Fig 5

Fig 5

A-B) Box-plots with the quantification of mitotic figures within the LRL atoh1a:GFP cells (A), and the total number of LRL atoh1a:GFP cells (B), in atoh1a^WT and atoh1a^fh282 embryos. C-D) atoh1a^WT and atoh1a^fh282 embryos in the Tg[atoh1a:GFP] background were analyzed for apoptotic figures by TUNEL. Note that no differences between wild type and mutant embryos were observed (Table 2 for values and statistical analysis). E-G) atoh1a^WT (n = 8) and (H-J) atoh1a^fh282 (n = 10) embryos in the Tg[atoh1a:GFP] background were concomitantly analyzed for atoh1a (E, H), atoh1a-derivatives visualized with anti-GFP staining (F, I) and ptf1a (G, J) expression. E’-J’) Reconstructed transverse views of dorsal views displayed in (E-J) at the level of the otic vesicle. Note that the atoh1a:GFP cells in the atoh1a^fh282 mutant did not migrate towards the differentiation domain and did not display ptf1a (see white arrow in H’-J’), indicating that progenitor cells did not switch fate. K-L) Time-lapse stills showing delamination from the LRL of tracked atoh1a:GFP cells (indicated with white asterisk) in atoh1a^WT (n = 28) and atoh1a^fh282 (n = 12) embryos in the Tg[atoh1a:GFP] background. Dorsal views of hemi-neural tubes (dashed white line indicates the apical region of the hindbrain), with anterior to the left and lateral at the top. Numbers at the top-right indicate the minutes after the beginning of the movie. Note that in wild type embryos, the cell delaminates and migrates towards ventral, allocating in the corresponding neuronal differentiation zone (see the first three dorsal frames and then the following ventral ones), whereas in atoh1a^fh282 embryos the indicated cell remains within the dorsal epithelium (see that there are four dorsal and two medial frames because the cell never reaches ventral). M) Box-plot indicating the time of delamination from the LRL of atoh1a:GFP cells in atoh1a^WT and atoh1a^fh282 embryos. Note that cells from wild type embryos exit the LRL much earlier than the cells from mutant siblings. Since the atoh1a^fh282 mutant allele only caused a deleterious phenotype in homozygosity, wild type and heterozygous conditions showed identical phenotypes and they were displayed as single wild type condition. nt; neural tube lumen; ov, otic vesicle. Scale bars correspond to 50 μm. ns, non-statistically significant; *** p<0.001.