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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
  • Tsadok Cohen, PhD, Postdoctoral Fellow
  • Albana Davidhi, BS, former Postbaccalaureate Fellow
  • Ipsita Dey-Guha, PhD, former Postdoctoral Fellow
  • Kevin Francis, PhD, Postdoctoral Fellow
  • Marat Gorivodsky, PhD, former Postdoctoral Fellow
  • Alexander Grinberg, DVM, Senior Research Assistant
  • Alexander Holtz, BA, former Postbaccalaureate Fellow
  • Eric J Lee, DVM, Senior Research Assistant
  • Evgeny Makarev, PhD, Postdoctoral Fellow
  • Mahua Mukhopadhyay, PhD, former Research Fellow
  • Ginat Narkis, PhD, Postdoctoral Fellow
  • Elizabeth Newman, BS, Postbaccalaureate Fellow
  • William Olson, BS, Postbaccalaureate Fellow
  • Matthew Phillips, PhD, Postdoctoral Fellow
  • Andreas Teufel, MD, PhD, former Postdoctoral Fellow
  • Itai Tzchori, PhD, former Postdoctoral Fellow
  • Eric Wier, BS, Postbaccalaureate Fellow
  • Lisa William-Simons, BS, AAS, Senior Research Assistant
  • Yangu Zhao, PhD, Staff Scientist

Our group studies the function of members of the Lhx gene family, which encode LIM-homeodomain transcription factors. Transcription of target genes is regulated by oligomeric complexes involving individual Lhx gene products plus members of the Ldb and Ssdp families of transcriptional co-regulators. 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 continue this work with a main focus on the involvement of Lhx genes in development of the mouse brain. A novel aspect of our work concerns the reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state. We have set up collaborations with the aim of generating patient-specific iPS cell clones pertaining to a number of patient cohorts under current study by the NIH intramural research community. Neuronal cells differentiated from these iPS clones will be of preeminent importance for subsequent experiments aimed at studying the basis for neurological deficiencies observed in these patients.

Functions of LIM-homeobox genes and their transcriptional co-regulator Ldb1 in mouse development

Lhx6 and Lhx8 are two closely related LIM-homeobox genes that share strong sequence homology and overlapping expression patterns in the developing mouse ventral telencephalon. Our previous published studies have shown that Lhx8 is required for the generation of several major groups of cholinergic neurons in the telencephalon while Lhx6 contributes to the proper positioning and differentiation of GABA-ergic interneurons in the neocortex and hippocampus. Both Lhx6 and Lhx8 are prominently expressed in the medial ganglionic eminence (MGE) of the developing ventral telencephalon. However, the globus pallidus, a major structure of the basal ganglia derived from the MGE, develops normally in either the Lhx6 or Lhx8 single mutant. This raised the possibility that the two genes share redundant functions in the development of the globus pallidus. To address this issue, we generated and analyzed mutants lacking the function of both Lhx6 and Lhx8. Indeed, the combined functional ablation of these two genes disrupts the formation of the globus pallidus. Furthermore, we detected severe defects in the tangential migration of GABAergic interneurons from the MGE to the cortex in Lhx6/Lhx8 double mutants that are more pronounced than those previously observed in the Lhx6 single mutant. We conclude that Lhx6 and Lhx8 play important, if partially redundant, roles in controlling the development of the telencephalon. Ldb1 encodes a co-regulator known to mediate the function of a number of Lhx genes. In an effort to show that Lhx6 and Lhx8 are also dependent on Ldb1, we generated mice carrying a floxed Ldb1 allele and crossed them with a transgenic line that expresses the Cre recombinase under the control of the 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 MGE. Moreover, they display a defect in the development of cholinergic or GABAergic neurons similar to that observed in either Lhx8 or Lhx6 single mutants. Likewise, the functional ablation of Ldb1 phenocopies impairment in the development of the globus pallidus observed in Lhx6/Lhx8 double mutants.

Our previous studies revealed important roles of the LIM-homeobox genes Lhx3 and Lhx4 in the development of the anterior and intermediate lobes of the mouse pituitary gland, which are derived from the oral ectoderm. However, little is known about the development of the posterior lobe of the pituitary, which is derived from neuroectoderm of the ventral diencephalon. The signaling molecule FGF8 is detected in the neuroectoderm of the ventral diencephalon. To analyze the role of FGF8 in pituitary development, we crossed a floxed Fgf8 mouse line (kindly provided by Mark Lewandoski, NCI) with the Nkx2.1-Cre line to inactivate Fgf8 in the ventral diencephalon. Our analysis of this Fgf8 conditional mutant at the embryonic stage E15.5 of development revealed that FGF8 is required for the proper formation of the posterior lobe of the pituitary. We are currently investigating the mechanism underlying the regulation of posterior pituitary development by FGF8.

Another set of LIM-homeobox genes, Lhx 2, Lhx9, and Lmx1b, together with the gene encoding the Ldb1 co-regulator of transcription, play prominent roles in the development of the forelimbs and the hind limbs of the mouse. With the help of loss-of-function analysis of various combinations of these genes, we were able to show that simultaneous loss of Lhx2 and Lhx9 resulted in patterning and growth defects along the proximodistal (PD) and anteroposterior (AP) limb axes. Similar, but more severe, phenotypes were observed when the activities of all three of these LIM-homeobox genes were significantly reduced by Cre-mediated knockdown of the gene encoding their obligatory cofactor Ldb1. This reveals that the dorsal limb–specific factor Lmx1b can partially compensate for the function of Lhx2 and Lhx9 in regulating AP and PD limb patterning and outgrowth. Mechanistically, the action of these three LIM-homeobox genes is tied in with signaling events that precede subsequent limb development. We demonstrated that Lhx2 and Lhx9 can fully substitute for each other, and that Lmx1b is partially redundant in controlling the production of output signals in mesenchymal cells of the early limb bud in response to FGF8 and Shh signaling.

In a previous annual report, we presented an outline of our initial experiments on Ssdp1, considered to be another co-regulator of Lhx-encoded transcription factors in mouse development. Its ablation causes severe developmental defects in the embryo. Ssdp1 is predominantly found in the cytoplasm of cells, and the molecule does not contain a nuclear localization domain. The work by Dey-Guha et al. (see below) has shown that phosphorylation involving N-terminal tyrosine residues of Ssdp1 facilitates the transfer of this molecule to the nucleus where it can bind to Lhx/Ldb1 transcription complexes. This suggests a fast and efficient means of SSDP-mediated transcriptional regulation of Lhx gene products. We are currently examining, in vivo, a possible functional interaction of Ldb1 and Ssdp proteins during lens development, a known site of Lhx gene action. To this end, we generated mutant embryos in which loss of Ldb1 or Ssdp1 function is targeted to early stages of lens differentiation. Direct interaction of these two regulators of transcription would predict similar phenotypes in both scenarios.

In a separate study, the microtubule-associated Ste20 kinase SLK was found to interact with Ldb gene products. The Ldb factors bind directly to the SLK carboxy-terminal ATH domain in vitro and in vivo. They co-localize with SLK in migrating cells. We find that both loss and gain of function of these factors result in increased cell motility. Conditional mutation of Ldb1 increases focal adhesion turnover and enhances migration in fibroblasts. Ldb proteins thus appear to assert negative control on SLK activity, thereby contributing an important function to the control of cell migration.

Pleiotropic functions of the cilia component IFT172 in mouse embryo development

IFT172 is a structural component of primary cilia of the cell. We used a knockout approach to study its function in mouse development. Mutants exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema, and extensive hemorrhages. They seldom survive mid-gestation. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Global brain-patterning defects occur along the dorsal–ventral (DV) and anterior–posterior (AP) axes of the embryo. We were able to show that Ift172 plays multiple roles, such as involvement in early regulation of FGF8 at the midbrain–hindbrain boundary, maintenance of the isthmic organizer, and mediating the function of the node (the early embryonic organizer) and the formation of head-organizing center (the anterior mesendoderm, or AME). Our findings suggest that forebrain and mid–hindbrain growth and AP patterning depend on the early function of Ift172 at gastrulation. All these important early controls of embryonic development appear to depend on cilia morphogenesis and cilia-mediated signaling.

Reprogramming of somatic cells to a pluripotent state

The reprogramming of somatic cells to an early embryonic state of development is a research goal of utmost biomedical importance. This is because such pluripotent cells could potentially give rise to virtually any cell of the body via specialized multistage differentiation programs. Shinya Yamanaka’s seminal work, demonstrating that pluripotency can be induced in mammalian somatic cells via viral transfection of four transcription factors (c-Myc, Sox2, Oct4 and Klf4), has revolutionized current research. Work in a rapidly increasing number of laboratories worldwide focuses on the potential of induced pluripotent stem (iPS) cells to serve as diagnostic tools and as a source for treatment of human disorders.

Reprogramming experiments did not start with the generation of iPS cells. Efforts have been going on for years to find suitable ways of turning back the natural course of events that lead from a pluripotent cell to a fully differentiated end product. Best known among the results of these earlier efforts are the generation of embryonic stem cells via transfer of somatic nuclei into oocytes and the reprogramming of somatic cells via fusion with embryonic stem (ES) cells. Our own reprogramming studies began with the latter approach. Previous work by others had shown that fusion of somatic cells with ES cells can yield pluripotent cell-cell hybrids, albeit at low fusion and reprogramming efficiencies. In our own experiments, briefly outlined in last year’s report, we examined the ability of undifferentiated ES cell lines to reprogram the nuclei of mouse embryo fibroblasts (MEF) through cell-cell fusion. Activated baculovirus induced fusion events in 70–85% of the cells, resulting in efficient reprogramming. The resulting MEF/ES cell hybrids, although nearly tetraploid, exclusively expressed ES markers and exhibited characteristics of normal ES cells. When comparing the reprogramming potency of four well-known ES cell lines (R1, J1, E14, C57BL/6), we noticed that E14 cells stood out as significantly less potent in their reprogramming ability. We analyzed histone modifications and were able to demonstrate that low reprogramming potency was correlated with reduced H3 lysine 9 acetylation (H3K9ac) levels. Treatment of E14 cells with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) significantly increased H3K9 acetylation levels, as well as the cells' reprogramming capacity to a level observed in R1 cells. Furthermore, treatment of E14 cells with HDAC inhibitors increased the size and differentiation state of teratomas resulting from injection of the cells into immune tolerant mice. This allowed us to conclude that H3K9 acetylation levels are correlated with ES cell pluripotency and reprogramming efficiency.

More recently, our laboratory has undertaken systematic efforts to reprogram both mouse and human somatic cells to an iPS state via viral transfection of the four transcription factors c-Myc, Sox2, Oct4, and Klf4. The mouse iPS cells thus generated show ES-like morphology and marker profiles and can be differentiated into various germline derivatives. At present, our main focus is on the reprogramming of human somatic cells. We are in the process of adapting published procedures for the purpose of generating iPS cells from normal human fibroblasts and keratinocytes. We will use expression analysis of relevant pluripotency genes and the ability of these cells to form teratomas in mice to assess the degree of reprogramming achieved. Karyotyping will ensure that these human iPS cells have maintained their chromosomal integrity. Supported by the Director’s Challenge Award Program of the NIH, we have established a collaboration with a number of NIH research groups who study the epigenetics of the reprogramming process (Sartorelli), the “stemness” of leukemia stem cells (Liu), as well as the brain-specific pathophysiology of patient cohorts with Parkinson’s disease (Hoffer), Smith-Lemli-Opitz syndrome and Niemann-Pick Type C disease (Pavan, Porter), and Chediak-Higashi disorder (Gahl). The generation of patient-specific neuronal cells differentiated from the disease-specific 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.

Outside projects performed with mouse mutants generated by our laboratory

Collaborating laboratories continue to rely on the expertise of our research team in generating gene-altered mice. The publications cited below report on two such mutants, one containing a conditional allele for glucose-6-phosphatase-alpha (Peng et al.), the other serving as a tool allowing for tetracycline-mediated Cre excision events (Bäckman et al.).

Additional Funding

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

Publications

  • Dey-Guha I, Malik N, Lesourne R, Love PE, Westphal H. Tyrosine phosphorylation controls nuclear localization and transcriptional activity of Ssdp1 in mammalian cells. J Cell Biochem 2008 103:1856-1865.
  • Tzchori I, Day TF, Carolan PJ, Zhao Y, Wassif CA, Li L, Lewandoski M, Gorivodsky M, Love PE, Porter FD, Westphal H, Yang Y. LIM homeobox transcription factors integrate signaling events that control three-dimensional limb patterning and growth. Development 2009 136:1375-1385.
  • Gorivodsky M, Mukhopadhyay M, Wilsch-Braeuninger M, Phillips M, Teufel A, Malik N, Huttner W, Westphal H. Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain. Dev Biol 2009 325:24-32.
  • Peng WT, Pan CJ, Lee EJ, Westphal H, Chou JY. Generation of mice with a conditional allele for G6pc. Genesis 2009 47:590-594.
  • Bäckman CM, Zhang Y, Malik N, Shan L, Hoffer BJ, Westphal H, Tomac AC. Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice. J Neurosci Methods 2009 176:16-23.

Collaborators

  • Rafael Casellas, PhD, Molecular Immunology and Inflammation Branch, NIAMS, Bethesda, MD
  • Janice Y. Chou, PhD, Program on Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
  • William Gahl, MD, PhD, Clinical Director, NHGRI, Bethesda, MD
  • Edit Hermesz, PhD, University of Szeged, Szeged, Hungary
  • Barry Hoffer, MD, PhD, Director, Intramural Research Program, NIDA, Baltimore, MD
  • Wieland Huttner, MD, Director, Max Planck Institute, Dresden, Germany
  • Mark Lewandoski, PhD, Cancer and Developmental Biology Laboratory, NCI at Frederick, Frederick, MD
  • Pu Paul Liu, MD, PhD, Genetics and Molecular Biology Branch, NHGRI, Bethesda, MD
  • Paul Love, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
  • Eran Meshorer, PhD, Institute of Life Sciences, Hebrew University, Jerusalem, Israel
  • William Pavan, PhD, Genetic Disease Research Branch, NHGRI, Bethesda, MD
  • Forbes D. Porter, MD, PhD, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
  • John L. Rubenstein, MD, PhD, University of California San Francisco, San Francisco, CA
  • Luc Sabourin, PhD, Ottawa Hospital Research Institute, Ottawa, Canada
  • Vittorio Sartorelli, PhD, Laboratory of Muscle Biology, NIAMS, Bethesda, MD
  • Yingzi Yang, PhD, Genetic Disease Research Branch, NHGRI, Bethesda, MD

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