The Section on Mammalian Molecular Genetics (SMMG) is focused on the molecular genetics of embryonic development. Transcriptional control is a key element of developmental regulation, involving an elaborate repertoire of cis-regulatory target gene sequences as well as the cooperation and physical interaction of multiple factors that regulate gene expression. Core elements, present in many cell contexts, are thought to form complexes with cell- or tissue-specific factors to establish positive and negative control of gene expression and to bring about cell specification and tissue identity in the developing embryo. We study the molecular genetics of this process. Much of our work deals with the functional evaluation of members of the LIM class of homeobox genes (termed Lhx genes) during mouse development. During the course of these experiments, we identified two novel classes of proteins, encoded by the Ldb and Ssdp gene families, respectively. These are co-factors whose interaction with the Lhx-encoded LIM-homeodomain factors and with other transcriptional regulators is essential for embryonic development. Additional topics of our studies are members of the Dkk family of Wnt inhibitors that form a link between the action of Lhx genes and pattern formation in the developing central nervous system. The closely related Lhx1 and Lhx5 genes are topics of our ongoing work. The two genes are prominently expressed in many regions of the developing central nervous system, including the spinal cord and the cerebellum. Earlier work had shown that Lhx1 null embryos lack anterior head structures and that Lhx5 null mutants are impaired in hippocampal development. Lhx1 null embryos die at an early stage of development, precluding a thorough analysis of possible brain defects. We were also concerned that functional redundancies may exist between Lhx1 and Lhx5. We therefore generated conditional Lhx1 mutants as well as Lhx1/5 double mutants to be able to examine more closely the individual or combined roles of both genes in brain development. The analysis is focused on the developing cerebellum in these mutants. The cerebellar phenotype of embryos that lack either Lhx1 or Lhx5 is not remarkable. However, the examination of Lhx1/5 double mutants revealed that the Purkinje cells of the primordial cerebellum are largely missing, as defined by the absence of the Calbindin marker. Our experiment shows that both Lhx1 and Lhx5 are essential for the generation of Calbindin-positive Purkinje cells and that there is redundancy in this functional aspect. Another aspect of our current work concerns functions of Lhx2 in the context of the development of the pituitary gland. Our previous studies had established a role for this gene in brain and eye development and in hematopoiesis. Null embryos were anophthalmic because of a developmental arrest of the eye anlagen prior to the formation of the optic cup. In addition, deficient cell proliferation in the forebrain resulted in hypoplasia of the neocortex and aplasia of the hippocampal anlagen. The Lhx2-deficient mutants died in utero, possibly because of a cell non-autonomous defect of definitive erythropoiesis that caused severe anemia.Here we describe an additional phenotype. Deletion of Lhx2 impairs formation of the posterior lobe of the pituitary gland. We observe over-proliferation and a lack of proper differentiation of precursor cells in this tissue. Loss of Lhx2 function also leads to a disorganization of the anterior and intermediate lobes of the pituitary gland, possibly secondary to defects in the formation of the posterior pituitary. Our earlier work had identified Lhx3 and Lhx4 as essential regulators of pituitary development. The present work shows that Lhx2 plays an important role in the process as well. Members of the Dkk family of Wnt inhibitors are widely expressed in the developing embryo. We previously reported on Dkk1, a mediator of Lhx and other transcriptional activity during head induction. Severe rostral defects are the result of Dkk1 functional ablation. Our recent work has dealt with the phenotype of Dkk2 knockout mice. These mutants are characterized by a profound defect in cornea cell turnover, indicating that Wnt pathway regulation is essential for the maintenance of ocular surface integrity. The cornea is a very ordered, transparent structure whose epithelium is replaced every three or four weeks by derivatives of stem cells that reside in the limbus, a transitional zone between the corneal and the abutting conjunctival epithelium. The cornea of young adult Dkk2 null mice is opaque and contains cells normally found in skin and conjunctiva, including hair follicles, sebaceous glands and goblet cells. Wnt signals are upregulated in the mutant limbus region. Dkk2 has therefore been identified as a Wnt inhibitor that regulates the turnover of corneal epithelia.
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