William C. Smith

Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos

We have cloned a cDNA encoding a novel polypeptide capable of inducing dorsal development in Xenopus embryos. RNA transcripts from this clone rescue normal development when injected into ventralized embryos and result in excessive head development at high doses. Therefore, we have named the cDNA noggin, noggin cDNA contains a single reading frame encoding a 26 kd protein with a hydrophobic amino-terminal sequence, suggesting that it is secreted. In Northern blot analysis this cDNA hybridizes to two mRNAs that are expressed both maternally and zygotically. Although noggin transcript is not localized in the oocyte and cleavage stage embryo, zygotic transcripts are initially restricted to the presumptive dorsal mesoderm and reach their highest levels at the gastrula stage in the dorsal lip of the blastopore (Spemann organizer). In the neurula, noggin is transcribed in the notochord and prechordal mesoderm. The activity of exogenous noggin RNA in embryonic axis induction and the localized expression of endogenous noggin transcripts suggest that noggin plays a role in normal dorsal development.

Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center

Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in midgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development.

A nodal-related gene defines a physical and functional domain within the Spemann organizer

A functional screen for gene products that rescue dorsal development in ventralized Xenopus embryos has yielded Xenopus nodal-related 3 (Xnr3), a diverged member of the TGF beta superfamily. Xnr3 is specifically expressed in the Spemann organizer and is only expressed in the epithelial layer of the organizer immediately preceding and extending through gastrulation. Like noggin, Xnr3 can induce muscle in ventral mesoderm explants, consistent with a role in patterning the gastrula. In other ways, the activity of Xnr3 is different from noggin. Embryos receiving injections of Xnr3, particularly in the animal pole, send out tube-like extensions of tissue from the site of injection. These protrusions usually contain no axial mesoderm and only occasionally are positive for neural markers. It has previously been proposed that the epithelial layer of the organizer initiates and coordinates the morphogenetic movements at gastrulation. The protrusions observed may reflect an activity of Xnr3 in promoting morphogenesis.

A retinoic acid response element is present in the mouse cellular retinol binding protein I (mCRBPI) promoter.

Genomic and cDNA sequences for the mouse cellular retinol binding protein I (mCRBPI) are presented. A specific cis‐acting element responsible for retinoic acid (RA) inducibility of the mCRBPI promoter was identified and characterized. Deletion mapping of a CRBPI promoter‐‐chloramphenicol acetyltransferase reporter gene construct localized this element to a 259 bp restriction fragment located approximately 1 kb upstream from the transcription start‐site. A sequence closely resembling the previously characterized RA response element (RARE) of the RA receptor beta 2 (RAR‐beta 2) promoter, and consisting of a direct repeat of the motif 5′‐GGTCA‐3′ separated by three nucleotides, was found within this restriction fragment. Mutation of these 5′‐GGTCA‐3′ motifs to GGAGC and GGGGC abolished RA‐inducible transcription whereas a mutation to a direct repeat of the GTTCA motif found in the RARE of the RAR‐beta 2 promoter resulted in enhanced inducibility. Oligonucleotides containing the direct repeat of the GGTCA motif were able to confer RA‐dependent transcriptional enhancement to the herpes simplex thymidine kinase promoter, as well as to bind directly all three retinoic acid receptors (RARs) alpha, beta and gamma, as determined by gel retardation/shift assays. The control of CRBPI gene transcription by RA‐RAR complexes interacting with the RARE characterized here may correspond to a feedback mechanism important in regulating retinoid metabolism and action.

Self- and Cross-Fertilization in the Solitary Ascidian Ciona savignyi

Solitary ascidians are hermaphrodites that release sperm and eggs simultaneously. However, many species are self-sterile, owing to a self/non-self recognition system operating at the outer surface of the chorion during sperm-egg interaction. In Ciona intestinalis, self-incompatibility is thought to have a genetic basis. Here, we report a survey of the self-fertilization potential of a Santa Barbara, California, population of Ciona savignyi, a close relative of C. intestinalis. We found that, in contrast to reports on C. intestinalis, C. savignyi is highly self-fertile. However, using two nonlethal recessive mutant strains, aimless (aim) and immaculate (imc), and a stable transgenic strain that expresses green fluorescent protein (GFP) in the notochord to follow offspring genotype, we demonstrate that non-self sperm outcompete self-sperm in fertilization competition assays. When the chorion was removed, both self- and non-self sperm performed equally well in the competition assay. Thus the non-self/self gamete recognition in C. savignyi is not absolute but relative, and is mediated by one or more components in the chorion. We discuss the significance of this finding in the context of natural populations in the wild, where individuals of C. savignyi are typically found growing in large groups that spawn in unison and where self-fertilization would be expected to be very rare.