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The Cannabinoid Receptor Interacting Protein 1 (Cnrip1) was discovered as an interactor with the intracellular region of Cannabinoid Receptor 1 (CB1R, also known as Cnr1 or CB1). Functional assays in mouse show cannabinoid sensitivity changes and Cnrip1 has recently been suggested to control eye development in Xenopus laevis. Two Cnrip1 genes are described in zebrafish, cnrip1a and cnrip1b. In situ mRNA hybridisation revealed accumulation of mRNA encoding each gene primarily in brain and spinal cord, but also elsewhere. For example, cnrip1b is expressed in forming skeletal muscle. CRISPR/Cas9 genome editing generated predicted null mutations in cnrip1a and cnrip1b. Each mutation triggered nonsense-mediated decay of the respective mRNA transcript. No morphological or behavioural phenotype was observed in either mutant. Moreover, fish lacking both Cnrip1a and Cnrip1b both maternally and zygotically are viable and fertile and no phenotype has so far been detected despite strong evolutionary conservation over at least 400 Myr.
Figure 1. Cnrip1 genes in zebrafish. (a) Amino acid sequence alignment of Cnrip1a and Cnrip1b with the two splice variants of the single human CNRIP1 gene, named CRIP1a and CRIP1b. Distinct exons are shown in alternating black and blue font (red indicates amino acids coded by two exons). Comparing both zebrafish genes and CRIP1a asterisk indicates identity and colon similarity between all three, full stop similarity between any two. Bold indicates identity between CRIP1a and CRIP1b. (b) Synteny of adjacent genes in vertebrate CNRIP1 loci. Colours link homologous genes.
Figure 2. Accumulation of mRNAs from cnrip1a and cnrip1b genes. Whole mount in situ mRNA hybridisation of embryos at the indicated stages for antisense probes to cnrip1a (a) and cnrip1b (b). Lateral views (first two images in each panel) are anterior to top dorsal to left, except 24 hpf in which anterior is to left and dorsal to top. Dorsal views (remaining images) are anterior to left flatmounts (a) or wholemount (b), except panel a top right, which is a wholemount montage with anterior to top right. Sense control in inset in panel a top right is anterior to right dorsal to bottom. Quantitative evidence of reproducibility is given in Table S1. tel telencephalon, FB forebrain, MB midbrain, HB hindbrain, SC spinal cord, pf pectoral fin. Barsâ=â100âµm.
Figure 3. Mutagenesis of cnrip1a and cnrip1b. (a) Alignments of wild type with each of three mutant alleles showing the predicted expressed mutant polypeptide beneath. Bold underline indicates the gRNA target, red font the protospacer motif recognised by Cas9, hyphens deleted bases, blue highlight inserted bases and asterisks novel stop codons. In the mutant protein sequences, bold text indicates the residual wild type fragment and normal font the aberrant polypeptide tail. (b,c). To test for nonsense-mediated decay, about 50 siblings from in-crosses of cnrip1a
+/kg98 (b) and cnrip1b
+/kg101 (c) mutant carriers were subjected to in situ hybridisation for the cognate mRNA at the indicated stages, photographed, DNA extracted, PCR performed across the mutant locus and genotype confirmed by DNA sequencing as indicated beneath each panel. Quantitative evidence of reproducibility is given in Table S1.
Figure 4. Maternal zygotic double cnrip1a
kg98
;cnrip1b
kg101 mutant fish develop normally. (a) Schematic of crosses to generate a cnrip1 double mutant. (b) Darkfield image of 1 dpf double mutant indicating normal complex CNS folds (white arrowhead). (c) Differential interference contrast image of 1 dpf double mutant with arrowheads indicating normal eye (red), ear (purple), notochord (cyan) and haematopoetic tissue (black). (d) Dorsal, lateral and oblique (inset) views of 8 dpf larvae with arrowheads indicating pectoral fins (blue), jaw (turquoise), food traversing gut (green), swim bladder (orange) and xanthophores (yellow). Quantitative evidence of reproducibility is given in Table S1. All fish shown with anterior to left and dorsal up. Barsâ=â200 μm.
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