Overview
Our laboratory studies genes important for embryonic development of mice, and the connections between mutations in these genes and congenital human disease syndromes. Our analyses focus on the Notch pathway, an evolutionarily conserved cell communication and signaling system, and on genes of the Snail superfamily, which encode transcriptional repressor proteins. We have created and analyzed numerous genetically engineered mouse models to understand the essential functions of individual components of these pathways. We have also generated models for inherited human disease syndromes such as Alagille syndrome.
Scientific report
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. We have constructed many targeted null and conditional mutations of genes encoding Notch pathway components, and our analyses of these mutants have demonstrated an essential requirement for Notch signaling during multiple stages of mouse embryogenesis. This work has also provided insight into inherited human disease syndromes caused by mutations of Notch pathway components. JAG1 haploinsufficiency in humans causes Alagille syndrome, an inherited disorder characterized by developmental abnormalities of the liver, heart, eye, skeleton and kidney. We showed that while Jag1+/- heterozygous mice did not exhibit most phenotypes characteristic of humans with Alagille syndrome, mice doubly heterozygous for Jag1 and Notch2 targeted mutations exhibit multiple defects similar to human Alagille syndrome patients. Our work was the first to implicate a critical role for the Notch2 gene in the pathogenesis of Alagille syndrome. This role was validated by the finding that heterozygous NOTCH2 mutations were present in a subset of Alagille syndrome patients who lack JAG1 mutations.
Bile duct defects in the liver are a characteristic feature of Alagille syndrome. We have specifically studied the role of the Notch2 gene in bile duct formation in the liver by disrupting Notch2 function utilizing mice expressing Cre recombinase under the control of a liver-specific promoter and an allele permitting conditional inactivation of Notch2 gene function using the Cre-loxP system. The bile duct defects exhibited by liver-specific Notch2-deficient mice were very similar to those exhibited by Jag1+/- Notch2+/- double heterozygous mice. However, Jag1+/- Notch2+/- mice exhibit defects in many organs other than the bile ducts of the liver, such as the heart and the kidney. Liver-specific deletion of the Notch2 gene represents a more specific model for studying the role of Notch signaling during bile duct morphogenesis and remodeling.
We have shown that the Jag1 gene also plays an important role during inner ear development. Jag1 expression regulates the ability of progenitor cells to differentiate into hair cells, the sensory cells of the inner ear. Mice with conditional deletion of the Jag1 gene in the inner ear exhibit defects in formation of all six sensory patches in the inner ear. The Jag1 gene was required by the sensory precursors, the progenitor cells that give rise to both the hair cells and supporting cells. By understanding how the sensory areas develop normally, it may be possible to develop molecular tools that will aid in sensory cell regeneration in the mammalian inner ear. We have also found that phenotypes exhibited by Notch pathway mutants are dependent on the genetic background of the mutant mice. Jag1+/- heterozygous mice exhibit semicircular canal defects on a C3H genetic background, but not on a C57BL/6 background. We identified a significant modifier locus on Chromosome 7, as well as a suggestive locus on Chromosome 14. Jag1+/- heterozygous mice also exhibit eye dysmorphologies that are modified by genetic background, and these modifiers are independent of the ear phenotype modifiers.
We also have been examining the role of the Notch signaling pathway during vascular development in mice. We and others have shown that, on some inbred genetic backgrounds, haploinsufficiency for the Dll4 gene leads to embryonic lethality at about 10 days of gestation due to defects in vascular development. Several different Notch pathway loss- of- function mutant embryos exhibit defects in arterial specification of nascent blood vessels, and developleading to arteriovenous malformations. We have now generated gain-of-function Notch mutant embryos that express the activated Notch1 intracellular domain specifically in vascular endothelial cells. These mutants exhibit vascular remodeling defects and form arteriovenous malformations. However, utilizing intracardiac ink injections we have shown that the type of arteriovenous malformation formed by the Notch pathway loss-of-function mutants is very distinct from that formed by Notch1 gain-of-function mutants.
Analysis of Snail superfamily genes
Phylogenetic analysis of Snail family genes has revealed that these genes form a superfamily consisting of two independent families, termed Snail and Scratch. In mice, there are three Snail family genes (Snai1, Snai2 and Snai3) and two Scratch family genes (Scrt1 and Scrt2). We are studying the roles during mouse development of genes of the Snail superfamily during mouse development. These genes encode DNA binding zinc finger proteins that act as transcriptional repressors.
Snail family genes are key regulators of epithelial-mesenchymal transitions in vertebrates, including the transitions that generate the mesoderm and neural crest. We have shown that Snai1-/- mutant mouse embryos exhibit early lethality that precludes the analysis of Snai1 gene function later in embryogenesis. Therefore, we generated an allele permitting conditional inactivation of Snai1 gene function using the Cre-loxP system. Utilizing this Snai1-flox allele, we demonstrated that, contrary to observations in frog and avian embryos, the Snail family genes Snai1 and Snai2 are not required for formation and delamination of the neural crest in mice. However, embryos with conditional inactivation of Snai1 function exhibit defects in left-right asymmetry determination. This work demonstrates that while some aspects of Snail family gene function, such as their role in left-right asymmetry determination, appear to be evolutionarily conserved, their role in neural crest cell formation and delamination is not. This work also demonstrates that species-specific differences in the regulation of neural crest formation and migration are more profound than previously appreciated.
Cleft palate is one of the most common human birth defects. We demonstrated that Snai2-/- mice exhibit partially penetrant cleft palate, which is made completely penetrant on a Snai1-heterozygous genetic background. Cleft palate in Snai1+/- Snai2-/- embryos is due to a failure of the elevated palatal shelves to fuse. Furthermore, while tissue-specific deletion of the Snai1 gene in neural crest cells does not cause any obvious defects, neural crest-specific Snai1 deletion on a Snai2-/- genetic background results in multiple craniofacial defects, including a cleft palate phenotype distinct from that observed in Snai1+/- Snai2-/- embryos. In embryos with neural crest-specific Snai1 deletion on a Snai2-/- background, palatal clefting results from a failure of MeckelÕs cartilage to extend the mandible and thereby allow the palatal shelves to elevate, defects similar to those seen in a human disease termed the Pierre Robin Sequence.
Lab staff
Principal Investigator: Thomas Gridley, Ph.D.Associate Research Scientist: Luke Krebs, Ph.D.
Research Assistant III: Christine Norton, B.S.
Graduate Student: Ying Chen, B.S., M.S.
Publication listings
Publications 2003-Present
Xu K, Nieuwenhuis E, Cohen B, Wang W, Canty AJ, Danska J, Coultas L, Rossant J, Wu MY, Piscione TD, Nagy A, Gossler A, Hicks GG, Hui CC, Hemkelman RM, Yu LX, Sled JG, Gridley T, Egan SE. 2009. Lunatic fringe-mediated Notch signaling is required for lung alveogenesis. Am J Physiol Lung Cell Mol Physiol (Epub Nov 6, 2009).
Rodilla V, Villanueva A, Obrador-Hevia A, Robert-Moreno A, Fernandez-Majada V, Grilli A, Lopez-Bigas N, Bellora N, Alba MM, Torres F, Duanch M, Sanjuan X, Gonzalez S, Gridley T, Capella G, Bigas A, Espinosa L. 2009. Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer. Proc Natl Acad Sci USA 105(15):6315-6320.
Lomeli H, Starling C, Gridley T. 2009. Epiblast-specific Snai1 deletion results in embryonic lethality due to multiple vascular defects. BMC Res Notes 2:22.
Rodriguez S, Sickles HM, DeLeonardis C, Alcaraz A, Gridley T, Lin DM. 2008. Notch2 is required for maintaining sustentacular cell function in the adult mouse main olfactory epithelium. Dev Biol. 314(1):40-58.
Robert-Moreno A, Guiu J, Ruiz-Herguido C, L—pez ME, InglŽs-Esteve J, Riera L, Tipping A, Enver T, Dzierzak E, Gridley T, Espinosa L, Bigas A. 2008. Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1. EMBO J. 27(13):1886-95.
Lozier J, McCright B, Gridley T . 2008. Notch signaling regulates bile duct morphogenesis in mice. PLoS ONE 3(3):e1851.
Hashimoto-Torii K, Torii M, Sarkisian MR, Bartley CM, Shen J, Radtke F, Gridley T, Sestan N, Rakic P. 2008. Interaction between Reelin and Notch signaling regulates neuronal migration in the cerebral cortex. Neuron 60(2):273-84.
Escriva M, Peiro S, Herranz N, Villagrasa P, Dave N, Montserrat-Sent’s B, Murray SA, Franc’ C, Gridley T, Virtanen I, Garc’a de Herreros A . 2008. Repression of PTEN phosphatase by Snail1 transcriptional factor during gamma radiation-induced apoptosis. Mol Cell Biol 28(5):1528-40.
Murray SA, Oram KF, Gridley T. 2007. Multiple functions of Snail family genes during palate development in mice. Deveopment 134:1789-1797.
Gridley T. 2007. Notch signaling in vascular development and physiology. Development 134:2709-2718.
Kiernan AE, Li R, Hawes NL, Churchill GA, Gridley T. 2007. Genetic background modifies inner ear and eye phenotypes of Jag1 heterozygous mice. Genetics 177:307-311.
Amsen D, Antov A, Jankovic D, Sher FA, Radtke F, Souabni A, Busslinger M, McCright B, Gridley T, Flavell RA. 2007. Direct regulation of Gata3 expression determines the T helper differentiation potential of Notch. Immunity 27:89-99.
Gridley T. 2007. Vessel guidance. Nature 445:722-723.
Gridley T, Woychik RP. 2007. Laser surgery for mouse geneticists. Nat Biotec 25:59-60.
Murray SA, Gridley T. 2006. Snail family genes are required for left-right asymmetry determination, but not neural crest formation, in mice. Proc Natl Acad Sci USA 103:10300-10304.
McCright B, Lozier J, Gridley T. 2006. Generation of new Notch2 mutant alleles. Genesis 44:29-33.
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.
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.
Gridley T. 2006. The long and short of it: Somite formation in mice. Dev Dyn 235:2330-2336.
Casey LM, Lan Y, Cho ES, Maltby KM, Gridley T, Jiang R. 2006. Jag2-Notch1 signaling regulates oral epithelial differentiation and palate development. Dev Dyn 235:1830-1844.
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.
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.
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.
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.
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.
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.
Gridley T. 2004. Kick it up a Notch: Notch1 activation in T-ALL. Cancer Cell 6:431-432.
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.
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.
Gridley T. 2003. Notch signaling and inherited disease syndromes. Hum Mol Genet 12 Spec No 1:R9-13.
