Normal Endoderm Research Articles

XenopusNanos1 is required to prevent endoderm gene expression and apoptosis in primordial germ cells

Nanos is expressed in multipotent cells, stem cells and primordial germ cells (PGCs) of organisms as diverse as jellyfish and humans. It functions together with Pumilio to translationally repress targeted mRNAs. Here we show by loss-of-function experiments that Xenopus Nanos1 is required to preserve PGC fate. Morpholino knockdown of maternal Nanos1 resulted in a striking decrease in PGCs and a loss of germ cells from the gonads. Lineage tracing and TUNEL staining reveal that Nanos1-deficient PGCs fail to migrate out of the endoderm. They appear to undergo apoptosis rather than convert to normal endoderm. Whereas normal PGCs do not become transcriptionally active until neurula, Nanos1-depleted PGCs prematurely exhibit a hyperphosphorylated RNA polymerase II C-terminal domain at the midblastula transition. Furthermore, they inappropriately express somatic genes characteristic of endoderm regulated by maternal VegT, including Xsox17α, Bix4, Mixer, GATA4 and Edd. We further demonstrate that Pumilio specifically binds VegT RNA in vitro and represses, along with Nanos1, VegT translation within PGCs. Repressed VegT RNA in wild-type PGCs is significantly less stable than VegT in Nanos1-depleted PGCs. Our data indicate that maternal VegT RNA is an authentic target of Nanos1/Pumilio translational repression. We propose that Nanos1 functions to translationally repress RNAs that normally specify endoderm and promote apoptosis, thus preserving the germline.

Repression of mesodermal fate byfoxa, a key endoderm regulator of the sea urchin embryo

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

Evidence for dual mechanisms of mesoderm establishment in Xenopus embryos.

In Xenopus embryos, the maternally encoded transcription factor VegT (also known as Xombi, Antipodean, Brat, and Xtbx6) is essential for normal endoderm and mesoderm formation. This finding and the localization of VegT mRNA in the vegetal hemisphere of the oocyte are consistent with several models of germ layer patterning. Specific models have been proposed in which (1) combinations of cytoplasmic determinants, (2) inductive signals, or (3) intracellular concentration (morphogen effects) predominate. We test predictions of these models. We show that contrary to previous proposals, FGF does not suppress endoderm formation and so cannot be an anti-endoderm mesodermal determinant. We further show that, at the right dose, VegT can induce mesodermal marker expression cell autonomously and that it induces mesoderm at concentrations below those that induce endoderm. These results are consistent with a dual mechanism of mesoderm establishment in which both VegT-initiated inductive signals and an intracellular VegT morphogen gradient play a part.

Expression and functional analysis of Cititf1, an ascidian NK-2 class gene, suggest its role in endoderm development.

In solitary ascidians the fate of endoderm is determined at a very early stage of development and depends on cytoplasmic factors whose nature has not been determined. We have isolated a member of the NK-2 gene family, Cititf1, from the ascidian Ciona intestinalis, showing high sequence homology to mammalian TITF1. The Cititf1 gene was expressed in all endodermal precursors at the pregastrula and gastrula stages, and is thus the first specific regulatory endodermal marker to be isolated from an ascidian. Cititf1 expression was downregulated at the end of gastrulation to reappear at middle tailbud and larval stages in the most anterior and ventral parts of head endoderm, regions which give rise, after metamorphosis, to the adult endostyle, where Cititf1 mRNA was still present. Microinjection of Cititf1 mRNA into fertilized eggs resulted in tadpole larvae with abnormalities in head-trunk development consequent to the formation of excess endoderm, perhaps due to recruitment of notochord precursors to an endodermal fate. These data suggest that Cititf1 plays an important role in normal endoderm differentiation during ascidian embryogenesis.

Xsox17α and -β Mediate Endoderm Formation in Xenopus

We have isolated two Xenopus relatives of murine Sox17 expressed in gastrula presumptive endoderm. Xsox17α and -β expression can be induced in animal caps by activin, but not by FGF. Ectopic expression of these genes in animal caps induces the expression of endoderm markers; this induction is blocked by overexpression of a fusion of the Xsox17β HMG domain to the Drosophila Engrailed repressor domain, as is induction of endoderm markers by activin and the expression of endodermal markers in whole embryos and isolated vegetal poles. These experiments, as well as the effects of the mRNAs on embryo phenotypes, suggest that the Xsox17 genes mediate an activin-induced endoderm differentiation pathway in animal caps and are involved in normal endoderm differentiation in embryos.

Induction of myofibrillogenesis in cardiac lethal mutant axolotl hearts rescued by RNA derived from normal endoderm

A strain of axolotl, Ambystoma mexicanum, that carries the cardiac lethal or c gene presents an excellent model system in which to study inductive interactions during heart development. Embryos homozygous for gene c contain hearts that fail to beat and do not form sarcomeric myofibrils even though muscle proteins are present. Although they can survive for approximately three weeks, mutant embryos inevitably die due to lack of circulation. Embryonic axolotl hearts can be maintained easily in organ culture using only Holtfreter's solution as a culture medium. Mutant hearts can be induced to differentiate in vitro into functional cardiac muscle containing sarcomeric myofibrils by coculturing the mutant heart tube with anterior endoderm from a normal embryo. The induction of muscle differentiation can also be mediated through organ culture of mutant heart tubes in medium 'conditioned' by normal anterior endoderm. Ribonuclease was shown to abolish the ability of endoderm-conditioned medium to induce cardiac muscle differentiation. The addition of RNA extracted from normal early embryonic anterior endoderm to organ cultures of mutant hearts stimulated the differentiation of these tissues into contractile cardiac muscle containing well-organized sarcomeric myofibrils, while RNA extracted from early embryonic liver or neural tube did not induce either muscular contraction or myofibrillogenesis. Thus, RNA from anterior endoderm of normal embryos induces myofibrillogenesis and the development of contractile activity in mutant hearts, thereby correcting the genetic defect.

Properties of cells from inverted embryos ofXenopus laevis investigated by scanning electron microscopy.

Xenopus embryos held inverted from the one cell stage show a partial reversal of the pattern of cleavage: the blastocoel forms towards the new upper pole, and the non-pigmented cells forming the blastocoel roof are smaller than normal endoderm cells. Two properties of the cells from inverted embryos have been studied: their capacity to form cilia when cultured for 48 h, normally a property of ectoderm cells; and their scanning electron microscopical appearance when isolated and cultured for shorter periods, which differs for normal ectoderm and endoderm cells. Groups of the upper, non-pigmented cells from inverted embryos do not form cilia in a longerterm culture, whereas groups of the lower, pigmented cells do. In contrast, the scanning electron microscopical appearance of the upper, non-pigmented cells of inverted embryos is more like that of normal ectoderm cells; the appearance of lower, pigmented cells is more like that of normal endoderm. Thus the determination to form cilia is not reversed by inversion, whereas the control of cell morphology is.

Studies of testosterone-induced involution of the bursa of Fabricius

The present investigation deals with the effect of testosterone on each of the tissue components of the bursa of Fabricius: the endodermal epithelium, the mesenchyme, and the hemopoietic stem cells. Tissue combination experiments between testosterone-treated endoderm and normal mesenchyme and vice versa have shown that the androgen damages irreversibly the bursal epithelium. The latter is not seeded by hemopoietic stem cells and cannot undergo follicle formation when treated with high doses of testosterone. This occurs even if it is associated with a nontreated bursal mesenchyme. On the contrary, associations of testosterone-treated mesenchyme with normal endoderm result in normal bursa histogenesis. By using an original test of viability for lymphoid cells based on the application of the quail-chick marker system, we demonstrate that disappearance of hemopoietic cells in the endoderm results from their expulsion from the bursa and not from their death in situ. The conspicuous effect of testosterone on the bursa of Fabricius can be related to the levels of androgen receptors found in the organ. Typical cytosol androgen receptors are demonstrated in both bursal endoderm and mesoderm, although the amount in the former is higher. The concentration of binding sites in the bursa is >10 times higher than that in other organs such as lung and small intestine whose development is not affected by testosterone, contrasting with glucocorticosteroid receptor (measured by labeling with dexamethasone) found in the same concentration in all tissues.

379. Effect of androgen on the development of the bursa of fabricius in the duck embryo

The present investigation deals with the effect of testosterone on each of the tissue components of the bursa of Fabricius: the endodermal epithelium, the mesenchyme, and the hemopoietic stem cells. Tissue combination experiments between testosterone-treated endoderm and normal mesenchyme and vice versa have shown that the androgen damages irreversibly the bursal epithelium. The latter is not seeded by hemopoietic stem cells and cannot undergo follicle formation when treated with high doses of testosterone. This occurs even if it is associated with a nontreated bursal mesenchyme. On the contrary, associations of testosterone-treated mesenchyme with normal endoderm result in normal bursa histogenesis. By using an original test of viability for lymphoid cells based on the application of the quail-chick marker system, we demonstrate that disappearance of hemopoietic cells in the endoderm results from their expulsion from the bursa and not from their death in situ. The conspicuous effect of testosterone on the bursa of Fabricius can be related to the levels of androgen receptors found in the organ. Typical cytosol androgen receptors are demonstrated in both bursal endoderm and mesoderm, although the amount in the former is higher. The concentration of binding sites in the bursa is >10 times higher than that in other organs such as lung and small intestine whose development is not affected by testosterone, contrasting with glucocorticosteroid receptor (measured by labeling with dexamethasone) found in the same concentration in all tissues.