Calcitonin (CT) was originally identified as a peptide hormone released by parafollicular cells of the thyroid gland in response to hypercalcemic stimuli. In mammals, it was found that CT secretion produces a rapid lowering of extracellular calcium levels by inhibition of bone resorption and stimulation of renal calcium excretion, effects mediated by high affinity receptors located on osteoclasts and certain renal tubular cells. Subsequently, it was shown that CTRs are widely distributed, suggesting that CT may have more complex and diverse functions. CT may also be important in the early differentiation of the vertebrate embryo.
The aims of this proposal are to complete the determination of the structure of the murine CTR gene, introduce targeted mutations into the gene by homologous recombination in embryonic stem cells and create transgenic mice that express these mutations in order to define the role of CT and the CTR gene in embryonic development. The zebrafish system will also be used to understand the role of the CTR in early embryonic development.
In Specific Aim 1, determination of the structure of the murine CTR gene will be completed and the 5'-regulatory sequences identified. The murine brain CTR cDNA, sequenced in this laboratory, will be used to identify exons in the CTR gene (see Project 4).
In Specific Aim 2, restriction fragments prepared from the murine CTR cDNA or the genomic CTR clones will be subcloned into vectors to produce CTR- specific cRNA probes. These will be utilized to localize CTR gene transcripts during various stages of murine development with techniques of in situ hybridization. The pattern of CTR expression determined by in situ hybridization will be correlated with that obtained by autoradiography with radioiodinated CT and immunohistochemistry with CTR antibodies.
In Specific Aim 3, homologous recombination in embryonic stem (ES) will be used for targeted disruption of the CTR gene; the ES cells will be used to produce transgenic mice. This will permit direct determination of the consequences of disruption of the CTR gene in murine embryonic development.
In Specific Aim 4, the lacZ gene will be placed just downstream from the translation initiation site in the CTR gene by means of homologous recombination of ES cells to produce mutant mice. This approach will permit the determination in vivo of the spatial and temporal expression of the CTR gene and the lineage of cells that express the gene during development.
In Specific Aim 5, zebrafish CTR cDNA will be cloned and vectors incorporating these sequences or sequences from the CTR gene will be utilized to examine the consequences of ectopic production or overexpression of CT or the CTR in early embryonic development.
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