Benjamin R. Troutwine, Paul Gontarz, Mia J. Konjikusic, Ryoko Minowa, Adrian Monstad-Rios, Diane S. Sepich, Ronald Y. Kwon, Lilianna Solnica-Krezel, Ryan S. Gray
]. For example, the expression of AIS-associated variants of the centrosomal protein gene POC5 led to cilia defects in cell culture and spine deformities in zebrafish [
]. How motile cilia and CSF flow contribute to the pathogenesis of scoliosis in these models remains unresolved.
]. The fiber is largely composed of the glycoprotein subcommissural organ (SCO)-spondin [
], which is expressed and secreted from the SCO of the brain and from the floor plate at the ventral midline of the spinal cord [
]. In zebrafish, scospondin-null mutants fail to secrete and assemble a RF and fail to develop a straight body axis during embryonic development [
]. The failure to straighten the body axis or “curled tail down” phenotype has long been observed in zebrafish mutants with disrupted motile cilia physiology [
], many of which also exhibit defects in RF assembly [
]. Despite the clear association of the RF with morphogenesis of a straight body axis, it is not yet clear how disruptions of these processes regulate spine morphogenesis during larval development and in adults.
Hypomorphic Mutations of Scospondin Lead to Progressive Scoliosis in Zebrafish
]. To further determine the nature of the scospondinstl297 and scospondinstl300 alleles, we crossed scospondinstl297/+ heterozygous mutants to heterozygous scospondinhsc105/+-null mutants, which phenocopy the axis straightening defect reported for scospondinicm13/icm13 mutant (scospondinhsc105 described in [
] in this issue of Current Biology). For the progeny resulting from these crosses, we observed no defects in straightening the body axis in embryos. Instead, putative transheterozygous scospondinhsc105/stl297 mutant larvae displayed only mild axial curvatures at 15 dpf (27.1%; n = 236), which was identical to phenotypes observed in both zygotic and maternal zygotic scospondinstl297/stl297and scospondinstl300/stl300 mutants (Figures 1D and S1E). Altogether, these results confirm that scospondinstl297 and scospondinstl300 are independent hypomorphic missense mutations of scospondin.
]. First, we segmented 16 distinct vertebral bodies along the spine and each vertebra into the three elements: the centrum; the haemal arch; and the neural arch (Figure S2N’). Second, we quantified (1) vertebral mass, as total volume and thickness (Figures S2E–S2G, S2K–S2M, S2E’–S2G’, and S2K’–S2M’), and (2) bone quality, as tissue mineral density in comparison to a hydroxyapatite (HA) standard (mgHA/cm3; Figures S2H–S2J and S2H’–S2J’). These data demonstrated an overall increase in vertebral mass and mineralization in both scospondinstl297/stl297 and scospondinstl300/stl300 mutant fish (Figure S2). We observed no change in centrum length comparing homozygous scospondin mutants and heterozygous controls (Figures S1D and S1D’), indicating that decreased body lengths observed in the mutant fish are attributable to spine curvature rather than shortened or compressed vertebrae. Analysis of Z scores for each of these morphometric measures of the vertebral bodies and spine demonstrated that scospondinstl297/stl297 mutants display a more severe scoliosis (measured as sagittal and lateral displacement of the spine), although scospondinstl300 mutants exhibited increased bone volume and mineralization of the spine (Figures S2O and S2P). We suggest that this increase in bone deposition is a response to increased mechanical loading of the spine as the deformity progresses.
Disassembly of the Reissner Fiber Due to Defects in Secretion from the Floor Plate Is Correlated with the Onset of Axial Curvatures in Zebrafish
]. scospondinstl297 mutant phenotype segregates with a T6784A (ENSDART00000097773.4) mutation (Figure S1J), predicted to alter an evolutionarily conserved cysteine 2262 to serine (C2262S) in one of the low-density lipoprotein (LDL) receptor domains of the SCO-spondin protein (Figures 1H and S1L). scospondinstl300 phenotype segregates with a T2635A mutation (Figure S1K), predicted to alter cysteine 879 to serine (C879S), which is a highly conserved cysteine adjacent to a trypsin inhibitor like cysteine-rich domain (Figures 1J and S1L). Homology modeling of the Danio rerio SCO-spondin protein sequence partially maps onto a crystal structure of very low-density lipoprotein receptor (PDB: 6byv). Analysis of the model suggests that the scospondinstl297 mutation could disrupt an evolutionarily conserved disulfide bond (CysIV-CysVI) of the LDL receptor type A motif (Figures 1H and 1I), shown to be involved in protein stability of the LDL receptor [
]. Homology modeling for the region containing the scospondinstl300 mutation was unsuccessful; however, this cysteine is also neighboring many other well-conserved cysteine residues in SCO-spondin, suggesting it may also have a role in disulfide bonding.
], which labels the RF, the floor plate, and terminal ampulla region at the base of the spinal cord in zebrafish (Figures 2A and 2C ). In contrast to scospondinicm13/icm13 mutants, which fail to form a fiber at 3 dpf [
], scospondinstl297/stl297 mutant embryos displayed no obvious defects in the assembly of the RF (Figure 2B; n = 8). However, at 5 dpf, several scospondinstl297/stl297 mutants displayed a variety of defects of the RF (Figure 2F), including irregular punctate pattern (Figure 2E’), disassembled fiber with an occasional bolus of Reissner material (Figure 2E’’), or disassembled fiber with diffuse AFRU staining (Figures 2D, 2D’, and 2E’’’). We also observed several scospondinstl297/stl297 mutants displaying a normal fiber at 5 dpf (Figure 2E). scospondinstl297/+ larvae showed no changes in RF expression at 5 dpf (n = 16; Figures 2C and 2C’). Interestingly, the presence of an intact RF in scospondinstl297/stl297 mutants was directly correlated with a straight body axis at 5 dpf; in contrast, defects in the RF were directly correlated with the mild to severe axial curvatures in these mutants. At 10 dpf, scospondinstl297/+ heterozygous mutant larvae (100%; n = 8) displayed a straight body axis with an intact RF (Figures 2G and 2H). In contrast, scospondinstl297/stl297 mutants displayed diffuse AFRU staining, without a RF (100%; n = 8; Figures 2I and 2J). Interestingly, we observed apical localization of AFRU-stained Reissner material in floor plate cells in heterozygous scospondinstl297/+ (n = 8) and scospondinstl300/+ (n = 7) larvae at 10 dpf (Figures 2G’, 2H, and S1F). In contrast, we consistently observed the Reissner material localization at the basal surface of floor plate cells in both scospondinstl297/stl297 (n = 8) and scospondinstl300/stl300 (n = 6) mutants at 10 dpf (Figures 2I and S1G). These data suggest that disassembly of the RF in the two hypomorphic scospondin mutants may be in part due to disrupted secretion of Reissner material from the floor plate.
]. We have shown that mutation of two independent, evolutionarily conserved cysteine residues in SCO-spondin led to disassembly of the RF, axial curvatures in larval fish, and AIS-like scoliosis in adults. We hypothesize that the apical to basal switch in Reissner material polarity in the floor plate cells is the result of disrupted disulfide bonding within the protein leading to unfolded mutant SCO-spondin, which impedes SCO-spondin/Reissner material secretion from the SCO and floor plate, preventing normal RF assembly. Altogether, these data suggest that the RF has a continuous and instructive role in axial straightness and spine morphogenesis in zebrafish.
Dynamic Properties of the Reissner Fiber Revealed in Scospondin-GFPut24 Knockin Zebrafish
]. In our hands, SCO-spondin-GFP expression observed in scospondin-GFPut24, knockin zebrafish displayed tight colocalization (Pearson’s R value, 0.98) with the AFRU antiserum labeling [
] (Figures S3F–S3H). High-magnification confocal time-lapse imaging of the floor plate in scospondin-GFPut24 embryos demonstrated active secretion of SCO-spondin-GFP from the floor plate to merge with the RF (Figure S3I; Video S1), which supports a critical role for the floor plate in RF assembly during larval development. These observations also suggest that defects in apical localization of the AFRU-labeled Reissner material in scospondinstl297 mutants (Figure 2I’) may be driving progressive disassembly of the RF. In the head, we observed SCO-spondin-GFP expression in the SCO and in the flexural organ, with the RF joining these two organs (Figures 3A, 3A’ , S3C, and S3C’). In the tail, we detected SCO-spondin-GFP expression in the floor plate and the RF ending as a coiled mass within the terminal ampulla at the base of the spinal cord (Figures 3C, 3C’, and S3B’). We observed SCO-spondin-GFP-labeled RF and terminal ampulla in young adult fish (60 dpf; Figures S3D and S3D’), suggesting that the RF functions through the life cycle in zebrafish.
]. We speculate that the combination of bulk CSF secretion and its flow within the central canal is helping to push the bolus of RF material and the RF along the central canal. After the fiber is formed, during active axis elongation (between 2 and 3 dpf), we observed the dynamic breakdown of the RF in the terminal ampulla and distribution of SCO-spondin-GFP signal outward into the developing fin fold (Video S3).
], which is supported by the observations of rostral-caudal transport of radiolabeled-monoamines along the fiber in rat [
] and our observations of RF movement during embryonic development in scospondin-GFPut24/+ embryos (Figures 3F–3L). To directly quantify RF motility during zebrafish development, we photobleached the RF in the head and tail regions in scospondin-GFPut24/+ embryos (Figures 3B and 3D; Video S4). At all stages of development that we assayed, the photobleached regions of the RF consistently traveled in a continuous rostral to caudal direction in the brain (Figure 3B) and tail regions (Figure 3D). In the brain, we observed that the average speed of RF motility at 3 dpf was 58.6 ± 14.3 and at 5 dpf was 40.3 + 5.6 nm/s (Figures 3B and 3E). Within the tail region, we found the average speed of RF motility at 3 dpf was 220 ± 87 nm/s, although, at 5 and 7 dpf, the speed was markedly slower (70 ± 14 nm/s and 70 ± 24 nm/s, respectively; t test; p = 1.3 × 10−5; Figures 3D and 3E). Using high-speed imaging (10 Hz) at higher magnification, we also detected rapid movement of SCO-spondin-GFP-labeled puncta moving sporadically down the RF in a rostro-caudal direction, some of which occasionally extended away from the fiber toward the floor plate and retracted back into the bulk RF (Figure S3E; Video S5).
Our in vivo analyses of endogenous SCO-spondin-GFP expression and dynamics demonstrate several new properties of the RF, including (1) the initial secretion of Reissner material travels as punctate material from the brain, which precedes the elaboration of the fiber; (2) we directly confirm the hypothesis that the RF continually moves in a rostral-caudal direction at multiple stages of development; (3) we evidenced active breakdown of the RF at terminal ampulla; (4) the RF is a conduit for the rapid migration of substances in the CSF in a rostral-caudal direction; and (5) SCO-spondin secretion from the floor plate contributes to RF assembly. The scospondin-GFPut24 knockin zebrafish line, which allows for analysis of dynamic properties of the RF in vivo, sets the stage for future studies aimed at defining molecular interactions of the Reissner fiber and the dynamics of central canal components with which the RF interacts to regulate axial morphogenesis.
Loss of the Reissner Fiber Is Associated with Scoliosis in Additional Independent Scoliosis Mutant Zebrafish Strains
]. For this reason, we hypothesized that the loss of the RF may be a common phenotype associated with the onset of scoliosis in independent scoliosis mutant zebrafish in our collection. To test this, we first crossed scospondin-GFPut24 to a dominant enhancer-trap transgenic scoliosis mutant, Et(druk-GFPdut26/+) (R.S.G., A.R. McAdow, L.S.-K., and S.L. Johnson, unpublished data). This mutant was generated by a fortuitous, Tol2-GFP integration on Danio rerio chromosome 11, landing between the MON1 secretory trafficking family member A and macrophage stimulating 1 receptor b genes. Et(druk-GFPdut26/+) fish also display a unique GFP-expression pattern in the brain and spinal cord (data not shown; Figure 4C’ ), which is tightly linked with the onset of axial curvatures around 15–18 dpf (Figure 4C) and adult-viable scoliosis (98%; n = 981). At 5 dpf, the majority of Et(druk-GFPdut26/+);scospondin-GFPut24/+ larvae showed a fully assembled RF (n = 6/6; Figures 4A and 4A’). At the onset of axial curvatures in these mutants, we observed a consistent loss of the SCO-spondin-GFP-labeled RF (100% of fish at 6.1–7.0 mm; n = 9; Figures 4D and 4D’). In contrast, similarly sized scospondin-GFPut24/+ knockin larvae always displayed a typical RF (100% of fish sized 6.3–6.8 mm; n = 9; Figures 4B and 4B’). Although the molecular genetics of the dominant Et(druk-GFPdut26/+) scoliosis mutant remains to be defined, we do provide direct evidence that the disassembly of the SCO-spondin-GFP-labeled RF in real time, in a living animal, is coincident with the onset of axial curvatures and scoliosis in these mutants.
]; concurrently, we observed the complete disassembly of the RF (Figure 4H) within the central canal (Figure 4H’). In contrast to the absent or diffuse staining observed in scospondinstl297 mutants (Figures 2E’’’ and 2F), we consistently detected the AFRU-stained Reissner material filling up the entire central canal in kif6sko mutants at both 3 and 5 dpf (Figures 4H’’ and 4J), suggesting that the secretion of the material is not affected; rather, its ability to polymerize in the central canal is disrupted. Defects in motile cilia components give rise to defects in RF formation in zebrafish embryos [
], and the motile ciliated ependymal cell cilia are lost in adult kif6 mutants [
]. This suggests that alterations in CSF flow may underlie the loss of RF polymerization in kif6sko mutants. However, bulk CSF flow is grossly unaffected in kif6 mutant embryos [
], suggesting alternative models of kif6 regulation for RF assembly are possible. Altogether, our observations of RF disassembly in three independent scoliosis mutants strongly support the critical role of the RF structure to regulate the homeostasis of the straight body axis and for spine morphogenesis in zebrafish. Additional studies focused on the elucidation of cellular and molecular differences related to RF disassembly in these three independent scoliosis mutant strains are warranted.
], neurogenesis during early brain development [
], and through its direct interaction with the ciliated CSF-contacting neurons lining the central canal, as a mechanosensory organ controlling the “flexure of the body” [
]. Early work in amphibians demonstrated that the resection of the SCO disrupted RF assembly and led to scoliosis in some animals [
]. Here, we used forward genetics and cell biology approaches in zebrafish to demonstrate that two evolutionally conserved cysteine residues in the SCO-spondin protein are critical for stability of the RF during larval development. One of these cysteines (C2262) is predicted to form a disulfide bridge in one of several canonical LDL receptor A domains found in SCO-spondin. Interestingly, the LDL protein apolipoprotein B has been directly visualized in the central canal in zebrafish [
] and is found in the CSF of rat and humans by proteomic analysis [
]. Apolipoprotein B is also an important neurogenic factor in vitro [
], is important for brain development in mice [
], and forms a complex with SCO-spondin in the CSF, which can synergistically modulate neurodifferentiation in organotypic brain culture [
]. For these reasons, it is tempting to speculate that the C2262S mutation may also disrupt important LDL interactions with the Reissner fiber, causing alterations in neuronal differentiation in scospondinstl297 mutant zebrafish.
Our results using a variety of genetic models of scoliosis in zebrafish, a novel scospondin-GFP knockin strain, and analysis of cell biology and time-lapse imaging approaches to assay the RF in vivo now demonstrate that the intact fiber and its dynamic properties are required for maintaining a straight body axis and spine morphogenesis. Our study opens up a new field of exploration of dynamic properties of the Reissner fiber assembly, of molecular interactions of the fiber and CSF components for axial morphogenesis, and whether this physiology is driving scoliosis in humans.