MURINE GASTRULATION REQUIRES HNF-4 REGULATED GENE-EXPRESSION IN THE VISCERAL ENDODERM - TETRAPLOID RESCUE OF HNF-4(- -) EMBRYOS/

Immediately prior to gastrulation the murine embryo consists of an out er layer of visceral endoderm (VE) and an inner layer of ectoderm. Dif ferentiation and migration of the ectoderm then occurs to produce the three germ layers (ectoderm, embryonic endoderm and mesoderm) from whi ch the fetus is derived. An indication that the VE might have a critic al role in this process emerged from studies of Hnf-4(-/-) mouse embry os which fail to undergo normal gastrulation. Since expression of the transcription factor HNF-4 is restricted to the VE during this phase o f development, we proposed that HNF-4-regulated gene expression in the VE creates an environment capable of supporting gastrulation. To addr ess this directly we have exploited the versatility of embryonic stem (ES) cells which are amenable to genetic manipulation and can be induc ed to form VE in vitro. Moreover, embryos derived solely from ES cells can be generated by aggregation with tetraploid morulae. Using Hnf-4( -/-) ES cells we demonstrate that HNF-4 is a key regulator of tissue-s pecific gene expression in the VE, required for normal expression of s ecreted factors including alphafetoprotein, apolipoproteins, transthyr etin, retinol binding protein, and transferrin. Furthermore, specific complementation of Hnf-4(+) embryos with tetraploid-derived Hnf-4(+/+) VE rescues their early developmental arrest, showing conclusively tha t a functional VE is mandatory for gastrulation.