Fig. 3-S1

Mutations in gas6, mpp6, and plec do not affect CNS Mbp expression.

(A–E) CRISPR-Cas9 technology was used to generate mutations in zebrafish gas6 (A), mpp6a (B), mpp6b (C), pleca (D), and plecb (E). (A) A CRISPR/Cas9 system guide RNA (gRNA) designed to disrupt exon 2 (targeted sequence highlighted blue and SexAI restriction enzyme site used for genotyping denoted) of

gas6 generated gas6^stl228, a 4 bp deletion predicted to result in a frameshift and premature STOP. Alignments of wt and mutant nucleotide sequences (top), and predicted wt (706 aa) and mutant (78 aa) protein sequences (bottom) shown. (B) A gRNA designed to disrupt the 3^rd coding exon 5 (targeted sequence highlighted purple, and HpyCH4IV restriction enzyme site used for genotyping denoted) of mpp6a generated mpp6a^stl233, a 1 bp deletion predicted to result in a frameshift and premature STOP. Alignments of wt and mutant nucleotide sequences (top), and predicted wt (550 aa) and mutant (69 aa) protein sequences (bottom) shown. (C) A gRNA designed to disrupt exon 2 (targeted sequence highlighted green and BstNI restriction enzyme site used for genotyping denoted) of mpp6b generated mpp6b^stl234, a 6 bp deletion predicted to result in a two amino acid deletion and a single amino acid change within the receptor targeting domain. Alignments of wt and mutant nucleotide sequences (top), and predicted wt (539 aa) and mutant (537 aa) protein sequences (bottom) shown. (D) A gRNA designed to disrupt exon 3 (targeted sequence highlighted orange, and DdeI restriction enzyme site used for genotyping denoted) of pleca generated pleca^stl261, a 20 bp deletion predicted to result in a frameshift and premature STOP. Alignments of wt and mutant nucleotide sequences (top), and predicted wt (4752 aa) and mutant (103 aa) protein sequences (bottom) shown. (E) A gRNA designed to disrupt the 3^rd coding exon (targeted sequence highlighted yellow, and Hpy188III restriction enzyme site used for genotyping denoted) of plecb generated plecb^stl236, a 13 bp deletion predicted to result in a frameshift and premature STOP. Alignments of wt and mutant nucleotide sequences (top), and predicted wt (4530 aa) and mutant (111 aa) protein sequences (bottom) shown. (F– O) Whole mount ISH showing mbp expression (CNS denoted by black arrows) at 5 days post-fertilization (dpf) in (F) gas6 control larva (wt and gas6^stl228/+, N = 11/12), (G) gas^{stl228/stl228} mutant larva (N = 5/8), (H) mpp6a control larva (wt and mpp6a^stl233/+, N = 17/18), (I) mpp6a^{stl233/stl233} mutant larva (N = 5/7), (J) mpp6b control larva (wt and mpp6b^stl234/+, N = 24/24), (K) mpp6b^{stl234/stl234} (N = 4/4), (L) pleca control larva (wt and pleca^stl261/+, N = 18/18), (M) pleca^{stl261/stl261}mutant larva (N = 4/4), (N) plecb control larva (wt and plecb^stl236/+, N = 36/38), and (O) plecb^{stl236/st’236} mutant larva (N = 10/10). Dorsal views are shown for all, anterior to the left. No gross changes in mbp expression were observed in putative ligand mutants relative to sibling controls.