THE FORMATION AND MAINTENANCE OF THE DEFINITIVE ENDODERM LINEAGE IN THE MOUSE - INVOLVEMENT OF HNF3 FORKHEAD PROTEINS/

Little is known about genes that govern the development of the definit ive endoderm in mammals; this germ layer gives rise to the intestinal epithelium and various other cell types, such as hepatocytes, derived from the gut. The discovery that the rat hepatocyte transcription fact or HNF3 is similar to the Drosophila forkhead gene, which plays a crit ical role in gut development in the fly, led us to isolate genes conta ining the HNF3/forkhead (HFH) domain that are expressed in mouse endod erm development. We recovered mouse HNF3beta from an embryo cDNA libra ry and found that the gene is first expressed in the anterior portion of the primitive streak at the onset of gastrulation, in a region wher e definitive endoderm first arises. Its expression persists in axial s tructures derived from the mouse equivalent of Hensen's node, namely d efinitive endoderm and notochord, and in the ventral region of the dev eloping neural tube. Expression of the highly related gene, HNF3alpha, appears to initiate later than HNF3beta and is first seen in midline endoderm cells. Expression subsequently appears in notochord, ventral neural tube, and gut endoderm in patterns similar to HNF3beta. Microsc ale DNA binding assays show that HNF3 proteins are detectable in the m idgut at 9.5 days p.c. At later stages HNF3 mRNAs and protein are expr essed strongly in endoderm-derived tissues such as the liver. HNF3 is also the only known hepatocyte-enriched transcription factor present i n a highly de-differentiated liver cell line that retains the capacity to re-differentiate to the hepatic phenotype. Taken together, these s tudies suggest that HNF3alpha and HNF3beta are involved in both the in itiation and maintenance of the endodermal lineage. We also discovered a novel HFH-containing gene, HFH-E5.1, that is expressed transiently in posterior ectoderm and mesoderm at the primitive streak stage, and later predominantly in the neural tube. HFH-E5.1 is highly similar in structure and expression profile to the Drosophila HFH gene FD4, sugge sting that HFH family members have different, evolutionarily conserved roles in development.