Aims of the Project
This project will develop the pipid frog Silurana (Xenopus) tropicalis as a model system for vertebrate developmental genetics. Its close relative, Xenopus laevis, has been a highly productive embryological system, but despite a recent breakthrough in the generation of transgenic frogs1, 2, genetic analyses have been hindered by its polyploid genome and long generation time. Transfer of techniques developed in X. laevis to the diploid, fast-breeding S. tropicalis should permit combining transgenesis and conventional genetic approaches with the advanced embryology and molecular biology presently available in X. laevis.
To establish an infrastructure for performing genetic research on S. tropicalis, by:
A. continuing to optimize husbandry, reproduction, and manipulation of S. tropicalis
B. creating a polymorphic panel of isogenic strains of S. tropicalis by generating gynogenetic homozygous diploids or conventional inbreeding
C. characterizing naturally-occuring recessive mutations which are uncovered in the process of creating isogenic strains for use as genetic markers.
D. generating a basic molecular genetic map of the S. tropicalis genome.
To generate stable transgenic S. tropicalis lines with the goal of streamlining current embryological procedures or creating novel experimental paradigms, specifically
A. to facilitate preparation of genetic mosaics and supplant laborious gene expression assays with rapid in vivo reporter detection, by establishing transgenic reporter lines tissue-specifically expressing Green Fluorescent Protein (GFP) driven by characterized X. laevis regulatory elements (brachyury, cardiac actin, N-tubulin, and goosecoid promoters) with useful domains of expression;
B. to simplify tissue labeling for transplantation, by establishing transgenic lines expressing GFP ubiquitously using the cytoskeletal actin and cytomegalovirus promoters
C. to exert temporal control expression over transgenic gene function, by evaluating inducible systems
D.. to combine transgenesis with transplantation techniques in order to implement genetic mosaic assays
To use transgenic reporter assays to identify, dissect, and compare cis-acting genomic regulatory regions in sets of genes with overlapping temporal and spatial regulation, specifically
A. genes expressed early (Six-3) and late (g-crystallin) in eye development
B. genes expressed in the Spemann organizer (xNR3, noggin, and goosecoid)
Specific aim #4:
To identify and characterize genes involved in axial extension, morphogenetic movements, axial patterning, and eye development via