Research in Dr. Murray's laboratory focuses on two major areas: dissecting the genetic mechanisms of craniofacial development and dysmorphology, and developing new genetic tools and resources for the scientific community. We have a longstanding interest in determining the role of Snail family genes in orofacial development, particularly the mechanism by which they regulate fusion of the mammalian secondary palate. We are also interested in identifying novel genes and pathways that affect craniofacial development through positional cloning of spontaneous and ENU-induced mouse mutants.
Supporting this basic research interest, a significant portion of the lab effort is dedicated to developing new mouse genetic resources for the scientific community. This includes the development of a high-throughput, comprehensive pipeline for characterization of Cre driver strains distributed by the JAX Cre Repository (cre.jax.org) and the development of novel Cre strains with specific utility to the orofacial clefting research community. We are also developing ES cell resources to facilitate development of new mouse genetic tools and collaborating to produce disease-specific mouse ES cell lines for in vitro research. Finally, we are working with a number of collaborators to develop better animal models for human conditions, such as cystic fibrosis and preterm birth.
Dissecting the mechanism of secondary palate fusion
Cleft lip with or without cleft palate is seen at a frequency of approximately 1 in 700 newborns, making it one of the most common congential birth defects in humans. In addition to disabilities in speech, feeding and breathing, these malformations have a dramatic impact on the physical appearance of the affected individual, often with devastating psychological and social consequences. The etiology of orofacial clefting is known in only a minority of cases, emphasizing the need for better understanding of the genes responsible for the normal development of the lip and palate.
Genetic regulation of secondary palate formation can be divided into three stages: vertical palate shelf growth and patterning, palate shelf elevation, and palate shelf fusion. Defects in any of these processes can result in a cleft palate phenotype, and the mouse has proven to be a critical tool in identifying some of the genes that regulate these events. Fusion of the paired palate shelves requires a series of specific changes in the epithelium. Initially, the periderm cells of the bilayered epithelium bulge along the medial aspects of the shelves and extend lamellipodia on their surface. The events following fusion are a matter of some controversy, but current evidence suggests migration of the periderm cells to the oral and nasal aspects of the fusing shelf allows for intercalation of the underlying basal epithelial layer, forming the medial epithelial seam (MES), which subsequently undergoes apoptosis to yield a fully fused secondary palate. Defects in this process are evident in both Snai2-/- mice and Snai2-/-;Snai1+/- compound mutant mice, where the palate shelves appear to approach and contact, but fail to form an epithelial seam despite normal formation of lamellipodia on the surface of the shelves.
Our current work aims to understand how interactions between the secondary palate shelf epithelium and mesnechyme interact to regulate shelf fusion. In particular, we are currently exploring the mechanism by which Snai1 and Snai2 regulate this process. To address this, we are asking three basic questions. First, is the cleft palate phenotype due to a failure in normal migration of the periderm cell layer? Second, is the cleft palate phenotype due to Snai1/Snai2 deletion cell autonomous to the mesenchyme, basal epithelium or periderm cell layer? Finally, what are the downstream pathways affected by Snai1/Snai2 deletion?
Novel models of cleft lip and palate
To extend our understanding of the genetic mechanisms that govern craniofacial development, we employ a forward genetic approach to identify novel genes and pathways, including positional cloning of spontaneous and ENU-induced craniofacial mutants. In collaboration with Dr. Leah Rae Donahue and the Craniofacial Mutant Resource, we have been characterizing a number of semi-dominant spontaneous mutants that, when rendered homozygous, display robust developmental abnormalities. Using both regional and exome capture, combined with Next-Gen sequencing, we have dramatically increased the speed with which candidate genes can be identified and validated. Several new developmental mutants have been identified as part of this screen, including models of cleft palate and holoprosencephaly.
As part of an ENU mutagenesis screen, we have recently identified a novel mouse model of cleft palate, clfp4, which displays cleft secondary palate, omphalocele and skeletal malformations with high penetrance. Preliminary histological analysis has revealed that the cleft palate in these animals results from a failure of palate shelf elevation. The skull of mutant embryos is misshapen and displays defects in calvarial ossification. Defects in ventral body wall fusion are accompanied by a failure of the sternum to fuse. Overall, mutant embryos show some degree of axial truncation and display widespread sub-epidermal hemorrhaging.
Preliminary mapping data placed the mutation at the distal end of Chromosome 10, a region where this combination of phenotypes has not been reported. Through targeted array capture of the exome within our critical region and Illumina sequencing, we identified a single non-synonymous coding mutation in a gene that has not previously been reported to play a role in craniofacial development. Due to the early lethality of mice null for this gene, its role in the development of the palate and craniofacial complex has not been investigated. However, significant evidence suggests the mutant gene function as a co-receptor for several signaling molecules known to play a role in craniofacial development. Indeed, our amino acid change is predicted to disrupt a highly conserved motif within the receptor interaction domain. We are currently exploring the impact the clfp4 mutation has on these pathways during neural crest migration, development of the facial structures and growth of the palate shelves, and will present our current findings. The identification of this novel gene function will provide important insight into the molecular networks that regulate craniofacial and palate development.
The Jackson Laboratory Repository distributes a large, diverse collection of Cre driver strains to scientists around the world, including over 200 strains as live colonies. The utility of existing Cre driver strains critically depends upon careful characterization of their function. Characterization data for most Cre strains is limited to that provided by the donating investigators, which is often incomplete and focused on a specific tissue of interest to their research question. Thus, complementing the published data with a standardized, unbiased, and extensive characterization pipeline promises to maximize the value of these resources for the scientific community.
To address this issue, our group has embarked on an ambitious project to add value to these strains by comprehensively characterizing Repository Cre lines. Despite the best efforts of those developing new Cre lines, the fidelity of Cre activity is not always ideal. Many difficulties have been reported in various Cre lines, including mosaic or incomplete deletion in a target tissue/cell type, inconsistent activity, expression in non-target tissues, and/or Cre-related toxicity. In many cases, however, this data is not reported or available to the potential user. Our preliminary results indicate the vast majority of Cre driver strains exhibit unexpected recombinase activity in a number of tissue types, highlighting the need for extended analysis. We have standardized our data annotation scheme to include 11 broad organ systems, 30 individual organs/structures and 89 substructures, all of which are consistent with the mouse Anatomical Dictionary. Slide-scanned images and associated annotations are published on a dedicated website (cre.jax.org) and submitted to Creportal.org. This information will allow users to make informed judgments about the suitability of a particular line for their experiments, and enhance the power of large-scale mouse gene targeting projects.
The overall goal of the JAX FaceBase project is to facilitate orofacial clefting research by generating new mouse genetic tools (Cre strains) and by providing a repository of mouse strains critical for clefting research community (linky to sites). Our initial set of planned Cre drivers includes four BAC transgenic drivers, deltaNp63-CreERT2, Krt6a-Cre, Tbx22-CreERT2, Lhx7/8-Cre. These strains are designed to target the oral basal epithelium, the oral peridem cell layer, the posterior secondary palate shelf, and the mesenchyme adjacent to the fusing facial prominences, respectively. We are also employing a novel approach to Cre driver generation, using highly conserved enhancer sequences identified by our collaborator, Dr. Axel Visel at Lawrence Berkley National Laboratory (http://enhancer.lbl.gov/). Individual enhancer elements that have been empirically validated to direct expression to the midface and palate will be used to drive Cre expression from the ROSA26 locus. Our first three elements all target different parts of the fusing facial prominences at E11.5, and future drivers will use new elements identified by Dr. Visel that are expressed during secondary palate development. All of these strains will be characterized for orofacial-specific activity and examined for specificity using our Cre characterization pipeline. Together, these activities will provide both FaceBase members and the general scientific community with new research tools, novel genetic models, and comprehensive mouse repository services to enhance research in orofacial clefting.
Principal Investigator: Steve Murray, Ph.D.
Research Project Managers: Rick Bedigian, Ph.D.; Leslie Goodwin, Ph.D.
Research Assistant II: Brianna Caddle, Caleb Heffner, Jocelyn Sharp
Research Assistant II: Larry Bechtel, Polyxeni Gudis
Executive Assistant: Aimée Picard
Adams D, Baldock R, Bhattacharva S, Copp AJ, Dickinson M, Greene ND, Henkelman M, Justice M, Mohun T, Murray SA, Pauws E, Raess M, Rossant J, Weaver T, West D. 2013. Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening. Mar 18[Epub ahead of print]. PMID: 23519032
Heffner CS, Pratt CH, Babiuk RP, Sharma Y, Rockwood SF, Donahue LR, Eppig JT, Murray SA. 2012. Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource. Nat Commun. Nov 20;3:1218. doi: 10.1038/ncomms2186. PMCID: PMC3514490Xu Y, Wang Y, Besnard V, Ikegami M, Wert SE, Heffner C, Murray SA, Donahue LR, Whitsett J. 2012. Transcriptional Programs Controlling Perinatal Lung Maturation. PLoS ONE 7(8): e37046. doi:10.1371/journal.pone.0037046.
Murray SA, Eppig JT, Smedley D, Simpson EM, Rosenthal N. 2012. Beyond knockouts: cre resources for conditional mutagenesis. Mamm Genome. 2012 Aug 29. [Epub ahead of print]. PMID: 22926223
Besnard V, Wert SE, Ikegami M, Xu Y, Heffner C, Murray SA, Donahue LR, Whitsett JA. 2011. Maternal synchronization of gestational length and lung maturation. PLoS One 2011; 6(11):e26682. PMCID: PMC3212521
Murray SA. 2011. Mouse resources for craniofacial research. Genesis 49(4): 190-9. PMCID: PMC3610317
Hochheiser H, Aronow BJ, Artinger K, Beaty TH, Brinkley JF, Chai Y, Clouthier D, Cunningham ML, Dixon M, Donahue LRD, Fraser SE, Iwata J, Marazita ML, Murray JC, Murray SA, Postlethwait J, Potter S, Shapiro L, Spritz R, Visel A, Weinberg SM, Trainor PA for the Facebase Consortium. 2011. The FaceBase Consortium: A Comprehensive Program to Facilitate Craniofacial Research. Developmental Biology 355(2): 175-82. PMCID: PMC3440302
Varlakhanova NV, Cotterman RF, Devries WN, Morgan J, Donahue LR, Murray S, Knowles BB, Knoepfler PS. 2010. Myc maintains embryonic stem cell pluripotency and self-renewal. Differentiation 80:9-19. PMCID: PMC2916696.
Murray SA, Morgan JL, Kane C, Sharma Y, Heffner CS, Lake J, Donahue LR. 2010. Mouse gestation length is genetically determined. PLoS ONE 5(8):e12418. PMCID: PMC2928290.
Escriva M, Peiro S, Herranz N, Villagrasa P, Dave N, Montserrat-Sentis B, Murray SA, Franci C, Gridley T., Virtanen I, Garcia de Herreros A. 2008. Repression of PTEN phosphatase by Snail 1 transcriptional factor during gamma radiation-induced apoptosis. Mol Cell Biol 28(5):1528-40. PMCID: PMC2258777.
Murray SA, Oram KF, Gridley T. 2007. Multiple functions of Snail family genes during palate development in mice. Development 134:1789-97.
Howell GR, Shindo M, Murray S, Gridley T, Wilson LA, Schimenti JC. 2007. Mutation of a ubiquitously expressed mouse transmembrane protein (Tapt1) causes specific skeletal homeotic transformations. Genetics 175:699-707.
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 U S A 103:10300-4.
Murray SA, Gridley T. 2006. Snail1 gene function during early embryo patterning in mice. Cell Cycle 5:2566-70.
Murray SA, Carver EA, Gridley T. 2006. Generation of a Snail1 (Snai1) conditional null allele. Genesis 44:7-11.
Murray SA, Yang S, Demicco E, Ying H, Sherr DH, Hafer LJ, Rogers AE, Sonenshein GE, Xiao ZX. 2005. Increased expression of MDM2, cyclin D1, and p27Kip1 in carcinogen-induced rat mammary tumors. J Cell Biochem 95:875-84.
Collin GB, Cyr E, Bronson R, Marshall JD, Gifford EJ, Hicks W, Murray SA, Zheng QY, Smith RS, Nishina PM. 2005. Alms1-disrupted mice recapitulate human Alstrom syndrome. Hum Mol Genet 14:2323-33.
Murray SA, Zheng H, Gu L, Jim Xiao ZX. 2003. IGF-1 activates p21 to inhibit UV-induced cell death. Oncogene 22:1703-11.
Gu L, Ying H, Zheng H, Murray SA, Ziao ZX. 2003. The MDM2 RING finger is required for cell cycle-dependent regulation of its protein expression. FEBS Lett 544:218-22.
Zheng H, You H, Zhou XZ, Murray SA, Uchida T, Wulf G, Gu L, Tang X, Lu KP, Xiao ZX. 2002. The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response. Nature 419:849-53.
You H, Zheng H, Murray SA, Qiang Y, Uchida T, Fan D, Xiao ZXJ. 2002. IGF-1 induces Pin1 expression in promoting cell cycle S-phase entry. J Cell Biochem 84:211-216.
Gallo-Hendrikx E, Murray SA, Vonderhaaar BK, Xiao ZXJ. 2001. Vanadate disrupts mammary gland development in whole organ culture. Dev Dynamics 222:354-367.
Gacheru SN, Thomas KM, Murray SA, Csiszar K, Smith-Mungo LI, Kagan HM. 1997. Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: effects of TGF-beta 1 and serum deprivation. J Cell Biochem 65:395-407.