Fig. 5
Ubiquitous and cell-type specific Cre-mediated conditional rescue with rbbp4-off. (A) Diagram of expected Cre mediated inversion of rbbp4off to on orientation. (B) Gross morphological phenotype of microcephaly and microphthalmia in 5 dpf transheterozygous rbbp4off/?larva. (C) Cre injected 5 dpf transheterozygous rbbp4off/?4larva shows rescue of gross phenotype and loss of mRFP expression. (D) Activated caspase-3a labeling throughout midbrain and retina section from 2 dpf transheterozygous rbbp4off/?4embryo. (E) Absence of activated caspase-3a labeling in midbrain and retina of 2 dpf transheterozygous rbbp4off/?4embryo after Cre injection. (F) Quantification of caspase-3a labeling in control rbbp4off/?4(n=3) and Cre injected rbbp4off/?4 (n=3) midbrain (** p<0.01) and retina (**** p<0.0001). (G) Genomic DNA qPCR quantification of rbbp4off original orientation 5? and 3 junctions in control rbbp4off/?4 (n=3) and Cre injected rbbp4off/?4 (n=3). Cre injection reduced the level of rbbp4off original orientation 5? (>93%) and 3? junctions (>93%). (H ? J?) Activated caspase-3A and Cre labeling in sectioned head tissue from 2 dpf rbbp4off/?4 (H-H"), ascl1b-2A-Cre; rbbp4off/?4 (I-I"), and neurod1-2A-Cre; rbbp4off/?4 (J-J") embryos. (K) Quantification of caspase-3a labeling in rbbp4off/?4(n=3), ascl1b-2A-Cre; rbbp4off/?4 (n=3) and neurod1-2A-Cre; rbbp4off/?4 (n=3).rbbp4off/?4vs. ascl1b-2A-Cre; rbbp4off/?4 midbrain (n.s. p=0.3248) and retina (n.s. p=0.8153), and neurod1-2A-Cre; rbbp4off/?4 midbrain (n.s. p=0.7794) and retina (n.s. p=0.9365). OT, optic tectum; R, retina; Th, thalamic region. Error bars represent mean ± s.e.m. with two-tailed t-test. Scale bars: 200 ?m (B, C), 50 ?m (D, E, H ? J). 10 ?m (H? ? J?).
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