Rescue of early embryonic lethality reveals multiple essential roles for the zinc finger transcription factor Gata3 in murine embryonic development.

摘要

Gata3 belongs to a family of transcription factors that are indispensable to development. Gata3 deficient mice die at embryonic day 10.5 with severe deformities in the central nervous system and fetal liver hematopoiesis. Here, we describe a method to study the role of Gata3 in late embryogenesis. In order to experimentally address these goals, we isolated two yeast artificial chromosomes (YAC) that represent large contiguous, non-chimeric segments of the Gata3 locus on chromosome 2. Then, as a reference point we targeted a lacZ reporter gene to the initiation codon of the chromosomal Gata3 gene in embryonic stem cells, generating Gata3lacZ/+ “knock-in” mice. When we asked whether a Gata3 YAC encoding lacZ (Gata3 lacZ YAC) could reproduce Gata3 lacZ/+ expression profile, even the largest 625 kbp YAC lacked the patterning element(s) that are critical for Gata3 expression in sympathetic ganglia and the adrenal gland. However, break point mapping of Gata3 lacZ YAC transgenics allowed us to localize distant urogenital-, CNS-, and endocardial-specific transcriptional regulatory elements in the Gata3 gene locus.; In complementation experiments, the 625 kbp YAC was able to rescue several of the mutant phenotypes and prolong fetal life, but it was unable to overcome the embryonic lethality of Gata3 homozygous gene targeted mutants. Subsequent investigations revealed that Gata3 −/− embryos had low levels of noradrenaline in the sympathetic nervous system. Feeding catecholamine intermediates to pregnant dams partially averted the Gata3 mutation-induced embryonic lethality, providing us the opportunity to examine the function of Gata3 in late embryonic development. In the cardiovascular system, the older Gata3−/− embryos revealed non-compaction of the ventricular myocardium, cardiac outflow obstruction due to persistence of endothelial cells in the endocardial cushions and excessive systemic neovascularization due to increased secretion of Vegf.; Together, Gata3 YAC transgenic mice and pharmacologically rescued Gata3−/− embryos should help to precisely position putative developmental effectors, and determine the epistatic relationships in the regulatory cascades that lead to Gata3 function in other organs.