This is a proposal to investigate the genetic pathways underlying the development of the mammalian skull vault and the pathogenesis of calvarial foramina. From human genetics and directed mutagenesis in the mouse, FGF receptors 1-3, Twist, Msx2, and Alx4 have emerged as key regulators of skull morphogenesis. Gain of function mutations in Msx2 and FGFR receptors 1-3 are associated with craniosynostosis; loss of function mutations in Msx2 and Alx4 with calvarial foramina, and the loss of function mutation in Twist with both the craniosynostosis and calvarial foramina. Two key questions remain unanswered (i) what are the roles of these genes in the morphogenetic processes underlying skull vault development? (ii) do these genes function in the same or distinct regulatory pathways? In addressing these questions, we have found a striking overlap in cranial defects caused by loss of function mutations in Msx1, Msx2 and Twist. Each mutant exhibits calvarial ossifications defects in the frontal bone, which, from lineage tracing experiments with the Wnt1-cre/R26R neural crest marking system, is derived from neural crest. These findings suggest, first, that Msx and Twist genes are associated in a regulatory pathway, and, second, that this pathway controls calvarial neural crest development. Here we propose to test the overall hypothesis that Msx genes are downstream effectors of Twist in skull vault development.
Our first aim will entail asking whether mutations in Msx1, Msx2 and Twist cause aberrant neural crest development (including migration, and appropriate spatial arrangement in the calvarial analage). We will also test the hypothesis that each gene regulates calvarial osteoblast development. In our second aim, we will investigate functional and regulatory relationships between genes associated with calvarial foramina. A preliminary analysis of Msx1 Msx2 and Twist Msx2 compound mutants has led us to three specific hypotheses (i) that Msx1 and Msx2 function redundantly in frontonasal neural crest and in the appositional growth phase of frontal bone development; (ii) that Msx2 and Twist cooperate in these processes, and (iii) that Msx1 and Msx2 are downstream effectors of Twist in frontonasal neural crest and frontal bone development. We will test these hypotheses rigorously through an analysis of Msx1-Msx2, Twist-Msx1, and Twist-Msx2 compound mutant mice. In our third aim, we will investigate the molecular mechanism by which the Twist regulates a potential downstream target--Msx2. Our results suggest that loss of Twist function causes downregulation of Msx2 in the frontal bone and frontal suture, and that this downregulation is mediated by a direct effect of the Twist protein on the Msx2 promoter. We will use transgenic and gene targeting approaches to continue to test this hypothesis. In addition, we will test the hypothesis that loss of Msx2 function resulting from loss of regulability of Msx2 by Twist contributes to the calvarial foramen defect in Twist mutant mice.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
2R01DE012450-14
Application #
6472067
Study Section
Special Emphasis Panel (ZRG1-OBM-2 (01))
Program Officer
Small, Rochelle K
Project Start
1989-02-01
Project End
2007-01-31
Budget Start
2002-02-15
Budget End
2003-01-31
Support Year
14
Fiscal Year
2002
Total Cost
$365,625
Indirect Cost
Name
University of Southern California
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Sun, Jingjing; Ting, Man-Chun; Ishii, Mamoru et al. (2016) Msx1 and Msx2 function together in the regulation of primordial germ cell migration in the mouse. Dev Biol 417:11-24
Ishii, Mamoru; Sun, Jingjing; Ting, Man-Chun et al. (2015) The Development of the Calvarial Bones and Sutures and the Pathophysiology of Craniosynostosis. Curr Top Dev Biol 115:131-56
Ting, Man-Chun; Wu, Nancy L; Roybal, Paul G et al. (2009) EphA4 as an effector of Twist1 in the guidance of osteogenic precursor cells during calvarial bone growth and in craniosynostosis. Development 136:855-64
Maxson, Robert; Ishii, Mamoru (2008) The Bmp pathway in skull vault development. Front Oral Biol 12:197-208
Fu, Hualin; Ishii, Mamoru; Gu, Ying et al. (2007) Conditional alleles of Msx1 and Msx2. Genesis 45:477-81
Chen, Yi-Hui; Ishii, Mamoru; Sun, Jingjing et al. (2007) Msx1 and Msx2 regulate survival of secondary heart field precursors and post-migratory proliferation of cardiac neural crest in the outflow tract. Dev Biol 308:421-37
Merrill, Amy E; Bochukova, Elena G; Brugger, Sean M et al. (2006) Cell mixing at a neural crest-mesoderm boundary and deficient ephrin-Eph signaling in the pathogenesis of craniosynostosis. Hum Mol Genet 15:1319-28
Chai, Yang; Maxson Jr, Robert E (2006) Recent advances in craniofacial morphogenesis. Dev Dyn 235:2353-75
Ishii, Mamoru; Han, Jun; Yen, Hai-Yun et al. (2005) Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest. Development 132:4937-50
Brugger, Sean M; Merrill, Amy E; Torres-Vazquez, Jesus et al. (2004) A phylogenetically conserved cis-regulatory module in the Msx2 promoter is sufficient for BMP-dependent transcription in murine and Drosophila embryos. Development 131:5153-65

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