The mouse t-complex on chromosome 17 has long been a focus of interest for developmental biologists. Because many embryonic lethal mutations are localized in the region, corresponding genes of importance during development are inferred to reside there. It is thus a prime target for high priority analysis. We have completed and verified a Sequence Tagged Site (STS) /Bacterial artificial chromosome (BAC)-based physical map. It represents about half of the estimated 30 Mb t-complex in 450 BACs fitted with 650 STSs and integrating many of the short tandem repeat markers used in genetic mapping. Forty-three selected BACs were sequenced, most in conjunction with the Mouse Genome Consortium. The sequences independently verify and augment whole genome shotgun sequencing, including the provision of sequences that fill otherwise open gaps. We have analyzed and provided a curated annotation that provides candidate genes and clones to 1) identify various t-complex embryonic lethal mutations in conjunction with collaborating laboratories, and 2) investigate critical processes in early development. We are currently focusing on a candidate for the tw5 embryonic lethal. Histological studies have demonstrated that mice homozygotic for tw5 die at the gastrulation stage due to extensive death of the embryonic ectoderm cells, which fail to aggregate. Several metalloproteases have critical roles in cell-cell signal transduction, and the gene ADAMTS-10, a zinc metalloendopeptidase with thrombospondin domains, maps to the genetically defined critical region (about 600 Kb) where the embryonic lethal mutation maps. This gene is a potential candidate for an important function during development, and is one of few especially plausible candidates for involvement in tw5 lethality that stand out from the genomic analysis. We have found that ADAMTS-10 is alternatively spliced in a tissue-specific manner, and alternative splicing results in protein isoforms that either include or exclude the thrombospondin domains. The expression pattern indicates that it is widely transcribed in embryos, and continues to be expressed at elevated levels in adult brain, lung, kidney, spleen, and ovary. The current focus of the work includes cloning and characterization of major isoforms, and generation of antibodies to specific epitopes that fall within or outside the thrombospondin domains, in order to characterize the nature of expression of different protein isoforms using immunohistochemistry. At the same time, we will examine the effect of ablation of the gene on early development, including direct attempts to see if a transgenic large insert (BAC) clone containing the gene can complement the tw5 mutation in mice. Finally, as a technological approach to systematic studies of the localization and protein partners of this and possibly many other genes, we have initiated pilot screening for phage-display antibodies.
|Abe, Kuniya; Yuzuriha, Misako; Sugimoto, Michihiko et al. (2004) Gene content of the 750-kb critical region for mouse embryonic ectoderm lethal tcl-w5. Mamm Genome 15:265-76|
|Kargul, G J; Nagaraja, R; Shimada, T et al. (2000) Eleven densely clustered genes, six of them novel, in 176 kb of mouse t-complex DNA. Genome Res 10:916-23|