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Supplementary figure 3: Suppression of otud3 function interfered with neurogenesis and BMP-signaling. A) Embryos injected with 2.5pmol otud3 morpholino developed smaller heads with smaller and dysplastic eyes, accompanied by a generally non-regular expression of pax6 and n-beta-tubulin within remaining neural territories. B-D) Basal and bmp4-induced expression of neural marker genes in animal cap explants. A) Semi-quantitative RT-PCR analysis of relative ncam1 expression after injection of synthetic RNA encoding bmp4 and otud3. Expression of xbra was monitored to exclude mesoderm induction. RNA input was controlled by analyzing odc1 expression. C) Quantification of relative ncam1 and foxd3 expression by sqRT-PCR. Increasing doses of bmp4 RNA led to decreased expression of ncam1 (1, 2). Co-injection of otud3 RNA opposed bmp4 effects (3, 4). Untreated caps (5) and whole embryos (WE) were included as controls. D) Raw data. The effect was followed in three independent experiments. Each set showed a clear suppression of bmp4 signaling upon otud3 co-expression as monitored by the induction of the neural marker gene ncam. Comparable bands (a-d) were analyzed by ImageJ and evaluated using TTEST (Excel). In two experiments bra expression was analyzed to exclude an indirect effect on neural induction by mesoderm formation. Shown are the original images taken with a BioRad2000. Bands outside the spotted rectangle represent unrelated controls. The only adjustment was to inverse the color for a better illustration. The calculation revealed significance (2-sided, heteroskedastic; <0,05=*, <0,01=**; <0,001=***). |
