FIGURE 8.

Subset of nuclearized YAP positive cells are actively proliferating cells and are actively recruited to the developing bud. Immunofluorescence assay of Hvul_YAP performed using anti-CheYki antibody and proliferating cell population using Ki67 marker showing colocalization of a subset of YAP positive cells. The images shown here are maximum intensity projections. (A,A’) This panel shows the head and shoulder region of the polyp showing brightly stained nuclearized YAP expressing cells are not actively proliferating. The individual cells expressing YAP in the outer hypostomal region are also non-proliferative. The white square in figure (A′) is enlarged in the insets shown on the right of the figure. The top inset shows the tissue from a X-Y plane in the centre with orthogonal slices of Y-Z and X-Z plane indicated in right and below respectively. The region of interest wherein one of the cells showing colocalization is confirmed on the X-Y-Z axes. Middle inset shows the same image in green channel (YAP) and lower inset shows the red channel (Ki67). (B,B’) The budding polyps showing active incorporation of proliferative cells into the developing bud. These cells co-stain with Hvul_YAP suggesting their colocalization. Panel (B) clearly shows small and dense localization of actively proliferating cells being recruited into the newly forming bud in early stages. The panel (B′) shows a mature bud where the tentacles have started forming and the active recruitment of cells into the bud body has reduced. Hence, the Ki67 expressing cells are larger in size and less dense. (N = 3), Red: Ki67 & Green: YAP. (Scale bar: A and A’, 50 μm, B, 100 µm and B’, 90 µm).