This is a first competitive renewal to study transcriptional regulation of mammalian brain development. The focus of the proposed studies is a transcription factor named BF-1 which is a member of the Winged Helix family of proteins. During the previous funding period the PI's lab generated a targeted deletion of this gene. Heterozygotes were healthy and fertile; homozygotes died within minutes of birth due to anomalous brain development. Results suggested that BF-1 was essential for growth of the telencephalon because at early stages, no significant differences in size of the telencephalic vesicles were apparent. BrdU labeling and in situ hybridizations suggest reduced cell proliferation, precocious expression of neuronal marker gene, and reduced expression of sonic hedgehog (shh). These data led to the PI's hypothesis that BF-1 regulates cell cycle and thus controls number of neurons in telencephalon and as assayed in Mink cell culture system. Therefore, the PI proposes that BF-1 serves normally to promote growth of neuronal precursor cells in the neuroepithelium of the telencephalon and thereby prevents precocious differentiation. This grant is aimed at understanding the mechanism by which BF-1 regulates cell proliferation/differentiation. Working on the assumption that BF-1 might antagonize the TGF-b signal transduction pathway leading to neuronal differentiation, the PI has shown, using a mink lung cell line where BF-1 expression is under control of tetracycline withdrawal, that the presence of BF-1 promotes entry into S phase in the presence of TGF-b. He has also shown that downstream TGF-b events are blocked by the presence of BF-1- hyperphosphorylation of retinoblastoma, block of induction of p15 mRNA levels, and block of activation of reporter genes driven by a TGF-b- responsive promoter. It is further shown that the DNA-binding domain of BF-1 is not required for these """"""""antagonistic"""""""" activities. In another set of studies, the PI collaborated to perform a targeted deletion of another WH gene called TWH. The expression of this gene is complementary to that of BF-1 in that it is expressed within the neural tube where BF-1 is not. The phenotype of this mutant mouse is complex and appears to be most interesting from the point of view of spinal cord development.
Seven specific aims are proposed that extend these observations. They are to: 1) delineate the functional domains of BF-1 in terms of its ability to antagonize cell cycle arrest mediated by TGF-b and to transform chick embryo fibroblasts, since a chick homolog of BF-1 has been shown by others to be a proto-oncoprotein 2) to examine the function of BF-1 in neuroepithelial cells by generating immortalized neuroepithelium using a ts T antigen or v-myc under control of the tet activator. Uptake of 3H and expression of neuronal markers such as MAP2 will be used to measure cell proliferation and differentiation. Since BMPs have been suggested to be inhibit proliferation of neural progenitors in forebrain, and since some of these factors are expressed in the developing neuroectoderm, the PI will examine the effects of treating the immortalized neuroepithelial cells lacking BF-1 with these BMPs. Experiments examining the effects of other neural inducers such as follistatin are also proposed, as well as tests of the dominant negative BMP receptor. As an alternative to the stable telencephalic progenitor cell lines, the PI proposes to examine the effects of BF-1 in PC12 lines where BF1 is under control of tet transactivation. Cell cycle effects as well as the effects with BMP will be examined if expression of BF-1 blocks differentiation by BMP. 3) gain-of-function experiments in which BF-1 is expressed ectopically in the neuroepithelium and effects on the developing brain are examined. The PI will generate transgenic mice in which the BF-1 cDNA is under control of the TWH promoter which is expressed throughout the telencephalon neuroepithelium except where the BF1 gene is normally expressed. Effects on BrdU incorporation (cell number ) and expression of neuronal markers (shh, Dlx-2, etc) will be measured. The general prediction is that BF1 should cause more proliferation than normal and delay neuronal differentiation compared to controls, although the PI also proposes that apoptosis might result. 4 and 5 identify the steps in the TGF-b pathway which are modulated by BF-1 by examining interactions of BF-1 with FAST-1 and Smad proteins which are known mediators of the activator pathway of TGF-b in the mink lung cells. Co-immunoprecipitation/Western blot studies with tagged intermediates will be used to examine potential interactions between BF-1 and these other proteins. The two-hybrid genetic system will also be used. 6) identify mouse homologs of the FAST-1 gene which are expressed in the mouse neuroepithelium and demonstrate their function in TGF-b signaling pathways. Studies will be performed to determine the developmental time course and location of expression of these new FAST forms. Antibodies will be generated and studies will be done to determine the interactions between the FAST proteins and other members of the TGF b pathway such as Smad. 7) study the potential roles of BF-1 and sonic hedgehog in the development of the ventral telencephalon.
This aim i s premised on the finding that shh is missing in the ventral telencephalon in the mutant BF-1 mouse. Forebrain explants will be dissociated and cultured. Rate of proliferation will be quantitated by measuring BrdU incorporation and it will be determined if cells recapitulate expression or lack of expression of certain markers, such as shh. If so, the PI will expose cells to ectopic shh and determine effects on proliferation and expression of markers. Comparisons will be made between wild type and BF1 mutant cell cultures +/- shh.
