High resolution gene expression analysis detected dynamic spatiotemporal localization of rspo2 and rspo3 transcripts in zebrafish cranial mesenchyme. (A) Whole-mount RNA in situ hybridization detected rspo2 and rspo3 transcripts in the midbrain (mb), forebrain (f), otic vesicle (o), and Meckel’s cartilage (arrowhead) at 24 and 48 hpf in lateral and dorsal views. Transcript of rspo3 was additionally detected at 24 and 48 hpf in the forebrain (f) and at 48 hpf in the ethmoid plate (open arrowhead). Scale bar: 100 um. (B) Maximum projections of z-stacks of coronal sections of zebrafish embryos, section in plane with eyes. Labeled schematic at left. At 48 hpf, rspo2 and rspo3 transcripts were highly co-localized in the mesenchyme surrounding the condensing trabeculae cartilage mesenchyme, marked by runx2a expression. rspo2 is also detected within the condensing mesenchyme at this timepoint. At 5 dpf rspo2 and rspo3 continue to be co-expressed in mesenchyme and perichondrium surrounding cartilage elements, specifically the ethmoid plate and Meckel’s cartilage. Rspo3 expression is particularly high in runx2a expressing osteogenic precursor cells associated with Meckel’s cartilage, as well as where the palatoquadrate meets the ethmoid plate (white arrowhead). Abbreviations: e: eye, o: olfactory organ, s: stomodeum, t: trabeculae. Scale bar: 100 μm.

RNAscope gene expression analysis of Rspo2 and Rspo3 in mouse embryos. Coronal sections of wild-type mouse embryos at E13.5 and E15.5. Labeled schematic at left. (A) RNAscope revealed diffuse expression of Rspo3 throughout the cranial mesenchyme at E13.5. Col1a1 in situ hybridization and Runx2 immunofluorescence were used to identify osteogenic precursor cells and Rspo3 expression was detected within these cells. Of particular note is Rspo3 expression within Meckel’s cartilage (m) and the developing mandible (arrow; mn). (B) At E15.5, Rspo3 expression is detected diffusely thorough the mesenchyme, including in Meckel’s cartilage (m) and within osteogenic precursor cells (co-expressed with Runx2). Rspo2 expression is isolated to discrete cells within the developing tooth germ (tg). (B’) Higher magnification images (boxes depict location) of Rspo3 and Runx2 co-expression. (C) Within the developing molar at E15.5, Rspo2 and Rspo3 transcripts were detected in distinct non-overlapping regions, with Rspo3 expression in Meckel’s cartilage (m) and the dental follicle (df), while Rspo2 is expressed exclusively within the dental pulp (dp). Scale bar: 100 μm.

rspo2 and rspo3 are differentially expressed in zebrafish pharyngeal teeth. Representative images of maximum projections of z-stacks of sagittal sections of 180 dpf zebrafish. rspo2 (red) and rspo3 (white) transcripts were detected by RNAscope in situ hybridization. rspo3 is diffusely expressed throughout the supporting mesenchyme and highly expressed in enamel epithelium (open arrow) and some tooth pulp (closed arrow). Often within the tooth pulp, rspo3 expression is restricted to the outermost odontoblasts (*). Meanwhile, rspo2 expression is highest in enamel epithelium (open arrow) and very low within the tooth pulp. Scale bar: 100 μm.

Synergistic effect of rspo2 and rspo3 ablation on zebrafish limb development and craniofacial morphology. (A) Schematic illustrating experimental design. Targeted mutagenesis of rspo3^−/− in zebrafish was carried out using CRISPR/Cas9 gene editing. A – 20 bp deletion was bred to homozygosity. Intercross or rspo3^+/− were injected with 4 gRNAs against rspo2 and the resulting larvae were genotyped and analyzed for phenotype. (B) Whole mount ventral and lateral images of Alcian blue/Alizarin red S stained 9 dpf larvae. rspo3^−/− embryos that were rspo2 gRNA/Cas9 injected (rspo2^Δ) larvae were similar to wild-type except that rspo2^Δ larvae exhibited disrupted development of the pectoral fin. Impaired fin development was exacerbated with decreasing genetic dosage of rspo3 (black arrows, dotted yellow lines delineate fins). While craniofacial development in rspo3^−/− and rspo2^Δ larvae were largely normal, rspo3^−/−; rspo2^Δ double mutants exhibited a dysmorphic lower jaw (white arrow). Scale bar: 100 μm. (C) Quantification of pectoral fin developmental disruption. rspo2^Δ larvae tended to have disrupted development of a single pectoral fin. This effect was significantly exacerbated with decreasing genetic dosage of rspo3, as rspo3^−/−; rspo2^Δ double mutant larvae failed to develop pectoral fins altogether. (D) Quantification of angle measurements between Meckel’s cartilage (m) and palatoquadrate (pq). While rspo3^−/− and rspo2^Δ mutants had normal lower jaw morphology, rspo3^+/−; rspo2^Δ and rspo3^−/−; rspo2^Δ mutants displayed a significantly decreased angle at the Meckel’s/palatoquadrate joint. N = 10–16. p < 0.01. *Indicates significance relative to wild-type.

Synergistic effect of rspo2 and rspo3 ablation on zebrafish tooth development and Meckel’s cartilage. (A) Flat-mount images of Alcian blue/Alizarin red S stained 9 dpf zebrafish ventral cartilages. Zoom of pharyngeal teeth to right. rspo3^−/− larvae displayed anterior shortening of Meckel’s cartilage, which was exacerbated with rspo3^−/−; rspo2^Δ gRNA disruption (black bars). *indicate absent teeth. Scale bar: 200 μm. (B) Alizarin red S staining of pharyngeal teeth shows that rspo3^−/− are generally normal relative to wild-type while rspo2^Δ larvae have a reduced number of teeth (average of 2 versus 8). Tooth number in rspo2^Δ larvae decreased further with decreasing wild-type alleles of rspo3 (zero teeth detected in rspo3^−/−; rspo2^Δ mutant). (C) Quantification of the anterior–posterior/rostral length of Meckel’s cartilage shows a primary effect in rspo3^−/− larvae, which is exacerbated in rspo3^−/−; rspo2^Δ mutants. (D) Quantification of the anterior–posterior length of ceratohyal cartilage shows no effect in rspo3^−/− larvae, suggesting a cartilage element-specific effect of rspo3 and rspo2. N = 10–16. p < 0.01. *Indicates significance relative to wild-type.

rspo3 ablation does not impact initial bone mineralization but increases osteoclast area of activity. (A) Representative images of maximum intensity projections of confocal z-stack of whole-mount 10 dpf zebrafish. Zebrafish express sox10:kaede transgene allowing fluorescent imaging of cartilage elements. Zebrafish were live-stained with Alizarin red S to visualize mineralized structures. No differences in intensity of Alizarin staining, elements stained, or cartilage architecture were noted in the rspo3^−/− fish. Scale bar: 100 μm. (B) Lateral view of 14 dpf showing increased osteoclast activity (red stain in the black dotted box) in rspo3 mutant as compared to wild-type. At 21 dpf, more areas of osteoclast activity in the dentary, hyomandibular, pharyngeal teeth and jaws (solid arrow) were observed in rspo3^−/− compared to wild-type. Quantification of total area of red staining. P < 0.05. N = 5. Scale bar: 100 μm.

rspo3 mutants exhibited midface deficiency, frontal bossing and decreased body length. Reduced body length, midface deficiency and frontal bossing were observed in adult rspo3^−/− (180 dpf). (A) Lateral image of adult zebrafish showing midface depression in rspo3^−/− (solid arrow) compared to wild-type (open arrow). (B) Body length was significantly decreased in rspo3^−/− mutants relative to wild-type. (C) Oblique micro-CT image of rspo3^−/− and wild-type fish at 180 dpf. Individual bone elements are color coded (blue: parashenoid, pink: maxilla, yellow: premaxilla, green: dentary and red: anguloarticular). Scale bar: 10 μm. (D) Bone volume of the skull and of specific bones in rspo3^−/− and wild-type individuals demonstrates element-specific differences in volume. Parasphenoid and anguloarticular bone volume were significantly reduced in rspo3^−/− compared to wild-type fish. Abbreviations: aa: anguloarticular, d: dentary, m: maxilla, p: parasphenoid, pm: premaxilla. (E) 2D cephalometric analysis obtained from micro-CT of rspo3^−/− and wild-type fish. The angle formed by parasphenoid line and a line tangent to frontal bone identified frontal bossing, with increased angle in rspo3^−/− compared to wild-type. Diagram of lateral view of adult zebrafish showing the angular measurement. Bar chart showing statistical differences in the angular measurement between rspo3^−/− and wild-type. (F) 2D cephalometric analysis of rspo3^−/− and wild-type fish. The distance between nasal bone and a line drawn between dentary and frontal bone landmarks were measured. Diagram of lateral view of adult zebrafish showing the linear measurement from nasal bone to a line tangent to the frontal bone and dentary. The linear measurement value was significantly greater in rspo3^−/− mutants than in wild-type indicating the presence of midface hypoplasia. *p ≤ 0.05. Scale bar: 100 μm.

Adult rspo3 mutant zebrafish have reduced teeth number. (A) Micro-CT of 180 dpf zebrafish reveals that the adult rspo3^−/− animals exhibit decreased tooth number with several sockets missing teeth that are present in the wild-type. (B) rspo3^−/− have significantly fewer teeth than wild-type on both right and left sides. N = 9.7. *p < 0.05. (C) Summary diagram illustrating that rspo2 and rspo3 both regulate pharyngeal tooth development as well as have roles in morphogenesis of the craniofacial skeleton.