Genes That Control Mouse Development

Heiner Westphal, MD, Head, Section on Mammalian Molecular Genetics
Yangu Zhao, PhD, Staff Scientist
Ipsita Dey-Guha, PhD, Visiting Fellow
Evgeny Makarev, PhD, Visiting Fellow
Ginat Narkis, PhD, Visiting Fellow
Itai Tzchori, PhD, Visiting Fellow
Rui Lin, BA, Technical Training Fellow
Alexander Grinberg, DVM, Senior Research Assistant
Eric J.M. Lee, DVM, Senior Research Assistant

Our group uses a loss-of-function approach to study fundamental controls exerted by transcriptional regulators of the LIM-homeodomain (LIM-HD) proteins and associated co-factors. Oligomeric complexes involving individual members of the LIM-HD, Ldb, and Ssdp families together with additional transcriptional regulators regulate transcription of target genes, thereby allowing for a multifaceted temporal and spatial control of gene activity. Over the years, we have gained insights into the individual or combined action of individual LIM-HD factors in setting the premises for tissue patterning and organ formation in the developing mouse embryo. We have continued our work with a detailed analysis of LIM-HD involvement in the development of the mouse embryo. A novel aspect of our work concerns the role of the LIM-HD transcriptional machinery in embryonic stem (ES) cell activation, in the reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state, and in the maintenance and activation of stem cells in the adult organism.

Functions of the LIM-homeobox genes Lhx6 and Lhx8, and their transcriptional co-regulator Ldb1 in mouse brain development

De la Cuesta,¹ Westphal, Zhao; in collaboration with Rubenstein

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 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 GA BA-ergic interneurons and their migration from the medial ganglionic eminence (MGE) to their ultimate positions in the neocortex and hippocampus. Given that the structures MGE and globus pallidus—the latter is a major component of the basal ganglia derived from the MGE—show no noticeable defects in single Lhx6 or Lhx8 mutants, the two genes may conceivably share redundant functions in the development of these two structures. To address this issue, we generated and analyzed mutants lacking the function of both Lhx6 and Lhx8. For our initial analysis of the E18.5 double mutants, we used Er81, a marker specific for the developing MGE and globus pallidus, as well as the PLAP reporter gene, which expresses placental alkaline phosphatase and is present in the Lhx6 mutant locus. We are now examining 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 also identified Ldb1 as an obligatory co-factor of Lhx genes in the development of the cerebellum. To show that the function of the transcriptional regulators encoded by Lhx6 and Lhx8 mutants is equally dependent on Ldb1, we crossed the Ldb1-floxed mice with a transgenic line that expresses 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 showed a loss of Ldb1 immunostaining in the GE. We are analyzing the mutants by using the various markers for the MGE and for cholinergic and GA BAergic neurons, similar to those used in the analysis of Lhx6 and Lhx8 mutants.

Zhao Y, Flandin P, Long JE, de la Cuesta MD, Westphal H, Rubenstein JL. Distinct molecular pathways for development of telencephalic interneuron subtypes revealed through analysis of Lhx6 mutants. J Comp Neurol 2008;510:79-99.

Reprogramming of somatic cells to a pluripotent state

Davidhi,² Tzchori, Westphal; in collaboration with Casellas, Meshorer

Our recent experiments have shown that somatic nuclei can be reprogrammed to a pluripotent state when fused with ES cells, giving rise to pluripotent hybrids. However, the resulting fusion and reprogramming efficiencies have thus far been particularly 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 to 85 percent of the cells, causing efficient reprogramming. The resulting ES/MEF mouse hybrids, although nearly tetraploid, exhibited characteristics of normal ES cells. We further showed by reverse transcriptase- PCR (RT-PCR) that the ME F/ES hybrids express ES markers while losing MEF markers. Comparing the potency of four ES cell lines, we found that the E14 line was significantly less potent than the R1, J1, and C57BL/6 lines in its ability to reprogram MEFs. This low reprogramming potency correlated with reduced H3 lysine-9 acetylation (H3K9ac) levels. Treatment of E14 cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate resulted in a statistically significant increase of H3K9 acetylation levels and raised the cells’ 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. Baculovirus induction can substantially enhance fusion and subsequent reprogramming of somatic cells while the acetylation level of pluripotent stem cells directly correlates with their reprogramming efficiency.

Embryonic and adult stem cells

Dey-Guha, Geum,³ Gorivodsky,⁴ Makarev, Mukhopadhyay,⁵ Narkis, Westphal; in collaboration with Niehrs, Tam

Lhx and Ldb genes are known to play important roles in stem cell maintenance and differentiation. ES cells are well suited for investigating the targets of Ldb1-mediated transcriptional events because their differentiation under controlled in vitro conditions is clearly established. In this scenario, ES cells give rise to a well-documented arsenal of transcripts, including all known Lhx transcription factors. To determine specific targets of Ldb-mediated transcription events, we have implemented neuronal differentiation protocols. We work with ES cells that contain a null deletion of the Ldb2 gene and a floxed Ldb1 gene; the 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 the cells’ transcriptional profiles and differentiation potentials in the presence or absence of Ldb1 activity. We are establishing the validity of the approach by using RT-PCR or qRT-PCR to compare predicted marker gene expression, such as the standard neuronal markers and GATA and Lhx genes. Subsequently, we will use whole-genome expression analysis to identify batteries of downstream targets that are positively or negatively regulated by Ldb1. Furthermore, using an Ldb1 antibody, we will perform chromatin immunoprecipitation assays to identify regulatory elements in select candidate genes.

To determine the role of the Ldb1 gene in stem cell niches in adult tissues, we used our floxed mutant allele to interfere with Ldb1-mediated transcriptional activity in the small intestine and skin. Using tissue-specific differentiation markers and a set of tools that allow us to measure cell proliferation and cell apoptosis, we noticed that Ldb1 ablation had a profound effect on the maintenance of stem cell niches of the gut and skin. We are investigating the mechanism by which the Ldb-mediated transcriptional apparatus controls stem cell maintenance in these tissues, with special emphasis on Dkk and Kremen genes and their function in regulating Wnt activity in the stem cell niches.

Ellwanger K, Saito H, Clément-Lacroix P, Maltry N, Niedermeyer J, Lee WK, Baron R, Rawadi G, Westphal H, Niehrs C. Targeted disruption of the Wnt regulator Kremen induces limb defects and high bone density. Mol Cell Biol 2008;28:4875-82.
Hwang M, Gorivodsky M, Kim M, Westphal H, Geum D. The neuronal differentiation potential of Ldb1-null mutant embryonic stem cells is dependent on extrinsic influences. Stem Cells 2008;26:1490-5.
Lewis SL, Khoo PL, De Young RA, Steiner K, Wilcock C, Mukhopadhyay M, Westphal H, Jamieson RV, Robb L, Tam PP. Dkk1 and Wnt3 interact to control head morphogenesis in the mouse. Development 2008;135:1791-801.

¹Melissa de la Cuesta, BS, former Postbaccalaureate Fellow
²Albana Davidhi, BS, former Postbaccalaureate Fellow
³Dongho Geum, PhD, former Postdoctoral Fellow
⁴Marat Gorivodsky, PhD, former Postdoctoral Fellow
⁵Mahua Mukhopadhyay, PhD, former Research Fellow

Collaborators

Rafael Casellas, PhD, Molecular Immunology and Inflammation Branch, NIH, Bethesda, MD
Eran Meshorer, PhD, Institute of Life Sciences, Hebrew University, Jerusalem, Israel
Christof Niehrs, PhD, Deutsches Krebsforschungszentrum, Heidelberg, Germany
John L.R. Rubenstein, MD, PhD, University of California San Francisco, San Francisco, CA
Patrick P.L. Tam, PhD, Children’s Medical Research Institute, Sydney, Australia

For further information, contact westphah@mail.nih.gov or visit http://westphal.nichd.nih.gov.