The goal of this proposal is to determine whether the three novel candidate transcription factors (members of the HMG box and homeobox gene families) we have recently identified play key roles in epithelial tubule formation relevant to kidney development and cystic disease. This proposal seeks to further explore the candidate transcription factors identified through the use of two novel approaches first developed in the PI's lab. 1) We have recently demonstrated that ureteric bud cell lines can be induced to undergo branching tubulogenesis in vitro in three dimensional matrix gels in the presence of serum free conditioned medium secreted by metanephric mesenchyme-derived cells. This represents perhaps the simplest system for studying ureteric bud branching tubulogenesis and relics on cell lines derived directly from day 11.5 embryos. 2) We have also recently developed a modification of the differential display technique that employs statistically designed primers. This published technique is known as CODD (Codon Optimized Differential Display). The method allows for the analysis of differentially expressed coding regions. Using CODD and other approaches to target homeobox genes differentially expressed in the in vitro tubulogenesis system, we have identified a number of candidate transcription factors that are likely to be involved in branching tubulogenesis. One such gene, tentatively called TRF, has been found to have very high homology to High Mobility Group (HMG) proteins that function in the assembly of the transcriptional unit. TRF expression is increased by all factors favoring in vitro branching tubulogenesis. Two candidate homeobox gene products have also been identified, one of which is virtually kidney-specific (H108) and another of which is expressed mainly in day 11 embryos (H111). The experiments proposed will: a) analyze the spatial and temporal expression patterns of these genes during in vitro tubulogenesis and in the developing kidney (SA1); b) determine their function in in vitro tubulogenesis through stable transfection of antisense-expressing constructs (SA2); and c) further examine the role of what appears to be the most interesting of these genes (based on results from SA1 and SA2) in kidney organogenesis in organ culture and through creation of a knockout animal. A wide array of cellular and molecular techniques will be employed.
| Drummond, Iain A (2011) Polycystins, focal adhesions and extracellular matrix interactions. Biochim Biophys Acta 1812:1322-6 |
| Mangos, Steve; Lam, Pui-ying; Zhao, Angela et al. (2010) The ADPKD genes pkd1a/b and pkd2 regulate extracellular matrix formation. Dis Model Mech 3:354-65 |
| Moller, Clemens C; Mangos, Steve; Drummond, Iain A et al. (2008) Expression of trpC1 and trpC6 orthologs in zebrafish. Gene Expr Patterns 8:291-6 |
| Mangos, Steve; Liu, Yan; Drummond, Iain A (2007) Dynamic expression of the osmosensory channel trpv4 in multiple developing organs in zebrafish. Gene Expr Patterns 7:480-4 |
| Pathak, Narendra; Obara, Tomoko; Mangos, Steve et al. (2007) The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation. Mol Biol Cell 18:4353-64 |
| Obara, Tomoko; Mangos, Steven; Liu, Yan et al. (2006) Polycystin-2 immunolocalization and function in zebrafish. J Am Soc Nephrol 17:2706-18 |
| Kramer-Zucker, Albrecht G; Olale, Felix; Haycraft, Courtney J et al. (2005) Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis. Development 132:1907-21 |
| Drummond, Iain A (2005) Kidney development and disease in the zebrafish. J Am Soc Nephrol 16:299-304 |
| Li, Zhixing; Stuart, Robert O; Eraly, Satish A et al. (2003) Debt91, a putative zinc finger protein differentially expressed during epithelial morphogenesis. Biochem Biophys Res Commun 306:623-8 |
| Xu, G Mark; Gonzalez-Perrett, Silvia; Essafi, Makram et al. (2003) Polycystin-1 activates and stabilizes the polycystin-2 channel. J Biol Chem 278:1457-62 |
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