Sea-urchin RNAs displaying differences in developmental regulation and in complementarity to a collagen exon probe

Sea-urchin embryo RNAs of 9 kb and 7 kb hybridise with a collagen-coding probe. The delta Tm of the hybrids indicates a 70% sequence identity between these RNA regions. Both RNAs are localised in the pluteus endomesoderm, but accumulate over different developmental periods: the 9 kb RNA first appears in the blastula and reaches a maximum concentration during the gastrula stages, while the 7 kb RNA is first detected in the gastrula and is at maximal concentration in the pluteus larva. Animalization by transient exposure of the early stage embryo to Zn2+ alters the developmental profile of the 9 kb collagen mRNA in a way that is clearly different from responses of other mRNAs whose accumulations are initiated during the blastula stage (Nemer, M. (1986) Dev. Biol. 114, 214-224).     

Stage-specific keratins in Xenopus laevis embryos and tadpoles: the XK81 gene family

This report describes the isolation and characterization of genomic and cDNA clones which define a subfamily of type I keratins in Xenopus laevis whose expression is restricted to embryonic and larval stages. The XK81 subfamily, named after the prototype cDNA clone DG81, contains four members arranged in two pairs of closely homologous loci; they were named 81A1, A2, B1, and B2. Genomic clones were obtained representing all of these regions. The A1 gene has been completely sequenced together with approximately 1 kb of flanking sequences at each end; this gene corresponds to the previously reported cDNA clone 8128 (Jonas, E., T. D. Sargent, and I. B. Dawid, 1985, Proc. Natl. Acad. Sci. USA, 82:5413-5417). The B2 gene is represented by a partial cDNA clone, DG118. Upstream sequences and about half of the coding regions have been sequenced for the B1 and B2 genes, whereas the A2 locus has been identified on the basis of hybridization data and could be a gene or pseudogene. Genomic Southern blotting indicates that all members of the subfamily have been isolated. The keratin proteins encoded by the B1 and B2 genes are 96% homologous in the central rod domain, whereas A/B gene homology in this region is 81%. During development mRNAs derived from A and B genes accumulate coordinately during gastrula and neurula stages; in the tadpole, 81A mRNA decays rapidly, whereas 81B mRNA shows a second abundance peak, persists for most of tadpole life, and decays by metamorphosis. RNAs derived from the XK81 keratin subfamily are undetectable in the adult, where different type I keratin genes are expressed.     

The role of gsc and BMP-4 in dorsal-ventral patterning of the marginal zone in Xenopus: a loss-of-function study using antisense RNA.

The dorsal-specific homeobox gene goosecoid (gsc) and the bone morphogenetic protein 4 gene (BMP-4) are expressed in complementary regions of the Xenopus gastrula. Injection of gsc mRNA dorsalizes ventral mesodermal tissue and can induce axis formation in normal and UV-ventralized embryos. On the other hand, BMP-4 mRNA injection, which has a strong ventralizing effect on whole embryos, has been implicated in ventralization by UV, and can rescue tail structures in embryos dorsalized by LiCl. The above-mentioned putative roles for BMP-4 and gsc are based on gain-of-function experiments. In order to determine the in vivo role of these two genes in the patterning of the Xenopus mesoderm during gastrulation, partial loss-of-function experiments were performed using antisense RNA injections. Using marker genes that are expressed early in gastrulation, we show that antisense gsc RNA has a ventralizing effect on embryos, whereas antisense BMP-4 RNA dorsalizes mesodermal tissue. These loss-of-function studies also show a requirement for gsc and BMP-4 in the dorsalization induced by LiCl and in the ventralization generated by UV irradiation, respectively. Thus, both gain- and loss-of-function results for gsc and BMP-4 support the view that these two genes are necessary components of the dorsal and ventral patterning pathways in Xenopus embryos.     

Localized synthesis of the Vg1 protein during early Xenopus development

The Xenopus Vg1 gene encodes a maternal mRNA that is localized to the vegetal hemisphere of both oocytes and embryos and encodes a protein related to the TGF-beta family of small secreted growth factors. We have raised antibodies to recombinant Vg1 protein and used them to show that Vg1 protein is first detected in stage IV oocytes and reaches maximal levels in stage VI oocytes and eggs. During embryogenesis, Vg1 protein is synthesized until the gastrula stage. The embryonically synthesized Vg1 protein is present only in vegetal cells of an early blastula. We find that Vg1 protein is glycosylated and associated with membranes in the early embryo. Our results also suggest that a small proportion of the full-length Vg1 protein is cleaved to give a small peptide of M(r) = approximately 17 x 10(3). These results support the proposal that the Vg1 protein is an endogenous growth-factor-like molecule involved in mesoderm induction within the amphibian embryo.     

A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity.The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.     

Refinement of gene expression patterns in the early Xenopus embryo

During blastula and gastrula stages of Xenopus development, cells become progressively and asynchronously committed to a particular germ layer. We have analysed the expression of genes normally expressed in ectoderm, mesoderm or endoderm in individual cells from early and late gastrula embryos, by both in situ hybridization and single-cell RT-PCR. We show that at early gastrula stages, individual cells in the same region may express markers of two or more germ layers, and 'rogue' cells that express a marker outside its canonical domain are also observed at these stages. However, by the late gastrula stage, individual cells express markers that are more characteristic of their position in the embryo, and 'rogue' cells are seen less frequently. These observations exemplify at the gene expression level the observation that cells of the early gastrula are less committed to one germ layer than are cells of the late gastrula embryo. Ectodermal cells induced to form mesendoderm by the addition of Activin respond by activating expression of different mesodermal and endodermal markers in the same cell, recapitulating the response of marginal zone cells in the embryo.     

Early expressed genes showing a dichotomous developing pattern in the lancelet embryo

Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3beta class genes. The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region, respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling. The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.