This proposal focuses on investigation of the function of genes expressed during mammalian gastrulation. The ultimate goal of this laboratory is to integrate more than a century of experimental embryology with an understanding of gastrulation on a molecular level. Little is known in mammals about the genes that direct anteroposterior axis formation early in embryo genesis or of the signals that initiate their expression. Gastrulation, the period during which they act, is a critical time for development. A large number of poorly understood human genetic and epigenetic embryological abnormalities arise during this time, including renal, cardiac, CNS, and assorted midline malformations. Several immediate goals are addressed here. First, functional studies win be performed on two homeobox-containing genes that in preliminary experiments have been found to become expressed during gastrulation in an intriguing manner. These studies will include (i) isolating full length cDNAs and genomic clones, (ii) performing high resolution mapping of the position of one of the genes which is located near a known developmental mutation, (iii) characterizing expression of the genes in embryonic stem cells to develop an in vitro model for their expression, (iv) characterizing their expression in embryos homozygous for known developmental mutations, and (v) performing loss-of-function and gain-of-function experiments accompanied by morphological, histochemical, and molecular analyses. Subsequent experiments are designed to address the effort and the length of time required to perform loss-of-function experiments using homologous recombination to inactivate genes.
The aim of this section of the proposal to develop a simple and rapid assay to determine if inactivation of a gene specifically during gastrulation would be likely to result in an informative phenotype. The first approach attempted win be to use modified antisense oligonucleotides to inhibit gene expression in embryos cultured transiently in vitro. The feasibility of inhibiting genes known or presumed to be required for gastrulation (cyclin, beta-actin, and T) will be determined. Finally, genes acquired from a variety of sources including this laboratory that are known to be expressed during gastrulation will be screened using this protocol, to identify ones critically required for normal development. The information gained from these studies will lead, ultimately, to an understanding of the functions of these genes in early development, and to new insights into gastrulation.
Showing the most recent 10 out of 12 publications
