Fig 1.
CRISPR/Cas9 strategy to reduce Pcdhg isoform diversity.
A) A schematic of the clustered Pcdh loci with Pcdha, Pcdhb and Pcdhg arrayed in tandem. Constant exons for Pcdha are in gray and those for Pcdhg in black. The homologous C variable (V) exons are indicated by purple boxes. B) The Pcdhg cluster is comprised of 22 V exons and 3 constant exons. The V exons are subdivided into γA, γB and γC groups based on sequence homology. sgRNA were designed to target each V exon (red asterisks). As shown for Pcdhgb2 as an example, each V exon has its own promoter, and upon transcription, is spliced to the three constant exons. In the resulting protein, the extracellular domain (cadherin repeats EC1-EC6), the transmembrane domain (TM) and variable cytoplasmic domain (VCD) are encoded by the single V exon, while the C-terminal constant domain (Con) is encoded by the three constant exons. C) Examples for Pcdhgb2 and Pcdhga11 indicate the general design strategy for sgRNA, targeting downstream of and proximal to the start codon. An example mutation in Pcdhga11 (generated in the Pcdhgem6 allele) leads to a premature stop codon. D) The workflow for generating an allelic series of mutants with reduced Pcdhg isoform diversity. Values indicate the number of mice–total or from each sgRNA injection concentration–screened at each step.
Table 1.
Summary of Pcdhg exons disrupted in CRISPR/Cas9 targeted strains.
Individual mouse lines are summarized with the allele name along with the name used to describe the lines analyzed here. Red boxes indicate disrupted exons–"X” for discrete frame-shifting indel and dashed lines for large scale rearrangements or deletions between the indicated exons. “IF” indicates an in-frame indel predicted to result in an expressed protein.
Fig 2.
Three new Pcdhg alleles with reduced isoform diversity via CRISPR/Cas9 genome editing.
Schematics of the mutated Pcdhg alleles verified or identified by whole genome sequencing in three strains illustrate the range of mutations induced by CRISPR/Cas9 targeting. A) In 13R1 mutants, frame-shifting indels disrupted 6 exons (upward triangle indicates insertion, downward indicates deletion, red indicates frame-shift) and an inversion and deletion disrupted exons A9, B6, and A10. B) In 3R1 mutants, there was a small frame-shifting insertion in exon B2 along with multiple deletions and rearrangements between exons. For example, genomic DNA between the exon A5 and exon A6 guide sites was inverted and inserted at the B6 guide site, followed by coding sequence from the gene Anp32a (purple box) and a transposable element (green box). Only exons C3, C4, and C5 remain intact. C) In 3R2 mutants, a single large deletion resulted in a fusion between guide sites at exon A1 and exon A11. This was accompanied by small, frame-shifting deletions in exons A12 and C5 and a small in-frame deletion in exon C4 (downward pointing green triangle).
Fig 3.
Pcdhg isoform expression reflects genomic locus status in CRISPR/Cas9 mutant lines.
A) Quantitative RT-PCR analysis of cerebral cortex cDNA from 13R1 mutants (red), 3R1 mutants (blue) and 3R2 mutants (green) verified that intact isoforms were expressed at expected levels. Expression of constant exons was significantly reduced in 3R2 mutants only. 13R1 mutants were analyzed at P0, while 3R1 and 3R2 animals were analyzed at P14, each with littermate controls (white). * = p < 0.05; ** = p < 0.01; *** = p < 0.001 by Tukey post-hoc test comparing the indicated genotype with wild type. n = 3–9 animals per genotype. Box plots represent the median, first and third quartile, range, and outliers. B) Western blots of brain lysates from 13R1 (at P0), 3R1, and 3R2 mutants (at 1 month of age) with littermate controls confirm the protein isoform expression predicted by allele sequencing. Arrowhead indicates the specific γB2 band at the predicted size; asterisk indicates non-specific background band of incorrect size present in adult samples only. C) Total γ-Pcdh protein levels were quantified using Li-Cor quantitative western blotting, normalizing to GAPDH. Values from three samples per genotype were compared to littermate controls using student’s t-test with the indicated p-values.
Fig 4.
Spinal interneurons undergo excessive apoptosis in 13R1, but not 3R1 mutants.
Cryosections from P0 spinal cords were immunostained for the indicated markers (green; DAPI counterstain for nuclei, blue). NeuN staining in cryosections from P0 spinal cords of (A) wild type, (B) 13R1 mutants, and (C) 3R1 mutants revealed that 13R1 mutant cords were smaller with substantial loss of ventral interneurons. (D-F) Comparison of GFAP-positive astrocytes within the ventral spinal cord showed reactive gliosis in (E) 13R1, but not in (D) wild type or (F) 3R1. Specific interneuron populations were quantified, including (G-I,P) FoxP2+ and (J-L,Q) Pax2+ ventral interneurons. Both populations were significantly reduced in 13R1 mutants compared to wild type or 3R1 animals (P, Q). The horizontal white line in J indicates the divide used to quantify ventral interneurons. M-O) Staining for cleaved caspase 3 (CC3) reveals more apoptotic cells in (N) 13R1 than in (M) wild type or (O) 3R1 mutants (quantified in R). Scale bar is 200 μm. ** = p < 0.01; *** = p < 0.001 by Dunnett’s multiple comparison test comparing the indicated genotype with wild type. n = 32–48 hemicords total from 3 animals per genotype. Box plots represent the median, first and third quartile, range, and outliers.
Fig 5.
Substantial loss of retinal thickness without layer disorganization in 13R1 mutants.
A-C) Cryosections taken in cross section through retinas of (A) wild type, (B) 13R1/cRKO, and (C) 3R2 mutants at two weeks of age were immunostained for SV2 to label the synaptic layers (outer and inner plexiform layers; OPL, IPL) and DAPI to label the inner and outer nuclear layers (ONL, INL) and retinal ganglion cell layer (RGL). The IPL and INL were both notably thinner in 13R1/cRKO than in wild type or 3R2 mutants. This was not accompanied by disorganization of neuronal subtype stratification within the IPL, as revealed by (D-F) Melanopsin and TH immunolabeling of ipRGCs and dopaminergic amacrine cells, respectively, and (G-I) VGLUT3 and ChAT immunolabeling of glutamatergic amacrine cells and starburst amacrine cells, respectively. Scale bar is 50 μm. Images are representative of at least 6 retinas per genotype analyzed.
Fig 6.
Reduced retinal neuron numbers in 13R1 mutants.
Whole mount retinas from two-week-old mice were immunostained to label neuronal subtypes, imaged en face by confocal microscopy, and cell densities quantified. Analyzed cell types include (A-E) VGLUT3+ amacrine cells, (F-J) TH+ dopaminergic amacrine cells, (K-O) Melanopsin+ ipRGCs, and (P-T) Brn3a+ RGCs. For all four of the subtypes assayed, densities were significantly reduced in 13R1/cRKO retinas compared to wild-type. Densities were not reduced in either 3R1 or 3R2. Scale bar in D is 100 μm (for A-D); scale bar in S is 300 μm (for F-S). ** = p < 0.01; *** = p < 0.001 by Tukey post-hoc test comparing the indicated genotype with wild type. n = 6 retinas per genotype. Box plots represent the median, first and third quartile, range, and outliers.
Fig 7.
Two further mouse alleles that indicate γC4 is the sole γ-Pcdh isoform necessary and sufficient for postnatal survival.
A) A schematic of the 1R1 allele illustrates a large deletion from the sgRNA guide site in exon A1 to that in exon C3, as well as a frame-shifting insertion in exon C5 (red upward triangle). Only exon C4 remained intact. All mutations were verified by whole-genome sequencing. B) C4KO mutants were made by targeting exon C4 only for CRISPR/Cas9 genome editing, resulting in a 13 base pair deletion (red downward triangle). C) Western blot analyses on P0 cortex confirmed the protein expression expected from sequence analysis. Particularly, γC4 was present in 1R1 mutants (arrowhead, specific lower band of expected size; asterisk indicates larger non-specific band) but absent in C4KO animals, while all other isoforms analyzed were absent in 1R1 but present in C4KO. D) Total γ-Pcdh protein levels were quantified using Li-Cor quantitative western blotting, normalizing to GAPDH. Values from three samples per genotype were compared to wild type controls using a Dennett’s multiple comparison test with the indicated p-values.
Fig 8.
The isoform γC4 is necessary for normal neuronal survival.
Cryosections from P0 spinal cords were immunostained for the indicated markers (green; DAPI counterstain for nuclei, blue) A-B) Immunostaing for NeuN revealed grossly smaller spinal cords and neuronal loss in C4KO mutants, but not 1R1 animals. C-D) This was accompanied by reactive gliosis in C4KO. E-H) Interneuron subtypes were analyzed as in Fig 4 (E-F, FoxP2 and G-H, Pax2). K-L) Ventral interneurons were drastically reduced in C4KO mutants; decreases in 1R1 mutants were statistically significant, but substantially more modest. I-J) Apoptosis was increased in C4KO animals, but not in 1R1 mutants, as demonstrated by CC3 immunolabeling (quantified in M). N-Q) Whole mount retinas from 1R1 mutants were assayed for neuronal loss at 2 weeks of age and in adult as in Fig 6. There were no reductions in (N) VGLUT3+ amacrine cells, (O) TH+ dopaminergic amacrine cells, or (P) Melanopsin+ ipRGCs. Q) At 2 weeks of age, Brn3b+ RGCs were also not reduced, but their density was significantly lower in adult retinas. R) Summary of phenotypes in the analyzed strains. Mutations that disrupted Pcdhgc4 resulted in high levels of cell death (+++) and postnatal lethality, while mutations that spared Pcdhgc4 resulted in little to no cell death and animal survival. Scale bar is 200 μm. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 by Dunnett (spinal cord) or Tukey (retina) test comparing the indicated genotype with wild type. Wild type values in K-M are represented from Fig 4. n = 36 hemicords total from 3 animals per genotype or n = 6 retinas per genotype. Box plots represent the median, first and third quartile, range, and outliers.
Fig 9.
PCDHGC4 is constrained in humans.
The gnomAD database (https://gnomad.broadinstitute.org/; [58]) was queried for each cPcdh isoform to determine the extent to which loss-of-function (LoF) mutations were tolerated within the human population. A) The ratio of observed to expected (o/e) LoF mutations (y-axis) surrounded by its 90% CI is plotted for each PCDHG isoform (x-axis). PCDHGC4 (red circle) is the only isoform in which the upper bound of the 90% CI falls below 0.35 (red line), indicating constraint. B) LoF o/e (y-axis) is plotted for each isoform from the PCDHA and PCDHB cluster (x-axis). PCDHAC2 is the only constrained isoform (red circle).