The parent grant (Procollagen C-proteinase Enhancers: In vivo Roles, R01 AR53815) for this Revision Application involves in depth characterization of PCOLCE1-null, PCOLCE2-null, and PCOLCE1/PCOLCE2 doubly null knockout mice to study in vivo roles of the two procollagen C-proteinase enhancer proteins PCOLCE1 and PCOLCE2 which enhance the ability of BMP1-like extracellular proteinases to biosynthetically cleave the C-propeptides from the major fibrillar procollagens. The parent grant is a continuation of work from previous grants of the principal investigator that focused on the BMP1-like proteinases, their substrates, biological functions, and modulators of those functions. However, the present parent grant does not investigate additional biological roles of BMP1-like proteinases, other than those affected by PCOLCE1 and 2. Here we propose direct studies of additional functions of the BMP1-like proteinase, that will be leveraged by use of novel reagents created in this lab, and skills obtained in our previous studies. Specifically, although knockout of the Tll1 gene, which encodes the BMP1-like proteinase, mammalian tolloid-like 1 (mTLL1), is embryonic lethal, we have recently been successful in creating mice with floxed Tll1 alleles, which will allow tissue-specific Tll1 knockout. We've shown that Tll1 is highly expressed in specific structures of the developing and adult central nervous system (CNS). Thus, we've crossed our floxed Tll1 mice to mice in which Cre recombinase is driven by the CNS-specific enhancer sequences of the nestin gene, and demonstrate herein CNS-specific knockout of Tll1. We propose using these mice to determine in vivo Tll1 CNS functions. We request salary support for a lab member to conduct such studies. In addition, we have begun using the powerful zebrafish system to further define functions of the BMP1-like metalloproteinases. However, our previous work in this system employed the equipment and fish of another lab, greatly delaying our studies. We propose acquiring equipment that will enable us to perform zebrafish work in our lab. We propose first using such equipment to determine in vivo roles of the uncharacterized protein zebrafish Chordin-like (zCHL), which we have found to be a maternal factor in earliest embryos, and hypothesize to be a substrate of BMP1-like proteinases, involved in regulating BMP signaling. Obtaining the equipment will not only greatly enhance ability to successfully complete the zCHL study, but will greatly enhance our ability to use the powerful zebrafish system to further explore the BMP1-like proteinases, and ECM components that have also been the focus of research in this laboratory.
We've found that removing a particular gene in mice results in hearts that are in the wrong place, and which don't work sufficiently well to keep the embryo alive. We also have found that this gene is expressed at high levels in the developing and adult central nervous system. We propose using a method that will inactivate this gene only in the central nervous system, so that animals in which this is done will not die of heart defects in the embryonic stage, but will develop into adults in which we can study the effects of loss of this gene in the structure of the brain and its functioning in behavioral and physiological tests. In addition, we propose using a small tropical fish, known as the zebrafish, to examine the role in early development of a gene we've discovered. We believe that this protein is important in determining what will be the front and what will be the back of the embryo. Zebrafish were chosen for this study, as they generate eggs that are fertilized externally and develop outside the mother. The embryos are therefore accessible for observation and genetic manipulation at all stages of development without requiring any invasive procedures on the mother. Zebrafish embryos are optically transparent and develop rapidly, allowing one to analyze developmental events as they occur in the intact embryo. We believe that insights obtained from study in the mouse and zebrafish systems will provide important insights into the functions of the related human genes, and thus into how defects in such genes might be involved in human disease.
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