Proliferation in the palatal shelf epithelium. (A) For three embryos at three consecutive stages during ruga 5 addition (ED13.5), a graph is given correlating epithelium thickness and mitotic index along the anterior/posterior (A/P) axis of the palatal shelf. The individual measures of epithelium thickness correspond to black points, joined with a thick yellow line. The mitotic index of the epithelium was calculated for different regions as figured in grey (ruga region), white (gap region) or light grey (region of interest where ruga 5 appears). It is indicated with black bars. For embryo II, an extra mitotic index was also calculated for the anterior third (two red stars) and the posterior two-thirds (red star) of the region of interest and is indicated with red bars. (B) Proliferation along the A/P axis of the palatal shelf as visualized on sagittal cryosections with the KI67 antibody at ED13.5 (ruga 5 formed) for an embryo of similar weight as embryo III in A. Ki67 postive cells (pink) are proliferating cells, while Ki67 negative cells (blue, dapi nuclear staining) are quiescent cells. White brackets point the ruga3-ruga4 gap and the region between the last formed ruga (5) and ruga 8. (C) For an equivalent embryonic stage and sectioning plane as in B, sagittal section of an embryo after whole mount in situ hybridization with the Shh probe. (D) A dissected palatal shelf at ED13.5 (ruga 5 in formation) was stained with the nuclear marker Draq5TM. The cells are more densely packed in well-formed rugae, allowing their visualization. (E) The same palatal shelf was immunolabeled with Ki67 to show proliferation in the epithelium. Note that Ki67 negative cells are found mainly in rugae (especially in more mature rugae 1–3) and in the region immediately anterior to ruga 8 (compare labeling in the regions pointed by the two white arrows). (F) A littermate embryo of similar weight was hybridized with the Shh probe to help staging. The large space between ruga 4 and 8 confirmed that ruga 5 is already developing, as for embryo II in A.