Mammalian Developmental Genetics And Animal Models Of Human Diseases

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Transcription of target genes is regulated by oligomeric complexes involving individual members of the LIM-HD transcription factors form oligomeric complexes with LIM domain binding (Ldb) factors, thereby allowing for a multifaceted temporal and spatial control of gene activity. One of three separate projects under current investigation concerns functions of the LIM-homeobox genes Lhx6, Lhx8 and their transcriptional co-regulator Ldb1 in mouse brain development. Lhx6 and Lhx8 share high sequence homology and overlapping expression patterns in the developing mouse ventral telencephalon. Our previous analysis of the Lhx8 knockout mutant had revealed a defect in the generation of several major groups of cholinergic neurons in the telencephalon. Subsequently, our analysis of the Lhx6 mutant showed that this gene is required for the differentiation of cortical and hippocampal GABA-ergic interneurons and their migration from the medial ganglionic eminence (MGE) to their ultimate positions in the neocortex and hippocampus. Since the MGE and the globus pallidus, a major component of the basal ganglia derived from the MGE, show no noticeable defects in single Lhx6 or Lhx8 mutants, these two genes may conceivably share redundant functions in the development of these structures. To address this issue, we decided to generate and analyze mutants lacking the function of both Lhx6 and Lhx8. For our initial analysis of the E18.5 double mutants, we utilized Er81, a marker specific for the developing MGE and globus pallidus structures, as well as the PLAP reporter gene, expressing placental alkaline phosphatase, that is present in the Lhx6 mutant locus. Presently, we examine cell proliferation and apoptosis over a broader developmental time course in an effort to study details of the control exerted by Lhx6 and Lhx8 on interneuron patterning in the ventral telencephalon. In a previously published experiment, we demonstrated that a conditional inactivation of Ldb1 leads to defects in development of the cerebellum similar to those observed in the Lhx1/Lhx5 double mutant. The experiment identified Ldb1 as an obligatory cofactor of Lhx genes in the development of the cerebellum. In an effort to show that the function of the transcriptional regulators encoded by Lhx6 and Lhx8 mutants is equally dependent on Ldb1, we have crossed the Ldb1 floxed mice with a transgenic line that expresses the Cre recombinase under the control of regulatory elements of the Nkx2.1 gene. Nkx2.1 acts as an upstream regulator for both Lhx6 and Lhx8 in the ventral telencephalon. The Ldb1/Nkx2.1-Cre conditional mutants show a loss of Ldb1 immunostaining in the GE. These mutants are being analyzed using the various markers for the MGE and for cholinergic and GABAergic neurons, similar to those used in the analysis of Lhx6 and Lhx8 mutants.[unreadable] [unreadable] Reprogramming of somatic cells to a pluripotent state is the topic of a second project of our research team. Recent experiments have shown that somatic nuclei can be reprogrammed to a pluripotent state when fused with embryonic stem (ES) cells, giving rise to pluripotent hybrids. However, the resulting fusion and reprogramming efficiencies have thus far been very low. We examined the ability of undifferentiated ES cell lines to reprogram the nuclei of murine embryonic fibroblasts (MEF) through the cell-cell fusion method. Activated baculovirus induced fusion events in 70-85% of the cells and resulted in efficient reprogramming. The resulting ES/MEF mouse hybrids, although nearly tetraploid, exhibited characteristics of normal ES cells. We further showed by RT-PCR that the MEF/ES hybrids express ES markers while loosing MEF markers. By comparing the potency of four different ES cell lines we found that the E14 line was significantly less potent than R1, J1 and C57BL/6 lines in its ability to reprogram MEFs. This low reprogramming potency was correlated with reduced H3 lysine 9 acetylation (H3K9ac) levels. Treatment of E14 cells with histone deacetylase (HDAC) inhibitors, Trichostatin A (TSA) and sodium butyrate, resulted in a statistically significant increase of H3K9 acetylation levels, and raised their reprogramming capacity to a level observed in the R1 ES cell line. Furthermore, we found that induced pluripotent stem (iPS) cells can also reprogram MEFs , albeit at low efficiency. However, addition of TSA did not increase the number of reprogrammed colonies. In line with these findings, we observed that iPS incubation with TSA did not increase the acetylation levels of the resulting hybrids. Fusion and subsequent reprogramming of somatic cells can be substantially enhanced through baculovirus induction, and the acetylation level of pluripotent stem cells is directly correlated with their reprogramming efficiency.[unreadable] [unreadable] The third project of the group deals with genes that maintain stem cells and control their differentiation in the embryo and in the adult organism. Lhx and Ldb genes are known to play important roles in stem cell maintenance and differentiation. Embryonic stem (ES) cells are well suited to investigate targets of Ldb1-mediated transcriptional events because their differentiation under controlled in vitro conditions is well established. In this scenario, ES cells give rise to a well documented arsenal of transcripts, among them all known Lhx transcription factors. We have implemented neuronal differentiation protocols in an effort to determine specific targets of Ldb-mediated transcription events. We work with ES cells that contain a null deletion of the Ldb2 gene and a floxed Ldb1 gene. These cells are comparable to wild type ES cells as mice corresponding to this genotype are fully viable. Cre-mediated conditional ablation of Ldb1 in the mutant ES cells enables us to study their transcriptional profiles and differentiation potentials in the presence or absence of Ldb1 activity. The validity of the approach is being established by comparing predicted marker gene expression, such as standard neuronal markers, GATA genes, and Lhx genes, via RT-PCR or qRT-PCR. Subsequently, whole genome expression analysis will be used to identify batteries of downstream targets that are positively or negatively regulated by Ldb1. Furthermore, using an Ldb1 antibody, chromatin immunoprecipitation assays will be used to identify regulatory elements in select candidate genes. [unreadable] [unreadable] In an effort to determine the role of the Ldb1 gene in stem cell niches present in adult tissues, we used our floxed mutant allele in an effort to interfere with Ldb1-mediated transcriptional activity in the small intestine and the skin. Using tissue-specific differentiation markers and a set of tools that allow us to measure cell proliferation and cell apoptosis, we have noticed a profound effect of Ldb1 ablation on the maintenance of the stem cell niches of the skin and the gut. The mechanism by which the Ldb-mediated transcriptional apparatus controls stem cell maintenance in these tissues is under current investigation, with special emphasis on Dkk and Kremen genes and their function in regulating Wnt activity in the stem cell niches.