Our previous published studies have revealed important roles of the two closely related LIM-homeodomain genes Lhx1 and Lhx5 in the development of parts of the mouse central nervous system including the hippocampus and the cerebellum. We since discovered an additional involvement of these two genes in the development of Cajal-Retzius (C-R) cells in the telencephalon. C-R cells are well known for their essential role in the formation of the laminar organization of the mammalian cortex via secretion of the glycoprotein reelin. The mechanisms underlying the generation and distribution of these neurons have just begun to be unraveled. We addressed this question in a collaborative study with the laboratory of Dr. Alfredo Varela-Echavarra who spent a sabbatical research year with us. Both Lhx1 and Lhx5 are expressed in reelin-positive C-R cells and also in cells close to the origin of C-R cells in the nascent telencephalon. Deletion of Lhx5 results in a drastic reduction of the number of C-R cells in the cortex and the appearance of ectopic reelin-positive cell clusters in the caudal telencephalon. Using fluorescent dye in cultured embryos we were able to show that reelin-positive cells exiting from the caudomedial telencephalon migrate abnormally in the absence of Lhx5 function. Our study thus revealed a new and complex role for Lhx5 in the regulation of differentiation and migration of C-R cells in the developing telencephalon. A separate effort was directed toward the elucidation of Lhx6/Lhx8 gene function in the development of cortical interneurons that derive from the medial ganglionic eminence (MGE) in the ventral telencephalon. In collaboration with Dr. John Rubensteins laboratory we discovered that co-expression of Lhx6 and Lhx8 is required for induction of the Sonic Hedgehog (SHH) signaling molecule in the MGE. Targeted conditional inactivation of Shh in MGE neurons revealed that Sonic Hedgehog controls Nkx2.1, Lhx6 and Lhx8 and regulates the specification and survival of interneurons emanating from the MGE. With the help of a floxed allele of the obligatory Ldb1 coregulator of Lhx gene function we extended our analysis of transcriptional controls exerted by LIM-homeodomain factors on the development of a diverse array of structures in the nascent forebrain. Our previously published studies had shown that deletion of Ldb1 using Cre driven by the Nestin gene enhancer led to defects in the development of Purkinje cells in the cerebellum. Likewise, Nkx2.1-Cre mediated deletion of Ldb1 in the MGE affected development of cortical interneurons and the basal ganglia, a phenotype similar to that observed in Lhx6 and Lhx8 single or double mutants. We since extended our analysis of brain functions exerted by Ldb1 by examining the hypothalamus of the Ldb1/Nkx2.1-Cre mutan and observed that Ldb1 plays an essential role in the formation of several important nuclei in the hypothalamus including the arcuate nucleus, the ventromedial nucleus, and the paraventricular nucleus. Lhx gene products and their Ldb co-regulators also play important roles in the transcriptional regulation of limb development. Our current experiments try to elucidate the involvement of Lhx/Ldb complexes at very early stages of this process. Limbs develop from small buds consisting of a core of mesenchymal precursor cells covered by a layer of ectoderm. Development of the vertebrate limb bud depends on reciprocal interactions between FGF10 in the mesenchyme and FGF4 and FGF8 in the apical ectodermal ridge (AER). Several LIM-homeodomain genes, together with those encoding the Ldb1 and Ldb2 co-regulators, are expressed in the limb buds. Our attention focused on their role in the regulation of FGF signal exchange in the early hind limb buds. We compared expression profiles obtained from mesenchymal cells that we isolated from the hind limb buds of Ldb1 conditional knockdown mouse embryos with those obtained from control litter mates. Using microarray analysis followed by in situ hybridization we were able to demonstrate that distinct signaling pathways are controlled by Ldb1 and interacting transcription factors during hind limb bud development. Shh and FGF signaling pathways as well as the activity of genes involved in the differentiation of muscle precursor cells are down-regulated after Ldb knockdown at early stages of hind limb development. By contrast, biological processes affecting cell fate and cell death are up-regulated when Ldb1 function is impaired. A loss of both Ldb1 and Ldb2 function in the hind limb bud halts subsequent limb development. Very similar phenotypes were observed after ablation of the Lhx gene Isl1 in the bud mesenchyme. Fgf10 expression in the bud mesenchyme is lost if Ldb or Isl1 functions are curtailed, whereas the expression of Tbx4, a transcription factor associated with early hind limb development, is not affected. These observations allow us to conclude that Isl1 in conjunction with the Ldb co-regulators of transcription act downstream, or in conjunction with, Tbx4 to orchestrate the earliest stages of hind limb development. Transcriptional regulation of embryonic development in invertebrates and vertebrates involves a variety of protein complexes composed of transcription factors and nuclear co-regulators. Among these are the Single-stranded DNA-binding proteins (SSDPs) and the LIM-domain binding (Ldb) proteins. They can physically interact with each other and are recognized as essential co-regulators of embryonic development. We collaborated with Dr. Ruth Ashery-Padan to test if both types of regulators are obligatory members of transcription complexes in vivo. A conditional transgene was generated that can be activated by Cre action. This transgene encodes the N-terminal end of SSBP1 which is responsible for the binding to Ldb proteins. Activation of the conditional transgene in the developing lens of mouse embryos has Ldb-independent tissue-specific consequences which severely affect early lens development. To identify proteins that interact with SSBP1 and might mediate its Ldb-independent activity, we performed a yeast two-hybrid analysis using the SSBP1 N- terminus as bait and detected several potential binding partners. Among these are the products of other members of the SSBP gene family that can interact with each other in the form of homodimers and heterodimers. This suggests the possibility that activation of the N-terminal end of SSDP1 interferes with essential, albeit Ldb-independent, action of SSBP proteins in early lens development. Our findings suggest hitherto unknown functions of SSBP proteins during embryonic development. Supported by a Directors Challenge Award Program, our laboratory has been able to establish the premises for generating human induced pluripotent stem (iPS) cells from fibroblasts transduced by the transcription factors Sox2, Oct4 and Klf4. Extensive marker analysis identified our reprogrammed cells as iPS cells whose embryonic stem cell-like properties remain stable after multiple rounds of in vitro propagation. We have since gained access to fibroblasts derived from NICHD cohorts of patients with rare childhood diseases. Our short term goal is directed toward generating a number of iPS cell lines from children diagnosed with Smith-Lemli-Opitz Syndrome (SLOS) that are being treated by Dr. F.D. Porter at NICHD. The generation of neuronal cells differentiated from patient-specific SLOS iPS cell clones will be of preeminent importance for subsequent experiments aimed at studying gene defects underlying the neurological deficiencies observed in these patients, at establishing drug screening protocols, and at developing novel avenues of therapy.
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