Modelling adipose tissue expansion in zebrafish

Obesity has been classified as a global epidemic by the World Health Organisation, with 13% of the world’s adult population estimated to be obese in 2016. An increased risk of multiple diseases is associated with obesity, including cardiovascular disease and type two diabetes. Obesity itself is defined as an excess of white adipose tissue (AT) and a body mass index of over 30 kg/m2. The excess adipose tissue present in obesity can become dysfunctional and display increased hypoxia, fibrosis, and necrotic adipocyte death. It is AT dysfunction which is thought to be responsible for obesity associated disease and the primary cause of AT dysfunction is a limited capacity of adipose tissue to expand. Obesity and by proxy adipose tissue expansion are known to be partially genetically regulated, with heritability estimates ranging from 40% to 70%. In order to identify the genetic variation underlying obesity, genome wide association studies (GWAS) have been utilised. Multiple GWAS studies, including a study into BMI by Kichaev et al (2019), have found that genetic variation at the FOXP1 locus is associated with adiposity levels. However, whether FOXP1 directly regulates adipose levels is entirely unknown. Here I generated a series of predicted loss of function foxp1 alleles in zebrafish. My goal was to determine whether foxp1 influenced AT growth and expansion in vivo. To this end a CRISPR/Cas9 system was employed to generate stable foxp1 mutant zebrafish. Changes in AT expansion were then assessed in the F2 generation, with live imaging of total AT area providing information on changes in growth dynamics between wild type and mutant fish. Mutant fish were found to have reduced levels of adipose tissue during normal development and also accumulated less AT in response to a high fat diet. In addition to generating foxp1 mutants I also generated a novel transgenic line which labels both adipocytes and adipose progenitor cells. Using the transgenic line, I performed high resolution in vivo imaging of zebrafish AT and compiled novel information about adipose progenitor cell behaviour. Adipose progenitor cell behaviour was also assessed in foxp1 mutant zebrafish. In summary, I have tested the role of foxp1 in adipose tissue expansion and found evidence to suggest that foxp1 is required for adipose tissue expansion. I have also generated a novel transgenic line which has allowed for unprecedented in vivo imaging of adipose tissue expansion. This is the first data to suggest that foxp1 plays an in vivo role in adipose tissue expansion. Further investigations into the mechanisms by which foxp1 influences AT expansion could lead to the creation of prediction strategies to prevent the development of unhealthy obesity

Mapping and Phenotyping Genes in the Del(13)Svea36H Region of Mouse Chromosome 13

Del(13)Svea36H (also known as Del36H) is a 12.7Mb gene-rich region that has been deleted in mouse chromosome 13. It contains numerous disease loci as well as showing conserved synteny to two regions of human chromosome 6p which can be missing in some deletion syndromes. The mouse deletion causes homozygous embryonic lethality but is hemizygously viable. It has therefore been used as a tool to screen for lethal recessive mutations that could help to identify developmental genes that are responsible for the symptoms of the associated human diseases. A Physical map of the region was constructed in order to facilitate the mapping of mutations arising from the lethal screen. Eleven lethal lines have been mapped to varying degrees of accuracy, including one lethal mutation that exhibited circulation defects which localised to Sox4. The forkhead transcription factor Foxf2 was considered a potential candidate for embryonic lethal mutations within the interval. To complement the lethal recessive screen, an attempt was made to identify an allelic series of mutations in the gene. ENU mutagenised mouse archives were screened for novel Foxf2 mutations. Three potential functional mutations were discovered - two coding mutations (Foxf2W174R and Foxf2V412F), and a splice site mutation. Foxf2W174R heterozygotes exhibited thinning of the iris stroma, alongside hypoplasia of the trabecular meshwork and canal of Schlemm and a reduction in the iridocorneal angle. Homozygote eyes at E18.5 show no signs of the developing ciliary body which were seen in wildtype littermates. Homozygote carriers survive for several days, significantly longer than the 18hr survival time observed in the knockout, suggesting a hypomorphic mutation. In contrast to Foxf2 knockouts, homozygotes for the W174R mutation do not possess a cleft palate or malformed tongue