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Fig. 5. Loss of function of Dlx1/Dlx2 results in a loss of late-born RGCs in the developing retina. (A,B) Expression of BRN3b in 7-day-old explant cultures of wild-type and Dlx1/Dlx2 mutant retinas collected at E18.5. BRN3b immunoreactivity is detected in wild type (A, box, insert) but not in mutant explant cultures. (C-T) BrdU birthdating assays identify retinal cells generated at the time of pulsing. (C-E,L-N) BrdU pulses at E12.5 label RGCs as identified by BRN3b protein expression in mutant (C, arrows) and wild-type (L, arrows) retinas collected at E18.5. (F-H,O-Q) E13.5 BrdU pulses also readily identify RGCs in both mutant (F, arrows) and wild-type (O, arrows) retinas. (I-K,R-T) BrdU pulses at E16.5 in mutant and wild-type retinas. Co-expression with BRN3b can be seen only in the wild-type retina (R, arrow). (U) Quantification of BRN3b-expressing RGCs born at E12.5, 13.5 and 16.5. RGCs born at E12.5 (asterisk) represent 10% of RGCs labeled in E18.5 wild-type retinas, but 16% of the RGCs in Dlx1/Dlx2 mutants yielding a significantly larger proportion of the population at E18.5 (t=8.8, P<0.05, n=5). RGCs born at E13.5 (cross) form a significantly larger proportion of the population of RGCs in wild-type retinas compared with mutants (15 versus 13.3%; t=3.32, P<0.05, n=5). RGCs born at E16.5 (#) form a significantly larger proportion (~3 fold) of the population of wild-type retinas than mutant retinas (1.5 versus 0.6% of E18.5 population; t=4.85, P<0.05, n=5).





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