Hugh R. Woodland

Xsox17α and -β Mediate Endoderm Formation in Xenopus

We have isolated two Xenopus relatives of murine Sox17 expressed in gastrula presumptive endoderm. Xsox17alpha and -beta 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 Xsox17beta 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.

The synthesis and storage of histones during the oogenesis of Xenopus laevis.

Abstract Further data, including two-dimensional gel electrophoresis and peptide mapping of newly synthesized proteins, confirms the view that oocytes make several types of histone. The newly synthesized histone is present in both nucleus and cytoplasm, but at a higher concentration in the oocyte nucleus and in great excess over the DNA binding sites. The unfertilized egg seems to contain a pool of histones detectable on two-dimensional electrophoretograms. The peptide maps of these proteins are consistent with their identification as histones. The egg contains enough histone to support nuclear replication through most of cleavage.

Development of the ectoderm in Xenopus: Tissue specification and the role of cell association and division

When do ectodermal cells become specified to form epidermis, that is, become committed to form epidermis when isolated? Animal pole explants of Xenopus cultured in saline from the 8-cell stage onwards express a specific epidermal antigen, whereas vegetal explants do not. The isolated outer layer of ectoderm formed by stage 7 is almost completely specified, the inner layer is only partially so. When are cell associations and cell divisions necessary for epidermal differentiation? Embryos that were either disaggregated or incubated in cytochalasin B after the midblastula stage do not require cell interactions, Ca2+, or cell divisions for epidermal differentiation to occur. Inhibition of mRNA transcription with actinomycin D shows that the epidermal antigen is certainly transcribed by the late gastrula stage (stage 12).

Histone synthesis during the development of Xenopus

The ~~omatin of animal cells contains approximately equal masses of DNA and histone. In the division cycle of a typical eukaryotic somatic cell this equivalence is achieved by close coupling of histone and DNA synthesis. The first aspect of the coupling is temporal: almost all histone is made during the S-phase. Accordingly, histone mRNA is present during the S-phase and most appears to be degraded at its end [l-5]. However, small amounts of histone, particularly ID, may be made at other stages, and the amount may be significant in non-dividing cells (e.g. [6-81) and especially in late eryt~opoiesis [9]_ Since chromatin contains equal masses of DNA and histones this represents turnover, particularly of the Hl fraction. The second aspect of the coupling is quantitative; because very little free histone can be detected in S-phase cells, the S-phase cell must make the same mass of DNA and histone [lo,1 1). This tight coupling does not apply to the embryos of the anuran amphibian Xenopus laevis. At 23°C its zygote divides to form 30 000 cells in the 9 h period between fertilization and the gastrula stage. After the first cell cycle of 1.5 h, in which the S-phase lasts 20 min and there are recognisable Gl and G2 periods, the cell cycle may be as short as 12 min, with an S-phase of as little as 10 min and no recognisable Gl or G2 [ 12,131. Tight regulation of histone synthesis by transcriptional control is not possible over such a short time scale without enormous reiteration of the histone genes, The embryo solves this problem in a different way. Xenopus embryos has been reported by a number of authors ] 14171. The first qu~titative study was made by Adamson and Wiled [l S]. When absolute rates of histone synthesis were computed, they showed that at early stages the rate of histone synthesis is far in excess of that of DNA (fig.lA). In the fertilized egg histones are made at -2500 pg/h, whereas the cell replicates only 6 pg of nuclear DNA during the first 1.5 h cell cycle. There is also temporal uncoupling of DNA and histone synthesis, since histones are made at an approximately constant rate throughout this cell cycle, even during mitosis (191. By the late blastula stage the rate of DNA synthesized per embryo is -10 OOO-fold greater than at the single cell stage, but the rate of histone synthesis has only increased 2-3-fold (fig.1). The pattern described above applies to the nucleosomal histones. In contrast the Hl group of histones are made at a low rate until the early blastula stage (500 cells), but then increase to normal levels by the gastrula stage (30 000 cells) {18-201.

Modulations of histone messenger RNA during the early development of Xenopus laevis

Abstract The amount of translatable histone mRNA in Xenopus oocytes, eggs, and embryos was found to be roughly similar, as judged by a cell-free translation assay. This observation provides further evidence that posttranscriptional control mechanisms bring about the 50-fold increase in the rate of histone synthesis during oocyte maturation. About three-quarters of the oocyte histone mRNA was found to be polyadenylated. However, polyadenylated histone mRNA was scarcely detectable in the egg and embryo. Our data are consistent with the speculation presented here that the disappearance of the poly(A) + histone mRNA component at the time of conversion of oocyte to egg (maturation) is due to deadenylation.

Utilization of stored mRNA in Xenopus embryos and its replacement by newly synthesized transcripts: Histone H1 synthesis using interspecies hybrids

We studied H1 gene expression in hybrids of Xenopus laevis (female) x Xenopus borealis (male) and saw paternal H1 synthesis in mid-blastulae, which indicates that the H1 genes are active by this stage. The behavior of the maternal store of H1 histone mRNA was studied in androgenetic haploids. In which all stored mRNA is of the laevis type and all new transcripts are borealis. This showed that the initial activation of H1 synthesis occurs entirely by mobilizing maternal transcripts and that these are then unstable, disappearing in a few hours, by the early gastrula stage.

Tissue-specific expression of actin genes injected into Xenopus embryos.

We have isolated a complete Xenopus borealis cardiac actin gene, which is normally expressed in the myotomes and heart of the embryo and tadpole. After injection into the zygote, this cloned gene becomes distributed throughout the embryo, but it is expressed almost wholly in the myotomes. The same wide distribution of injected DNA but spatially restricted pattern of expression is found with a fusion between the first two actin gene exons and the last exon of a mouse beta-globin gene. By contrast, a histone-globin fusion gene is expressed fairly uniformly in all regions. We discuss the special advantages of using Xenopus in studies of tissue-specific gene expression from injected, cloned genes in early development.

Global analysis of the transcriptional network controlling Xenopus endoderm formation

A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc-finger factors and Sox17 HMG domain proteins. Although a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here, we have used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators and over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodal genes, Mixer and Sox17 have both shared and distinct sets of downstream targets, and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, between Sox17 and Bix1/Bix2/Bix4, and between Sox17 and Xnr4. Furthermore, we find that Mixer does not function primarily via Sox17 as previously proposed. These data provides new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network.

The synthesis of histone H1 during early amphibian development

Abstract There are at least three H1 histones in Xenopus . These change in synthetic rates during development, but there is no evidence for stage-specific histones. H1 is made at a low rate in oogenesis, but since the other histones are also made at low rates, the relative synthetic rate of H1 is comparable to the rates at which each of the other histones is made. The non-H1 histones show a large, abrupt increase in their synthetic rate during oocyte maturation (Adamson, E. D., and Woodland, H. R. (1977). Develop. Biol. 57, 136–149), but the rate of H1 synthesis remains relatively low into cleavage. It increases during cleavage, until H1 and other histones again have comparable rates of synthesis by the late blastula stage. Evidence is presented that the low rate of H1 synthesis measured in eggs and cleaving embryos is not an artifact produced by poor recovery of H1 from these developmental stages. Previous work has shown that at these early stages the synthesis of all histones is under posttranscriptional control. The mechanisms responsible must therefore be wholly or partly separate for H1 and the other histones.

The possible role of mesodermal growth factors in the formation of endoderm inXenopus laevis

We have raised a monoclonal antibody, 4G6, against gut manually isolated from stage 42Xenopus laevis embryos. It is specific for endoderm and recognises an epitope that is first expressed at stage 19 and which persists throughout subsequent development. The antibody maintains gut specificity through metamorphosis and into adulthood. The epitope is conserved in the mouse, where it is also found in the gut. Isolated vegetal poles fromXenopus blastula stage embryos express the epitope autonomously after culturing to the appropriate stage. This shows that certain aspects of endoderm differentiation do not require germ layer interactions. Animal cap cells from stage 9 blastulae cultured in the presence of the mesodermal growth factors FGF, XTC-MIF and PIF form both endodermal and mesodermal tissues, assessed by the binding of tissue-specific monoclonal antibodies. Endoderm is typically found in those caps which form intermediate and ventral forms of mesoderm, that is muscle and lateral plate.