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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 {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.

Investigating the developmental processes of the human fetal heart is a challenging task. Few reports describe the morphological features during the first stage of heart maturation and consecutive developmental periods after cardiogenesis. Reasons for this include the difficulty of collecting suitable samples and the limitations of investigating modalities. This research was proposed to clarify the detailed morphological features of normal human fetal hearts in early-stage maturation, using post-mortem samples. These samples were analysed using high-resolution episcopic microscopy (HREM), and compared with the latest clinical imaging taken by 3D fetal echocardiography and compared with mouse samples. HREM, a newly-developed high-quality image modality, produces a computerbased 3D reconstruction which enables us to visualize detailed spatial structures of small specimens. HREM includes several procedural steps, which may affect the histological or morphological structures of samples, so I explored the potential effects. I found 12% shrinkage due to dehydration and polymerization. Therefore, while the general appearance of 3D reconstructed images looked identical to the pictures of the original heart samples, it is important to consider the effects of shrinkage when interpreting the morphological assessment by HREM. Normal human fetal hearts from the 9th to 11th weeks of postmenstrual gestation demonstrated unique morphological findings. Ventricular walls and trabeculations showed thick and random cellular structures. Atrioventricular and semilunar valves were also thick but histological maturation was observed within a few weeks after cardiogenesis. The great arterial walls were thick and comprised of dense cellular matrix. Morphologically, several characteristic findings, such as large atrial appendages, the developmental process of formation of the membranous ventricular septum and prominent coronary arteries, were recognised during this period. Heart size increased linearly with gestation. Normal human fetal hearts demonstrate geometrical development and histological and morphological maturation after the period of cardiogenesis. In comparison with human fetal hearts, mouse hearts demonstrate dramatic morphological alterations during a short maturation period. Fetal mouse hearts show some similar morphological findings to the human fetal heart, such as large atrial appendages, lack of formation of the membranous septum, and thickened great arterial walls. This suggests a shared mechanism of fetal heart maturation in mammals. Detailed clinical information regarding cardiac morphology is vital for accurate prenatal heart diagnosis in the first trimester. Fetal echocardiography in early gestation has become routine practice. However, the technical limitations of image acquisition and picture resolution make it difficult to visualize clear 3D images for fetal cardiac diagnosis. Current modalities for clinical investigation by 3D echocardiography do not have sufficient resolution to enable detailed morphological investigation of the human fetal heart between 10th to 12th weeks of postmenstrual gestation. Only the original data of the four-chamber view demonstrated no offsetting of the atrioventricular valves as seen on HREM. Further technical advances in 3D echocardiography will be required to enable precise cardiac diagnosis in the first trimester. This thesis describes morphological development in normal human fetal hearts for the first few weeks after cardiogenesis and contributes to a better understanding of the normal appearances in the first trimester which is vital for future investigation into the origin of congenital heart disease.