Risebro, CA; (2006) The role of Hand1 in cardiac morphogenesis. Doctoral thesis , UCL (University College London).

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Abstract

Handl encodes a bHLH transcription factor, which is essential for both placentation and cardiac morphogenesis during murine development. Extraembryonic defects in homozygous mutant embryos have precluded detailed analysis of Handl function in the heart. To circumvent the Handl-null early lethality and gain insight into the precise function of Handl during vertebrate heart development the bimodal tetracycline (Tet- Off) system has been employed to generate temporally controlled loss of function and gain of function models for Handl. The Tet-Off system comprises a tet-off transactivator and tet response element (responder) whereby transactivation of the responder and gene of interest can be switched off following addition of doxycycline. The transactivator has been targeted to the endogenous Handl locus {lcub}Tet-Off Handl) ensuring it is expressed in a spatial and temporal pattern consistent with the endogenous gene and generated transgenic lines for the responder (Tre2-Handl). Compound heterozygotes in either Handl-null or wild type backgrounds enable us to temporally switch off or over-express Handl respectively. Transactivator and responder constructs for gene targeting and transgenics respectively have been tested extensively in vitro, and sent to a commercial company to generate the mouse strains. Germline transmission was achieved for both Tet-Off-Handl and two transgenic Tre2-Handl strains. The Tre2-Handl responder lines failed to express Handl at a level suitable to rescue the mutant phenotype when crossed with the Tet-Off- Handl driver on a Handl-null background thus preventing further manipulation via dox administration to pregnant mothers to turn off Handl at different embryonic stages. However, a third line suitably expressed Handl under the tet-system and is currently being crossed with the driver for determining rescue as a first step towards validating the loss of function model. The transactivator and responder lines have also been crossed to generate mice that over-express Handl. At E9.5, embryos that over-express Handl have an extended outflow tract, convoluted looping of the linear heart tube and a small, thick-walled ventricle, which lacks a defined lumen. Marker analysis reveals inappropriate cardiomyocyte differentiation and an over-proliferation in the affected areas with a lack of ballooning in the presumptive left ventricle. In vitro differentiation studies have also been performed in ES cell lines that stably and precociously over-express Handl to complement the in vivo model.

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