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’’).
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).
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 (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).
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.
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.
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.
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).
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).
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.
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.
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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