Gibson, A.; (2007) Characterisation of three novel genes and their role in neural induction. Doctoral thesis , University of London.

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Abstract

In 1924, Spemann and Mangold (Spemann and Mangold, 1924) demonstrated the instructive effect of the vertebrate organiser, located in the dorsal lip of the blastopore in amphibia, in the acquisition of neural character during early development. This discovery led to much interest in the process of neural induction. More recently, a "default model" was proposed to account for neural induction, which postulates that ectoderm cells acquire a neural fate autonomously, if they receive no signal. However, this neural fate is inhibited by BMPs, which induce epidermis. The organiser in turn secretes BMP antagonists, which release neighbouring cells from inhibition. However, other results suggested that neural induction is more complex, comprising several sequential and/or parallel signals, which cooperate to induce neural fates. One such finding was that chick epiblast cells do not respond to BMP antagonists unless they are first exposed to an organiser for at least 5 hours, suggesting that other signals are required upstream of BMP inhibition. To identify the differences between cells that have or have not received such signals, a differential screen was performed. Among 15 genes isolated, only 3 correspond to previously known genes: Defender Against Cell Death (Dad1), Polyubiquitin II (Ubll) and Heavy chain ferritin (hcf). All 3 of these had been implicated in Programmed Cell Death (PCD). This project was designed to investigate whether PCD is important in neural induction and to study the role of these three proteins in both processes. First, the distribution of apoptotic cells was examined at different stages of development using TUNEL staining. It was found that PCD is random at first, but by the time the neural plate starts to be established PCD becomes concentrated at its lateral and anterior border. The expression patterns of the three genes were studied: all three are expressed in the neural plate around the time of neural induction, however they are not exclusive markers of the neural plate. The ability of the organiser (Hensen's node) to induce their expression within 5 hours was confirmed, and the ability of FGF signals to mimic this was also tested. The effects of the overexpression of the genes outside their normal expression domain were then examined, scoring both for changes in the distribution of PCD and for the expression of markers for neural, epidermal and border (prospective neural crest) territories. This revealed that overexpression of hcf leads to an increase in the number of cells undergoing PCD, but this effect is not cell autonomous. Finally, the expression patterns of the pro-apoptotic gene Cas9 and of the death effector Cas3 were tested and compared to the apoptotic patterns. An increase in PCD was observed in the region where expression of Cas3 and Cas9 overlaps and which is free from Dad1, consistent with the notion of an amplification loop between Caspase 3 and Caspase9, and with a possible involvement of DAD1 in the regulation of this process.

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