GENES THAT CONTROL MOUSE DEVELOPMENT
, Head, Section on Mammalian Molecular Genetics
Yangu Zhao, PhD, Staff Scientist
Marat Gorivodsky, PhD, Research Fellow
Mahua Mukhopadhyay, PhD, Research Fellow
Matthew David Phillips, PhD, Postdoctoral Fellow
Tsadok Cohen, PhD, Visiting Fellow
Ipsita Dey-Guha, PhD, Visiting Fellow
Evgeny Makarev, PhD, Visiting Fellow
Ginat Narkis, PhD, Visiting Fellow
Itai Tzchori, PhD, Visiting Fellow
Alexander Grinberg, DVM, Senior Research Assistant
Eric J.M. Lee, DVM, Senior Research Assistant
Lisa Williams-Simons, BS, AAS, Senior Research Assistant
Anna Davidhi, BS, Postbaccalaureate Fellow
Melissa de la Cuesta, BS, Postbaccalaureate Fellow
Barbara Do, BS, Postbaccalaureate Fellow
Rui Lin, BA, Technical Training Fellow
We focus on key developmental controls exerted by LIM-homeodomain (LIM-HD) transcription factors as the mouse embryo enters the phase of postgastrulation assembly of organ systems. Mounting evidence suggests that the LIM-HD proteins play a key role in lineage specification by activating downstream genes that define the ground state of organ identity. The assembly of newly formed cell lineages into complex tissue structures depends on precise patterns of cell migration and cell-cell communication that involve all major signaling pathways. The transcriptional activity of LIM-HD proteins is mediated by two families of obligatory cofactors, termed Ldb and Ssdp, that become part of the LIM-HD complex controlling target gene transcription. We have analyzed various scenarios of tissue assembly in the developing mouse embryo and discovered an intricate interplay between transcriptional regulation and secreted signal exchanges that accompanies organogenesis.
Role of Lhx genes in neuronal differentiation
Purkinje cells are major components of the neuronal network of the cerebellum and are essential for fine control of movement and posture. During embryonic development, these cells play an important role in controlling the proliferation of progenitors of granule cells, the other major type of neuron in the cerebellum. In this study, we describe the process that controls differentiation of Purkinje cells from earlier precursors. We noted that two closely related LIM-homeobox genes, Lhx1 and Lhx5, are expressed in developing Purkinje cells soon after they exit the cell cycle and migrate out of the cerebellar ventricular zone. Double mutant mice lacking function of both Lhx1 and Lhx5 show a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes an obligatory cofactor for all LIM-homeodomain proteins, results in a similar phenotype. Our experiment provides conclusive evidence that the transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum.
In a separate collaborative project, we investigated the role of the Lhx1/5 transcriptional apparatus in generating and maintaining the functional identity of a group of interneurons in the developing spinal cord. We found that the establishment of a dorsal inhibitory neurotransmitter program in these interneurons was critically dependent on an interaction of Lhx1/5 with members of the Pax family of homeobox genes.
Pillai A, Mansouri A, Behringer R, Westphal H, Goulding M. Lhx1 and Lhx5 maintain the inhibitory neurotransmitter status of interneurons in the dorsal spinal cord. Development 2007;134:357-66.
Zhao Y, Kwan KM, Mailloux C, Lee WK, Grinberg A, Wurst W, Behringer RR, Westphal H. LIM-homeodomain proteins Lhx1, Lhx5, and their cofactor Ldb1 control Purkinje cell differentiation in the developing cerebellum. Proc Natl Acad Sci USA 2007;104:13182-6.
Role of Lhx genes in limb development
To study the role of Lhx genes in early limb development, we targeted the genes’ obligatory cofactor Ldb1. We generated a conditional knockout allele by inserting two LoxP fragments into the Ldb1 locus–flanking coding exons. We used transgenes that target the Cre enzyme to the developing limb bud to delete Ldb1 function at the earliest possible stage of limb development. Three signaling centers, which respectively regulate limb patterning and growth along the proximo-distal, antero-posterior, and dorso-ventral limb axes, control patterning and growth during vertebrate limb development. Interactions among the signaling centers coordinate limb development. We found that Ldb1 plays a central role in the mechanism that coordinates these interactions via its interaction with distinct LIM homeodomain transcription factors encoded by the Lhx genes Lhx2, Lhx9, and Lmx1b. The resulting transcription complexes integrate the signaling events that link limb patterning and outgrowth along all three axes. Simultaneous loss of Lhx2 and Lhx9 function resulted in patterning and growth defects along the anterior-posterior and proximal-distal limb axes. We observed similar but more severe phenotypes when removal of required cofactor Ldb1 significantly reduced the activities of all three factors, Lmx1b, Lhx2, and Lhx9. We have thus uncovered a previously unappreciated function of the dorsal limb–specific Lmx1b gene in regulating anterior-posterior and proximal-distal limb patterning and outgrowth. A detailed analysis of signaling events during early limb development revealed that Lhx2, Lhx9, and Lmx1b play redundant roles in regulating the responsiveness of mesenchymal cells to Fgf8 and Shh ligands by maintaining their progenitor cell states.
Role of Lhx genes and their cofactors in development of the telencephalon
Lhx6 and Lhx8 are two closely related LIM-homeodomain genes whose patterns of expression often overlap in the developing ventral telencephalon. We previously generated an Lhx8 null allele. The phenotype of mutant embryos that lack Lhx8 function revealed an important role for this gene in the development of cholinergic neurons in the telencephalon. More recently, we studied embryos that lack Lhx6 function. Their phenotype allowed us to conclude that Lhx6 is essential for the generation and positioning of major subtypes of GABAergic neurons present in the neocortex, hippocampus, and several subcortical areas of the telencephalon. The Lhx6 loss-of-function allele expresses placental alkaline phosphatase (PLAP), permitting us to follow the development and fate of Lhx6-competent interneurons in Lhx6 null embryos. To analyze whether Lhx6 and Lhx8 cooperate during development of the telencephalon, we generated mutants lacking the function of both genes. Our analysis of the double mutants revealed a specific defect in the formation of the globus pallidus, indicating that Lhx6 and Lhx8 indeed play additional and redundant roles in the assembly of the ventral telencephalon. In addition to the analysis of Lhx6/Lhx8 double mutants, we generated an Ldb1/Nkx2.1-Cre conditional mutant. In this mutant, Ldb1—and thus the function of all Lhx genes that depend on this cofactor—is inactivated in cells that normally express Lhx6 and Lhx8. The phenotype of this conditional mutant resembles that of the Lhx6/Lhx8 double knockout mutant. We concluded that Lhx6 and Lhx8, in conjunction with Ldb1, are essential regulators of forebrain development.
Role of Lhx genes and their cofactors in kidney development
Mutations in the Lhx gene Lmx1B have been detected in patients with a genetic disorder called nail-patella syndrome. The patients suffer from renal failure associated with podocytes, which are essential components of the glomerular filtration apparatus. In a collaborative study, we generated a mouse model via a podocyte-specific knockdown of Ldb1, the above-mentioned obligatory cofactor for all LIM-homeodomain proteins. The renal phenotype of these mutant mice identifies Ldb1 is a candidate modifier gene of the human disorder.
Suleiman H, Heudobler D, Raschta AS, Zhao Y, Zhao Q, Hertting I, Vitzhum H, Moeller MJ, Holzman LB, Rachel R, Johnson R, Westphal H, Rascle A, Witzgall R. The podocyte-specific inactivation of Lmx1b, Ldb1 and E2a yields new insight into a transcriptional network in podocytes. Dev Biol 2007;304:701-12.
Intracellular trafficking of the Ssdp1 cofactor
Ssdp1 is another essential cofactor that mediates the action of LIM-HD proteins in mouse development. Its ablation causes severe developmental defects in the embryo. However, Ssdp1 is predominantly found in the cytoplasm of cells, and the molecule does not contain a nuclear localization domain. The question thus arises as to how Ssdp1 regulates transcriptional events in the nucleus. We therefore analyzed the intracellular transport of Ssdp1 in established cell lines grown in vitro. We observed that Ssdp1 localizes predominantly to the cytoplasm of 293T cells but is translocated to the nucleus when co-transfected with Lck, a member of the Src family of non-receptor tyrosine kinases. The Src tyrosine kinase inhibitor PP2 blocks the nuclear translocation of Ssdp1. Western blot analysis showed that co-expression of Ssdp1 and Lck in 293T cells induces Ssdp1 phosphorylation. Mutation of the N-terminal tyrosine residues 23 and 25 of Ssdp1 markedly reduces both phosphorylation and the nuclear localization of Ssdp1. Lck enhances the transcriptional activity of Ssdp1 in the context of known components of a LIM-HD/cofactor complex. We propose that phosphorylation involving N-terminal tyrosine residues of Ssdp1 is a fast and efficient means of regulating the transcriptional activity of LIM-HD complexes.
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 Cellular Biochem 2007;103 [E-pub ahead of print].
Dkk and the control of the canonical Wnt pathway in mouse development
In recent years, our laboratory has become interested in the action of canonical Wnt signals in embryo patterning and lineage stem cell determination. We studied mutant mice lacking the function of one or the other member of the Dkk gene family that encodes negative regulators of the canonical Wnt pathway. In two earlier publications, we reported on severe defects in early mouse development, notably in head induction, caused by ablation of Dkk1 function. Given that Dkk1 is co-expressed with the transcription factor goosecoid (Gsc) in the prechordal mesoderm, we studied mice that lacked Gsc as well as Dkk1 function and observed severe head truncation that is not apparent if only one or the other of the two genes is ablated. Like Dkk1, Gsc appears to act as a negative regulator of Wnt activity in this context. No such genetic interaction exists between Dkk1 and Sfrp5, a gene encoding an extracellular factor that can modulate Wnt signaling and that, like Dkk1, is expressed in the anterior visceral endoderm of the early mouse embryo. Our laboratory is currently investigating the role of Dkk family members during mouse embryo organogenesis. To this end, we have generated Dkk1 and Dkk2 double null-mutant mice in order to circumvent possible functional redundancy of the two genes. Phenotypic characterization of several interesting developmental defects in these mutants is under way.
Leaf I, Tennessen J, Mukhopadhyay M, Westphal H, Shawlot W. Sfrp5 is not essential for axis formation in the mouse. Genesis 2006;44:573-8.
Lewis SL, Khoo PL, De Young A, Bildsoe H, Wakamiya M, Behringer RR, Mukhopadhyay M, Westphal H, Tam PPL. Genetic interaction of Gsc and Dkk1 in head morphogenesis of the mouse. Mech Dev 2007;124:157-65.
Outside projects performed with mouse mutants generated by our laboratory
Collaborating laboratories continue to rely on our expertise in generating targeted mutations in specific genes of interest. The publications cited below report on the phenotypic analysis of two such mutants, one lacking the function of the multidrug resistance protein 4 and the other that of the enzyme glucose-6-phosphatase-beta.
Belinsky MA, Guo P, Lee K, Zhou F, Kotova E, Grinberg A, Westphal H, Shchaveleva I, Klein-Szanto A, Gallo JM, Kruh GD. Multidrug resistance protein 4 (Mrp4) protects bone marrow, thymus, spleen and intestine from nucleotide analog-induced damage. Cancer Res 2007;67:262-8.
Cheung YY, Kim SY, Yiu WH, Pan CJ, Jun HS, Ruef RA, Lee EJ, Westphal H, Mansfield BC, Chou JY. Impaired neutrophil activity and increased susceptibility to bacterial infection in mice lacking glucose-6-phosphatase-beta. J Clin Invest 2007;117:784-93.
1 Woon-Kyu Lee, PhD, former Postdoctoral Fellow
2 Christina Mailloux, BS, former Postbaccalaureate Fellow
3 Qi Zhao, PhD, former Postdoctoral Fellow
COLLABORATORS
Richard R. Behringer, PhD, Baylor College of Medicine, Houston, TX
Janice Chou, PhD, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Martyn D. Goulding, PhD, Salk Institute for Biological Studies, San Diego, CA
Gary D. Kruh, MD, PhD, Fox Chase Cancer Center, Philadelphia, PA
Mark Lewandoski, PhD, Cancer and Developmental Biology Laboratory, NCI, Frederick, MD
Paul Love, MD, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Nasir Malik, PhD, Cellular Neurophysiology Research Branch, NIDA, Baltimore, MD
Forbes D. Porter, MD, PhD, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
John L.R. Rubenstein, MD, PhD, University of California San Francisco, San Francisco, CA
William Shawlot, PhD, University of Minnesota, Minneapolis, MN
Patrick P.L. Tam, PhD, Children’s Medical Research Institute, Sydney, Australia
Ralph Witzgall, MD, Universität Regensburg, Regensburg, Germany
Yingzi Yang, PhD, Genetic Disease Research Branch, NHGRI, Bethesda, MD
For further information, contact westphah@mail.nih.gov.
