Extended Data Fig. 4: Carbonic anhydrase orthologue expression in zebrafish.
From: Carbonic anhydrase inhibition ameliorates tau toxicity via enhanced tau secretion
a. Images of agarose gels with PCR products amplified from cDNA generated using RNA from 9 d.p.f. rho:eGFP–tau-WT fish. The endogenous expression of carbonic anhydrases ca2a, cahz, ca4a, ca4b, ca4b, ca5, ca9 and ca14 was assessed in whole fish versus dissected eyes (100 bp DNA ladder used). All isoforms except ca4b and ca4c could be detected in eyes and hence suitable to target in tauopathy retinal models (see green mark for isoforms band detected and a red cross for absence of PCR product at the right size indicated by a red arrowhead). b. Images of agarose gels with PCR products amplified from cDNA using RNA from FAC-sorted neurons from fish with pan-neuronal expression of Dendra-tau-A152T. The endogenous expression of carbonic anhydrases in ca2a, cahz, ca4a, ca5, ca9 and ca14 was assessed by rt-PCR (1 Kb DNA ladder used). All isoforms were detected in neurons and hence suitable to be targeted in the pan-neuronal Dendra-tau model. c. qPCR results showing the decreased expression of cahz, ca2a, ca4a, ca5, ca9 and ca14 in whole fish after injection of CRISPR guide RNAs targeting those genes (RNA collected from pools of 10 embryos from 5 independent clutches run in triplicate; *p≤ 0.05, **p≤ 0.01; ***p≤ 0.001 and ****p≤ 0.0001 vs. uninjected siblings analysed by two-tailed unpaired t-test). d. Images of western blots showing the decrease in ca4 and ca9 protein after CRISPR/Cas9 injection in wild-type fish. A minimum of 3 independent clutches were run for each group in a single gel. e. Representative images of cryosections across the central retina of transgenic rho:eGFP–tau-WT injected with CRISPRs targeting carbonic anhydrases cahz, ca2a, ca4a, ca5, ca9 and ca14 quantified in Fig. 2d. The knockdown of cahz, ca4a, ca9 and ca14 resulted in an increased number of fluorescent photoreceptors that is, reduced degeneration. Scale bar represents 50 μm.
