Fig. 7

tbx2a functions upstream of tbx2b during pronephros segment and CS development. (A, B) Whole mount in situ hybridization was performed with tbx2a probe on 28 ss wild-type embryos and embryos injected with tbx2b MO. The tbx2a domain remained the same somite length despite the loss of tbx2b. In contrast, loss of tbx2a caused a notable decrease in the tbx2b domain. (C) The length of tbx2a and tbx2b expression domains of five representative embryos was measured (uM). While tbx2a expression is not significantly different wild-type and tbx2b morphants, the size of the tbx2b domain is significantly smaller than their wild-type siblings. (D) Fluorescent in situ hybridization was used to examine the distal region of the pronephros of tbx2a morphant embryos at the 28 ss, which revealed that stc1+ CS cells lacked tbx2b transcripts. DAPI (blue) was used to label nuclei. (E, F) A portion of embryos injected with tbx2b MO and tbx2a cRNA exhibited a normal number of CS cells, but the average number of CS cells was significantly larger than the wild-type controls. A more robust rescue was seen in embryos injected with tbx2a MO and tbx2b cRNA, which on average had a similar number of CS cells compared to wild-type embryos. (G, H) DL lengths were rescued in some embryos by tbx2a cRNA in tbx2b morphants, and by tbx2b cRNA in tbx2a morphants, such that the average DL lengths were not significantly different from wild-type controls. (F, H) CS cells and DL lengths were counted in each nephron in 10 individuals in each treatment. (*p<0.05, **p<0.001, N.S.=not significant). Abbreviations: MO (morpholino), cRNA (capped RNA), DL (distal late), CS (corpuscle of Stannius).