ABSTRACT
The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity–aγ-Pcdh hallmark–is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, as it is the only γ-Pcdh that cannot independently engage in homophilic trans-interactions, we find that γC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved γC4 function that likely involves distinct molecular mechanisms.
INTRODUCTION
Cell-cell recognition via transmembrane cell adhesion molecules is essential for neural circuit formation. With trillions of exquisitely specific synapses in the human brain, it has been suggested that molecular diversity–achieved either via alternative gene splicing or combinatorial expression of large adhesion molecule families–plays an important role (Zipursky and Sanes, 2010). In Drosophila, this is exemplified by Dscam1, a single gene capable of generating 38,016 distinct protein isoforms through alternative splicing (Schmucker et al., 2000). Dscam1 isoform diversity is essential for neurodevelopmental processes including axon guidance, synapse specificity, and neurite self-avoidance (Hattori et al., 2009, 2007; Zhan et al., 2004, reviewed in Hattori et al., 2008). Mammalian Dscams, despite important roles in neurodevelopment, do not generate such isoform diversity (Fuerst et al., 2009, 2008; Garrett et al., 2018, 2016). In this sense, the clustered protocadherins (cPcdhs) are the mammalian analogue to fly Dscam1, although their diversity is generated by differential isoform expression via promoter choice rather than alternative splicing (Zipursky and Sanes, 2010).
The cPcdhs, expressed broadly throughout the developing central nervous system (CNS), are cadherin superfamily molecules that engage in strictly homophilic trans-interactions. The cPcdh isoforms are encoded by three gene clusters arrayed in tandem at human chromosome 5q31 (encoding 53 cPcdh proteins) and on mouse chromosome 18 (encoding 58 cPcdh proteins) (Wu et al., 2001; Wu and Maniatis, 1999). In mouse, the Pcdha cluster encodes 14 α-Pcdhs, the Pcdhb cluster encodes 22 β-Pcdhs, and the Pcdhg cluster encodes 22 γ-Pcdhs (Figure 1A). While all three Pcdh gene clusters contribute to neural development to some extent (Chen et al., 2017; Emond and Jontes, 2008; Hasegawa et al., 2017, 2016; Ing-Esteves et al., 2018; Katori et al., 2009; Meguro et al., 2015; Mountoufaris et al., 2017), the Pcdhg locus is the only one required for postnatal viability, and disruption of this locus results in the strongest phenotypes (reviewed in Peek et al., 2017).
The Pcdhg clusteris comprised of 22 variable (V) exons, divided according to sequence homology into A, B, and C subgroups, and three constant exons. Each V exon is regulated by an individual promoter and, upon transcription, is spliced to the constant exons (Tasic et al., 2002; Wang et al., 2002)(Figure 1B). Each V exon encodes one γ-Pcdh isoform’s extracellular domain, including 6 cadherin (EC) domains, a transmembrane domain, and a membrane-proximal variable cytoplasmic domain (VCD), while the constant exons encode a C-terminal cytoplasmic domain common to all 22 γ-Pcdh isoforms (Figure 1B). Single-cell RT-PCR from cerebellar Purkinje neurons indicated that each neuron expressed all three γC isoforms (Pcdhgc3, Pcdhgc4, and Pcdhgc5) from both alleles while stochastically expressing ∼4 of the 19 γA and γB isoforms monoallelically (Kaneko et al., 2006). Interestingly, this does not hold in all neurons, as single-serotonergic neuron transcriptomics indicated that, of the γ-Pcdhs, many neurons expressed only γC4, and few if any expressed γC3 or γC5 (Chen et al., 2017); additionally, few olfactory sensory neurons expressed any of the C-type Pcdha or Pcdhg isoforms (Mountoufaris et al., 2017). Clustered Pcdh isoforms interact strictly homophilically in trans, engaging in anti-parallel interactions involving EC1-EC4, while EC5 and EC6 mediate promiscuous cis dimer formation between γ isoforms, as well as with α- and β-Pcdhs (Goodman et al., 2016b, 2016a; Rubinstein et al., 2015; Schreiner and Weiner, 2010; Thu et al., 2014). These two types of interactions result in a multimeric lattice of dimers between cell membranes sharing the same isoform composition (Brasch et al., 2019; Rubinstein et al., 2017), and indeed, homophilic specificity is observed at the multimer level (Schreiner and Weiner, 2010; Thu et al., 2014). In this way, the 58 cPcdh isoforms generate thousands of distinct recognition signals.
While α, β and γ isoforms can all contribute to multimer formation (Rubinstein et al., 2017), the γ-Pcdhs are particularly critical for neural development (Peek et al., 2017). Mice lacking either the entire Pcdhg cluster or the γC3-C5 V exons exhibited neonatal lethality, with excessive apoptosis of neuronal subtypes in the spinal cord and hypothalamus (Prasad et al., 2008; Su et al., 2010; Wang et al., 2002b). Pcdhg mutants also exhibited reduced synapse number and disorganized synaptic terminals within the spinal cord, which were separable from the increased cell death (Garrett and Weiner, 2009; Prasad and Weiner, 2011; Weiner et al., 2005). When neonatal lethality was circumvented with a conditional Pcdhg allele, distinct phenotypes were observed in other parts of the CNS, including reduced dendrite arborization in forebrain neurons (Garrett et al., 2012; Keeler et al., 2015; Molumby et al., 2016; Suo et al., 2012) accompanied by an increase in morphologically immature dendritic spines (Molumby et al., 2017). In retina-restricted mutants, many neuronal subtypes exhibited excessive cell death without separable synaptic disorganization (Ing-Esteves et al., 2018; Lefebvre et al., 2008). Starburst amacrine cells (SACs), however, survived in normal numbers, but exhibited clumping of their dendritic fields, indicative of a failure of self-avoidance (Kostadinov and Sanes, 2015; Lefebvre et al., 2012).
The parallel molecular and phenotypic diversity of cPcdhs could, a priori, indicate three potential models, all of which may be correct for distinct subsets of neurons or distinct functions. First, isoform diversity per se may be required. Second, there may be a high level of isoform redundancy such that any one (or few) isoform(s) may suffice. Third, there may be unique roles for individual isoforms, such that no other isoform can compensate for their loss. Some extant evidence exists for each of these possibilities. The γC3-5 isoforms, but not γA1-3, were required for postnatal viability in mice (Chen et al., 2012). As γC5 is expressed later in the postnatal period (Frank et al., 2005), and γC4 is unable to localize to the cell membrane alone (Rubinstein et al., 2015; Thu et al., 2014), it seemed most likely that this reflected a crucial role for γC3. While re-expressing a single γ-Pcdh isoform (γA1 or γC3) could rescue self-avoidance in SACs, it disrupted self/non-self recognition required for proper receptive field overlap and retinal circuit performance (Kostadinov and Sanes, 2015; Lefebvre et al., 2012). Similar re-expression of γA1 or γC3 in cortical neurons on a Pcdhg null background led to aberrantly increased or decreased dendrite arborization, depending on whether surrounding cells also expressed the same single isoform and could thus presumably engage homophilically (Molumby et al., 2016). A requirement for cPcdh diversity per se was discovered for the convergence of olfactory sensory neuron axons on particular glomeruli in the olfactory bulb: disruption of olfactory circuitry was mild in single gene cluster mutants, but devastating in mice lacking all three clusters, and it was not rescued by re-expressing a triad of single α-, β-, and γ-Pcdh isoforms (Hasegawa et al., 2016; Mountoufaris et al., 2017). Support for unique roles for individual cPcdh isoforms comes from the demonstration that the disrupted axonal branching observed in Pcdha mutant serotonergic neurons (Katori et al., 2009) is due entirely to the role of one isoform, αC2 (Chen et al., 2017), and from the demonstration of a unique role for γC3 in regulating Wnt signaling through Axin1 (Mah et al., 2016).
Thus, no single model is likely to encompass all of the γ-Pcdhs’ diverse functions. Additionally, most prior studies have relied on mis-or over-expression of individual isoforms, which could in some cases result in new, distinct phenotypes. Paralleling studies establishing the necessity of Dscam1 molecular diversity for specific neurodevelopmental roles in Drosophila, we used CRISPR/Cas9 genome editing to simultaneously target the 22 Pcdhg variable exons in an unbiased manner, and created a new allelic series of mouse mutants with reduced isoform diversity from the endogenous gene cluster. We find, surprisingly, that only one isoform – γC4, which uniquely cannot mediate homophilic trans- interactions independently – is strictly required for postnatal viability and survival of the many neuronal subsets shown previously to depend on the γ-Pcdhs. Our results: 1) show that some γ-Pcdh functions do not require molecular diversity; 2) confirm that at least some γ-Pcdh isoforms have unique roles; and 3) suggest that the regulation of neuronal survival may require novel mechanisms of cPcdh interaction and/or signaling involving γC4.
RESULTS
CRISPR/Cas9 strategy for reducing Pcdhg isoform diversity
To assess the importance of γ-Pcdh isoform diversity to postnatal viability and neurodevelopmental functions, we used a shotgun CRISPR/Cas9 genome editing screen to disrupt varying numbers of Pcdhg variable exons (Figure 1B-D). We reasoned that by injecting pooled individual single guide RNAs (sgRNAs) targeting each V exon into many zygotes, we could generate a number of unique mutant mouse lines, each of which harbored distinct patterns of reduced isoform diversity due to variability in which sgRNAs efficiently bound and directed mutations. We designed sgRNAs to target within ∼100-150 base pairs downstream of the start codon of each Pcdhg V exon, with the goal of creating frame-shifting mutations through non-homologous end joining (NHEJ) repair (Figure 1B-C)(Cong et al., 2013; Mali et al., 2013). A total of twenty guides were designed; the PcdhgB4 and PcdhgB5 V exons shared enough homology to allow a single guide to target both, as did PcdhB6 and PcdhgB7. Pooled sgRNAs were concentrated, then combined with Cas9 mRNA and microinjected into C57BL/6J mouse zygotes at three different concentrations (each individual guide at 50 ng sgRNA/μl, 10 ng sgRNA/μl, or 5 ng sgRNA/μl: HI, MED, and LO, respectively), as described in Materials and Methods (Figure 1D). Microinjected zygotes were transferred into a total of 20 pseudopregnant females, resulting in 100 live-born mice (16 from HI, 34 from MED, and 50 from LO). All 100 founders were screened at 7 Pcdhg exons by PCR with Sanger sequencing to detect frame-shifting mutations. From this initial screen, 15 founders exhibited some disruption (10 from HI and 5 from MED) and were designated for breeding. Most disruptions were found in pups from HI or MED injections, so the 50 founders resulting from the LO injections were not pursued further. The remaining 35 founders were further screened by PCR and Sanger sequencing at the remaining 15 Pcdhg variable exons. In this way, a total of 31 founders were identified that carried some constellation of mutations at the guide-targeted sites (14/16 HI, 17/34 MED).
Due to the likely mosaicism of the founders and the uncertainty of germline transmission of any given mutation, we did not characterize the 31 identified founders more extensively. Rather, each was crossed with wild-type C57BL/6J animals to generate G1 offspring for further analysis (Figure 1D). Sperm from male G1 mice was cryopreserved while somatic tissue was used for genotyping to identify lines of interest carrying reduced diversity of Pcdhg variable exons. Ninety-four G1 offspring were screened for heterozygous mutations using a custom amplicon assay from Illumina and Illumina MiSeq sequencing (see Materials and Methods for details, target coordinates are listed in Supplementary File 1). The amplicon assay was designed to sequence the 22 Pcdhg sgRNA target regions, all the analogous regions in the untargeted Pcdha and Pcdhb clusters, and the top 95 predicted potential off-target sites (Figure 1D). We screened for missense and nonsense mutations, small indels and, because our approach was intended to produce up to 22 double-stranded breaks within 160 kilobases, larger rearrangements between breakpoints. Smaller indels were identified using the Genome Analysis Toolkit (GATK) (McKenna et al., 2010), while larger rearrangements were identified with BreaKmer (Abo et al., 2015). Twenty-sixdistinct lines carrying unique constellations of mutations are represented in Table 1, derived from 12 different founders. Most mouse lines (20 lines from 9 founders) resulted from microinjection of the highest concentration of total sgRNAs (50 ng/μl/guide). Mutations ranged from only one disrupted V exon (leaving 21 intact) to 21 disrupted V exons (leaving only 1 intact; Table 1). Each V exon was disrupted in at least one mouse line, either by discrete indels, or through involvement in a rearrangement between breakpoints.
Whole genome sequencing reveals rearrangements undetected by amplicon sequencing
Based on initial results from the custom amplicon assay, we chose three lines for cryo-recovery and further analysis – Pcdhgem5, Pcdhgem12, and Pcdhgem35 (Table 1). Mice from each recovered line were intercrossed to generate homozygous mutants. Once obtained, we used these homozygous mutants to verify by PCRV-exon indels and rearrangements. We found that fewer exons were successfully amplified by PCR than expected (Figure 1-figure supplement 1A), indicating that amplicon sequencing failed to detect all the mutations harbored by the heterozygous G1 mutants. Therefore, we performed whole genome linked-read sequencing on homozygous mutants, using the Chromium Genome Sequencing Solution from 10X Genomics to detect large scale rearrangements (Figure 1-figure supplement 1, Figure 2-figure supplement 1). Paired-end reads from the whole genome sequencing were used to reconstruct the rearrangements (Figure 2). We found that Pcdhgem5 contained frame-shifting indels in V exons A1, B1, A5, A7, B7, and C4, all of which were accurately called by the prior amplicon sequencing. However, there was an inversion and deletion that disrupted exons A9, B6, and A10 that was undetected by the previous analysis (Figure 2-figure supplement 1A). Thus, Pcdhgem5 retained 13 intact V exons (Figure 2A), and we renamed the allele Pcdhg13R1; “13R” for the number of intact exons remaining and “1” as it was the first allele identified with this number (referred to as 13R1 hereafter for simplicity).
Sequencing of Pcdhgem12 revealed extensive rearrangements. A fusion between exons A5 and B8 identified by the amplicon sequencing was confirmed, but additional junctions were found between A1 and A4, between A12 and A9, and between B6 and A6 (Figure 2A, Figure 1-figure supplement 1C). Furthermore, linked reads revealed an insertion including a transposable element and coding sequence from Anp32a which aligns to exons 4-7 of transcript Anp32a-201 without the intervening introns (Figure 2B, Figure 1-figure supplement 1D). This phenomenon of the insertion of a transposable element along with coding sequence from an early expressed gene has been previously described in CRISPR genome editing (Ono et al., 2015). As this sequence was inserted 3’ to the inverted exon Pcdhga5, there is no associated transcription start site, and no protein product is expected. Altogether, at least 9 double-stranded breaks occurred, resulting in a frame-shifting 52 bp insertion into exon B2, and larger deletions and rearrangements disrupting all the other γA and γB isoforms. As only the 3 γC V exons remained intact in Pcdhgem12, we renamed this allele Pcdhg3R1 (3R1 hereafter; Figure 2B).
Whole genome sequencing from Pcdhgem35 mutants confirmed small frame-shifting deletions at exons A12 and C5, as well as an in-frame deletion of 9 bp in exon C4. However, this more exhaustive sequencing also revealed a large rearrangement undetected by the prior amplicon sequencing analysis. Here, there was a ∼94 kb deletion spanning the breakpoint from exon A1 to that of exon A11 (Figure 2-figure supplement 1B). Only exons B8 and C3 were unaffected by any mutation. As exon C4 still encoded a nearly full-length protein lacking only 3 amino acids (residues 27-29 in the signal peptide), we renamed the Pcdhgem35 allele Pcdhg3R2 (3R2 hereafter, Figure 2C), as it represented the second allele identified with 3 isoforms remaining.
In all three mutants analyzed thus far, rearrangements were identified by whole genome sequencing that were undetected by the amplicon analysis (Figure 2, Table 1 – Table supplement 1). With this information, we re-analyzed the amplicon sequencing data by visual inspection of the paired reads within the Integrative Genomics Viewer (IGV). We were able to find many, but not all, of the junctions identified by the whole genome sequencing, but missed by BreaKmer analysis of the amplicon sequencing (Figure 1-figure supplement 1B). Therefore, we manually inspected the alignments from each of the other frozen lines. Additional rearrangements were identified where each of the paired ends of multiple reads mapped to different exons (Table 1 – Table supplement 1).
Isoformdiversity perse is not required for postnatalviability
The complete deletion of the Pcdhg cluster results in neonatal lethality (Wang et al., 2002b), as does the deletion of the three γC V exons, whereas mice with deletion of the first three γA exons (A1, A2, A3) had no reported phenotypes (Chen et al., 2012). Of the three lines initially characterized, 3R1 and 3R2 homozygous mutant mice were born at expected Mendelian ratios and survived into adulthood without any overt differences from their wild-type or heterozygous littermates. In contrast, 13R1 homozygous mutants died within hours of birth with the hunched posture, tremor, and inability to right themselves or nurse that is characteristic of Pcdhgdel/del mutants lacking the entire Pcdhg cluster (Wang et al., 2002b; Weiner et al., 2005). Whole genome sequencing from all three lines confirmed that there were no off-target mutations in the Pcdha or Pcdhb locus, or any disruptions in the Pcdhg constant exons. However, it remained formally possible that the particular rearrangements within 13R1 mutants disrupted the expression of the other isoforms or V exon splicing to the constant exons, creating a functional null mutation.
To exclude this possibility, we performed quantitative real-time PCR using cDNA from the cerebral cortices of homozygous mutants and wild-type littermates (Figure 3A). To detect specific isoform transcripts, forward primers targeting the 3’ end of each V exon were used with a reverse primer in constant exon 1 (product spans 1 intron) or exon 2 (product spans 2 introns). To monitor total levels of Pcdhg locus transcription, a forward primer in constant exon1 was used with a reverse primer in constant exon 2 (spanning 1 intron, primer sequences in Figure 3 – figure supplement 2). Total locus expression was significantly reduced in 3R2 homozygous mutants, but in both 3R1 and 13R1 mutant cortex, expression levels were indistinguishable from controls. Furthermore, individual isoform transcription was reduced only when mutations completely disrupted the exon by deletion or inversion. Smaller indels generally had no effect on transcript expression level (e.g., A1, B1, A5, A7, B7, and C4 were not significantly reduced in 13R1 homozygous mutants, but A9, B6, and A10 were undetectable). Additionally, transcription of several V exon fusions was detected, including A4 (fusion with A1) and A9 (fusion with A12) in 3R1 (predicted protein products encoded by these fused transcripts are listed in Supplementary File 2). We also asked the extent to which mutations within the Pcdhg locus altered isoform transcription from the Pcdha or Pcdhb clusters. While there were no significant changes detected in 13R1 mutants, isoforms from the 3’ end of the Pcdhb locus were expressed at significantly higher levels in 3R1 and 3R2 homozygous mutants compared to controls (β11 in 3R1, β15 and β22 in both 3R1 and 3R2). Expression of Pcdha cluster genes appeared to be unchanged (Figure 3-figure supplement 1).
We also verified that these mRNA expression levels were reflected at the protein level, utilizing a series of antibodies specific for particular γ-Pcdh protein isoforms (Lobas et al., 2012). Western blot analysis of brain lysates from 13R1 neonates and 3R1 and 3R2 adults confirmed the presence of the expected isoforms at the appropriate molecular weights: 13R1 brains expressed γA isoforms, γB2, and γC3, but not γC4, while 3R1 and 3R2 brains expressed γC3 and γC4, but not γB2 or any γA isoforms (Figure 3B). Based on these analyses, we concluded that 13R1 homozygous mutants are not, in fact, complete Pcdhg functional nulls, and that the similarity of their neonatally lethal phenotype to that of nulls likely reflects the essential nature of a particular isoform lost in this line but present in both 3R1 and 3R2; that is, γC4, as described below. Before testing this conclusion in detail, we first asked whether the neonatal lethality of 13R1 was accompanied by cellular phenotypes previously described in Pcdhgdel/del null mice, and whether the viable 3R1 and 3R2 lines lacked these phenotypes.
Excessive developmental neuronal apoptosis occurs in mutants exhibiting neonatal lethality
Pcdhgdel/del mutants exhibit neonatal lethality with excessive cell death of interneurons in the ventral spinal cord and brainstem (Prasad et al., 2008; Wang et al., 2002b; Weiner et al., 2005). To ask if lethality in reduced diversity mutants was also accompanied by increased developmental apoptosis, we analyzed cryosections from spinal cords at P0. Indeed, we found significant cell death in 13R1 homozygous mutants, but not 3R1 or 3R2 mutants (Figure 4). Staining of transverse sections for the pan-neuronal marker NeuN revealed that 13R1 mutant spinal cords were grossly smaller, with obvious reductions in cell number primarily in the ventral spinal cord, as reported previously for Pcdhgdel/del null mutant neonates (Figure 4A-C)(Prasad et al., 2008; Wang et al., 2002b). This was accompanied by an increase in reactive astrocytes, revealed by GFAP labeling within the gray matter (Figure 4D-F). Spinal interneurons derive from 6 dorsal (dI1-6) and 4 ventral (V0-3) domains (reviewed by Lewis, 2006); we used antibodies against two transcription factors, FoxP2 and Pax2, that label distinct subsets of interneurons. We found significantly fewer FoxP2-positive ventral interneurons (derived from dI2, dI6, and V1) in 13R1 mutants than in wild type littermates or 3R1 homozygous mutants (Figure 4G-I,P). Pax2 labels a broader subset of interneurons, derived from dI4, dI6, V0, and V1, that settle in both the dorsal and ventral spinal cord. As we found previously for Pcdhg null animals (Prasad et al., 2008), 13R1 mutants had far fewer Pax2-positive ventral interneurons (Figure 4J-L,Q). To confirm that cell loss resulted from excessive apoptosis as observed in Pcdhg nulls, we assayed for cleaved caspase 3 (CC3), a marker of apoptotic cell death, and found significantly more CC3-labeled profiles in 13R1 mutants than in wild type littermates or 3R1 (Figure 4M-O,R). An additional phenotype previously described in Pcdhg null mutants is the clumping of parvalbumin-positive Ia afferent axon terminals around their motor neuron targets in the ventral horn; this phenotype is worsened, though not entirely due to, increased interneuron apoptosis (Prasad and Weiner, 2011). Again, we found that 13R1 mutants exhibited a null mutant-like phenotype while 3R1 mutant Ia afferent projections appeared similar to those of controls. (Figure 4-figure supplement 1).
Increased cell death of many neuronal subtypes is also a hallmark of Pcdhg loss of function in the retina, with retinal cell death occurring largely postnatally (Ing-Esteves et al., 2018; Lefebvre et al., 2008). To circumvent the neonatal lethality of 13R1 mutants, we made compound heterozygous mutants with the conditional loss of function allele Pcdhgfcon3 (Prasad et al., 2008) crossed with Pax6α-Cre to restrict recombination of this allele to the retina (Marquardt et al., 2001). These mutants are referred to as 13R1/cRKO (for conditional retinal knockout). Examination of immunostained retinal cross sections from 13R1/cRKO mutants at P14 revealed substantial thinning of the inner retina, including both cellular and synaptic layers, compared to wild type (Figure 5A-C). Neither 3R1 nor 3R2 mutant retinas were notably thinner (Figure 5C and data not shown). As reported for Pcdhg null mutants (Lefebvre et al., 2008), the retinal thinning in 13R1/cRKO mice was not accompanied by obvious disorganization of neurite stratification within the inner plexiform layer (Figure 5D-I). To verify that this resulted from cell loss, we measured the density of two amacrine cell types (tyrosine hydroxylase (TH)+ dopaminergic amacrine cells and VGLUT3+ amacrine cells) and two retinal ganglion cell types (Melanopsin+ RGCs and Brn3a+ RGCs) in whole-mount retinas from P14 animals. All four cell types were significantly less numerous in 13R1/cRKO mutants than in wild-type littermates or in 3R1 or 3R2 homozygous mutants, neither of which exhibited any abnormalities (Figure 6).
Pcdhgc4 is the necessary and sufficient Pcdhg cluster gene for postnatal viability
Having confirmed that the cellular phenotypes of 13R1 resemble the complete deletion of the Pcdhg cluster, we next turned to utilizing the novel CRISPR mutant mouse lines to confirm which γ-Pcdh isoforms were critical. We noted that homozygous 3R1 mutants survived and did not exhibit exacerbated apoptosis despite lacking expression of any functional γA or γB isoforms, indicating that one or more of the C3-C5 isoforms must be critical. Furthermore, 3R2 mutants survived and were phenotypically normal without a functional Pcdhgc5 gene, whilst 13R1 homozygous mutants, which exhibited neonatal lethality and exacerbated neuronal apoptosis uniquely harbored frame-shifting mutations in PcdhgC4. In a separate study, we have derived and are analyzing a CRISPR-targeted mouse line that specifically generated a Pcdhgc3 loss-of-function allele, and have found that they are viable and fertile as adults (Figure 7-figure supplement 1, D. Steffen, K.M. Mah, P.J. Bosch, A.M. Garrett, R.W. Burgess, and J. A. Weiner, in preparation). Together with prior data indicating that mice lacking the entire Pcdha and Pcdhb clusters are viable (Hasegawa et al., 2016; Mountoufaris et al., 2017), this suggests that Pcdhgc4 encodes the sole cPcdh isoform essential for organismal survival. We generated two additional mouse lines to confirm this conclusion.
First, we chose Pcdhgem8 from our list of mutants for cryorecovery (Table 1). This line harbored a large deletion from within exon A1 to within exon C3 identified by visual inspection of the aligned reads from the original amplicon sequencing, as well as a 1 bp frame-shifting deletion in exon C5. Upon cryorecovery, we generated homozygous mutants and verified these mutations by linked-read whole genome sequencing (Figure 7A, Figure 7-figure supplement 2A). As only V exon C4 was left intact, this strain was renamed Pcdhg1R1 (1R1 hereafter). These homozygous mutants survived into adulthood and were fertile, with no overt differences from their wild type litter mates. Both quantitative RT-PCR and western blot analyses reflected the expected isoform expression (Figure 7C, Figure 7-figure supplement 3).
Second, we generated an entirely new mutant mouse line by specifically targeting Pcdhgc4 for CRISPR/Cas9-directed gene disruption. This resulted in a 13 bp frame-shifting deletion 3’ to the start codon (Figure 7B; Figure-figure supplement 2B). We named this new allele PcdhgC4KO (C4KO hereafter). Homozygous C4KO mutants exhibited the same hunched posture, tremor, and inability to nurse or right themselves observed in Pcdhgdel/del and 13R1 mutants and died shortly after birth. Sanger sequencing confirmed that the analogous regions of the other Pcdhg V exons were not disrupted in this line (Figure 7-figure supplement 2B), while Western blot analyses verified that the other isoforms were produced as expected (Figure 7C). Together, these two additional mouse lines show that mutation of Pcdhgc4 alone recapitulated the overt phenotype of losing the entire cluster, whilst expression of this single γ-Pcdh isoform was sufficient to rescue viability, even when all 21 other Pcdhg isoforms were absent.
Pcdhgc4 is the crucial isoform for neuronal survival
To ask if these overt phenotypes of death vs. survival in C4KO and 1R1 mutants extended to neuronal cell survival, we again analyzed spinal cords from P0 neonatal mutants. As expected from their outward appearance, 1R1 homozygous spinal cords appeared grossly normal in overall size and neuronal density (NeuN+ cells), with little if any reactive gliosis (GFAP+; Figure 8B,D). In contrast, C4KO mutant spinal cords were grossly smaller and exhibited clear ventral interneuron loss (Figure 8A) accompanied by reactive gliosis (Figure 8C) that was essentially identical to that observed in complete Pcdhg null mutants (Prasad et al., 2008; Wang et al., 2002b) and 13R1 mutants (Figure 4). Analysis of individual cell types revealed significant loss of FoxP2-labeled cells and Pax2-positive neurons in the ventral spinal cord of C4KO mutants (Figure 8E,G,K,L). Significant reductions in 1R1 mutant cell density were observed, though they were much more modest (Figure 8F,H,K,L). Consistent with this, there were significantly more CC3-positive neurons in C4KO mutants than in 1R1 or WT neonates (Figure 8 I,J,M). As expected, these patterns of spinal interneuron survival corresponded to the presence (C4KO; Figure 8-figure supplement 1A) or absence (1R1; Figure 8-figure supplement 1B) of aggregated Ia afferent terminals around motor neurons in the ventral horn.
As 1R1 mutants survive into adulthood, we analyzed neuronal survival in the retina at 14 days of age and in adult. There was no indication of retinal thinning at either age. As above, cell densities were calculated (in cells per mm2) for four cell types: dopaminergic amacrine cells (TH+), VGLUT3+ amacrine cells, ipRGCs (Melanopsin+), and Brn3a+ RGCs (Figure 8O-R). The means of the cell densities were compared across genotype and age with a two-way ANOVA followed by pairwise comparisons with a Tukey analysis. There was a significant genotype effect in Brn3a+ cell density (p = 0.034), and a significant pairwise difference between wild type animals at 2 weeks and 1R1 mutants in adult (p = 0.046). No other differences reached statistical significance. Therefore, neuronal survival was largely normal in 1R1 mutants, with any cell loss being very modest compared to mutants lacking γC4 (13R1/cRKO: Figure 5, Figure 6; Pcdhg nulls: Lefebvre et al., 2008).
PCDHGC4 is constrained in humans
With the number of human genomes and exomes that have been sequenced, it is possible to test if variation occurs at the rate expected from random mutation. If predicted loss of function (LOF) mutations (e.g., frame-shifts and premature stop codon insertions) within a given gene are observed at lower rates than expected, it indicates that that gene is essential, and that loss of function is not tolerated (referred to as “constraint”). To ask if specific cPcdh isoforms are constrained in the human population, we queried the Genome Aggregation Database (gnomAD, Broad Institute), an aggregation of genomic variation across 141,456 human exomes and whole genomes (Karczewski et al., 2019). For each cPcdh isoform, the ratio of observed to expected (o/e) LOF mutations was reported along with its 90% confidence interval (CI, vertical lines, Figure 9A-B). A gene is considered constrained if the upper bound of the 90% CI falls below 0.35 (red line, Figure 9A-B). Within the PCDHG locus, only PCDHGC4 met this criterion (o/e = 0.14, 90% CI = 0.07-0.31, Figure 9A). Amongst the PCDHA and PCDHB isoforms, only PCDHAC2, the specific isoform required in serotonergic neurons, was constrained (o/e = 0.16, 90% CI = 0.09-0.34). Thus, consistent with our analysis of the orthologous gene in our allelic series of mice, PCDHGC4 is likely also essential in humans.
DISCUSSION
The 22 γ-Pcdhs comprise a diverse family of cadherin superfamily adhesion molecules with multiple distinct functions in distinct cell types. Extant data suggest three possible models for the role of isoform diversity: (1) a model of diversity where many isoforms are required for a given function; (2) a model of redundancy in which any single isoform (or small subset of isoforms) can serve a given function; and (3) a model of isoform specificity where one specific isoform (or small subset of isoforms) is strictly required for a given function. Here, we used a CRISPR/Cas9 strategy to reduce γ-Pcdh isoform diversity in an unbiased fashion, creating a new allelic series of mouse strains. In the course of analyzing several new Pcdhg alleles, we discovered that the control of neuronal survival and postnatal viability is best described by the third model. We found–surprisingly, given its inability to reach the cell surface and mediate homophilic adhesion without other cPcdh cis-interaction partners– that γC4 is the sole necessary and sufficient isoform.
Our strategy was to inject Cas9 mRNA along with 20 individual sgRNAs targeting each of the Pcdhg V exons simultaneously. By screening G1 offspring from many founders, we collected an array of mutants with distinct V exon mutation patterns, ranging from a single isoform disrupted (21 intact) to 21 isoforms disrupted (1 intact), all of which were cryopreserved. Each isoform was disrupted in at least one mutant line (Table 1). Furthermore, there was evidence of double-stranded breaks at each sgRNA site identifiable as indels or junctions between guide sites. Our G1 screening was done by next generation sequencing of a custom amplicon array. This technique was effective for identifying indels within each individual amplicon (e.g., smaller indels at each guide site), but was not exhaustive in identifying rearrangements between guide sites, even when both sides of the junction were predicted to be recognized by the amplicon array primers. This could be due to the new DNA sequence: 1) being difficult to amplify within the array, and therefore not sequenced; 2) not being accurately aligned to the reference genome; or 3) not being identified as a new junction by the bioinformatic algorithm BreaKmer. Indeed, some junctions that were not recognized by BreaKmer were clearly identified by visual inspection of the sequence alignments within IGV as paired reads mapping to two different exons (Table 1 – Table supplement 1). Those identified in two different mutant lines – 3R1 and 1R1 – were confirmed by whole genome sequencing. It is thus likely that many of the cryopreserved lines not yet recovered and made homozygous harbor additional mutations to those described here from the initial analysis (Table 1, Table 1 – Table supplement 1). We suggest that future studies targeting multiple sites within a relatively small region should use other methods to identify rearrangements between guide sites, such as targeted long-read sequencing (Bennett-Baker and Mueller, 2017).
One concern when using a pooled sgRNA approach such as ours is the compounding of potential off-target mutations. To assess this, we sequenced the top 95 predicted off-target sites in G1 offspring by amplicon sequencing. We did not identify indels attributable to Cas9 activity at any of these sites. While not exhaustive, this analysis is consistent with recent studies suggesting that off-target mutations from CRISPR/Cas9 are relatively rare in mouse model production (Iyer et al., 2015; Mianné et al., 2016; Nakajima et al., 2016). To further confirm the specificity of our CRISPR/Cas9 targeting, we performed whole genome sequencing on homozygous mutants from four lines at least three generations after G1. Here, we were particularly concerned with the closely linked and highly similar protocadherins encoded by the two adjacent gene clusters (Pcdha and Pcdhb) as well as Pcdh1 and Pcdh12, non-clustered protocadherins located within 450 kb of the Pcdhg cluster. There were no indelsor rearrangements identified within any of these genes. We conclude that, at the resolution assayed here, off-target mutations do not contribute to our results. This conclusion is bolstered by the segregation of postnatal and neuronal survival phenotypes observed across multiple mouse lines: the two lines harboring frame-shifting mutations in Pcdhgc4 (13R1 and C4KO, generated separately with distinct methodologies) died and exhibited similarly exacerbated neuronal apoptosis, while the two mouse lines in which Pcdhgc4 was untouched (3R1) or harbored a small in-frame deletion (3R2) survived and exhibited normal neuronal survival. The fact that mice harboring an additional allele (1R1) in which Pcdhgc4 is the only remaining functional isoform are viable and exhibit only mild neuronal survival alterations further supports the specificity of CRISPR targeting.
Isoform choice in the Pcdh gene clusters is regulated by CCCTC-binding factor (CTCF) and cohesin, which bind directly to a conserved sequence element (CSE) in each individual V exon promoter (Golan-Mashiach et al., 2012; Kehayova et al., 2011; Monahan et al., 2012). These proteins organize DNA loops, bringing the promoters of expressed isoforms into proximity with an enhancer region 3’ to the constant exons (Jiang et al., 2017). CTCF/cohesin complex binding is restricted by promoter methylation laid down during embryogenesis by the methyltransferase Dnmt3b. Indeed, in the absence of Dnmt3b, many more cPcdh isoforms were expressed by each neuron (Toyoda et al., 2014); conversely, in CTCF knockout forebrain neurons, expression of nearly all cPcdh genes was markedly reduced (Hirayama et al., 2012). Here, we analyzed the isoform expression in cerebral cortex from four new Pcdhg mutant strains using qPCR. In the Pcdhg cluster, indels or other genomic DNA junctions that allowed splicing from V exons to the constant exons resulted in expressed transcripts, while expression from isoforms deleted from the genome or inverted were undetectable, as expected (Figure 3, Figure 7-figure supplement 3). The lone exception was the 3’-most isoform, γC5, which was significantly reduced in 1R1 mutants which harbored a single base pair insertion in the Pcdhgc5 variable exon (Figure 7-figure supplement 3). We also analyzed select isoforms from the Pcdha and Pcdhb clusters. Here, large deletions in the 5’ end of the Pcdhg cluster resulted in significantly higher expression from the 3’ Pcdhb isoforms (β11, β15, and β22, Figure 3-figure supplement 1, Figure 7-figure supplement 3). This was most pronounced in 1R1 mutants, which harbor the largest deletion in the Pcdhg cluster, but was not detected in 13R1 mutants, which have the smallest deletion of the mutants assayed. This is likely due to regulatory elements within DNaseI hypersensitive sites HS16-20, located 3’ to the Pcdhg cluster, that are essential for expression from the Pcdhb cluster (Yokota et al., 2011). The movement of these elements closer to the Pcdhb cluster in the 1R1, 3R1, and 3R2 alleles is likely responsible for the increased expression observed. Consistent with this interpretation, it has previously been reported that moving Pcdha isoforms closer to regulatory elements located between the Pcdha and Pcdhb cluster (HS7 or HS5-1) by deleting intervening exons resulted in their increased expression (Noguchi et al., 2009). Importantly, this increased expression of Pcdhb cluster genes cannot be responsible for the viability of the 1R1, 3R1, or 3R2 mouse lines: similar Pcdhb overexpression was reported previously in Pcdhgdel/del animals, which nevertheless die at birth (Chen et al., 2012).
Previous interrogations of Pcdhg isoform diversity either deleted two sets of three V exons (A1-A3 or C3-C5) (Chen et al., 2012) or drove overexpression of a single isoform from a transgene (Lefebvre et al., 2012; Molumby et al., 2017, 2016). Our new series of mutants, including both those initially analyzed and the many that remain cryopreserved, will allow for a finer dissection of the role of isoform diversity and the identification of potential isoform-specific functions. Chen et al. (2012) showed that mice lacking γC3-C5 exhibited neonatal lethality and exacerbated spinal interneuron apoptosis similar to that of Pcdhg null mutants; in contrast, mice lacking A1-A3 were viable and outwardly normal. Our new results indicate that γC4 is, in fact, the sole essential isoform for neuronal survival and postnatal viability, indicating that loss of this isoform was responsible for the lethality observed in ΔC3-5 mice (Chen et al., 2012). This is particularly surprising, as the γC4 isoform is peculiar in several ways. Cell aggregation experiments in the K562 suspension cell line indicate that γC4, uniquely among γ-Pcdhs, cannot mediate trans homophilic binding on its own. Like the α-Pcdhs, it requires interaction with carrier isoforms (either otherγ- or β-Pcdhs) to reach the cell membrane (Thu et al., 2014), possibly because EC6 of γC4 inhibits surface delivery and the formation of cis-homodimers (Rubinstein et al., 2015; Thu et al., 2014). This raises the novel possibility that the postnatal viability and largely (though not entirely) retained neuronal survival observed in 1R1 mutants, in which γC4 is the only functional γ-Pcdh isoform, may reflect functions for this protein in cellular compartments other than the plasma membrane. Alternatively, γC4 may reach the membrane, escorted by β-Pcdhs (several of which are upregulated in 1R1 mutants) and participate in homophilic interactions that trigger intracellular signaling pathways specific to the γC4 VCD, which remain to be identified. Precedence for this latter possibility comes from our recent finding that the VCD of γC3 can, uniquely amongst γ-Pcdhs, bind to and sequester Axin1 at the membrane, which leads to suppression of some components of the Wnt signaling pathway (Mah et al., 2016).
Thus, while the downstream mechanisms through which γ-Pcdhs promote neuronal survival remain unknown, our findings suggest that either protein interactions specifically mediated by γC4, or a unique localization for this isoform, will be involved. In 1R1 mutants, γC4 alone was not entirely sufficient for normal neuron number in the spinal cord or retina (Figure 8). This could be explained potentially by the significantly reduced expression levels of γC4 (and thus of total γ-Pcdh proteins) in these mutants (Figure 7C, Figure 7-figure supplement 1), or it could indicate that other cPcdh isoforms contribute to survival of some neuronal subsets in a collaborative, if not strictly essential, manner. Consistent with this second possibility, cell death phenotypes observed in Pcdhg mutants were made more severe by additional disruption of the Pcdha and Pcdhb clusters, despite the fact that Pcdha or Pcdhb disruption did not increase cell death when Pcdhg remained intact (Hasegawa et al., 2016; Ing-Esteves et al., 2018). In any case, our genetic interrogation of the Pcdhg gene cluster clearly demonstrates that γC4 is the only γ-Pcdh isoform strictly necessary for neuronal survival and postnatal viability. Consistent with this, the orthologous PCDHGC4 is the only PCDHG gene (and, along with PCDHAC2, one of only 2 clustered Pcdh genes overall) that is constrained in humans, thus indicating an essential role conserved throughout mammalian evolution that is of potential clinical relevance. In this respect, the critical next step will be to elucidate the unique protein-protein interactions and intracellular signaling pathways in which γC4 participates during the development of the nervous system.
MATERIALS AND METHODS
Mouse strains
All animals were housed in the research animal facility either at The Jackson Laboratory, The University of Iowa, or Wayne State University under standard housing conditions with a 12 h/12 h light/ dark cycle and food and water ad libitum. All procedures using animals were performed in accordance with The Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Institutional Animal Care and Use Committee at each respective institution. All experiments included a mix of male and female animals. Previously described animals include Pcdhgfcon3 (Prasad et al., 2008) and Pax6α-Cre (Marquardt et al., 2001). The newly generated reduced Pcdhg diversity mutants are cryopreserved at The Jackson Laboratory (Stock numbers listed in Table 2).
Generation of Pcdhg reduced diversity mutants
Guide RNA (sgRNA) sequences were designed to target the near 5’ regions to the start codons of each variable exon (Table 3). Guides were designed using the tool at crispr.mit.edu and were analyzed using RGEN tools to minimize off target sites and to maximize the likelihood of frameshifting mutations (Bae et al., 2014a, 2014b; Park et al., 2015). 20 total guides were synthesized (IDT), as exons B4 and B5 had a high level of 5’ homology, as did exons B6 and B7. Guides were received lyophilized, resuspended in water, mixed in equal parts, then lyophilized again. This mixture was diluted and resuspended with S. pyogenes Cas9 mRNA to generate three guide concentrations: 50 ng/μl for each guide, 10 ng/μl for each guide, or 5 ng/μl for each guide. In each dilution, Cas9 mRNA was present at a concentration of 100 ng/μl. These mixtures were microinjected into C57BL/6J zygotes, which were subsequently implanted into pseudopregnant female C57BL/6J mice.
The resulting live founders were screened by PCR and Sanger sequencing (primers in Table 4). There were three iterations of screening. First, we chose 7 exons distributed across the locus for PCR amplification and Sanger sequencing in 100 live founders. Heterozygous and homozygous indels were identified by analyzing the sequence traces, and 15 founders were found to harbor some mutation. 50 of the 100 founders were from the lowest concentration of sgRNA injection (5 ng/μl). No mutations were found in the first round of screening in these animals, and they were not analyzed further. In the second round of screening, the 35 remaining founders were analyzed for indels at the other 15 exons. In the third round, they were screened for rearrangements between exons by PCR with single forward primers mixed with pooled reverse primers. PCR products detected above background level were purified and sequenced using the forward primer. Mice carrying any mutation detected in any round of screening were bred for one generation with C57BL/6J animals. The resulting G1 offspring were screened by amplicon sequencing. Males were prioritized when available for ease of sperm cryopreservation.
To generate PcdhgC4KO mice, only sgRNA complimentary to exon C4 was microinjected (50 ng/μl, Table 3) along with Cas9 mRNA (100 ng/μl). Of the resulting 16 live founders, 9 contained mosaicindels. 3 founders were crossed with C57BL/6J, one of which transmitted a 13 bp deletion. These mice were crossed to homozygosity, and all 22 variable exons were screened by PCR and Sanger sequencing to verify that only the Pcdhgc4 exon was disrupted.
Screening by amplicon sequencing
A TruSeq Custom Amplicon 1.5 assay was designed with Illumina DesignStudio. The amplicon length was 250 bp, 201 bp of which corresponded to target sequence. 153 targets were covered by 225 amplicons for a cumulative target length of 31,721 bp. Target regions were chosen to be centered at the sgRNA sites, the analogous sites in the variable exons in Pcdha and Pcdhb, and 95 predicted potential off-target sites. The target coordinates are listed in Supplementary File 1. Genomic DNA was extracted using the DNeasy 96 Blood & Tissue Kit (Qiagen) from 94 G1 animals and processed along with a C57BL/6J wild type control and a negative control. Libraries were constructed using the TruSeq Custom Amplicon (Illumina) Library prep kit. Briefly, custom probes were hybridized to the flanking region of interest in gDNA, DNA polymerase extended across the target region, and unique barcodes and sequencing primers are added by PCR. Libraries were pooled and sequenced on a MiSeq instrument from Illumina with paired-end reads, 150 bp long.
Sequences were matched to samples by barcode and aligned to the target regions (BaseSpace, Illumina). Variants were called using Genome Analysis Toolkit (GATK) (McKenna et al., 2010). As CRISPR/Cas9 mediated NHEJ events result in small to medium sized indels, but not SNPs (Allen et al., 2018), we focused on indels that exceeded the following thresholds: QUAL ≥ 850; DP ≥ 90; QD ≥ 5. We also filtered out indels detected in the wild type C57BL/6J control, as these reflect discrepancies from the reference genome. Subsequent whole genome sequencing confirmed these mutations in the Pcdhg locus, but not potential off-target mutations expected to be linked (e.g., Pcdhb7). Furthermore, one mutation that did not exceed our threshold was detected by whole genome sequencing (A12 in Pcdhgem35). Therefore, Table 1 – Table supplement 1 summarizes mutations detected in the Pcdhg locus above and below the threshold, as indicated.
Because of the pooled amplicon reaction, we reasoned that large rearrangements between guide sites could be detected by amplicon sequencing if enough sequence remained on either side of the junction. To find these junctions we used BreaKmer (Abo et al., 2015). We were able to detect some of these junctions with BreaKmer, but subsequent whole genome sequencing revealed rearrangements missed by BreaKmer in each sequenced mouse line. In some cases, this could be because deletions extended beyond the amplicon site on one or both sides of the junction. However, many junctions were flanked by enough amplicon sequence to expect them to be detectible in this data set. Indeed, by visually analyzing the read alignments in the Integrative Genomics Viewer (IGV, Broad Institute) we were able to find some of these junctions when mating paired-end reads mapped to different exons (Figure 1-figure supplement 1B). We used this approach to identify additional junctions in the cryopreserved mouse lines as indicated in Table 1 – Table supplement 1.
Whole genome sequencing
Genomic DNA was isolated from homozygous mutants using the DNA Extraction from Fresh Frozen Tissue protocol (10X Genomics). Briefly, nuclei were isolated from tissue, lysed using proteinase K, then DNA purified using magnetic beads. Linked-read whole genome libraries were constructed using the Genome Chip Kit v2 (10X Genomics). Briefly, high molecular weight DNA was partitioned into Gel Bead-In-EMulsions (GEMs) where unique barcoded primers were added to individual molecules of DNA. After the GEMs were dissolved, Illumina specific sequencing primers and barcodes were added by isothermal amplification, then library construction was completed via end repair, a-tailing, adapter ligation, and amplification. Libraries were sequenced on a HiSeq X (Illumina) by Novogene (Sacramenta, CA) with 150 bp paired-end reads. Sequences were aligned using the Long Ranger analysis pipeline from 10X Chromium and visualized in Loupe (10X Genomics) or IGV (Broad Institute). Paired-end reads were used to find NHEJ junctions between guide sites, while reads spanning the junctions were analyzed to uncover the specific sequences of these junctions.
Data deposition
All Illumina sequencing data is available at the Gene Expression Omnibus (GEO) with the accession number XXXXX (in progress) or the Sequence Read Archive (SRA) with number XXXX. Quantitative RT-PCR
RNA was isolated from cerebral cortex of animals at P0 (13R1 mutants and littermate controls) or 2-12 weeks of age (other mutants and littermate controls) using Trizol reagent. Five μg of RNA per sample was used to make cDNA using Superscript III (Invitrogen) according to the manufacturers protocol. qPCR was performed in triplicate (technical replicates) using primers listed in Figure 3 – Figure supplement 2 with SYBR Green PCR Master Mix. The relative abundance of each transcript was calculated using the ΔΔCt method, normalized to GAPDH and littermate controls. The relative levels of each transcript in controls, 3R1, and 3R2 mutants were compared using an ANOVA with Tukey post-hoc tests. 13R1 and 1R1 mutants were compared with littermate controls for each transcript using a t-test.
Western blotting
Whole brains at P0 or adult ages (3-8 months) were homogenized in RIPA buffer (0.1% SDS, 0.25% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM NaF) plus protease inhibitors (Roche Mini cOmplete) using a Dounce homogenizer and a Wheaton overhead stirrer. The lysate was centrifuged at 16,000 X g for 15 min at 4°C to remove cell debris and proteins were quantified using a BCA assay kit (Pierce/Thermo Scientific). Thirty or 40 µg of protein was resolved on Mini-PROTEAN TGX precast 12% SDS-PAGE gels (Bio-Rad) and proteins were transferred to a nitrocellulose membrane using the Bio-Rad Trans-Blot Turbo Transfer System. Membranes were blocked using 10% skim milk for 1 hour and incubated with the primary antibody in buffer (2.5% BSA in TBS-T (Tris buffered saline with 0.1% Tween-20) overnight at 4°C. The following day, membranes were washed using TBS-T and incubated in HRP-conjugated secondary antibody for 1 hour. Membranes were washed, developed using the SuperSignal West Pico ECL reagents (Thermo Scientific), and imaged using a Li-Cor Odyssey Fc Imaging system.
Immunofluorescence
Tissues were processed and stained as described previously (Garrett et al., 2016; Prasad et al., 2008). Briefly, neonatal spinal columns were removed and fixed by immersion in 4% paraformaldehyde (PFA) for four hours at 4° C, followed by extensive washing in PBS and cryopreservation in 30% sucrose. Eyes were enucleated and dissected to remove the cornea and lens, then fixed overnight by immersion in 4% PFA at 4° C followed by cryopreservation in 30% sucrose. Tissue was embedded in OCT (Sakura-Finetek) and sectioned with a cryostat onto positively charged Superfrost Plus slides (Fisher Scientific). After blocking in 2.5% bovine serum albumin with 0.1% Triton-X-100 in PBS, primary antibodies were incubated on the slides overnight at 4° C in a humidified chamber, followed by secondary antibodies for 1 hour at room temperature. Whole mount retinas were stained free floating in primary antibody diluted in blocking solution with 0.5% Triton-X-100 for 48-72 hours, and in secondary antibody for 24 hours. Sections were counter-stained with DAPI (4’,6-diamidino-2-phenylindole), prior to mounting with Fluoro-Gel mounting media (Electron Microscopy Services #17985-11).
Antibodies
Primary antibodies used included the following: Guinea pig anti-VGLUT3 (1:10,000, Millipore), sheep anti-tyrosine hydroxylase (1:500, Millipore), rabbit anti-melanopsin (1:10,000, ATS), mouse anti-Brn3a (1:500, Millipore), goat anti-choline acetyltransferase (1:500, Millipore), mouse anti-NeuN (1:500, Millipore), mouse anti-GFAP (1:500, Sigma), rabbit anti-FoxP2 (1:4000, Abcam), rabbit anti-Pax2 (1:200, Zymed), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling Technologies), mouse anti-parvalbumin (1:500, Sigma), and mouse anti-GAPDH (1:500, Abcam). Mouse monoclonal antibodies against γ-Pcdh proteins used for western blots (1:500-1:1000) were generated by NeuroMab in collaboration with the Weiner laboratory (Lobas et al., 2012) and obtained from Antibodies, Inc.: N159/5 (detecting an epitope in constant exon 1 or 2 and thus all 22 γ-Pcdh isoforms); N144/32 (detecting all γA subfamily isoforms); N148/30 (specific for γB2); N174B/27 (specific for γC3). A rabbit polyclonal antibody raised at Affinity BioReagents against the peptide sequence VAGEVNQRHFRVDLD (within EC1) from murine γC4 was also used for western blotting (1:1000). Secondary antibodies were conjugated with Alexa-488, −568, or −647 (1:500, Invitrogen) or HRP (1:1000-1:5000, Jackson Immunoresearch).
Imagequantification
Control and mutant spinal cords were imaged using epifluorescence at equivalent thoraco-lumbar locations using a Leica SPE TCS confocal microscope and captured with Leica Application Suite software. From the resulting images, cell counts were performed using Cell Counter plugin in Fiji image analysis software (Schindelin et al., 2012). For FoxP2 and CC3 analysis, all immunoreactive cells were counted per hemicord from 12 hemicords per animal and at least 3 animals per genotype. For Pax2 analysis, all interneurons ventral to the central canal were quantified and compared. Values for wild type and mutant genotypes were compared using One-way ANOVA with Dunnett’s multiple comparison test. Sample size was based on prior studies, as effective group sizes were known (Prasad et al., 2008).
Whole mount retinas were imaged by confocal microscopy. In the resulting image stacks, cell density was measured using the Cell Counter plugin in Fiji image analysis software (Schindelin et al., 2012). Values from at least two fields per retina, sampled from different regions midway between the center and periphery (technical replicates), were averaged. These averaged values from at least six retinas per genotype (biological replicates) were compared using an ANOVA with Tukey post-hoc tests. Sample size was based on prior studies, as effective group sizes were known. Retinas were analyzed at 2 weeks of age, with the exception of 1R1 mutants and littermate controls, which were analyzed at 2 weeks and adult (3-6 months of age). Here, values were compared using a two-way ANOVA with age and genotype as independent variables, followed by Tukey pairwise comparisons.
All p-values are listed in Supplementary File 3 gnom AD database analysis
For each cPcdh isoform in human, the gnom AD browser was queried (gnomAD.broadinstitute.org; (Karczewski et al., 2019)) and the ratio of observed to expected (o/e) loss of function variants was recorded along with its 90% confidence interval.
COMPETING INTERESTS
The authors declare no financial or non-financial competing interests.
SUPPLEMENTARY INFORMATION
Supplemental File 1: Genomic coordinates of custom amplicon sequencing targets
Supplemental File 2: Primary protein sequences resulting from CRISPR/Cas9-mediated mutations
Supplemental File 3: p-values from all analyses
ACKNOWLEDGEMENTS
We would like to thank the scientific services at the Jackson Laboratory for assistance throughout this project, including Genetic Engineering Technologies, Microinjection, and Reproductive Sciences services for the production and preservation of new mutants, the Genome Technologies service for sequencing, and the Bioinformatics service for data analysis. We would also like to thank Kate Miers for assistance with the mouse colony. This work was supported by NIH Grant NS090030 to R.W.B. and J.A.W. and NIH Grant NS055272 to J.A.W. The Scientific Services at The Jackson Laboratory are supported by NIH Grant CA034196.