The laboratory has studied the molecular basis of embryogenesis in Xenopus laevis and the zebrafish, with special emphasis on axis determination and pattern formation. We have investigated the regulation of these events by cell-to-cell signaling and by the spatially and temporally regulated action of transcription factors in several related systems.

The Role of Xlim-1 in Gastrulation
Hukriede, Dawid in collaboration with D. Weeks,University of Iowa
The Xlim-1 gene encodes a LIM-homeodomain protein that is involved in the functions of the Spemann organizer in neural induction and mesoderm patterning; it also plays a role in kidney formation. While a mouse knockout has been reported and shown to lack anterior head, we believe that the Xenopus gastrula allows a more detailed study of Xlim-1's function. Therefore, we study Xlim-1 function through an oligonucleotide antisense strategy, using DEED (diethylethylenediamine) antisense oligonucleotides that are stable in the embryo and that have been successfully used before (see Veenstra et al.). We find that the antisense oligos greatly reduce Xlim-1 mRNA concentration and lead to gastrulation defects and consequently failure of head formation. Somewhat surprisingly, most organizer-specific genes are activated normally in Xlim1-depleted embryos, but gastrulation movements, critical for germ layer establishment, are impaired. Thus, we conclude that regulation of cell movements is a major function of the Xlim-1 gene in the early embryo.

A Gene Expression Screen in the Zebrafish
Kudoh, Tsang, Hukriede, Dedekian, Clarke, Kiang, Schultz, Toyama, Dawid in collaboration with X. Chen and J. Epstein, UBC, NICHD
With the aid of high-throughput in situ hybridization, we have screened for developmentally regulated genes in zebrafish by searching for genes with restricted expression patterns. Such screens have been carried out in other developmental systems and have revealed interesting information. We have screened about 2,700 randomly picked cDNA clones from a normalized library and selected about 350 genes for further analysis. By partial sequencing, we could classify many of the genes into functional groups, showing that the selected set contained a preponderance of regulatory genes that encode either transcription factors or components of signal transduction pathways. We put the resulting information to three different uses. First, we identified particularly interesting genes for further study, including the iro3 gene. Second, we took advantage of mapping information about many of the clones that we generated with radiation hybrid mapping (see Hukriede et al.). With a map position and expression pattern known, a cDNA clone becomes an excellent candidate gene for the many developmental mutations that exist in the zebrafish. In at least one case, we matched one of the cDNAs to a mutation (studied by others) that affects ear development; the gene is still under study. Third, we used cDNAs from the screen to generate a detailed molecular anatomy atlas of the embryo, in which functional domains are characterized by gene expression rather than by morphology alone.

The Role of the iro3 Gene during Gastrulation
Kudoh, Dawid
Iroquios genes represent a subgroup of homeobox genes that are known to have a role in neural development. In our studies, we observed that the iro3 gene is already expressed during the blastula and gastrula stages in the zebrafish embryo in a dorsal endodermal domain (Figure 1).

In overexpression and inhibition studies, we showed that the early expression of iro3 plays a role during gastrulation in the specification of the dorsal axis and the activation of other organizer-specific genes. It appears that iro3 acts in a distinct manner during the early stages, along with its later role in neural plate differentiation. Repeated use of the same gene during subsequent stages of development is a known feature of many regulatory genes.