Fig. 1. Expression of the lens induction marker Pax6 is dependent on Fgfr kinase activity. (A,B) Mouse half-heads from P6 5.0-lacZ embryos were explanted at E8.5 and cultured for 8 hours either in the absence (A) or presence (B) of 10 µM SU9597. At this stage, X-gal staining reveals only a modest reduction of reporter construct expression in SU9597-treated explants. (C-F) Mouse half-heads from P6 5.0-lacZ embryos were explanted at E8.5 and cultured for 24 hours either in the absence (C,D) or presence (E,F) of 10 µM SU9597. X-gal staining reveals that at this stage, there is a dramatic reduction in reporter construct expression levels in the lens placode in presence of SU9597 whether assessed in whole-mount (compare C with E) or in section (compare D with F, arrowheads). All explants shown in A-F were derived from the same litter of P6 5.0-lacZ embryos. (G-J) Mouse half-heads from wild-type embryos were explanted at E8.5 and cultured for 24 hours either in the absence (G,H) or presence (I,J) of 10 µM SU9597. Frozen sections of the explants were labeled with ant-Pax6 antibodies using indirect immunofluorescence. Both the green and yellow colors show Pax6 immunoreactivity, but the yellow shows the peak signal intensity. This indicates that Pax6 levels are reduced in explants treated with the Fgfr kinase inhibitor. This is true for the lens placode (arrowheads) but is also apparent in the optic vesicle (ov). (K,L) As in G-J, but explants were performed at E9.5 and allowed to proceed for 24 hours. In this case, it is apparent that SU9597 reduces the level of Pax6 immunoreactivity, particularly in the deepest epithelium of the lens pit (lp). The lens pit of the treated explant (L) is also narrower than in the control (K). Explants in A-C,E are shown at the same magnification. All panels are labeled with the approximate stage of development reached at the end of the explant period.
