MAMMALIAN DEVELOPMENTAL GENETICS
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Heiner Westphal, MD, Head, Section on Mammalian Molecular Genetics Yangu Zhao, PhD, Staff Scientist |
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| We carry out a functional analysis of genes that control critical steps of postimplantation development in the mammalian embryo. Transcription factors are major players in the cascade of molecular events that implement the body plan. We use a loss-of-function approach to study in detail the function of the LIM/homeodomain (Lhx) class of transcription factors and of factors that mediate their action. In addition, the group identifies novel genes involved in specific aspects of embryonic development and collaborates with other research teams on projects focusing on gene-altered mice. Transcription factors that regulate mammalian development and factors that mediate their action Bronstein, Chen, Crumity, Geum, Gorivodsky, Grinberg, Huang, E. Lee, W.K. Lee, Ma, Mailloux, Malik, Morales, Mukhopadhyay, Schindler, Teufel, Wang, Westphal, Williams-Simons, Wu, Zhao; in collaboration with members of the Agulnick, Bayarsaihan, Dawid, Downs, Hukriede, Kennison, Niehrs, Ogryzko, Oliver, Podtelejnikov, Rubenstein, Segal, and Yamashita laboratories Over the years, our studies have shown that members of the Lhx gene family, encoding LIM-homeodomain-containing transcription factors, are involved in early patterning events in the development of the nervous system and in orchestrating organ assembly. More recently, we identified two gene families, termed Ldb and Ssdp, that encode obligatory cofactors of Lhx gene action. Our current work focuses on functions of the LIM-homeodomain proteins Lhx8 and Lhx2 and the LIM-domain-binding protein Ldb1 in embryonic development. Previously, we generated a strain of knock-out mice with a null-deletion of the LIM-homeobox gene Lhx8. Our initial analysis of the mutant mice revealed an important role for Lhx8 in development of the palate. Given that the same gene is also abundantly expressed in the developing ventral forebrain, we recently examined the brain of our Lhx8 null mice. The expertise of the Rubenstein laboratory greatly aided our work. Development of cholinergic neurons in the ventral telencephalon is severely impaired in the knock-out mutants. As a result, the mice lack the nucleus basalis, a major source of cholinergic neuron input to the cerebral cortex. In addition, the number of cholinergic neurons in several other regions of the subcortical forebrain, including the caudate putamen, septum, and magnocellular preoptic nucleus, is reduced. Our marker analysis revealed that progenitor cells form in the absence of Lhx8 gene function. However, the gene is essential for terminal differentiation of the majority of cholinergic neurons in the ventral telencephalon. The loss of telencephalic cholinergic projection neurons is a hallmark of neurodegenerative disorders such as Alzheimer's disease and is likely to play a role in the cognitive impairments of these patients. For this reason, the Lhx8 null mutant mouse constitutes a valuable model for functional studies of cholinergic projection neurons in the context of memory and cognition. Another ongoing study concerns the function of the LIM-homeobox gene Lhx2 in brain development. Our initial Lhx2 knock-out study had shown that the gene is essential for eye development and the formation of the telencephalon. More recently, we observed that development of the ventral diencephalon and pituitary is also impaired in the null mutant embryo, indicating an important additional role of Lhx2 in forebrain development. We have also continued our functional evaluation of protein complexes formed by LIM-homeodomain proteins and their associated cofactors. The Ssdp cofactors that we discovered are thought to form complexes with many transcription factors that are active during early stages of embryonic development. Whereas the vertebrate Ssdp gene family has several closely related members, the Drosophila ssdp gene is unique. Moreover, the existence of several mutant alleles of the gene makes the Drosophila system attractive for a genome-wide search for downstream genes whose transcription is regulated by Ssdp and associated proteins. Given that loss-of-function mutations in the Drosophila ssdp gene cause lethality at early pupal stages, we chose to focus our search on the late third instar larval stage, which precedes the pupal stage. We carried out the analysis in collaboration with the Oliver laboratory by using Drosophila genomic amplicon microarrays; we are currently evaluating the resulting wealth of data in an effort to identify direct targets of ssdp-mediated transcriptional regulation. Earlier work, carried out in collaboration with the Niehrs laboratory, had shown that Dkk1, an inhibitor of canonical Wnt signaling, is an important mediator of transcriptional activity during vertebrate head induction. We have extended these observations by showing that head induction is based on a functional cooperation between Dkk1 and the BMP antagonist noggin. In an unrelated project, we have identified FoxP4, a forkhead transcription factor involved in embryonic development, cell cycle regulation, and oncogenesis. Chen L, Segal D, Hukriede N, Podtelejnikov A, Bayarsaihan D, Kennison JA, Ogryzko V, Dawid IB, Westphal H. Ssdp proteins interact with the LIM-domain binding protein Ldb1 to regulate development. Proc Natl Acad Sci USA 2002;99:14320-14325. 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. 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. Teufel A, Wong EA, Mukhopadhyay M, Malik N, Westphal H. FoxP4, a novel forkhead transcription factor. Biochim Biophys Acta 2003;1627:147-152. 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. Collaborative projects Grinberg, Hasuike, Huang, E.J. Lee, Miyamoto,* Westphal; in collaboration with members of the Chou, Enikolopov, Gold, Lamb, and Sibley laboratories In collaboration with the Lamb laboratory, we have identified in azoospermic patients a single nucleotide deletion in the SYCP3 gene that causes a truncation of a protein required for chromosomal pairing during meiosis. The study represents an important advance in understanding the molecular basis of early meiotic arrest as a cause of nonobstructive azoospermia. Our experiments suggest that the truncated protein dominantly interferes with the function of the wild-type allele and thus causes male infertility. Our group has also been involved in a diverse array of other collaborative projects aimed at revealing gene functions via knockout. The studies have shown that (1) the D5 dopamine receptor is involved in blood pressure regulation; (2) the vasopressin receptor 1a is a negative regulator of B cell receptor signaling; (3) nitric oxide acts as a negative regulator of cell proliferation in the adult mammalian brain; and (4) the glycose-6-phosphate transporter, deficient in glycogen storage disease type Ib, is an important immuno-modulatory protein. Chen LY, Shieh JJ, Lin B, Pan CJ, Gao JL, Murphy PM, Roe TF, Moses S, Ward JM, Lee EJ, Westphal H, Manseld BC, Chou JY. Impaired glucose homeostasis, neutrophil trafficking and function in mice lacking the glucose-6-phosphate transporter. Hum Mol Genet 2003;12:2547-2558. Hollon TR, Bek MJ, Lachowicz JE, Ariano MA, Mezey E, Ramachandran R, Wersinger SR, Soares-da-Silva P, Liu ZF, Grinberg A, Drago J, Young WS III, Westphal H, Jose PA, Sibley DR. Mice lacking D5 dopamine receptors have increased sympathetic tone and are hypertensive. J Neu-rosci 2002;22:10801-10810. Hu SB, Zhao ZS, Yhap C, Grinberg A, Huang SP, Westphal H, Gold P. Vasopressin receptor 1a-mediated negative regulation of B cell receptor signaling. J Neuroimmunol 2003;135:72-81. Miyamoto MT, Hasuike S, Yogev L, Maduro MR, Ishikawa M, Westphal H, Lamb LJ. Azoospermia in patients heterozygous for a mutation in SYCP3. Lancet 2003; in press. Packer MA, Stasiv Y, Benraiss A, Chmielnicki E, Grinberg A, Westphal H, Goldman SA, Enikolopov G. Nitric oxide negatively regulates mammalian adult neurogenesis. Proc Natl Acad Sci USA 2003;100:9566-9571. COLLABORATING LABORATORIES Alan Agulnick, PhD, CyThera, Inc., San Diego CA Dashzeveg Bayarsaihan, PhD, Yale University, New Haven CT John L. Rubenstein, MD, PhD, University of California, San Francisco CA Daniel Segal, PhD, Tel Aviv University, Israel David R. Sibley, PhD, Molecular Neuropharmacology Section, NINDS, Bethesda MD *Toshinobu Miyamoto, PhD, former Postdoctoral Fellow For further information, contact hw@helix.nih.gov |
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