In an attempt to understand better both the functions and evolution of the large family of steroid/thyroid (S/T) hormone receptor genes we have been investigating their presence in lower organisms. We were unable to find any in yeast bu have found them in cnidaria and nematodes. The nematode work with Caenorhabditis elegans has been carried on in collaboration with Dr. Mike Krause. We previously cloned three cDNAs from C. Elegans, chr3, cnr8 and cnr 14. Subsequently, we have cloned and sequenced the genes for these cDNAs. Chr 3 has eight exons and 7 introns. We also have the sequence of 3,500 bases upstream of the transcription start site. Using this we constructed a plasmid with Lac Z as a reporter. It was possible to obtain transgenic worms by injecting this construct into the ovary. Staining showed expression in hypodermal cells beginning early in embryogenesis at the 1.5 fold stage and extending into the adult. This was confirmed by colocalization of chr3 with antibodies to lin26. Lin26 is known to be expressed exclusively in hypodermal cells. Crossing of the chr3 transgenic worms with worms bearing the skin1 mutation further confirmed hypodermal cell localization since these worms showed chr3 localization in the aberrant hypodermal cells caused by this mutation. We are currently attempting to obtain localization with an antibody to the chr3 protein. We are also attempting to obtain worms transgenic for the cnr14 gene. In the other part of our work we utilize several forms of cnidaria. The first is Tripedalia cystophora. Here we have isolated and partially sequenced a single S/T gene which exists in two isoforms and belongs to the RXR family. We also examined another cnidarian, Aurelia aurita which can be grown in culture. In this organism we have isolated and partially sequenced three different S/Y cDNAs. These represent homologues of RAR-RXR, an orphan receptor and the ecdysone receptor. The latter is of interest because the process of development from polyp to medusa (strobilation) is a type of metamorphosis. We have successfully taken polyps and disaggregated them into single cells, reaggregated then and seen them form polyps again. We are currently attempting to make transgenic aurelia in this manner and investigating the role of the S/T receptors in the process of strobilation.
|Kostrouchova, Marta; Housa, Daniel; Kostrouch, Zdenek et al. (2002) SKIP is an indispensable factor for Caenorhabditis elegans development. Proc Natl Acad Sci U S A 99:9254-9|
|Kostrouchova, M; Krause, M; Kostrouch, Z et al. (2001) Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans. Proc Natl Acad Sci U S A 98:7360-5|