This is a renewal application requesting five years of funding to continue Dr. Camper s research program to positionally clone and functionally characterize the mouse Ames dwarf (df) gene. The df mutation arose in descendants of an X-irradiated male mouse at Iowa State University in Ames, Iowa. Df is a recessive mutation that produces severe proportional dwarfism, hypothyroidism and infertility. The growth insufficiency results from a lack of thyroid stimulating hormone (TSH) and growth hormone (GH), and a lack of prolactin (PRL) contributes to the infertility of females. The Ames dwarf mice, like the Snell (Pitdw) dwarf mice, exhibit severe hypocellularity due to a deficiency in the three pituitary cells that produce PRL, GH, and TSH: lactotropes, somatotropes, and thyrotropes, respectively. The simultaneous loss of these three cell types suggest that they derive from a common precursor cell, and the similarity of the phenotypes of the non-allelic df and dw mutants suggests that the two mutated genes are involved in the proliferation and/or differentiation of this precursor cell. The dw mutation has been shown to result from defects in the Pit-1 transcription factor gene.
In Specific Aim 1 Camper proposes to clone the df gene. The df critical region will be physically mapped and the 3.5 Mb YAC and P1 contig encompassing the 635 kb df critical region will be completed. Exon trapping and cDNA selection will be applied to identify transcripts within the df critical region. Full length cDNA clones will be generated for the identified transcripts and these clones will be used in Northern analyses, and if necessary RNase protection and RT-PCR assays, to determine the tissue distribution of the cloned transcripts. These studies will allow Camper to prioritize transcripts as products of possible candidate df genes. Candidate gene sequences will then be compared in normal and df mutant mice to detect mutations in the putative df gene.
In Specific Aim 2, Camper will characterize the intron/exon structure and 5 end of the candidate df gene. In addition, df gene expression gene will be characterized by immunostaining of tissue sections, RT-PCR, RNase protection, in situ hybridization, and Northern analysis of pituitary cell lines. Confirmation that a mutation in a candidate gene is responsible for the df phenotype will be obtained in Specific Aim 3 by transgene correction, the generation of gain of function alleles, and the production of loss of function mutations. These studies will also begin to probe df gene function. Camper proposes in Specific Aim 4 to clone and chromosomally map the human df gene and to assess the association of df with human pituitary tumors and families of short stature.
Specific Aim 5 will establish the role of the df gene within the hierarchy of genes regulating pituitary development by comparing the expression of early pituitary markers in df and dw and by examining df gene expression in other mouse mutants with pituitary defects.
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