A FORKHEAD GENE-RELATED TO HNF-3-BETA IS REQUIRED FOR GASTRULATION AND AXIS FORMATION IN THE ASCIDIAN EMBRYO

We have isolated a member of the HNF3/forkhead gene family in ascidian s as a means to determine the role of winged-helix genes in chordate d evelopment, The MocuFH1 gene, isolated from a Molgula oculata cDNA lib rary, exhibits a forkhead DNA-binding domain most similar to zebrafish axial and rodent HNF-3 beta, MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome, The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages, During gastrulation, MocuFH1 expression occurs in the prospective endoderm ce lls, which invaginate at the vegetal pole, and in the presumptive noto chord and mesenchyme cells, which involute over the anterior and later al lips of the blastopore, respectively, However, this gene is not exp ressed in the presumptive muscle cells, which involute over the poster ior lip of the blastopore, MocuFH1 expression continues in the same ce ll lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brai n and spinal cord, The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduc e MocuFH1 transcript levels during gastrulation, Embryos treated with antisense ODNs cleave normally and initiate gastrulation, However, gas trulation is incomplete, some of the endoderm and notochord cells do n ot enter the embryo and undergo subsequent movements, and axis formati on is abnormal. In contrast, the prospective muscle cells, which do no t express MocuFH1, undergo involution and later express muscle actin a nd acetylcholinesterase, markers of muscle cell differentiation. The r esults suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and ax is formation, The effects of inhibiting MocuFH1 expression on embryoni c axis formation in ascidians are similar to those reported for knocko ut mutations of HNF-3 beta in the mouse, suggesting that HNF3/forkhead genes have an ancient and fundamental role in organizing the body pla n in chordates.