Tuesday, June 6, 2023

A CRISPR/Cas9-generated mutation in the zebrafish orthologue of PPP2R3B causes idiopathic scoliosis – Scientific Reports

Expression of PPP2R3B/ppp2r3b in human foetuses and zebrafish

To begin to investigate the requirement for PR70 in vertebrates, we looked for expression of PPP2R3B in human foetuses. The orientation of tissue sections used is given in Supplementary Fig. 1. Using in situ hybridisation, we noted some locations of PPP2R3B expression that are potentially relevant to scoliosis. This included the neural tube, dorsal root ganglia and myotome (Fig. 1A). As a control, we used a GFP antisense probe with the same length and GC content as the PPP2R3B probe, which gave no signal (Fig. 1B). Expression was also noted within the vertebrae as well as Meckel’s cartilage (Fig. 1C–H). PPP2R3B transcripts were detected within cartilage condensations suggestive of a role in chondrogenesis, although it was not expressed within the perichondrium where osteoblast precursors reside prior to their migration into the cartilage matrix (Fig. 1C’, E’). In both of these locations, PPP2R3B expression was similar to that of SOX9 on adjacent sections (Fig. 1D’, G’, H’). Within Meckel’s cartilage, chondrocytes within cartilage condensations also expressed SOX10, as did the perichondrium (Fig. 1F’). SOX10 is a marker of neural crest stem cells confirming the contribution of this lineage to skeletal elements within the jaw. We note that PPP2R3B expression was quite broad, albeit with accentuation of signal in certain locations, including myotome and vertebral chondrocytes, as shown by intermediate power images (Fig. 1A’, C’’).

Figure 1

PPP2R3B is expressed at sites of chondrogenesis in normal human foetuses. Expression of PPP2R3B, SOX9 and SOX10 in normal human foetuses at Carnegie stages (CS) 17 (A, B) 23 (C, D) and 22 (E–H). (A, B) In situ hybridisation at low power showing expression of PPP2R3B within interneuron/motor neuron precursors (np), dorsal root ganglia (drg) and myotome (m) but no signal generated using a GFP negative control. (C, D) Expression of PPP2R3B in vertebral bodies. Insets show regions magnified in C’ and D’. Note expression in chondrocytes (c). PPP2R3B is also expressed in the notochord (n) whereas SOX9 is not. (E–H) Expression of PPP2R3B within Meckel’s cartilage (insets magnified in E’–H’). Note expression co-localises with SOX10 and SOX9 within chondrocytes (c) but expression is absent from perichondrium (p). nt, neural tube; g, gut; k, kidney; tel, telencephalon; e, eye; ns, nasal septum; t, tongue. Scale bars are 500 µm in (AD, A’, C’); 60 µm in (C’, D’); 1 mm (EH); 10 µm in (E’–H’).

By aligning the human PR70 protein sequence to the zebrafish translated genome, we identified only a single orthologue with significant similarity, and the genomic locus encoding Ppp2r3b showed conserved synteny with their mammalian counterparts. Orthologues of neither PPP2R3B nor the adjacent gene, SHOX, are found in rodents. We therefore analysed expression of the orthologous zebrafish ppp2r3b gene by in situ hybridisation (Fig. 2A, B). At 24 h post-fertilisation (hpf) we noted repeated chevron-shaped patterns of expression along the trunk of the embryo representing the somites which will go on to form axial muscle. Within the head region, we also noted rostral expression (Fig. 2C).

Figure 2
figure 2

Expression of ppp2r3b in zebrafish. (A–C) In situ hybridisation showing expression of ppp2r3b at 24 hpf. High-power view showing expression in somites (B) and rostally (C). (D) Transverse section of 36 dpf zebrafish stained with Mallory’s trichrome. Arrows indicate examples of myosepta separating bundles of muscles fibres. (E) Similar section to (D) showing expression of ppp2r3b at 36 dpf. Arrows indicate myosepta that define muscle bundles outlined in red (mb, F). (G, H) Higher magnification images whereby arrows indicate myosepta, and arrowheads indicate staining around individual muscle fibres. Dashed boxes show the location of the insets which individual muscle fibres at higher magnification. (I) Inset from F showing mineralised bone (b) within vertebral centra showing expression of ppp2r3b in squamous chordoblast cells (arrowheads). Asterisks label individually staining cells of distinct morphology. Scale bars are 200 µm in (A, B); 40 µm in (C); 150 µm in (DH); 12 µm in (I).

We also analysed expression in zebrafish at 36 dpf of age. We noted prominent expression throughout the region in which fast twitch muscle is thought to reside, adjacent to vertebral centra (Fig. 2D–H). At this stage, a number of distinct muscle bundles separated by myospeta can be seen on transverse tissue sections (Fig. 2D). ppp2r3b is seen to be expressed prominently in these (Fig. 2E), and it is notable that staining intensity may vary between bundles, although this may relate to tissue sectioning quality (Fig. 2E). Within these muscle bundles, higher power imaging shows that ppp2r3b expression appears to surround individual muscle fibres (arrowheads, Fig. 2G, H) which is consistent with nuclear staining of multinucleate muscle fibres. In contrast there was only limited expression in proximity to mineralised bone—based on their morphology and location on vertebral bone surfaces, we did identify expression in what could be squamous chordoblast (osteoblast) -like cells, as previously reported9, although it is noteworthy that these cells were very rare (Fig. 2I). It should be noted that to definitively identify these cells as chordoblasts it will be necessary to identify specific markers for these cells in co-localisation studies in future.

Generation of ppp2r3b mutant zebrafish using CRISPR/Cas9 gene-editing

To generate a genetic model of ppp2r3b loss-of-function, we used CRISPR/Cas9 gene-editing to target this gene using a sgRNA located within exon 2 (Fig. 3A). This sgRNA was located on the forward strand and had no self-complementarity or predicted off-target sites according to the chopchop tool (http://chopchop.cbu.uib.no/). An Mse I restriction site was located within the sgRNA binding site which allowed us to monitor the efficiency with which indels were introduced at this location. Direct sequencing of a selection of cloned mutations from mosaic F0 embryos at 24 hpf was consistent with many previous reports in zebrafish, showing that CRISPR/Cas9 typically produces complex indels involving deletions of between 2 and 18 nucleotides (Fig. 3A).

Figure 3
figure 3

Targeting ppp2r3b in zebrafish using gene-editing. (A) ppp2r3b gene structure showing the location of primers used for genotyping (red arrows) and sgRNA (green) used for gene-editing. Below, examples of mutant sequence reads cloned from pooled F0 embryos. The sgRNA site is highlighted in yellow within the wild-type sequence. Inserted and deleted nucleotides are highlighted in pink and blue, respectively. (B) Sequence chromatograms showing the homozygous wild-type reference and alternative alleles, and the homozygous and heterozygous mutant reads. (C, D) RT-PCR showing expression of ppp2r3b using primers located within (C, D) exons 1–3 (band at expected size, 491 bp) or (D) or exons 1–7 (band at expected size, 1000 bp) at the indicated stages.

We have now outcrossed these F0 mosaics and their progeny for more than 5 generations to achieve germline transmission and to avoid possible off-target mutations. We isolated a line of zebrafish carrying a 7 bp deletion in exon 2 of ppp2r3b which results in the frameshift mutation p.Ala31ValfsX150 (Fig. 3B). Homozygous mutants are hereafter referred to as ppp2r3b−/−. During the course of our breeding and genotyping, we noted a single nucleotide polymorphism (SNP) located within the sgRNA binding site which is not present on publicly available databases. This SNP encodes the single amino acid substitution p.Ser33Asn. In all subsequent analyses, we selected only heterozygotes whose wild-type allele encoded the reference SNP at this location in our breeding population. RT-PCR and direct sequencing of gel extracted products using primers located in exons 1–3 or 1–7 failed to identify gross alternative splicing within ppp2r3b transcripts generated from pooled 24 hpf embryos from a hetxhet incross or individual homozygous mutant animals at 48 dpf of age—these products were of the predicted size, as in wild-types, and qRT-PCR demonstrated that transcript levels in mutants were not different from wild-type (Fig. 3C, D, Table 1). We noted that there was no deviation from expected Mendelian ratios showing that this mutation does not affect viability (Fig. 4). We also endeavoured to generate a pool of homozygous mutant adults with which to breed maternal-zygotic mutant zebrafish. This was not possible, because homozygotes never produced any eggs, and thus we conclude that they are infertile. Notably, crosses of homozygous females with wild-type males readily produced viable fry, whereas the opposite did not.

Table 1 qPCR analysis of bone and muscle marker genes.
Figure 4
figure 4

ppp2r3b mutants are viable at all ages. Proportions of wild-type (WT), heterozygous (Het) and homozygous (Hom) mutant (ppp2r3bAla31ValfsX150) animals at the stated ages. Total numbers of embryos analysed are indicated.

ppp2r3b homozygous mutant zebrafish exhibit a fully penetrant scoliosis phenotype

We did not identify any phenotypic abnormalities in heterozygous or homozygous mutants at larval stages. At 48 dpf, we noted that homozygotes developed severe kyphoscoliosis (Fig. 5A, B). The pattern of kyphoscoliosis was very stereotypical, characterised by two ventral curves located within the precaudal and caudal vertebrae at numbers 7–9 and 25, respectively. These ventral curves flanked a dorsal curve located at approximately caudal vertebrae number 18. There was often also a sharp lateral bend within the caudal fin, although this was not as consistent. At this age, wild-type siblings never exhibited kyphoscoliosis and the spine exhibited a very gentle ventral curvature within the precaudal region only. Scoliosis is a common phenotype in old zebrafish, presenting in excess of 18 months of age in our aquatics facility but never earlier than this. This is often associated with Mycobacterium chelonae infection, however, ongoing microbiological testing confirms that this species is absent from our facility.

Figure 5
figure 5

(A) Bright field images and (B) microCT scans of representative wild-type or homozygous ppp2r3b−/− mutant zebrafish at 36 or 48 dpf. White arrows in (B) point to sharp lateral curvatures of the spine. (C) Quantification of the proportion of animals with mild, moderate or severe kyphoscoliosis. Numbers of animals are given. Scale bars are 5 mm. All animals were generation F4.

We monitored the onset and progression of scoliosis in ppp2r3b−/− mutants. Scoliosis was first seen at 36 dpf (Fig. 5)—no axial defects were observed before this as we stained animals for Alizarin red and Alcian blue as early as 15 dpf (Fig. 6). At 36 dpf, the typical presentation was moderate ventral curvature within the precaudal region, with relatively little curvature of the caudal vertebral regions. However, by 48 dpf, the final pattern consisting of two ventral curves and one dorsal curve was apparent. Quantification of the proportion of animals with moderate or severe kyphoscoliosis confirmed that this phenotype became worse with time (Fig. 5C). At 48 dpf, the kyphoscoliosis phenotype was present in all homozygous mutants, but not in any wild-type or heterozygous siblings. Therefore, ppp2r3b−/− mutant zebrafish exhibit adolescent onset and progressive kyphoscoliosis, which is fully penetrant and reminiscent of human IS.

Figure 6
figure 6

Temporal analysis of cartilage. Articular cartilage within the hypural bones is lost by 36 dpf (A, arrows) in ppp2r3b−/− mutants but forms normally at 15 dpf (B) and is maintained until 26 dpf (C). Within the vertebral bodies, cartilage is induced and maintained normally (D, E) and is replaced by mineralised bone by 36 dpf (A, and data not shown) in mutants. Scales bars are (A) 2.5 mm, (B) 0.5 mm, (C) 2.5 mm, (D) 150 µm, (E) 5 mm.

To confirm that this phenotype is not the product of off-target mutations we also generated a second frameshift mutant line following microinjection of a ribonucleoprotein complex consisting of sgRNAs targeting exon 1 in complex with Cas9. This generated a 19 bp deletion, causing an out-of-frame p.L82fsX24 mutation. Notably, homozygous zebrafish for this mutation also exhibited profound scoliosis (Fig. 7).

Figure 7
figure 7

Scoliosis replicated in a mutant line encoding the mutation p.L82fsX24. Schematic of ppp2r3b showing the genic location of two sgRNAs used to target exon 1, highlighted in blue and pink, respectively. Deletion of 19 bp is indicated by ‘-’ symbols and the confirmatory sequence chromatogram is shown. Representative examples of two 3 month old zebrafish (above) stained for Alizarin red (below). All animals are generation F4. Scale bars are 5 mm.

Kyphoscoliosis in ppp2r3b mutants is associated with reduced bone mineralisation of vertebrae

To investigate this phenotype further, we analysed bone mineralisation and cartilage formation in ppp2r3b-/- mutants. Precaudal vertebrae 5–13 include a neural spine, which projects dorsally, and two ventrally located ribs, while the caudal vertebrae include neural and haemal spines which mirror one another in size. Alizarin red staining showed that the ratio between the lengths of the neural and haemal spines within the caudal region were approximately equal in length in homozygous mutants as in wild-types and heterozygotes (Fig. 8A–C). Within the precaudal region, the ribs are approximately 2.5 times longer that the neural spines (dorsal:ventral ratio of 0.4), however, we found that the ribs were relatively shorter in ppp2r3b homozygotes as compared to wild-types or heterozygotes (Fig. 8C), suggesting a defect in patterning and/or ossification. We also noted a marked reduction in Alizarin red staining intensity throughout the vertebral body and spines/arches, which was uniform across all vertebrae in caudal and precaudal regions (Fig. 8B).

Figure 8
figure 8

Morphometric analysis of mineralised bone. (A, B) Alizarin red staining of vertebrae 36 dpf zebrafish of the indicated genotypes. (C) Quantification of the neural spine:hemal spine/rib length ratios for vertebrae 5–28. Data-points for individual animals are indicated by different colours. Mean ± standard deviation of values of these ratios averaged across all ribs for each animal, indicated by the brackets, and subsequently averaged over three animals are 0.401 ± 0.024, 0.423 ± 0.029 and 0.335 ± 0.005 for wild-type, heterozygous and homozygous mutant animals, respectively (n = 3 animals). P-values compared to wild-type are indicated. This represents a statistically significant difference in homozygotes versus each of the other two genotypes (t-test). Tail lengths taking into account spinal curvature were 12.694, 12.872 and 12.749 mm for the representative wild-type, ppp2r3b+/− and ppp2r3b−/− animals shown, respectively. All animals are generation F4. Scale bars are 5 mm in (A); 2 mm in (B).

To investigate bone formation in more detail, we performed microCT scanning to compare skeletal tissue parameters of vertebrae at 36 dpf which represents the onset of scoliosis. This Alizarin red staining suggested that the gross structure of all vertebrae was normal without the characteristic wedging of vertebrae that has been reported previously4, even at the sites of curvature (Fig. 6A, B). Indeed, whereas 3D renders and longitudinal sections through contiguous vertebrae showed that adjacent vertebrae were closely apposed in wild-types, with a uniform and narrow intervertebral space, the intervertebral spaces were wedge-shaped in mutants, corresponding with the direction of curvature (Fig. 9A, B). Remarkably, we also found that multiple holes were apparent throughout the mutant vertebrae (Fig. 9A, B). Measurement of tissue mineral density (TMD), which is specific to cortical bone and is appropriate for analysis of non-trabeculated vertebral bone, was significantly reduced (Fig. 9C). However, the overall dimensions of the vertebrae, including length and diameter, were not affected suggesting a specific effect on bone mineralisation rather than morphogenesis. qRT-PCR of RNA extracted from the trunk at 36 dpf did not reveal any changes in the expression of a panel of key bone cell markers (Table 1). Histological analyses of osteoclasts and osteoblasts did not reveal differences between wild-type and mutant (Fig. 10)—Mallory’s trichrome staining showed similar numbers of vacuolated chordoblasts (osteoblasts) and squamous chordoblasts within the notochord centra and sheath, respectively. Tartrate-resistant acid phosphatase (TRAP) staining labels osteoclasts and revealed similar staining in neural and haemal arches in both wild-type and mutant, although no staining was detected within the vertebrae centra which is where the reduced TMD was observed previously. These data are subject to technical limitations relating to the proportionally small number of bone cells in whole zebrafish tissues at this stage and the non-quantitative nature of histological methods and are therefore not conclusively negative. In future, it will be necessary to analyse these cell types in more detail.

Figure 9
figure 9

Reduced mineral density of vertebral cortical bone in ppp2r3b−/− mutants. (A, B) Representative 3D and cross-sectional images from microCT scans of vertebrae in wild-type and ppp2r3b−/− mutant zebrafish at 36 dpf. Note holes are apparent in the mutant vertebrae. Red arrows indicate intervertebral discs. (C) Quantification of a selection of cortical bone parameters measured in wild-type and ppp2r3b−/− mutant vertebrae (n = 3). BV/TV, bone volume/tissue volume; B.Th, bone thickness; TMD, tissue mineral density. p-values are given (t-test). NS, not significant. Average tail length (TL) for both mutant and wild-type animals was 12.6 mm with no difference between the two groups (p-value = 0.15, t-test). TLs of the two representative animals shown in (A) and (B) were 12.912 and 12.907 mm, respectively. Animals are generation F4. Scale bars are 5 mm in (A, B) left panel; 3.75 mm (A, B) right panels.

Figure 10
figure 10

No obvious changes in osteoclast or osteoblast staining in vertebrae. (A, C) Mallory’s trichrome staining of vertebra centra. Black arrows show examples of vacuolated chordoblasts (osteoblasts) within the vertebra centrum while red arrows show squamous chordoblasts within the notochord sheath. (B, D) tartrate-resistant acid phosphatase (TRAP) staining in transverse sections from wild-type and ppp2r3b−/− mutant zebrafish at 36 dpf. Red signal indicates TRAP staining in neural and haemal arches, but no staining was detected within the vertebrae centra. Scale bars are 50 µm in (A, C); 14 µm in (B, D).

Abnormal mitochondria associated with axial muscle in ppp2r3b mutants

Given the link between progressive spinal curvature and proprioception, and the expression of PPP2R3B/ppp2r3b that we detected in muscle and bone, we monitored muscle fibre and neuromuscular junction formation in our mutant fish. Initially we analysed skeletal muscle birefringence at 48hpf, an established indicator of muscle fibre integrity based on polarised light transmission through striated fibres. No obvious changes in birefringence signal were detected between ppp2r3b mutants and wildtype siblings (Fig. 11A). To challenge a role for PR70 in neuromuscular development, embryos were further stained for F-Actin and Acetycholine receptor (AChR), using fluorescently conjugated phalloidin and α-bungarotoxin. Muscle fibres and AChR localisation appeared organised and indistinguishable between ppp2r3b−/− embryos and wild-type siblings (Fig. 11B). Thus, PR70 is not required for normal neuromuscular development.

Figure 11
figure 11

ppp2r3b mutants display abnormal mitochondria formation during adult stages of development, whilst muscle formation remains intact. (A) Skeletal muscle birefringence at 48hpf shows comparable muscle integrity between ppp2r3b mutants and wildtype siblings. Scale bar 200 µm. (B) Muscle fibres and motor neuron synapses appear normal in mutants compared to wildtype siblings at 48hpf, analysed by staining for F-Actin (Red) and Acetylcholine receptors (AChR, green) respectively. Left panels show a z-stack projection of F-Actin with AChR, right panels show a single focal plane of F-Actin/AChR/DAPI. Scale bar 100 µm. (C) Transmission electron micrographs indicate normal sarcomeric assembly (brackets) in juvenile mutants + compared to wildtype controls, however mitochondria (arrows) are noticeably malformed and less abundant. Scale bar 1 µm. (D) High magnification electron micrographs showing detailed images of the sarcomeres (brackets) and mitochondria (arrows) in wildtype compared to mutant adolescent muscle samples. Scale bar 200 nm. (E) Quantification of mitochondrial area adjacent to sarcomeres showed a statistically significant reduction in mutants (t-test) (n = 22 and 17 biological replicates for wild-type and ppp2r3b−/− animals, respectively).

To evaluate muscle development during juvenile growth, at a point when scoliosis has manifested, transmission electron micrographs were produced from muscle biopsies. ppp2r3b−/− fish displayed normal myofibril organisation and sarcomeric assembly (Fig. 11C and D), and consistent with this, qRT-PCR analyses showed that expression of key muscle markers was unchanged (Table 1). However, a marked reduction in mitochondrial content was observed in ppp2r3b mutants (Fig. 11C). Closer examination of the mitochondria showed general dysmorphic character in the mutants with undefined cristae and smaller overall size (Fig. 11D). Quantification of mitochondrial area adjacent to sarcomeres showing a statistically significant reduction in mutants (Fig. 11E). Taken together, these data suggest that ppp2r3b, whilst not required for the formation of muscle, is required to maintain muscle mitochondrial abundance.

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