The Ret Gene in the Enteric Nervous System: Expression Analysis and Generation of Ret Deficient Mice

This dissertation, "The Ret Gene in the Enteric Nervous System: Expression Analysis and Generation of Ret Deficient Mice" by King-yiu, Lee, 李景耀, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled The Ret gene in the enteric nervous system: Expression analysis and generation of Ret deficient mice submitted by Lee King Yiu for the degree of Doctor of Philosophy at The University of Hong Kong in August 2004 The RET proto-oncogene encodes for a transmembrane receptor tyrosine kinase and plays crucial roles in kidney and enteric nervous system (ENS) development. Alternative splicing of the RET gene generates splicing variants that encode RET 9, RET 51 and RET 43 isoforms. It has been hypothesized that these isoforms perform distinct functions and that their expressions are differentially regulated during mammalian development. To gain an insight into the expression patterns of ret isoforms during embryogenesis, the temporal and spatial expressions of ret gene in mouse embryos and adult mice was investigated. Using 3'RACE and RT-PCR, ret 9 and ret 51 transcripts were identified in both mouse embryos and adult mouse tissues. The ret 43 transcript was not detected. In situ hybridization showed that ret 9 was the dominant ret transcript in mouse embryos. Transcripts of ret 9 were detected in all cranial ganglia, in the sensory and autonomic ganglia of the trunk, in a subset of neurons of the dorsal root ganglion (DRG), in the motor neurons of the spinal cord, in the developing lung and kidney, in the enteric neuroblasts of the ENS and in thyroid. In contrast, ret 51 expression was weak and restricted to the motor column of the spinal cord, the DRG, the enteric neuroblasts, the developing lung and the kidney. In adult mice, ret 9 expression was relatively widespread in many organs while that of ret 51 was rather restricted. Present findings indicate that ret isoforms are temporally and spatially regulated in mouse embryos and adult mice. Homozygous ret knockout mice have been generated and these mutants are born alive but die within the first 24 hours showing ENS and kidney defects. However, detailed functional analyzes of ret in ENS development in ret-/- gut is hampered by an early apoptosis of enteric neural crest (NC) precursors in these mutants. In order to define the roles of ret in ENS development, a transgenic mouse model carrying the ret R972G mutation was generated in this study. In humans, the R972G mutation was heterozygous and causes ENS abnormality. Immuno-histochemical and morphological analysis of the guts from the R972G heterozygous mutants revealed mild or no abnormalities in the ENS of these mice, which could be attributed to the low level of expression of the ret R972G mutant allele. However, functional analyzes of the peristalsis of the guts of the mutants revealed a slight delay in the evacuation of intestinal content, suggesting that their ENS were functionally compromised. No homozygous ret R972G mutant mice could be obtained. The present findings suggest that homozygous ret R972G mutation results in embryonic lethality, which is different from that in ret-/- mice where a lack of ret activity does not cause embryo death. Thus, the R972G mutation was likely to exhibit a dominant negative role in embryo development. The ret R972G mutant mice being generated in this study shall be a valuable model for the in vivo study of ret functions in the development of ENS as well as other organs where ret is expressed. (500 words) DOI: 10.5353/th_b3144966 Subjects: Oncogenes Gene ex