Research interests

Genetic Analysis of Mouse Development and Disease

Our laboratory studies genes important for embryonic development in mice, and the connections between mutations in these genes and congenital human disease syndromes. Our analyses focus on a developmental signaling pathway termed the Notch pathway, and on genes of the Snail family, which encode zinc finger transcriptional repressors.

Analysis of the Notch Signaling Pathway

The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism. Genes of the Notch family encode large transmembrane receptors. Notch receptors interact with membrane-bound ligands that are encoded by the Jagged (Jag1 and Jag2) and Delta-like (Dll1, Dll3, and Dll4) gene families. The signal induced by ligand binding is transmitted intracellularly by a process involving proteolytic cleavage of the receptor and nuclear translocation of the intracellular domain of the Notch protein. Notch pathway genes are essential for normal embryonic development, and mutations in genes encoding components of the Notch signaling pathway are found in several types of cancer and in three inherited disease syndromes.

We have been conducting an extensive genetic analysis of the requirements for components of the Notch signaling pathway during embryogenesis in mice. For example, we have shown recently that haploinsufficiency for the Dll4 gene leads to embryonic lethality due to defects in vascular development, and have documented vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels, and develop arteriovenous malformations. Taken together, these results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage, and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. In collaboration with Dr. Anne Joutel at the H?pital Lariboisi?re in Paris, we also have shown that the Notch3 gene is required postnatally to generate functional arteries in mice. The Notch3 gene regulates arterial differentiation and maturation of vascular smooth muscle cells, and is required for arterial specification of vascular smooth muscle cells but not of endothelial cells.

We have been studying the role of Notch signaling in regulating the differentiation of sensory hair cells in the inner ear. Our previous work had shown that mice mutants for the Notch ligand encoded by the Jag2 gene differentiate supernumerary hair cells in the cochlea of the inner ear. We have now shown that another Notch ligand, encoded by the Dll1 gene, functions synergistically with the Jag2 gene in regulating hair cell differentiation. Supernumerary hair cells in Dll1/Jag2 double mutants arise primarily through a switch in cell fate rather than through excess proliferation. While these results demonstrate an important role for Notch-mediated lateral inhibition during cochlear hair cell patterning, we also detected abnormally prolonged cellular proliferation that preferentially affected supporting cells in the cochlea. Our results demonstrate that the Notch pathway plays a dual role in regulating cellular differentiation and patterning in the cochlea, acting both through lateral inhibition and the control of cellular proliferation.

Analysis of Snail Family Genes

We are studying the roles during mouse development of genes of the Snail family. These genes are homologs of the Drosophila gene Snail, which is required for mesoderm formation during Drosophila embryogenesis. Snail family genes encode DNA-binding zinc finger proteins that act as transcriptional repressors. We have made and analyzed targeted null mutations of the Snail family genes Snail (Snai1) and Slug (Snai2). Embryos homozygous for a null mutation of the Snai1 gene die during gastrulation, while Snai2 mutant homozygotes survive until birth. Both Snai2-/- embryos and Snai1+/- Snai2-/- double mutant embryos exhibit cleft palate, one of the most common human birth defects. Approximately 50% of Snai2-/- embryos exhibit cleft palate, while in Snai1+/- Snai2-/- double mutants the incidence of cleft palate increases to 100%. We have set up palate organ cultures to assess whether the Snai2-/- and Snai1+/- Snai2-/- palates are unable to fuse, even when cultured in contact with one another. These experiments indicate that the palatal shelves from Snai1+/- Snai2-/- mutant embryos are indeed unable to undergo palate fusion.

Mouse Models of Craniosynostosis

Congenital malformations affecting the craniofacial region are among the most common inherited disease syndromes in humans. Craniosynostosis is the premature fusion of the bones at the top of the skull, which leads to an abnormal head shape. Craniosynostosis is a frequent developmental anomaly, and can occur either as an isolated abnormality or as a syndrome in association with other congenital defects. The abnormal head shape associated with craniosynostosis may result in a variety of serious problems, and generally must be corrected surgically.

Saethre-Chotzen syndrome is one of the most common disorders of craniosynostosis in humans, and is caused by haploinsufficiency for the TWIST1 gene, which encodes a basic helix-loop-helix transcription factor. Mice heterozygous for a null mutation of the Twist1 gene exhibit partially penetrant skeletal defects that replicate certain features of Saethre-Chotzen syndrome, including fusions of the coronal suture and other cranial suture abnormalities. The mouse and human TWIST1 genes are mammalian homologs of the Twist gene of Drosophila. During Drosophila embryogenesis, mutations in the Twist gene interact with mutations in the Snail gene. To assess whether genetic interactions between Twist and Snail family genes are evolutionarily conserved, we crossed Twist1 heterozygous mice to mice heterozygous for mutations of the Snai1 and Snai2 genes. Our work shows that the mouse Twist1 mutation interacts with null mutations of both genes. Double heterozygous mice exhibit an enhanced incidence and severity of craniosynostosis. These results demonstrate that mutations of Snail family genes act as dominant enhancers of Twist1 haploinsufficiency, and show that genetic interactions between genes of the Twist and Snail families have been conserved during evolution.

Publications

• Brinkmeier ML, Potok MA, Cha KB, Gridley T, Stifani S, Meeldijk J, Clevers H, Camper SA. 2003. TCF and groucho-related genes influence pituitary growth and development. Mol Endocrinol 17:2152-2161.
• Gridley T. 2003. Notch signaling and inherited disease syndromes. Hum Mol Genet 12 Spec No 1:R9-13.
• Krebs LT, Iwai N, Nonaka S, Welsh IC, Lan Y, Jiang R, Saijoh Y, O'Brien TP, Hamada H, Gridley T. 2003. Notch signaling regulates left-right asymmetry determination by inducing Nodal expression. Genes Dev 17:1207-1212.
• Krebs LT, Xue Y, Norton CR, Sundberg JP, Beatus P, Lendahl U, Joutel A, Gridley T. 2003. Characterization of Notch3-deficient mice: Normal embryonic development and absence of genetic interactions with a Notch1 mutation. Genesis 37:139-143.
• Oram KF, Carver EA, Gridley T. 2003. Slug expression during organogenesis in mice. Anat Rec A Discov Mol Cell Evol Biol 271:189-191.
• Domenga V, Fardoux P, Lacombe P, Monet M, Maciazek J, Krebs LT, Klonjkowski B, Berrou E, Mericskay M, Li Z, Tournier-Lasserve E, Gridley T, Joutel A. 2004. Notch3 is required for arterial identity and maturation of vascular smooth muscle cells. Genes Dev 18:2730-2735.
• Gridley T. 2004. Kick it up a Notch: Notch1 activation in T-ALL. Cancer Cell 6:431-432.
• Hadland BK, Huppert SS, Kanungo J, Xue Y, Jiang R, Gridley T, Conlon RA, Cheng AM, Kopan R, Longmore GD. 2004. A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development. Blood 104:3097-3105.
• Krebs LT, Shutter JR, Tanigaki K, Honjo T, Stark KL, Gridley T. 2004. Haploinsufficient lethality and formation of arteriovenous malformations in Notch pathway mutants. Genes Dev 18:2469-2473.
• Pan Y, Lin MH, Tian X, Cheng HT, Gridley T, Shen J, Kopan R. 2004. Gamma-secretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis. Dev Cell 7:731-743.
• Parent AE, Choi C, Caudy K, Gridley T, Kusewitt DF. 2004. The developmental transcription factor Slug is widely expressed in tissues of adult mice. J Histochem Cytochem 52:959-965.
• Takahashi E, Funato N, Higashihori N, Hata Y, Gridley T, Nakamura M. 2004. Snail regulates p21WAF/ClP1 expression in cooperation with E2A and Twist. Biochem Biophys Res Commun 325:1136-1144.
• Anthony TE, Mason HA, Gridley T, Fishell G, Heintz N. 2005. Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells. Genes Dev 19:1028-1033.
• Kiernan AE, Cordes R, Kopan R, Gossler A, Gridley T. 2005. The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear. Development 132:4353-4362.
• Mason HA, Rakowiecki SM, Raftopoulou M, Nery S, Huang Y, Gridley T, Fishell G. 2005. Notch signaling coordinates the patterning of striatal compartments. Development 132:4247-4258.
• Mitsiadis TA, Regaudiat L, Gridley T. 2005. Role of the Notch signalling pathway in tooth morphogenesis. Arch Oral Biol 50:137-140.
• Oram KF, Gridley T. 2005. Mutations in Snail family genes enhance craniosynostosis of Twist1 haplo-insufficient mice: implications for Saethre-Chotzen Syndrome. Genetics 170:971-974.
• Savagner P, Kusewitt DF, Carver EA, Magnino F, Choi C, Gridley T, Hudson LG. 2005. Developmental transcription factor Slug is required for effective re-epithelialization by adult keratinocytes. J Cell Physiol 202:858-866.
• Kiernan AE, Xu J, Gridley T. 2006. The Notch Ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet 2:e4.
• Mason HA, Rakowiecki SM, Gridley T, Fishell G. 2006. Loss of notch activity in the developing central nervous system leads to increased cell death. Dev Neurosci 28:49-57.
• McCright B, Lozier J, Gridley T. 2006. Generation of new Notch2 mutant alleles. Genesis 44:29-33.
• Murray SA, Carver EA, Gridley T. 2006. Generation of a Snail1 (Snail1) conditional null allele. Genesis 44:7-11.
• Xu J, Norton CR, Gridley T. 2006. Not all Lunatic fringe null female mice are infertile. Development 133:579.

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