Sonic hedgehog (SHH) pathway activation is required for expansion of cerebellar granule neuron precursors (CGNP) during development and is etiologic in the human cerebellar tumor, medulloblastoma. However, the molecular mechanisms underlying Hedgehog regulatory effects on the cell cycle apparatus are poorly understood. Preliminary data and recently published work suggests an important role for proto-oncogene N-myc during CGNP proliferation downstream of SHH signaling in promoting cell cycle progression via regulation of D-type cyclins. Additionally, the winged-helix transcription factor, FoxM1, a regulator of B-type cyclin expression, has been implicated in HH-associated skin cancer. Our key hypothesis that new insights into development and tumorigenesis will emerge from a detailed understanding of Hedgehog regulatory effects on the cell cycle machinery within CNS precursors. To test this hypothesis, we propose the following three Specific Aims:
Specific Aim 1 is to determine genetic requirements for N-myc during development of the CGNP lineage. This will be established using a conditional (""""""""floxed"""""""") allele to inactivate N-myc specifically (a) in proliferating post-natal CGNP treated with SHH in culture, and (b) through intercrosses with a granule cell specific (Math 1) cre transgenic line to assess the cerebellar anlagen in vivo. The objective of Specific Aim 2 is to identify tissue-specific, Hedgehog-responsive cis-acting DNA regulatory sequences for N-myc using a classic deletion analysis in transgenic mice. In parallel, we propose to develop and test novel genome-wide tools to gain perspective on other direct targets of hedgehog signaling in CGNP and the upstream events that initially activate N-myc expression.
Specific Aim 3 is to determine genetic requirements for FoxM1 during development of the CGNP lineage. Because of neonatal lethality found in FoxM1-/- neonates, we will employ the identical tissue-specific knockout strategy used in Aim 1 using Math 1-cre combined a floxed allele of FoxM1 to target the granule cell lineage. The proposed work is intended to establish a """"""""proliferative pathway"""""""" for Hedgehog signaling and its effects on the cell cycle machinery within neural precursors. High levels of N-MYC expression are a conserved feature of Hedgehog-associated cases of medulloblastoma, a tumor primarily affecting children. Thus, elucidation of novel intra- and intercellular interactions of Hedgehog signaling during CNS development could provide clues for therapeutic intervention in human tumors.
