The HNF3/fork head family includes a large number of transcription factors that share a structurally related DNA binding domain. Fork head factors have been shown to play important roles both during development and in the adult. We now describe the cloning of a novel mammalian fork head factor that we have named FHX (fork head homologous X (FHX), which is expressed in many adult tissues. In the embryo, FHX expression showed a very early onset during the cleavage stages of preimplantation development. Polymerase chain reaction-assisted site selection experiments showed that FHX bound DNA with a dual sequence specificity. Sites recognized by FHX could be classified into two different types according to their sequences. Binding of FHX to sequences of each type appeared to occur independently. Our data suggest that either different regions of the fork head domain or different molecular forms of this domain could be involved in binding of FHX to each type of site. In transfection assays, FHX was capable of activating transcription from promoters containing FHX sites of either type.The regulated expression of the genome is essential for the homeostatic maintenance and correct cell differentiation and morphogenesis of an organism. Transcription factors play a preponderant role in this regard. From an evolutionary standpoint, all transcription factors derive from a small set of primitive factors. These, upon successive gene duplication-divergence events and fusion with other genes or parts of genes, have given rise to the whole panoply of extant transcription factors. Consequently, current transcription factors may be sorted into different families according to their homology relationships. One of these families, which appears to have been highly successful in evolution, is the fork head/HNF3 family, with known members in species from yeast to humans, with the striking exception of green plants, where no fork head factor has been described so far (for a review, see Ref.

As reported previously, we have extensively studied FoxJ2, a member of the Fork Head transcription factors family. While the biochemical and functional structures of this transcription factor are well understood, its biological function remains unknown. Here, we present data that address this point using transgenic mouse technology. We found that the birth rate and the number of transgenic animals obtained when transferring embryos over-expressing the FoxJ2 protein were lower than those obtained with embryos over-expressing a control protein, suggesting FoxJ2 overexpression has a negative effect on embryonic development. Transient FoxJ2 transgenesis experiments have confirmed that FoxJ2 over-expression has a lethal effect on embryonic development from E10.5. Moreover, in vitro culture of FoxJ2-microinjected embryos demonstrated a significant developmental blockage, indicating that FoxJ2 could also have an effect on preimplantation stages. Most probably, these negative effects of FoxJ2 over-expression during development also explain the low percentage of adult transgenic mice obtained. Furthermore, most of the transgenic mice that lived to adulthood did not show transgene expression. In fact, the only two adult transgenic animals (one male and one female) in which FoxJ2 transgene expression was detected showed a mosaic expression and died prematurely as a result of cardio-respiratory failure. Postmortem analysis of these animals revealed a hypertrophic heart and abnormal testes in the male. In order to identify genes regulated by FoxJ2 consistent with the phenotypes observed for FoxJ2 transgenic mice, EMSA assays and co-transfection experiments were carried out. Our data indicate that the genes coding for the gap junction protein Connexin-43 and the cellcell contact protein E-Cadherin, may be good candidates for FoxJ2-regulated genes. Interestingly, Connexin-43 and E-Cadherin show expression patterns similar to FoxJ2, and the phenotypes of Connexin-43 and E-Cadherin mutants resemble those of our FoxJ2 transgenic animals. These data suggest that the lethal effect on embryonic development of FoxJ2 overexpression, as well as the alterations observed in the heart and testes of adult transgenic mice, could be determined by changes in the transcription of genes such as Connexin-43 and/or E-Cadherin.