Piątkowska, Agnieszka Małgorzata; (2021) Cellular Dynamics of Somite formation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In amniote embryos, the process of segmentation generates epithelialised somites sequentially, in head-to-tail order, at regular time intervals. The pattern of somites is fundamental for the organisation of the adult segmental body plan as it guides the associated pattern of peripheral nervous system elements (nerves, neural crest cells and ganglia) and generates the skeletal musculature as well as the vertebral column. Several models of somite formation have been proposed but surprisingly, most of these do not provide clear links between the molecular mechanisms and the cell behaviours that generate the segmental pattern. Here, the cellular dynamics of the pre-somitic mesoderm (PSM) is studied, including mesenchyme-to-epithelial transition, as indicated by changes in aspect ratio of the cells, and changes in expression of cell polarity markers. Sagittal and transverse sections of embryos examined by scanning electron microscopy revealed that epithelialisation within the PSM is gradual and begins much earlier than previously thought. Each edge of the PSM starts to epithelialize at a characteristic position in space and time. The dorsal domain epithelializes first at 40% of the length of the PSM (measuring from the caudal end), followed medially at 55% PSM and then by the ventral and lateral sides, which epithelialize at 70% PSM. After a somite buds off from the PSM, its cells continue to elongate. Initially the dorsal and ventral walls are epithelialized to different extents, and there is no significant difference between anterior and posterior domains. Then cells on each side continue to elongate until full epithelialisation is reached. The core cells of the PSM and somites never epithelialise, which suggests that the core cells could be ‘trapped’ within the somitocoele after cells in each PSM domain elongate fully. Whether or not core cells have rostro-caudal identity (as does the rest of the somite) was studied with in situ hybridisation using markers like Uncx4.1, Meso2, Eph4A, LFng, Hairy1. The core cells of the PSM express Meso2, EphA4, Hairy1 and LFng, but the core cells of somites do not, which suggests that core cells may have initial rostro-caudal identity but later lose it. The caudal somite marker Uncx4.1 is expressed by core PSM cells. 3D confocal analysis of cell polarity markers PKCζ, PAR3, ZO1, Golgi marker GM130 and apical marker N-cadherin revealed that the pattern of polarisation is 3 characteristic for each marker and each PSM domain and does not follow the pattern of cell elongation described above. This observation suggests that epithelialization and polarisation may be regulated independently of each other, but this requires further study. The epithelialisation and cell polarity patterns observed within the PSM challenges the most popular model for somite formation: ‘the clock and wavefront model’. This model proposes that a group of PSM cells which experience the ‘clock’ and passing ‘wavefront’ form a somite together in a catastrophic fashion by budding off from the PSM together. It was previously proposed that segmental information resides in the medial- PSM (M-PSM) and is passed to the lateral-PSM (L-PSM). By rotating M-PSM 180° no change in segmentation pattern of L-PSM was observed. Also, M-PSM removal did not cause loss of L-PSM segmentation, suggesting that both PSM halves contain segmental information and can form somites autonomously. Also, when a small posterior-M-PSM fragment was rotated 180° it formed somites, but its rostro-caudal identity was shifted to match the host’s somites rostro-caudal identity. This could mean that there are two independent mechanisms, one for segmentation and the other for somite polarity.

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