The Section on Mammalian Molecular Genetics (SMMG) studies the molecular genetics of embryonic development. Our interest is focused on developmental controls exerted by LIM-homeodomain transcription factors and their cofactors, and by Wnt pathway regulators. In a separate project we describe our current efforts to reverse the differentiated state of somatic cells to a state of pluripotency. Members of the Lhx gene family encode LIM-homeodomain transcription factors that control important aspects of embryonic development. Studies on the mechanism of Lhx gene function led to our discovery of two obligatory cofactors that mediate the action of the LIM-homeodomain proteins. The first of these, Ldb1, interacts physically and genetically with Lhx gene products. Each Lhx gene family examined so far is dependent on Ldb1 cofactor activity. We found that embryos carrying Ldb1 null mutations exhibit severe early developmental defects. Even the in vitro differentiation of embryonic stem cells is severely affected by the lack of Ldb1 activity. This prompted us to ask whether Ldb1 is an obligatory interaction partner of each of the diverse array of Lhx gene products during mammalian development. To this end, our current efforts are directed toward examining Ldb1 action in specific regions of the developing embryo. If Ldb1 is indeed required to render each of the individual Lhx gene products active, selective Ldb1 gene inactivation should allow us to investigate the concerted activity of Lhx gene products in targeted areas of the developing embryo that lack Ldb1 activity. The main tool of our current protocol is a mouse mutant in which we inserted a floxed Ldb1 gene that can be inactivated in cells targeted by the Cre recombinase. If, as postulated, Ldb1 is obligatory for all LIM-homeodomain factor activity, the resulting phenotypes are expected to provide much needed information on the combined action of individual Lhx genes in specific target fields of the developing embryo. We plan to target an array of organotypic regions. In the first experiment of this kind we used a transgenic line that expresses Cre under the control of an enhancer element of the Nestin gene to delete the Ldb1 gene in the developing central nervous system. Our analysis of the resulting mutant revealed a defect in the specification of Purkinje cells in the developing cerebellum. Two Lhx genes, Lhx1 and Lhx5, are prominently expressed in the cerebellum of wild type embryos. It thus seemed likely that the products of these two genes were inactive in cells lacking Ldb1 activity. And indeed, when we generated mouse mutants with null deletions of both Lhx1 and Lhx5, we were able to copy the Purkinje cell phenotype, indicating that specific aspects of cerebellar development are controlled by the interaction of Ldb1 with these two LIM-homeodomain transcription factors. Ssdp1 is a second cofactor of Lhx transcriptional activity originally detected in our laboratory. Like Ldb1 it has been physically and genetically linked to Lhx gene action. In a recent collaboration with Dr. Sasaki and his colleagues in Japan we showed that Ssdp1 is an essential component of Ldb1-Lhx1 transcriptional activity during head formation in the mouse embryo. We presently examine the spectrum of Ssdp target genes in an effort to describe the extent of developmental controls exerted by this cofactor. Members of the Dkk family act as inhibitors of the canonical Wnt pathway during embryogenesis. The importance of Dkk1 in mediating transcriptional activity of LIM-homeodomain proteins and other transcription factors during head induction was demonstrated in our previous experiments. Our studies of the past year have uncovered an exciting new aspect of Dkk action. We studied mutant mice that lack the function of Dkk2 and noted that this gene controls the integrity of the ocular surface epithelium via regulation of the canonical Wnt pathway. In the absence of Dkk2 function, the epithelium of the cornea that covers the ocular surface is completely transformed into a stratified epithelium containing hair follicles and sebaceous glands. Stem cells that maintain corneal integrity throughout life reside in the limbus of the eye. It is here that Dkk2 is prominently expressed. This suggests that the selection and fate of these stem cells is tightly controlled by Wnt ligands and their regulators. Novel efforts are directed toward reprogramming somatic cells to an embryonic state of unlimited growth and multifaceted differentiation potential. In a direct search for a suitable source of reprogramming activity several laboratories, including our own, fused mouse ES cells with somatic cells and were able to demonstrate that markers of pluripotency, notably the transcription factor Oct4, were reactivated in the somatic nucleus of the resulting hybrid cells. The task ahead is to create conditions whereby the ES cell nucleus changes the epigenetic state of the hybrid cell but is prevented from participating in the formation of daughter cell nuclei. Pretreatment of the ES cell with inhibitors of DNA synthesis prior to fusion or mechanical removal of the ES cell nucleus from the hybrid cell are among the most promising of a host of attempts to generate a cell hybrid that will selectively replicate the somatic cell genome in a reprogrammed epigenetic state so that multipotent progeny cells can be propagated. In terms of potential benefits of this approach for future avenues of stem cell therapy, the line of thinking is as follows. Expose a patient?s somatic cell to factors that bring it back to a pluripotent stage comparable to that of an embryonic stem cell. Unlike most cells in our body, this cell would replicate fast and could be propagated under suitable in vitro culture conditions to generate a large number of progeny cells, all retaining pluripotency. When subjected to suitable differentiation protocols, these cells would give rise to a wide variety of differentiated cells available for use in tissue replacement protocols. Most important, these reprogrammed cells carry the patient?s own set of histocompatibility antigens on their surface, thus drastically reducing the risk of graft rejection, and they are not derived from an embryo, hence not subject to ethical concerns that restrict present human embryonic cell research. Clearly, the importance of experiments aimed at restoring stemness to somatic cells cannot be overstated.
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