Digit formation during embryonic development of bats and mice

Master Thesis


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University of Cape Town

The evolution of a strikingly elongated and webbed FL in bats, which contrasts with a small, free-toed HL, has seen extensive research into bat wing development in an effort to determine the molecular mechanism driving limb development. A recent RNA-seq and ChIP-seq study carried out on M. natalensis showed differences in FL and HL activity for several genetic pathways known to be involved in bone formation during key bat development stages CS15-CS17. In this project the prediction made from the literature and the RNA-seq results was that the observed decreased Wnt/β-catenin signalling and increased BMP signalling in the bat FL may lead to elevated levels of Sox9 expression, and larger fields of mesenchymal condensations. This was tested by annotating Sox9 in the M. natalensis genome to further analyse the expression levels and associated ChIP-seq data. In addition the behaviour of condensing mesenchymal cells during bat and mouse limb development was observed by visualising the various stages of chondrogenesis, using H&E and PNA stains. In addition the RNA-seq study found 3000 genes to be differentially expressed. Thus, the project also set out to create an immortalised bat autopod cell line to facilitate future testing and predictions. The Sox9 gene was successfully annotated and revealed to not be differentially expressed between FL and HL as predicted. However downstream targets of Sox9 were further inspected as potential ideas for further investigation. The histological stains provided a set of data characterising mesenchymal condensation in both mouse and bat stages, revealing many interesting features such as the non-specific binding behaviours of PNA prior to digit formation. In addition, quantitative results demonstrated the bat FL digits are already longer than the HL digits at CS16. Cell line work established a working protocol for the storage, dissociation and plating of bat primary cells that retain their bat limb expression identity. Mouse cells were successfully immortalised and a cell line was established from a HL digit cell. This project has facilitated further studies in understanding extreme digit elongation in the bat FL autopod during development.