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Home > Section on Mammalian Molecular Genetics

Mouse Molecular Genetics and Stem Cell Research

Heiner Westphal, MD
  • Heiner Westphal, MD, Head, Section on Mammalian Molecular Genetics
  • Alexander J. Blood, BS, Postbaccalaureate Fellow
  • Tsadok Cohen, PhD, Postdoctoral Fellow
  • Kevin Francis, PhD, Postdoctoral Fellow
  • Alexander Grinberg, DVM, Senior Research Assistant
  • Eric J. Lee, DVM, Senior Research Assistant
  • Evgeny Makarev, PhD, Postdoctoral Fellow
  • Ginat Narkis, PhD, Postdoctoral Fellow
  • Elizabeth Newman, BS, Postbaccalaureate Fellow
  • Peter O'Halloran, BS, Postbaccalaureate Fellow
  • William Olson, BS, Postbaccalaureate Fellow
  • Matthew Phillips, PhD, Postdoctoral Fellow
  • Eric Wier, BS, Postbaccalaureate Fellow
  • Lisa William-Simons, BS, AAS, Senior Research Assistant
  • Yangu Zhao, PhD, Staff Scientist

A major effort of our group is directed toward the functional analysis of LIM-homeodomain transcription factors encoded by the Lhx gene family and of associated Ldb co-regulators in mouse embryonic development. Current studies are centered on their involvement in patterning of the nascent forebrain and the limb anlagen. A second project concerns the reprogramming of human somatic cells to an induced pluripotent stem (iPS) cell state. We gained experience in generating iPS cells from human fibroblasts and have set up collaborations with the aim of generating iPS cell clones pertaining to a number of patient cohorts under current study at NICHD. We expect that the cells and their differentiated derivatives will become valuable tools for probing disease mechanisms and for drug screening.

Roles of LIM-homeodomain factors and Ldb coregulators in forebrain patterning and hind limb development

Our previous published studies 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 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 Alfredo Varela-Echavarría, 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 in 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 John Rubenstein's 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 to 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 have since extended our analysis of brain functions exerted by Ldb1 by examining the hypothalamus of the Ldb1/Nkx2.1-Cre mutant 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 are designed 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. We 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 Is/1 in the bud mesenchyme. Fgf10 expression in the bud mesenchyme is lost if Ldb or Is/1 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 Is/1, in conjunction with the Ldb co-regulators of transcription, acts downstream of, or in conjunction with, Tbx4 to orchestrate the earliest stages of hind limb development.

Ldb genes play hitherto unrecognized roles in the control of cell motility

In collaboration with the laboratory of Luc Sabourin, we looked for functions of Ldb1 and Ldb2 that lie outside the realm of their well-established roles as essential co-regulators of the transcriptional control of development. We discovered a hitherto unknown involvement of Ldb proteins in the control of cell migration in vitro and in vivo. They bind to a carboxyl-terminal domain of the microtubule-associated Ste20 kinase SLK. Ldb1 and Ldb2 co-localize with SLK in migrating cells, and gain or loss of either of these factors results in increased cell motility. The Ldb proteins thus act in this context as negative regulators of SLK activity.

KAP13 is essential for cardiomyocyte differentiation and heart morphogenesis.

Our group collaborated with the laboratory of our NICHD colleague James Segars to study the role of the Rho guanine nucleotide exchange factor AKAP13 in mouse heart development. AKAP13 proteins are known to coordinate signals originating from the cell membrane. We generated mice that carry a null mutation of Akap13. The mutation results in an embryonic lethal phenotype characterized by early defects in the myocardium that reveal an essential role of AKAP13 in heart development. We found the action of AKAP13 to be closely linked to that of the myocyte-specific enhancer factor 2C.

SSBP action independent of Ldb is required for early lens 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 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 we 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 that activation of the N-terminal end of SSDP1 might interfere with essential, albeit Ldb-independent, actions of SSBP proteins in early lens development. Our findings suggest hitherto unknown functions of SSBP proteins during embryonic development.

Our stem cell project focuses on the generation of human induced pluripotent stem cells.

Supported by a Director's 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 to generate a number of iPS cell lines from children diagnosed with Smith-Lemli-Opitz syndrome (SLOS), who are being treated by Forbes 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 the gene defects underlying the neurological deficiencies observed in these patients, establishing drug screening protocols, and developing novel avenues of therapy.

Additional Funding

  • NIH Director's Challenge Award Program for the study of "Induced pluripotent stem cells for the study of human disorders"

Publications

  • Storbeck CJ, Wagner S, O'Reilly P, McKay M, Parks R, Westphal H, Sabourin LA. The Ldb1 and Ldb2 transcriptional co-factors interact with the Ste20-like kinase SLK and regulate cell migration. Mol Biol Cell. 2009;20:4174-4182.
  • Dey-Guha I, Mukhopadhyay M, Phillips M, Westphal H. Role of ldb1 in adult intestinal homeostasis. Int J Biol Sci. 2009;5:686-694.
  • Zhao Y, Mailloux CM, Hermesz E, Palkóvits M, Westphal H. A role of the LIM-homeobox gene Lhx2 in the regulation of pituitary development. Dev Biol. 2010;337:313-323.
  • Mayers CM, Wadell J, McLean K, Venere M, Malik M, Shibata T, Driggers PH, Kino T, Guo XC, Koide H, Gorivodsky M, Grinberg A, Mukhopadhyay M, Abu-Asab M, Westphal H, Segars JH. The Rho guanine nucleotide exchange factor AKAP13 (BRX) is essential for cardiac development in mice. J Biol Chem. 2010;285:12344-12354.
  • Miquelajáuregui A, Varela-Echavarría A, Ceci ML, García-Moreno F, Ricaño I, Hoang K, Frade-Pérez D, Portera-Cailliau C, Tamariz E, De Carlos JA, Westphal H, Zhao Y. LIM-homeobox gene Lhx5 is required for normal development of Cajal–Retzius cells. J Neurosci. 2010;30:10551-10562.
  • Bronstein R, Levkovitz L, Yosef N, Yanku M, Ruppin E, Sharan R, Westphal H, Oliver B, Segal D. Transcriptional regulation by CHIP/LDB complexes. PLoS Genet. 2010;6(8):e1001063.
  • Phillips MD, Mukhopadhyay M, Poscablo C, Westphal H. Dkk1 and Dkk2 regulate epicardial specification during mouse heart development. Int J Cardiol. 2010;[Epub ahead of print].
  • Teufel A, Maass T, Strand S, Thieringer FR, Kanzler S, Galante T, Biesterfeld S, Westphal H, Galle PR. Liver-specific Ldb1 deletion results in enhanced liver cancer development. J Hepatol. 2010;in press.

Collaborators

  • Ruth Ashery-Padan, PhD, Department of Human Genetics, Tel Aviv University, Israel
  • Forbes D. Porter, MD, PhD, Clinical Director, NICHD, Bethesda, MD
  • John L. Rubenstein, MD, PhD, Department of Psychiatry, UCSF, San Francisco, CA
  • Luc Sabourin, PhD, Ottawa Hospital Research Institute, Ottawa, Canada
  • James H. Segars, MD, Program on Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
  • Alfredo Varela-Echavarría, PhD, Universidad Nacional Autónoma de México, Querétano, México

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