Browsing by Author "Gill, Zoe"

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    Characterization of transcription factors and LncRNAs involved in the development of the bat wing
    (2016) Gill, Zoe; Illing, Nicola
    Mammals have evolved a vast myriad of limb morphologies adapted for a wide range of activities. One of the most remarkable evolutionary adaptations of a mammalian limb is that of the forelimb wing of a bat used for powered flight. This capability evolved ~ 51 Mya from its arboreal ancestor without any fossil record of intermediate forms. To reconstruct how this transition occurred, an Evolutionary Developmental approach can be applied to investigate altered mechanisms present in bat limb development. Similar genes and signalling centres are present in both mice and bats, making mice a good model organism for comparison. This study used a pre-existing set of RNA-seq transcriptomes from three pivotal developmental stages (CS 15, CS 16 and CS 17) of bat development, to compare FL and HL gene expression. Of the list of differentially expressed genes, a subset was selected to characterise spatial expression patterns within the developing bat limb compared to mouse limbs by whole-mount in situ hybridisation. Five transcription factors: Lef1, Lhx8, HoxA10, Mllt3 and Tbx5, as well as two Long non-coding RNAs: Hottip and Tbx5-as1 were selected. Novel expression of Mllt3 was detected in FL autopods at CS15, in a region slated to expand with digit elongation. Lef1 in situ signal was more robust in HL autopods of CS 15 embryos compared to FLs and equivalently staged mice. Lhx8 displayed a strong signal in CS 16 and CS17 wrist tissue, as well as a faint signal in interdigital tissue in the FL autopods. The LncRNA Hottip displayed vastly different expression pattern between FL and HL, with staining being reduced in the digit and interdigital regions of the FL at CS 16L, whereas expression in the HL was robust in the digit, and even more so in the interdigital regions. The LncRNA Tbx5-as1, displayed a similar expression pattern to the known FL initiation transcription factor Tbx5 at late stages (CS 16L -CS 17L). Isoform characterization to validate the two LncRNAs, was performed on cDNA a CS 18L embryo. The cloned transcripts identified a new set of alternatively spliced isoforms for both LncRNAs. Unusual RNA-seq tracks in the HoxA10 locus were investigated using qPCR. It was discovered this region is variable amongst biological samples; however there is a large reduction in expression in this region from CS 15 to CS 16.