The goal of this project is to understand the mechanisms by which information encoded in extracellular signals is converted complex patterns in the developing mammalian embryo. This proposal focuses on the role of Sonic hedgehog (Shh) signaling. The Shh signaling pathway is not only essential for the induction of clinically relevant neurons within the CNS, but inappropriate activation of Shh signaling has been linked to the development of several l of tumor, most notably basal cell carcinoma (BCC), the most common form of skin cancer, and medullablastoma, the most common brain tumor of children. Further, loss of Shh signaling underlies many cases of familial an spontaneous holoprosencephaly. Consequently, understanding how a Shh signal is received, transduced and modulated is likely to lead to new insights with direct relevance to human health. In view of its close relation. to the human embryo, and the availability of genetic approaches which can precisely modify gene activity, the mouse is used as an experimental system. An active Shh signal is generated by an unusual autocatalytic cleavage which leads to the covalent attachment of cholesterol to the l9 kcal signaling peptide. Cholesterol modification may play several roles in Shh-signaling, for example preventing free diffusion of ligand or participating in receptor recognition.
Aim 1 proposes to explore the biological significance of cholesterol modification by generating a non-tethered allele of Shh (N-Shh). The activity of this allele in CNS and limb patterning will be addressed in the presence of normal or reduced levels of two Hedgehog binding proteins, Hip and Ptc, negative modulators of Shh signaling.
Aim 2 proposes to characterize the Shh interaction domain of Hip using truncated forms of Hip produced by cultured cells, or by limited proteolysis of purified Hip. Hip's role in embryogenesis and its genetic interactions with Ptc-l, will be examined in mice carrying Hip and Ptc null mutations. A mouse strain (Shh') has been generated in which an essential exon of Shh is flanked by the target recognition sequences (loxP sites) of the P1 phage integrase, CRE. Intercrossing the conditional allele with transgenic lines express CRE allows the spatial and temporal removal of Shh function.
Aim 3 proposes to adopt this strategy to address Shh action in the limb and specific regions of the developing CNS, where its role cannot be assessed with existing null mutations.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS033642-10
Application #
6692669
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Mamounas, Laura
Project Start
1994-12-01
Project End
2004-11-30
Budget Start
2003-12-01
Budget End
2004-11-30
Support Year
10
Fiscal Year
2004
Total Cost
$520,207
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Finch, Caleb E; McMahon, Andrew P (2016) Stem cells for all ages, yet hostage to aging. Stem Cell Investig 3:11
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Lopez-Rios, Javier; Duchesne, Amandine; Speziale, Dario et al. (2014) Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs. Nature 511:46-51
Rajurkar, M; Huang, H; Cotton, J L et al. (2014) Distinct cellular origin and genetic requirement of Hedgehog-Gli in postnatal rhabdomyosarcoma genesis. Oncogene 33:5370-8
Junker, Jan Philipp; Peterson, Kevin A; Nishi, Yuichi et al. (2014) A predictive model of bifunctional transcription factor signaling during embryonic tissue patterning. Dev Cell 31:448-60
Junker, Jan Philipp; Noël, Emily S; Guryev, Victor et al. (2014) Genome-wide RNA Tomography in the zebrafish embryo. Cell 159:662-75
Hettmer, Simone; Teot, Lisa A; van Hummelen, Paul et al. (2013) Mutations in Hedgehog pathway genes in fetal rhabdomyomas. J Pathol 231:44-52
Holtz, Alexander M; Peterson, Kevin A; Nishi, Yuichi et al. (2013) Essential role for ligand-dependent feedback antagonism of vertebrate hedgehog signaling by PTCH1, PTCH2 and HHIP1 during neural patterning. Development 140:3423-34

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