| Summary: | The molecular complexity of cardiogenesis is revealed by the high incidence of heart phenotypes resulting from mutation to any of a vast number of genes across multiple pathways. Many proteins, in pathways critical for correct embryonic patterning, are thus also identified as playing a role in aspects of heart development. A developmental role for Sox4 has been shown in mouse cardiogenesis, as apparent by the phenotype of two published alleles. Both null and conditional null mutant alleles exhibit septation defects, oedema, and lethality by E14.5. However, the causative step in development, where a loss of functional Sox4 marks the beginning of the defect, has not been described; nor has it been uncovered whether mutation to Sox4 results in dysregulation of other aspects of embryonic patterning. This thesis documents the homozygous embryonic phenotype of two mouse lines, both harbouring an ENU-induced point mutation to the HMG-box of Sox4. In addition to the Sox4 mutation, the first mouse line examined also contains the wellcharacterised satin (sa) coat mutation to the Foxql gene, a mutation closely linked to Sox4 on chromosome 13. This mutation had been used as a phenotypic recessive marker in the ENU mutagenesis screen from which the Sox4 allele was isolated. Phenotypic analysis of the Sox4ENU/ENU Foxqlsa/sa phenotype reveals that the primary cardiac defect of Sox4 arises as early as E9.5. As the satin allele has been well characterised and does not result in cardiac dysmorphology, the cardiac phenotype observed can thus be attributed to the Sox4 point mutation. At this developmental stage, mutant embryos can be grouped into three phenotypic classes, which vary in severity. A phenotype is evident at early stages of development in the most severely affected embryos, such that only a proportion of homozygotes can be examined for the later cardiac phenotype. At E12.5-E13.5, the cardiac phenotype of embryos resembles that of the published Sox4 null allele. However, our analysis goes further to uncover abnormalities in the development of the atrial septum and atrioventricular structures. To expand on the understanding of this phenotype, a general analysis was conducted using key molecular markers, selected to reveal consequences of the phenotype. Following description of the phenotype a comprehensive analysis of the expression of Sox4 was carried out by wholemount and section in situ hybridisation. Since the mutant phenotype arises between gastrulation (for the most severe mutants) and E9.5 (for the mildest of mutants), Sox4 expression was examined from pre-gastrulation stages to E12.5 in wholemount, and E9.5 to E12.5 by section in situ to cardiac regions. The most significant expression of relevance to cardiogenesis is the identification of Sox4 transcripts in several domains. Early in development, expression is observed in the mesoderm lateral to the cardiac crescent, a region known to contribute to the posterior components of the heart tube, and the endoderm adjacent to the cardiac crescent and subsequent heart tube. Section analysis of the cardiac region of E9.5 embryos revealed the onset of specific expression of Sox4 in the endocardium of the outflow tract and atrioventricular canal, with expression also identified in the emerging cushion mesenchyme. Endocardial and mesenchymal expression in the AVC and OFT continue at stages E10.5, El 1.5 and E12.5. The spatiotemporal expression pattern of Foxql is also shown, and exhibits a degree of overlap with Sox4 in the endoderm and in anterior structures. Through an extensive breeding programme, it was possible to segregate the mutant Sox4 allele from satin. Phenotypic abnormalities evident in compound homozygotes but not in Sox4 homoygotes are convincing evidence of a genetic interaction between Sox4 and Foxql, although the precise nature of this remains unknown. The work carried out in this candidature provides novel evidence of the embryonic expression and function of Sox4. It has been possible to identify the origin of the cardiac defect caused by mutation to Sox4 as well as reveal additional aspects of an early phenotype resulting from loss of function of Sox4. Thus, this study provides a significant contribution to understanding the function of this gene.
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