Differential keratin gene expression during the differentiation of the cement gland of Xenopus laevis.

The expression of both epidermal and nonepidermal keratins has been detected in the cement gland of Xenopus laevis by antibody staining. Northern blot and in situ hybridizations with gene-specific probes indicated the expression of the nonepidermal keratin, XK endo B, and the embryonic epidermal keratin, XK70, in the cement gland. Furthermore, since explanted animal pole cells can be induced to differentiate into cement gland cells in vitro by incubation in NH4Cl, we have demonstrated the in vitro induction of XK endo B, maintenance of XK70, and repression of another embryonic epidermal keratin, XK81. This is the first report of keratin gene expression in the cement gland.     

A study of cell interactions involved in Pleurodeles waltlii epidermal differentiation

Summary A polyclonal antibody (SP-2) has been produced, which recognizes antigens expressed in epidermal cells of Pleurodeles waltlii embryos. The antigens appear first at the end of gastrulation in the external surface of the embryo and are selectively expressed in ectodermally derived epidermal structures. Ectodermal commitment was investigated using cell cultures and blastocoel graft experiments. The four animal blastomeres of the 8-cell stage as well as the animal cap explants of the early gastrula stage cultured in vitro differentiate into epidermis, and SP-2 antigens are expressed. The expression of SP-2-defined antigens is inhibited both in vivo and in vitro by the inductive interaction of chordomesoderm. Once dissociated, ectodermal cells do not react with SP-2. Conversely, the aggregation of ectodermal cells may restore the expression of SP-2 antigens. Transplantation of animal cap explants or isolated ectodermal cells into the blastocoel of a host embryo at the early gastrula stage shows that only cells integrated into the epidermis express the marker antigens. When vegetal cells were dissociated from donor embryos before the mid-blastula stage and implanted into the blastocoel of host embryos at the early gastrula stage, their progeny were found in all germ layers, cells that were found in the host epidermis were stained with SP-2, whereas those contributing to mesoderm and endoderm were not. Thus the acquisition of cell polarity in epidermal differentiation and the organization of cells into epithelial structures are essential for SP-2-defined antigen expression.     

Genesis of Epidermal Action Potentials in Cytokinesis Arrested Xenopus Embryos

When Xenopus embryos were treated continuously with cytochalasin B (3–10 μg/ml) from the 8 cell stage, cleavage arrested embryos in various degrees were observed. In 3–5 μg/ml cytochalasin B, cytokinesis was inhibited at the midblastula stage and pigment granules remained at the cell cortex of the animal pole. These cells showed epidermal like action potentials when the control embryos (St. 26/28) generated epidermal action potentials. In 5–7 μg/ml cytochalasin B, furrows, following their formation at early cleavage stages, regressed and no further cleavage from the 16 cell stage to morula stage took place. The pigment granules were dispersed throughout the interior of the cytoplasm. These cells showed no epidermal action potentials. Thus, it is considered that cytokinesis per sé , following the midblastula stage, is not a prerequisite for the genesis of epidermal action potentials, and that chronological times corresponding to the tailbud larva stage and a stable structure of the cellular cortex are required to bring about these potentials.     

VegT activation of Sox17 at the midblastula transition alters the response to nodal signals in the vegetal endoderm domain

In Xenopus, the prospective endoderm and mesoderm are localized to discrete, adjacent domains at the beginning of gastrulation, and this is made evident by the expression of Sox17 in vegetal blastomeres and Brachyury (Xbra) in marginal blastomeres. Here, we examine the regulation of Sox17alpha expression and the role of Sox17alpha in establishing the vegetal endodermal gene expression domain. Injection of specific inhibitors of VegT or Nodal resulted in a loss of Sox17alpha expression in the gastrula. However, the onset of Sox17alpha expression at the midblastula transition was dependent on VegT, but not on Nodal function, indicating that Sox17alpha expression is initiated by VegT and then maintained by Nodal signals. Consistent with these results, VegT, but not Xenopus Nodal-related-1 (Xnr1), can activate Sox17alpha expression at the midblastula stage in animal explants. In addition, VegT activates Sox17alpha in the presence of cycloheximide or a Nodal antagonist, suggesting that Sox17alpha is an immediate-early target of VegT in vegetal blastomeres. Given that Nodal signals are necessary and sufficient for both mesodermal and endodermal gene expression, we propose that VegT activation of Sox17alpha at the midblastula transition prevents mesodermal gene expression in response to Nodal signals, thus establishing the vegetal endodermal gene expression domain. Supporting this idea, Sox17alpha misexpression in the marginal zone inhibits the expression of multiple mesodermal genes. Furthermore, in animal explants, Sox17alpha prevents the induction of Xbra and MyoD, but not Sox17beta or Mixer, in response to Xnr1. Therefore, VegT activation of Sox17alpha plays an important role in establishing a region of endoderm-specific gene expression in vegetal blastomeres.     

Ascidian tail formation requires caudal function.

Although the tail is one of the major characteristics of animals of the phylum Chordata, evolutionary aspects of the molecular mechanisms involved in its formation are not clear. To obtain insights into these issues, we have isolated and investigated the caudal gene of an ascidian, one of the lower animal groups among chordates. Ascidian caudal is expressed from the midgastrula stage onward in the lateral walls of the posterior neural tube cell lineage and also in the posterior epidermal cells from the neurula stage. Thus, ascidian caudal expression is restricted to the ectoderm of a tail-forming region throughout embryogenesis. Suppression of caudal function by an antisense oligonucleotide or a dominant negative construct caused inhibition of the cell movement required for tail formation. Overexpression of wild-type caudal mRNA in an ascidian animal cap, an animal half explant prepared at the eight-cell stage, caused elongation of the cap. Furthermore, Xenopus embryos injected with dominant negative ascidian caudal exhibited defects in elongation, suggesting a conserved caudal function among chordates. These results indicate that caudal function is required for chordate tail formation and may play a key role in its evolution.     

Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis

We describe the cloning, expression pattern and functional overexpression analysis of Xotx5b, a new member of the Otx gene family in Xenopus laevis. Early expression of Xotx5b resembles that of Xotx2, being detected in the organizer region at early gastrula stage, and, shortly after, also in anterior neuroectoderm. During neurula stages Xotx5b exhibits a changing and dynamic pattern of expression. After neural tube closure, Xotx5b is expressed in the eye and pineal gland, both involved in photoreception. Overexpression of Xotx5b has a similar effect to that of Xotx2, producing posterior truncations and inducing ectopic cement gland and neural tissue in whole embryos. In animal cap assays, Xotx5b and Xotx2 are both able to activate XAG, to strongly suppress the expression of the epidermal marker XK81, and to reciprocally activate each other. Finally, in einsteck transplantation assays, Xotx5b is able to respecify a tail/trunk organizer to a head organizer.     

Regionalization of the tail-tip epidermis requires inductive influence from vegetal cells and FGF signaling in the development of an ascidian, Halocynthia roretzi

The epidermis of an ascidian larva derived from animal-hemisphere cells is regionalized along the anterior-posterior (AP) axis through inductive signals emanating from vegetal-hemisphere cells in early stages of the development. Previously, we showed by blastomere isolation and ablation experiments that the contact between the animal and vegetal hemispheres until the 32-cell stage is necessary for the proper AP patterning of the epidermis in the tailbud-stage embryo. We here addressed the patterning mechanism of the posteriormost epidermis using a tail-tip epidermis marker, HrTT-1. Employing blastomere isolation and ablation experiments along with knockdown of a master regulator gene for posterior mesoderm, we have demonstrated that presence of the posterior vegetal cells after the 32-cell stage is necessary for the expression of HrTT-1. To explore the timing and nature of the influence of the posterior vegetal cells, we treated the embryos with FGF signaling inhibitors at various developmental stages and found that HrTT-1 expression was lost from embryos treated with the inhibitors from stages earlier than the late neurula stage, just prior to the onset of HrTT-1 expression but not after the initial tailbud stage, at which the expression of HrTT-1 had started. In embryos lacking HrTT-1 expression, the expression domain of Hrcad, which would otherwise be localized anterior to that of HrTT-1, expanded to the tail-tip. These results suggest that FGF signaling from the neurula to initial tailbud stages is necessary for the initiation but not maintenance of HrTT-1 expression in the tail-tip epidermis. The contact with posterior vegetal cells until and after the 32-cell stage may be required for FGF signaling to occur in the posterior tail, which in turn regionalizes the tail-tip epidermal territory.     

Fates and states of determination of single vegetal pole blastomeres of X. laevis

Vegetal pole cells of Xenopus morulae contribute progeny to all three germ layers, but from the midblastula stage onward they contribute only to the endoderm. We have investigated whether this restriction in fate reflects cell determination by implanting labeled vegetal pole cells into the blastocoels of host embryos and asking which structures later include labeled progeny. Single vegetal pole cells from the morula and also from the midblastula stage can contribute progeny to all germ layers. At the early gastrula stage the cells can contribute only to the endoderm. Thus the restriction of fate in the midblastula does not reflect cell determination. However, the cells do become determined by the beginning of gastrulation.     

A demonstration of cellular interactions during the formation of mesoderm and primordial germ cells in Pleurodeles waltlii

Animal, vegetal, dorsal and ventral blastomeres of eight-cell embryos of the urodele Pleurodeles waltlii were isolated and cultured for 15 days. The four animal blastomeres produced vesicles delimited by an irregularly shaped epidermis. In all other explants, the formation of mesodermal structures occurred, which can be interpreted as the result of inductive interaction, occurring during segmentation, between the ectodermal animal cap and vegetal yolk mass. Primordial germ cells (PGCs), which formed in 78% of cases when the presumptive ventral half to the embryo was cultured, occurred in only 48% of cases when the two ventral vegetal blastomeres were cultured alone. The absence of PGCs in the explants emanating from the four vegetal blastomeres is thought to have been due to inhibition of differentiation by notochord. This hypothesis has been confirmed by culture experiments in which the addition of presumptive chordomesoderm of young gastrulae prevented the differentiation of PGCs under conditions in which they are normally formed. These observations suggest that, in urodeles, PGCs do not arise from cells segregated as early as the eight-cell stage, but are the product of later inductive interaction between ectoderm and endoderm.     

Expression of intermediate filament proteins during development of Xenopus laevis. III. Identification of mRNAs encoding cytokeratins typical of complex epithelia

A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55,454; Mr approximately 58,000 on SDS-PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3'-untranslated portion of the 2.4 kb mRNA encoding this cytokeratin, designated XenCK55(5/6), in Northern blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCK1(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the 'XK81 subfamily' previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3'-untranslated region in Northern blots, S1 nuclease and hybrid-selection-translation assays and found the approximately 1.6 kb XK81 mRNA and the resulting protein of Mr approximately 48,000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.     

Clonal analysis of mesoderm induction in Xenopus laevis

Acidic fibroblast growth factor (aFGF) has been used to induce mesoderm from single animal pole cells of midblastula stage Xenopus embryos. The cells are individually cultured in a completely defined medium and are able to differentiate as small clones in a high proportion of cases. FGF-treated cells can give rise to several mesodermal cell types, while untreated cells show only epidermal or neural differentiation. Mesodermal differentiation can occur in clones of as few as eight cells, indicating that any additional cell-cell interactions required for mesodermal differentiation can be met by the medium used.