Fig 4

Fig 4 pomp and psmd6 mutant zebrafish larvae show cardiac and extracardiac phenotypes.

A. Schematic illustrating pomp gene structure and CRISPR deletion. pomp exons are shown as boxes and the start codon is labeled. Guide RNA target sites are labeled in green and orange. DNA sequencing of the pomp^scm41 strain showed the pomp gene sequence between the two guide RNA sites has been deleted. B. Schematic illustrating psmd6 gene structure and CRISPR deletion. psmd6 exons are shown as boxes and the start codon is labeled. Guide RNA target sites are labeled in blue and red. DNA sequencing of the psmd6^scm40 strain showed the psmd6 gene sequence between the two guide RNA sites has been deleted. C.-D. qRT-PCR analysis of (C) pomp and (D) psmd6 expression in 4 dpf phenotyped pomp and psmd6 control (presumed +/+ and +/-) or mutant (mut, presumed -/-) larvae. Each dot represents a replicate batch of embryos. Bars represent mean +/- SEM. N = 5 (control) or 4 (mutant) replicates. Each replicate consists of 10-20 embryos. **** P < 0.0001. E. Western analysis using anti-Ubiquitin and anti-beta Tubulin (as a loading control). Larvae from pomp and psmd6 clutches were phenotyped as control (presumed +/+ and +/-) or mutant (presumed -/-) larvae at 4 dpf and collected for lysates. Lysates were prepared from 3 replicate pools of animals for each condition, with n = 20 animals per replicate. Larvae from bortezomib or DMSO control treatments were collected at 4 dpf, with n = 20 animals per lysate. Molecular weight markers are shown. F.-K. Images of live 4 dpf larvae. The asterisk in F marks the swim bladder. Arrowheads in H, K point to heart cavity swelling/edema. Black arrows in H, K point to reduced craniofacial structures. White arrows in H, K point to areas of blood pooling. Embryos were genotyped. N = 3 pomp^+/+, 12 pomp^+/-, 9 pomp^-/-, 4 psmd6^+/+, 8 psmd6^+/-, 6 psmd6^-/-. Scale bars = 500 μm. L.-Q. Ventral views of hearts labeled with myl7:EGFP in live 4 dpf larvae with pigment inhibited. R.-S. Ventral views of hearts labeled with myl7:EGFP in formaldehyde-fixed 4 dpf larvae with pigment bleached. Scale bars = 100 μm. V, ventricle. A, atrium. T. Graph showing frequencies of heart defect categories observed in 4 dpf larvae. The total number of larvae scored is given at the base of the columns. Embryos were genotyped. U. Survival plot of two independent clutches each of pomp and psmd6. Dead embryos were collected and fixed over the course of the experiment; all embryos were genotyped at the conclusion of the experiment. Control (+/+ and +/-) curves are shown for only one clutch of each line; similar results were seen with the second clutch of controls for each line. Numbers of animals per clutch: pomp clutch 1: 40 +/+ , 100 +/-, 52 -/-; pomp clutch 2: 44 +/+ , 93 +/-, 61 -/-; psmd6 clutch 1: 60 +/+ , 107 +/-, 55 -/-; psmd6 clutch 2: 27 +/+ , 61 +/-, 22 -/-.