Studying Hoxb5 Functions in Enteric Nervous System Development by Dominant-Negative Repressor Engrailed-Hoxb5

This dissertation, "Studying Hoxb5 Functions in Enteric Nervous System Development by Dominant-negative Repressor Engrailed-Hoxb5" by Wai-chun, Cheng, 鄭維俊, 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 "Studying Hoxb5 functions in enteric nervous system development by dominant-negative repressor engrailed-Hoxb5" Submitted by Cheng Wai Chun for the degree of Doctor of Philosophy at The University of Hong Kong, in Feburary 2008 Enteric nervous system (ENS) is the autonomic nervous system of the gut that regulates the peristalsis and secretion of the intestine. In mammals, vagal neural crest cells (NCC) migrate to the foregut and colonize the entire gut in a proximal-distal direction in embryos. NCC proliferate and differentiate into enteric neurons and glia of the ENS. Defective ENS development in human results in Hirschsprung's disease (HSCR) which is characterized by the absence of enteric neurons and glia (aganglionosis) in the distal intestine and patients develop intestinal obstruction. Development of ENS is regulated by various signaling pathways, such as the Ret/Gfrα1/Gdnf pathway. Mutations of the genes implicated in the ENS development only account for about 60% of all the cases, indicating the existence of unknown disease-causing loci. Hoxb5 encodes a transcription factor Hoxb5 and belongs to the Hox gene family. Hox genes are highly conserved developmental control genes critical for body patterning and tissue specification. Hoxb5 protein contains a DNA-binding homeodomain and a transactivation domain. The expression pattern of Hoxb5 in vagal NCC correlates with the ENS development, suggesting an important function of Hoxb5 in the process. In Hoxb5 knockout mouse model, phenotype in ENS has not been reported, which could be accounted by the functional redundancy among the Hox family members. To elucidate the role of Hoxb5 in ENS development, the engrailed-Hoxb5 (enb5) dominant-negative transcription repressor approach was employed in the current study. The transactivation domain of Hoxb5 was replaced by the engrailed repressor domain derived from Drosophila. This dominant-negative form of Hoxb5 competed with the wild type Hoxb5 for the binding sites of the Hoxb5 downstream targets and suppressed their transcriptions. enb5 was specifically expressed in vagal NCC by Cre-mediated recombination using the vagal NCC specific b3IIIa-Cre transgenic mouse. The phenotypes of the enb5/b3IIIa-Cre double transgenic embryos and adults were analyzed to identify developmental pathways in the ENS development that normally required Hoxb5. Current study indicates that Hoxb5 is required in the vagal NCC migration during ENS development. enb5-expressing NCC failed to migrate to the small intestine, leading to a 34%-37% reduction of enteric ganglia (hypoganglionosis) in the intestine, and transgenic mice display defective peristalsis. Occasionally complete absence of enteric ganglia (aganglionosis) in the intestine was observed in these transgenic mice. Ret expression was markedly reduced or absent in the enb5-expressing NCC and ganglia. Furthermore, it was demonstrated in vitro that Hoxb5 induced Ret transcription and enb5 completely abolished the induction effect of Hoxb5. To conclude, Ret is a downstream target of Hoxb5 in vagal NCC. Perturbed Hoxb5 signaling leads to a downregulation of Ret and retarded migration of vagal NCC, which results in hypoganglionosis and aganglionosis in mice. The discovery of the functional role of Hoxb5 in ENS development in this study not only enriches our current u