specification and early morphogenesis.
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The classically defined induction of the liver from the endoderm, elicited by the cardiac mesoderm, has recently been discovered to involve signaling by fibroblast growth factors (FGFs). Multiple FGFs induce hepatic gene expression independent of an effect on growth. A subset of these FGFs cooperates with other factors to promote morphogenesis of the newly specified hepatocytes. Subsequent to the formation of the liver bud, distinct mesenchymal signals and hepatic response pathways stimulate further growth and differentiation of the hepatic parenchymal cells and prevent apoptosis. The initial stages of hepatogenesis are therefore beginning to be understood, and serve as a paradigm for the development of other tissues from the endoderm. (+info)
Signals from trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm.
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We used Pax-2 mRNA expression and Lim 1/2 antibody staining as markers for the conversion of chick intermediate mesoderm (IM) to pronephric tissue and Lmx-1 mRNA expression as a marker for mesonephros. Pronephric markers were strongly expressed caudal to the fifth somite by stage 9. To determine whether the pronephros was induced by adjacent tissues and, if so, to identify the inducing tissues and the timing of induction, we microsurgically dissected one side of chick embryos developing in culture and then incubated them for up to 3 days. The undisturbed contralateral side served as a control. Most embryos cut parallel to the rostrocaudal axis between the trunk paraxial mesoderm and IM before stage 8 developed a pronephros on the control side only. Embryos manipulated after stage 9 developed pronephric structures on both sides, but the caudal pronephric extension was attenuated on the cut side. These results suggest that a medial signal is required for pronephric development and show that the signal is propagated in a rostral to caudal sequence. In manipulated embryos cultured for 3 days in ovo, the mesonephros as well as the pronephros failed to develop on the experimental side. In contrast, embryos cut between the notochord and the trunk paraxial mesoderm formed pronephric structures on both sides, regardless of the stage at which the operation was performed, indicating that the signal arises from the paraxial mesoderm (PM) and not from axial mesoderm. This cut also served as a control for cuts between the PM and the IM and showed that signaling itself was blocked in the former experiments, not the migration of pronephric or mesonephric precursor cells from the primitive streak. Additional control experiments ruled out the need for signals from lateral plate mesoderm, ectoderm, or endoderm. To determine whether the trunk paraxial mesoderm caudal to the fifth somite maintains its inductive capacity in the absence of contact with more rostral tissue, embryos were transected. Those transected below the prospective level of the fifth somite expressed Pax-2 in both the rostral and the caudal isolates, whereas embryos transected rostral to this level expressed Pax-2 in the caudal isolate only. Thus, a rostral signal is not required to establish the normal pattern of Pax-2 expression and pronephros formation. To determine whether paraxial mesoderm is sufficient for pronephros induction, stage 7 or earlier chick lateral plate mesoderm was cocultured with caudal stage 8 or 9 quail somites in collagen gels. Pax-2 was expressed in chick tissues in 21 of 25 embryos. Isochronic transplantation of stage 4 or 5 quail node into caudal chick primitive streak resulted in the generation of ectopic somites. These somites induced ectopic pronephroi in lateral plate mesoderm, and the IM that received signals from both native and ectopic somites formed enlarged pronephroi with increased Pax-2 expression. We conclude that signals from a localized region of the trunk paraxial mesoderm are both required and sufficient for the induction of the pronephros from the chick IM. Studies to identify the molecular nature of the induction are in progress. (+info)
Retinoid signaling is essential for patterning the endoderm of the third and fourth pharyngeal arches.
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The requirement of retinoic acid (RA) in the initial formation of the pharyngeal arches was investigated by treating headfold-stage mouse embryos with a pan-RAR antagonist in vitro and in vivo. This results in a complete absence of mesenchyme, arteries, nerves and epibranchial placodes of the 3rd and 4th pharyngeal arches, complete agenesis of the 3rd and 4th pouches and consistent lack of the 6th arch artery. Mesodermally derived endothelial cells are absent from the 3rd and 4th pharyngeal arch region and the distribution domain of EphA2 transcripts in mesodermal cells is shifted caudally. In situ hybridization with CRABPI, kreisler and EphA4 probes and the pattern of expression of a Wnt1-lacZ transgene show that neural crest cells (NCC) normally destined to the 3rd and 4th arches migrate ectopically. Most interestingly, the appearance of the 3rd and 4th arches is prevented by the antagonist only during a very narrow window of time, which does not correspond to the period of post-otic NCC migration. Both the timing of appearance and the nature of the defects in RAR antagonist-treated embryos indicate that migrating NCC and mesodermal cells destined to the caudal pharyngeal arches do not represent primary targets of RA action. Alterations in the endodermal expression pattern of Hoxa1, Hoxb1, Pax1, Pax9, Fgf3 and Fgf8 in response to the antagonist-induced block in RA signal transduction demonstrate for the first time that RA signaling is indispensable for the specification of the pharyngeal endoderm and suggest that this signaling is necessary to provide a permissive environment locally for the migration of NCC and mesodermal cells. Our study also indicates that the formation of the 2nd pharyngeal arch and that of the 3rd and 4th pharyngeal arches probably involve distinct RA-dependent developmental processes. (+info)