We study the molecular genetics of embryonic development, focusing on developmental controls exerted by LIM-homeodomain transcription factors and their cofactors and by Wnt pathway regulators. In a separate project, we aim to reverse the differentiated state of somatic cells to a state of pluripotency.
Embryonic development controlled by Lhx-Ldb1-Ssdp1 transcription complexes
Dey-Guha, Geum, Gorivodsky, Grinberg, Kim, Malik, Morales, Mukhopadhyay, Phillips, Varela-Echavarria, Westphal, Zhao; in collaboration with the laboratories of Palkovits, Rubenstein, and Sasaki
Members of the Lhx gene family encode LIM-homeodomain transcription factors that control important aspects of embryonic development. In the past, our analysis of mutants containing defective Lhx genes provided us with a host of data regarding the function of individual LIM-homeodomain factors during embryogenesis. An example is our recent study on Lhx8 function in the context of forebrain development. Studies on the mechanism of Lhx gene function led to our discovery of two obligatory co-factors that mediate the action of the LIM-homeodomain proteins. One of these, Ldb1, interacts physically and genetically with Lhx gene products. Each Lhx gene family examined so far is dependent on Ldb1 co-factor activity. We found that embryos carrying Ldb1 null mutations exhibit severe early developmental defects. Even the in vitro differentiation of embryonic stem cells is severely affected by the lack of Ldb1 activity. Accordingly, we asked whether, during mammalian development, Ldb1 is an obligatory interaction partner of each of the diverse arrays of Lhx gene products. Our current efforts are thus directed toward examining Ldb1 action in specific regions of the developing embryo. If Ldb1 is indeed required to render each of the individual Lhx gene products active, selective Ldb1 gene inactivation should allow us to investigate the concerted activity of Lhx gene products in targeted areas of the developing embryo that lack Ldb1 activity.
The main tool of our current protocol is a mouse mutant in which we inserted a floxed Ldb1 gene that can be inactivated in cells targeted by the Cre recombinase. If, as postulated, Ldb1 is obligatory for all LIM homeodomain factor activity, we would expect the resulting phenotypes to provide much needed information on the combined action of individual Lhx genes in specific target fields of the developing embryo. We plan to target an array of organotypic regions, as we did in the first experiment of this type, in which we used a transgenic line that expresses Cre under the control of an enhancer element of the Nestin gene to delete the Ldb1 gene in the developing central nervous system. Our analysis of the resulting mutant revealed a defect in the specification of Purkinje cells in the developing cerebellum. Two Lhx genes, Lhx1 and Lhx5, are prominently expressed in the cerebellum of wild-type embryos. It thus seemed likely that the products of the two genes were inactive in cells lacking Ldb1 activity. Indeed, when we generated mouse mutants with null deletions of both Lhx1 and Lhx5, we were able to copy the Purkinje cell phenotype, indicating that the interaction of Ldb1 with these two LIM-homeodomain transcription factors controls specific aspects of cerebellar development. To define more fully the role of Lhx1 and Lhx5 in development of the Purkinje cells, we analyzed the mutant phenotype at earlier development stages and found that the progenitors of the Purkinje cells are formed and proliferate normally in the ventricular zone in the developing cerebellum. However, after the cells leave the cell cycle and migrate out of the ventricular zone, they fail to differentiate and are gradually eliminated, probably through cell apoptosis. The results suggest that Lhx1 and Lhx5 are required for proper differentiation of the Purkinje cells in the development of the cerebellum.
Originally detected in our laboratory, Ssdp1 is a second co-factor of Lhx transcriptional activity. Like Ldb1, it has been physically and genetically linked to Lhx gene action. In a recent collaboration with the laboratory of Hiroshi Sasaki, we showed that Ssdp1 is an essential component of Ldb1-Lhx1 transcriptional activity during head formation in the mouse embryo. We are currently examining the spectrum of Ssdp target genes in an effort to describe the extent of developmental controls exerted by this co-factor.
Mukhopadhyay M, Teufel A, Yamashita T, Agulnick AD, Chen L, Downs KM, Schindler A, Grinberg A, Huang SP, Dorward D, Westphal H. Functional ablation of the mouse Ldb1 gene results in severe patterning defects during gastrulation. Development 2003;130:495-505.
Nishioka N, Nagano S, Nakayama R, Kiyonari H, Ijiri T, Taniguchi K, Shawlot W, Hayashizaki Y, Westphal H, Behringer RR, Matsuda Y, Sakoda S, Kondoh H, Sasaki H. Ssdp1 regulates head morphogenesis of mouse embryos by activating the Lim1-Ldb1 complex. Development 2005;132:2535-2546.
Zhao Y, Marin O, Hermesz E, Powell A, Flames N, Palkovits M, Rubenstein JL, Westphal H. The LIM-homeobox gene Lhx8 is required for the development of many cholinergic neurons in the mouse forebrain. Proc Natl Acad Sci USA 2003;100:9005-9010.
Dkk-mediated regulation of the canonical Wnt pathway in the mouse embryo
Grinberg, Gorivodsky, Mukhopadhyay, Poscablo, Westphal; in collaboration with the laboratories of Morasso and Niehrs
During embryogenesis, members of the Dkk family act as inhibitors of the canonical Wnt pathway. In our previous experiments, we demonstrated the importance of Dkk1 in mediating transcriptional activity of LIM-homeodomain proteins and other transcription factors during head induction. Over the past year, our studies have uncovered an exciting new aspect of Dkk action. We studied mutant mice that lack the function of Dkk2 and noted that the gene controls the integrity of the ocular surface epithelium by regulating the canonical Wnt pathway. In the absence of Dkk2 function, the epithelium of the cornea that covers the ocular surface is completely transformed into a stratified epithelium containing hair follicles and sebaceous glands. Stem cells that maintain corneal integrity throughout life reside in the limbus of the eye. It is here that Dkk2 is prominently expressed, suggesting that the selection and fate of these stem cells is tightly controlled by Wnt ligands and their regulators.
del Barco Barrantes I, Davidson G, Grone HJ, Westphal H, Niehrs C. Dkk1 and noggin cooperate in mammalian head induction. Genes Dev 2003;17:2239-2244.
Restoring “stemness”
Huang, Tzchori, Westphal
We are directing novel efforts toward reprogramming somatic cells to an embryonic state of unlimited growth and multifaceted differentiation potential. In a direct search for a suitable source of reprogramming activity, several laboratories, including our own, fused mouse embryonic stem cells with somatic cells and were able to demonstrate that markers of pluripotency, notably the transcription factor Oct4, were reactivated in the somatic nucleus of the resulting hybrid cells. The task ahead is to create conditions whereby the ES cell nucleus changes the epigenetic state of the hybrid cell but is prevented from participating in the formation of daughter cell nuclei. Pretreatment of the ES cell with inhibitors of DNA synthesis before fusion or mechanical removal of the ES cell nucleus from the hybrid cell are among the most promising of a range of attempts to generate a cell hybrid that will selectively replicate the somatic cell genome in a reprogrammed epigenetic state to enable propagation of multipotent progeny cells.
The potential benefits of this approach for future avenues of stem cell therapy are several. If a patient’s somatic cell is exposed to factors that bring it back to a pluripotent stage comparable to that of an embryonic stem cell, such a cell would, unlike most cells in the body, replicate quickly and could be propagated under suitable in vitro culture conditions to generate a large number of progeny cells, all retaining pluripotency. When subjected to suitable differentiation protocols, the cells would give rise to a wide variety of differentiated cells available for use in tissue replacement protocols. Most important, the reprogrammed cells would carry the patient’s own set of histocompatibility antigens on their surface, thus drastically reducing the risk of graft rejection. Furthermore, the fact that the cells would not be derived from an embryo would eliminate the ethical concerns that restrict present human embryonic cell research. Clearly, the importance of experiments aimed at restoring “stemness” to somatic cells cannot be overstated.
Westphal H. Restoring stemness. Differentiation 2005;73:1-5.
Additional and joint projects
Grinberg, Huang, Hasuike,1 Lee, Malik, Miyamoto,2 Teufel,3 Westphal, Wong4; in collaboration with the laboratories of Borrelli, Chou, Enikolopov, Germino, Gold, Kruh, Lamb, Stratakis, and Young
Our laboratory was the first on the NIH main campus to generate gene-altered (transgenic and knockout) mice. We were also the first to introduce CRE-mediated alterations of floxed genes in the mouse, now the worldwide standard as the most popular way to generate inducible mutations. During the past two decades, many colleagues have requested our involvement in various aspects of experiments that rely on gain- or loss-of-function approaches to address the role of specific genes in mouse development. The papers listed below describe some of the results of collaborations of the last two years. In most of the studies, our participation was limited to the design of transgenes and targeting constructs and the generation of mouse strains containing the corresponding gene alterations. However, reports by Teufel et al. (Biochim Biophys Acta 2003;1577:109) and Miyamoto et al. (Lancet 2003;362:1714) reflect our own research efforts, with the reported experiments carried out entirely in our laboratory. In the first of the two research efforts, we characterized a new member of the FoxP family of transcription factors. The second effort extended earlier observations on mouse mutants and described a mutation of the testis-specific SYCP3 in patients with azoospermia. Our findings suggest that SYCP3 has an essential meiotic function in human spermatogenesis that is compromised by the mutant protein via dominant negative interference.
Belinsky MG, Dawson PA, Shchaveleva I, Bain LJ, Wang R, Ling V, Chen ZS, Grinberg A, Westphal H, Klein-Szanto A, Lerro A, Kruh GD. Analysis of the in vivo functions of mrp3. Mol Pharmacol 2005;68:160-168.
Bielsky IF, Hu SB, Szegda KL, Westphal H, Young LJ. Profound impairment in social recognition and reduction in anxiety-like behavior in vasopressin V1a receptor knockout mice. Neuropsychopharmacology 2004;29:483-493.
Kirschner LS, Kusewitt DF, Matyakhina L, Towns WH, Carney JA, Westphal H, Stratakis CA. A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissues. Cancer Res 2005;65:4506-4514.
Kobayashi M, Iaccarino C, Saiardi A, Heidt V, Bozzi Y, Picetti R, Vitale C, Westphal H, Drago J, Borrelli E. Simultaneous absence of dopamine D1 and D2 receptor-mediated signaling is lethal in mice. Proc Natl Acad Sci USA 2004;101:1465-1470.
Piontek KB, Huso DL, Grinberg A, Liu L, Bedja D, Zhao H, Gabrielson K, Qian F, Mei C, Westphal H, Germino GG. A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo. J Am Soc Nephrol 2004;15:3035-3043.
1Shiga Hasuike, MD, PhD, former Postdoctoral Fellow
2Toshinobu Miyamoto, MD, PhD, former Visiting Fellow
3Andreas Teufel, MD, former Postdoctoral Fellow
4Eric Wong, PhD, former Special Volunteer
COLLABORATORS
Emiliana Borrelli, PhD, Institut de Génétique et Biologie Moléculaire et Cellulaire, Illkirch, France
Janice Chou, PhD, Heritable Disorders Branch, NICHD, Bethesda, MD
Grigori Enikolopov, PhD, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Gregory G. Germino, MD, The Johns Hopkins University School of Medicine, Baltimore, MD
Philip Gold, MD, Clinical Neuroendocrinology Branch, NIMH, Bethesda, MD
Gary D. Kruh, MD, PhD, Fox Chase Cancer Center, Philadelphia, PA
Dolores J. Lamb, PhD, Baylor College of Medicine, Houston, TX
Maria I. Morasso, PhD, Laboratory of Skin Biology, NIAMS, Bethesda, MD
Niehrs, PhD, Deutsches Krebsforschungszentrum, Heidelberg, Germany
Miklos Palkovits, MD, Semmelweis University, Budapest, Hungary
John L. Rubenstein, MD, PhD, University of California San Francisco, San Francisco, CA
Hiroshi Sasaki, PhD, RIKEN Center for Developmental Biology, Kobe, Japan
Constantine A. Stratakis, MD, DSc, Developmental Endocrinology Branch, NICHD, Bethesda, MD
Larry J. Young, PhD, Emory University, Atlanta, GA
For further information, contact hw@mail.nih.gov.