Ewsr1a Regulates the Development of the Axial Skeleton in Zebrafish

Abstract

The EWSR1 (EWS RNA binding protein 1) was originally discovered in Ewing sarcoma, a pediatric bone cancer. In a previous study, we identified zebrafish ewsr1a, the homologue of human EWSR1. Using a zebrafish mutant for ewsr1a, we demonstrated that Ewsr1a promotes differentiation of chondrogenesis in Meckel’s cartilage, a component of the facial skeletal elements. Furthermore, we demonstrated that Ewsr1a interacts with the transcription factor SRY-BOX transcription factor 9 (SOX9) and modulates the transcription of its target genes. In this study, the role of Ewsr1a was further investigated in axial skeletogenesis. The maternal zygotic (MZ) ewsr1a/ewsr1a zebrafish display curved spines derived from irregularly spaced intervertebral discs (IVDs) and aberrant differentiation of nucleus pulposus cells. Using an in vitro cell culture system, we further show that the N-terminus of Ewsr1a is required for the interaction with Sox9. In zebrafish, the Ewsr1a also interacts with a Sox9 target gene, specifically the first intron of col2a1a. Zebrafish with the MZ ewsr1a/ewsr1a genotype display an increased level of collagen type II protein in the notochord starting at 36 h post fertilization. We propose that Ewsr1a contributes to IVD formation by regulating the expression of col2a1a.

Keywords

skeletogenesis nucleus pulposus intervertebral disc

Introduction

The EWSR1 (EWS RNA binding protein 1) was originally discovered as part of the EWSR1/FLI1 fusion gene, which results from chromosomal translocation in a pediatric bone cancer, Ewing sarcoma.¹ The major function of the EWSR1/FLI1 is known as an aberrant transcription factor, while EWSR1 itself is a multifunctional protein.^2–4 EWSR1 regulates numerous biological activities. For example, it regulates transcription through interacting with RNA polymerase II and TFIID, facilitates DNA damage repair by inhibiting R-loop formation, and maintains centromere and RNA splicing.^5–9 We previously reported that EWSR1 interacts with Aurora B kinase and regulates midzone formation during mitosis and prevents induction of aneuploidy.^10–14 Using the zebrafish ewsr1a (a homologue of human EWSR1) mutant, we also demonstrated that Ewsr1a promotes chondrogenesis of Mekel’s cartilage, prevents the induction of aneuploidy, and prevents tumorigenesis in the presence of a wildtype tp53.^13,15

In this study, we expanded our research to characterize the role of Ewsr1a in the axial skeleton using ewsr1a zebrafish mutants. The formation of the axial skeleton starts with the notochord, an axial midline structure derived from the mesoderm.¹⁶ During this process, the notochord undergoes vacuolization and secretes extracellular matrix proteins (e.g., collagens and aggrecan) to form the notochord sheath, and this process is necessary for spine formation.^16–23 It is known that human vertebrae are formed through endochondral ossification, whereas zebrafish vertebrae are developed through intramembranous ossification. In mammals, the notochord sheath forms both condensed and non-condensed areas that are evenly distributed throughout the axis. The condensed area gives rise to the intervertebral disc (IVD), which is composed of nucleus pulposus cells and annulus fibrosus cells.^24,25 In contrast, the non-condensed area generates the cartilaginous primordia of the vertebral bodies (VB), which are derived from sclerotomal cells.²⁵ In teleost, vacuolation of notochordal cells generates hydrostatic pressure, allowing for the elongation of the axis. This region becomes distinct when the vacuoles undergo fragmentation in the subset of notochordal cells. Then, the segmentation of the notochord sheath, known as chordacentra, undergoes mineralization along the anterior-posterior (A-P) axis and will become IVD.^22,23 The remaining areas containing vacuolated cells become VB.²⁶

Collagen Type II is a protein that is essential for forming fibrils in the notochord sheath, and it plays a significant role in the development of the axial skeleton. Col2a1 null mice displayed high numbers of notochordal cells and fewer nucleus pulposus cell in the IVD.²⁰ This observation highlights the significance of the expression of collagen type II at the notochord sheath, suggesting that the proper organization of the extracellular matrix at the notochord sheath is required for the differentiation of the nucleus pulposus cells. Previously, we discovered that the Ewsr1a interacts with the chondrocyte master regulator Sox9, and this interaction modulates the expression of Sox9 target genes, including Col2a1a mRNA.¹⁵

Here, we demonstrate that Ewsr1a is required for the formation of IVD through regulating the expression of collagen type II. We show that the N-terminus of Ewsr1a is essential for its interaction with Sox9 in vitro, and it interacts with the first intron of col2a1a, which is a major target gene of Sox9 in zebrafish embryos. Our study uncovers a unique role of Ewsr1a in the development of the axial skeleton.

Materials and Methods

Zebrafish maintenance and ethics statement

The ewsr1a mutant line was obtained from Znomics, Inc. All embryos were maintained and staged as previously described.²⁷ Experiments were performed according to protocols approved by the University of Kansas, Lawrence, Institutional Animal Care and Use Committee (#197-02).

Alizarin red staining

Adult zebrafish were stained with Alizarin Red using a method described in the previous study.²⁸ The images were visualized using a Leica MZ FLIII dissecting microscope that was equipped with a Leica DFC320 camera.

Immunohistochemistry

Immunohistochemistry was performed on zebrafish embryos following the previously described method with minor modifications.¹⁰ The following antibodies were used in this study: anti-collagen II primary antibody (Developmental Studies Hybridoma Bank at the University of Iowa, IA) (Invitrogen, diluted at 1:250), and anti-mouse Alexa 594 antibody (Invitrogen, dilution at 1:250). The images were documented with an Exi Aqua camera (Q Imaging) mounted on a Nikon Eclipse Ti microscope.

Co-immunoprecipitation

Co-immunoprecipitation (Co-IP) was performed as previously described with minor modifications.¹² HeLa cells were transfected with 14 ug of pSG5-Sox9 and pCS2-ewsr1a or pCS2-ewsr1a-C using Lipofectamine 2000 (LifeTechnologies, MA). Lysates were immunoprecipitated with either anti-mouse IgG or anti-FLAG antibodies (Agilent Technologies) conjugated to DYNA-protein G beads, followed by western blot using anti-Sox9 (AbCam, MA, diluted at 1/500). To generate an antibody against Ewsr1a, the C-terminus of ewsr1a (763–1878 bp) was cloned into the pET28a vector. The Ewr1a recombinant protein was purified from Escherichia coli (E. coli) and used as an antigen to generate a polyclonal antibody in rabbits (Pacific Immunology, CA).

Chromatin immunoprecipitation

Thirty-five wt/wt embryos at 27 hour post fertilization (hpf) were euthanized and subjected to a chromatin immunoprecipitation (ChIP) assay following a previously reported protocol.²⁹ The following PCR primers were designed for the col2a1a gene (4620–4865) and were used in this study.

col2a1a: F (5′-TTGCTTAGCACTGTACAGCC-3′)

col2a1a: R (5′-GTACCAAATCAGCATCATTCC-3′)

Results

Ewsr1a regulates the development of IVD in zebrafish

We previously reported that the maternal zygotic (MZ) ewsr1a/ewsr1a mutant, a line established by intercrossing zygotic ewsr1a/ewsr1a homozygous mutants, displays craniofacial defects in Meckel’s cartilage.¹⁵ During the analysis, we discovered that the mutant also displays curved spines, prompting a study of the function of Ewsr1a during axial skeleton formation.

Alizarin red staining revealed that the neural arches were typically bent twice and projected dorsally in wt/wt zebrafish, while the trunk neural arches and spines were straight in the MZ ewsr1a/ewsr1a mutant zebrafish (Fig. 1A and B). One or more hemal spines were irregularly shaped with bifurcations or kinks in 75% (n = 20) of the MZ ewsr1a/ewsr1a zebrafish (Fig. 1B). In contrast, 9% of the wt/wt fish displayed either a bifurcated hemal spine or one bent hemal spine, resulting in a significant difference (n = 11, p = 0.0006). In addition, the MZ ewsr1a/ewsr1a mutants displayed curved spines (40%, n = 20). On the contrary, wt/wt fish did not display curved spines (0%, n = 11, p = 0.03) (Fig. 1A). In addition to the asymmetrical centrum of the caudal vertebrae and the curved spines, the IVD width was reduced in the MZ ewsr1a/ewsr1a (Fig. 1D and E). The average IVD width at the caudal vertebrae in the MZ ewsr1a/ewsr1a was 63 ± 16 mm (n = 19 fish), which was significantly less compared to 78 ± 22 mm in the wt/wt (n = 10 fish) (Fig. 1C and E). Note that between 6 and 15 IVDs were scored per fish, and the average score was presented as a single circle on the graph. The results suggest that the Ewsr1a plays an important role in IVD formation.

FIG. 1.

MZ ewsr1a/ewsr1a zebrafish have a reduced IVD width and curved spines. (A–D). The calcified bones were visualized by alizarin red staining. (A and B). Lateral views (anterior to the left) of wt/wt and MZ ewsr1a/ewsr1a zebrafish. The calcified bones were visualized by alizarin red staining. (C and D). Magnified views (anterior to the left) of wt/wt and MZ ewsr1a/ewsr1a zebrafish in areas indicated by black squares in (A) and (B). (E) The average width of the IVD was measured in wt/wt (n = 10 fish) and MZ ewsr1a/ewsr1a (n = 19 fish) (Welch’s t-test, p < 0.05). Note that between 6 and 15 IVDs were scored per fish, and the average score was presented as a single circle on the graph.

The notochordal cells differentiate into nucleus pulposus cells, which make up the IVD.^30,31 This differentiation process is initiated by the formation of the notochord sheath, a layer of extracellular matrix proteins surrounding the notochord.²⁶ Since the notochord plays a crucial role in the development of IVD, we first investigated whether notochord specification was impacted. In situ hybridization using the notochord marker ntl as a probe revealed that the expression along the notochord was unaffected in the mutant at 27 hpf (wt/wt; n = 23, MZ ewsr1a/ewsr1a; n = 15) (p = 0.35) (Fig. 2A and B). In addition, the growth of the notochord of embryos at 2 and 7 dpf was not significantly affected in the mutant compared to the wt/wt zebrafish (Table 1). To visualize the cell morphology in the spines, we performed H&E staining on 5 dpf embryos, juvenile fish (1 cm total body length), and adult zebrafish. The morphology of the notochordal cells in the 5 dpf MZ ewsr1a/ewsr1a mutants (n = 6) was similar to that of the 5 dpf wt/wt zebrafish (n = 6) (p = 0.001) (Fig. 2C and D). The cell margins of the notochordal cells in the wt/wt zebrafish were clear and distinct, while some cells in the MZ ewsr1a/ewsr1a mutants had thicker and less clear cell margins (Fig. 2D, indicated with an asterisk). The spines of the wt/wt juvenile fish (1 cm total body length) displayed major reorganization, including in the IVD, with a partially calcified VB (Fig. 2E). In the wt/wt fish, the original notochordal cells with large vacuole morphology were no longer present, but instead, the nucleus pulposus cells with smaller cell morphology clustered in the IVD region (n = 3) (Fig. 2E). In contrast, the notochordal cells were retained in the MZ ewsr1a/ewsr1a mutants (100%, n = 3) (p = 0.05) (Fig. 2F). This result suggests that notochordal cells failed to differentiate to nucleus pulposus cells in the MZ ewsr1a/ewsr1a mutants. Furthermore, the adult IVD region in the adult MZ ewsr1a/ewsr1a fish (100%, n = 3) displayed an irregular and asymmetrical shape compared with the symmetrically shaped IVD in the wt/wt zebrafish (0%, n = 3) (p = 0.05) (Fig. 2G and H). Together, Ewsr1a is required for vertebral column formation by promoting the differentiation of notochordal cells into nucleus pulposus cells.

FIG. 2.

MZ ewsr1a/ewsr1a zebrafish display aberrant differentiation of nucleus pulposus cells. (A and B) wt/wt (left) and MZ ewsr1a/ewsr1a (right) zebrafish embryos at 27 hpf visualized by in situ hybridization using ntl antisense RNA as a probe. (C and D) wt/wt (left) and MZ ewsr1a/ewsr1a (right) zebrafish embryos at 5 dpf visualized by H&E staining. nc: notochordal cells. *: area with less clear cell margins. (E and F) wt/wt (left) and MZ ewsr1a/ewsr1a (right) juvenile (total body length: 1 cm) zebrafish visualized by H&E staining. Arrowheads (<) represent the remaining notochordal cells. (G and H) wt/wt (left) and MZ ewsr1a/ewsr1a (right) adult zebrafish visualized by H&E staining. All images are lateral views (anterior to the left).

Table 1.

The notochord length of the MZ ewsr1a/ewsr1a mutant is not affected compared to the wt/wt embryos. The average length (Mm) of the notochord scored from the lateral views of the wt/wt and MZ ewsr1a/ewsr1a zebrafish embryos

Dpf	wt/wt	MZ ewsr1a/ewsr1a
2	2.73 ± 0.07, (n = 6)	2.71 ± 0.05, (n = 4)
3	3.43 ± 0.08, (n = 11)	3.27 ± 0.07, (n = 11)
4	3.63 ± 0.10, (n = 8)	3.52 ± 0.09, (n = 11)
6	3.69 ± 0.07, (n = 11)	3.62 ± 0.12, (n = 8)
7	3.74 ± 0.09, (n = 11)	3.70 ± 0.07, (n = 11)

Ewsr1a regulates the expression of collagen type II

A previous study demonstrated that the expression of the collagen type II in the notochord is essential for the differentiation of nucleus pulposus cells.^20,32 We previously showed that the MZ ewsr1a/ewsr1a mutant embryos (27 hpf) express higher levels of col2a1a and col2a1b mRNA compared to wt/wt using qPCR.¹⁵ In the same study, we showed that the col2a1a and col2a1b mRNA are upregulated in the axis of the MZ ewsr1a/ewsr1a mutant (27 hpf) compared to wt/wt (27 hpf) using in situ hybridization.¹⁵ Note that there were no signals in the axis of the 3 dpf MZ ewsr1a/ewsr1a and wt/wt embryos (data not shown). The study demonstrated that Ewsr1a regulates col2a by regulating Sox9, the master regulator of chondrocyte formation.¹⁵ To determine if the expression level and localization of collagen type II protein are altered in this mutant, we conducted immunohistochemistry with an anti-collagen type II antibody. At 36 hpf, the MZ ewsr1a/ewsr1a mutant exhibited a higher intensity of collagen II signals in the notochord extension (anterior end of the notochord) compared to the wt/wt (73%, n = 30 vs. 0%, n = 16, respectively; p < 0.0001) (Supplementary Fig. S1A and B). The mutants also displayed a higher intensity and number of collagen type II signals (93%; n = 55) compared to the wt/wt (16%, n = 25) (p < 0.0001) (Supplementary Fig. S1C and D). It is worth noting that there was unexpected ectopic expression of collagen type II in the yolk extension of the mutant, for reasons unknown (Supplementary Fig. S1D). The data suggest that Ewsr1a may play a role in inhibiting the expression of collagen II in the notochord of normal embryos. Furthermore, the collagen type II expressing cells cluster with a regularly spaced vertical stripe pattern in wt/wt (9% displayed a disorganized pattern, n = 11), whereas this pattern is disorganized in MZ ewsr1a/ewsr1a mutants (100%, n = 8) at 3 dpf (p < 0.0001) (Fig. 3). The results suggest that Ewsr1a is required for maintaining a low expression level of collagen type II and for the formation of the notochord sheath in normal embryos. It is noteworthy that the correlation between the high expression of collagen II and its pattern disorganization in the MZ ewsr1a/ewsr1a mutants remains unknown.

FIG. 3.

The MZ ewsr1a/ewsr1a mutants display increased expression of collagen II in the notochord. The trunk of wt/wt (left panel) and MZ ewsr1a/ewsr1a (right panel) 36 hpf zebrafish embryos at views (anterior to the left). All embryos were visualized with immunohistochemistry using an anti-collagen type II antibody (red), co-stained with wheat germ agglutinin (green). Col II, collagen type II; WGA, wheat germ agglutinin.

Because our previous study showed that Ewsr1a interacts with Sox9a, a co-IP experiment was performed to identify the essential domain of Ewsr1a required for interaction with Sox9a. A FLAG-tagged sox9a and either full-length or 3′-terminus of ewsr1a DNA constructs were co-transfected into HeLa cells, respectively (Fig. 4A). The lysates were immunoprecipitated using an anti-FLAG antibody and an anti-mouse IgG antibody as a negative control, followed by Western blot analysis using an anti-Ewsr1a antibody (Fig. 4A). Consistent with a previous report, the full-length Ewsr1a was co-precipitated with Sox9a.¹⁵ However, there was a significantly reduced interaction between Sox9a and the deletion construct containing only the C-terminus of Ewsr1a. This result suggests that the N-terminus of Ewsr1a is the domain required for interaction with Sox9a (Fig. 4A). Our previous data showed that Ewsr1a inhibits the expression of col2a1a mRNA at 27 hpf embryos.¹⁵ To further elucidate whether Ewsr1a interacts with the col2a1a gene, a ChIP assay was performed. From 27 hpf wt/wt zebrafish embryo lysates, Ewsr1a was immunoprecipitated, and the co-precipitated genomic DNA was amplified by PCR primers that were designed at the Sox9 consensus sequence of col2a1a located in the first intron (Fig. 4B). As a result, we discovered that Ewsr1a interacts with the first intron of the col2a1a gene (Fig. 4B). Together, Ewsr1a regulates the expression of col2a1a by interacting with the first intron of the col2a1a gene.

FIG. 4.

Ewsr1a interacts with col1a2a gene. (A) (Top) Schematics of Ewsr1a. (Middle) Co-IP of lysates obtained from untransfected (untransf.), empty vector-transfected (vector) and FLAG-SOX9-transfected HeLa cells using an IgG control and anti-FLAG antibody, followed by Western blot with anti- Ewsr1a antibody (Input: 1/250). (Bottom) Western blot with anti-SOX9 antibody. (B) (Left) Schematics of col2a1a locus. Black circle represents Sox9 binding site, and arrows represents PCR primers used for ChIP assay. (Right) ChIP assays using IgG (negative control), and anti-Ewsr1a in 27 hpf wt/wt zebrafish. Input: 4.5% of DNA from total lysates, dw: negative control for PCR reaction.

Discussion

This study aimed to characterize axis formation in the MZ ewsr1a/ewsr1a zebrafish mutant. We demonstrate that Ewsr1a regulates IVD formation, potentially by suppressing the expression of col2a1a during the differentiation from notochordal cells to nucleus pulposus cells. Because Ewsr1a regulates Sox 9 target genes through interaction, we further conducted a deletional analysis of Ewsr1a to identify the essential domain required for the interaction with Sox 9. We identified that the N-terminus of Ewsr1a is an essential domain required for the interaction with Sox 9 and showed that Ewsr1a regulates the first intron of the col2a1a gene.

Our results suggest that Ewsr1a plays a crucial role in the formation of the IVD by regulating the differentiation of notochordal cells into nucleus pulposus cells. Our previous study demonstrated that Ewsr1a interacts with Sox 9 and inhibits the expression of col2a1a during early embryogenesis.¹⁵ Because collagen type II is crucial for the formation of collagen fibrils in the notochord sheath, the vertebral body, located outside the notochord sheath, exerts a mechanical force that contributes to the elimination of the notochordal cells.²⁰ Another possibility is that the collagen fibril creates a scaffold that allows the clustering of as-yet unknown signaling molecules, triggering notochordal cell elimination and promoting nucleus pulposus cell differentiation. The identification of this signal will be critical for a better understanding of the differentiation of the nucleus pulposus. It is noteworthy that polycystin1a (pkd1)a/b- and pkd2-deficient zebrafish embryos induce overexpression of collagen type II, leading to a curved axis. Therefore, the study supports the importance of the expression level of collagen type II.³³

In addition to inhibiting the differentiation of the nucleus pulposus in MZ ewsr1a/ewsr1a mutant, our previous study also demonstrated that the chondrogenesis of endochondral ossification in Mackel’s cartilage was delayed in the mutants. It has been suggested that both phenotypes stem from compromised Sox 9 activities, as these phenotypes resemble in Sox 9 knockdown mice.^34,35 In this study, we discovered that the N-terminus of Ewsr1a is essential for its interaction with Sox9. The N-terminus of Ewsr1a contains a prion-like domain (PrLD).^36,37 This domain has self-assemble activity, and it leads to phase separation. Therefore, it is possible that the self-assembled Ewsr1a may prevent Sox9 from accessing its target locus due to phase-separated compartments. Because differentiation of all cell types is a dynamic process, Ewsr1a may serve as a temporal hub through its self-assembly mechanism, and this complex may inhibit the activity of Sox 9. To test this concept, it is essential to further generate mutation constructsfor PrLD of Ewsr1 and study its activities of self-assembly, the expression of the col2a1a gene, and the differentiation of notochordal cells into nucleus pulposus cells. Spinal deformity induced during developmental stages (e.g., congenital kyphosis, congenital scoliosis, and congenital lordosis) often compromises quality of life due to fatigue, stiffness of muscle, neurological defects, and cerebrospinal fluid leakage.^38,39 Given the phenotypes displayed in the MZ ewsr1a/ewsr1a zebrafish mutant, it is possible that the Ewsr1a (or human EWSR1) may play a critical role in spinal development. Here, we report a unique role of EWSR1 in axial development, and this zebrafish model should provide a platform for further dissecting the pathway.

Footnotes

Acknowledgments

Research reported in this publication was supported by the University of Kansas Graduate Research Fund (GRF).

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Supplemental Material

References

Delattre

, Zucman

, Plougastel

, et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature, 1992; 359(6391):162–165; doi: 10.1038/359162a0

Lessnick

, Ladanyi

. Molecular pathogenesis of Ewing sarcoma: New therapeutic and transcriptional targets. Annu Rev Pathol, 2012; 7:145–159; doi: 10.1146/annurev-pathol-011110-130237

Paronetto

. Ewing sarcoma protein: A key player in human cancer. Int J Cell Biol, 2013; 2013:642853; doi: 10.1155/2013/642853

Erkizan

, Uversky

, Toretsky

. Oncogenic partnerships: EWS-FLI1 protein interactions initiate key pathways of Ewing’s sarcoma. Clin Cancer Res, 2010; 16(16):4077–4083; doi: 10.1158/1078-0432.CCR-09-2261

Immanuel

, Zinszner

, Ron

. Association of SARFH (sarcoma-associated RNA-binding fly homolog) with regions of chromatin transcribed by RNA polymerase II. Mol Cell Biol, 1995; 15(8):4562–4571.

Bertolotti

, Melot

, Acker

, et al. EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: Interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Mol Cell Biol, 1998; 18(3):1489–1497.

Yang

, Chansky

, Hickstein

. EWS.Fli-1 fusion protein interacts with hyperphosphorylated RNA polymerase II and interferes with serine-arginine protein-mediated RNA splicing. J Biol Chem, 2000; 275(48):37612–37618; doi: 10.1074/jbc.M005739200

Kitagawa

, Niikura

, Becker

, et al. EWSR1 maintains centromere identity. Cell Rep, 2023; 42(6):112568; doi: 10.1016/j.celrep.2023.112568

Gorthi

, Romero

, Loranc

, et al. EWS-FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma. Nature, 2018; 555(7696):387–391; doi: 10.1038/nature25748

10.

Azuma

, Embree

, Sabaawy

, et al. Ewing sarcoma protein ewsr1 maintains mitotic integrity and proneural cell survival in the zebrafish embryo. PLoS One, 2007; 2(10):e979; doi: 10.1371/journal.pone.0000979

11.

Park

, Turkalo

, Nelson

, et al. Ewing sarcoma EWS protein regulates midzone formation by recruiting Aurora B kinase to the midzone. Cell Cycle, 2014; 13(15):2391–2399.

12.

Embree

, Azuma

, Hickstein

. Ewing sarcoma fusion protein EWSR1/FLI1 interacts with EWSR1 leading to mitotic defects in zebrafish embryos and human cell lines. Cancer Res, 2009; 69(10):4363–4371; doi: 10.1158/0008-5472.CAN-08-3229

13.

Park

, Galbraith

, Turner

, et al. Loss of Ewing sarcoma EWS allele promotes tumorigenesis by inducing chromosomal instability in zebrafish. Sci Rep, 2016; 6:32297; doi: 10.1038/srep32297

14.

Kim

, Park

, Schulz

, et al. EWSR1 prevents the induction of aneuploidy through direct regulation of Aurora B. Front Cell Dev Biol, 2023; 11:987153; doi: 10.3389/fcell.2023.987153

15.

Merkes

, Turkalo

, Wilder

, et al. Ewing sarcoma EWSA protein regulates chondrogenesis of Meckel’s cartilage through modulation of Sox9 in zebrafish. PLoS One, 2015; 10(1):e0116627; doi: 10.1371/journal.pone.0116627

16.

Williams

, Alkhatib

, Serra

. Development of the axial skeleton and intervertebral disc. Curr Top Dev Biol, 2019; 133:49–90; doi: 10.1016/bs.ctdb.2018.11.018

17.

Akiyama

, Lefebvre

. Unraveling the transcriptional regulatory machinery in chondrogenesis. J Bone Miner Metab, 2011; 29(4):390–395; doi: 10.1007/s00774-011-0273-9

18.

Goldring

, Tsuchimochi

, Ijiri

. The control of chondrogenesis. J Cell Biochem, 2006; 97(1):33–44; doi: 10.1002/jcb.20652

19.

Dale

, Topczewski

. Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene. Dev Biol, 2011; 357(2):518–531; doi: 10.1016/j.ydbio.2011.06.020

20.

Aszodi

, Chan

, Hunziker

, et al. Collagen II is essential for the removal of the notochord and the formation of intervertebral discs. J Cell Biol, 1998; 143(5):1399–1412.

21.

Bundy

, Rogers

, Hoffman

, et al. Segmental expression of aggrecan in the non-segmented perinotochordal sheath underlies normal segmentation of the vertebral column. Mech Dev, 1998; 79(1–2):213–217.

22.

Lleras Forero

, Narayanan

, Huitema

, et al. Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock. Elife, 2018; 7:e33843; doi: 10.7554/eLife.33843

23.

Wopat

, Bagwell

, Sumigray

, et al. Spine patterning is guided by segmentation of the notochord sheath. Cell Rep, 2018; 22(8):2026–2038; doi: 10.1016/j.celrep.2018.01.084

24.

Chan

, Au

, Tam

, et al. Coming together is a beginning: The making of an intervertebral disc. Birth Defects Res C Embryo Today, 2014; 102(1):83–100; doi: 10.1002/bdrc.21061

25.

Sivakamasundari

, Lufkin

. Bridging the gap: Understanding embryonic intervertebral disc development. Cell Dev Biol, 2012; 01(02)

26.

Bagnat

, Gray

. Development of a straight vertebrate body axis. Development, 2020; 147(21):dev175794; doi: 10.1242/dev.175794

27.

Kimmel

, Ballard

, Kimmel

, et al. Stages of embryonic development of the zebrafish. Dev Dyn, 1995; 203(3):253–310; doi: 10.1002/aja.1002030302

28.

Javidan

, Schilling

. Development of cartilage and bone. Methods Cell Biol, 2004; 76:415–436.

29.

Lindeman

, Vogt-Kielland

, Alestrom

, et al. Fish’n ChIPs: Chromatin immunoprecipitation in the zebrafish embryo. Methods Mol Biol, 2009; 567:75–86; doi: 10.1007/978-1-60327-414-2_5

30.

Choi

, Cohn

, Harfe

. Identification of nucleus pulposus precursor cells and notochordal remnants in the mouse: Implications for disk degeneration and chordoma formation. Dev Dyn, 2008; 237(12):3953–3958; doi: 10.1002/dvdy.21805

31.

Alkhatib

, Ban

, Williams

, et al. IVD development: Nucleus pulposus development and sclerotome specification. Curr Mol Biol Rep, 2018; 4(3):132–141; doi: 10.1007/s40610-018-0100-3

32.

Yan

, Hatta

, Riggleman

, et al. Expression of a type II collagen gene in the zebrafish embryonic axis. Dev Dyn, 1995; 203(3):363–376; doi: 10.1002/aja.1002030308

33.

Mangos

, Lam

, Zhao

, et al. The ADPKD genes pkd1a/b and pkd2 regulate extracellular matrix formation. Dis Model Mech, 2010; 3(5–6):354–365; doi: 10.1242/dmm.003194

34.

, Wang

, Bhattaram

, et al. Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes. Dev Cell, 2012; 22(3):597–609; doi: 10.1016/j.devcel.2011.12.024

35.

Henry

, Liang

, Akdemir

, et al. The postnatal role of Sox9 in cartilage. J Bone Miner Res, 2012; 27(12):2511–2525; doi: 10.1002/jbmr.1696

36.

King

, Gitler

, Shorter

. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease. Brain Res, 2012; 1462:61–80; doi: 10.1016/j.brainres.2012.01.016

37.

Selig

, Sohn

, Stoja

, et al. Phase separation of the oncogenic fusion protein EWS::FLI1 is modulated by its DNA-binding domain. Proc Natl Acad Sci U S A, 2025; 122(20):e2221823122; doi: 10.1073/pnas.2221823122

38.

Wang

, Thome

, Brook

, et al. Occult spinal CSF leak: To patch or not to patch-a case-based update. Childs Nerv Syst, 2025; 41(1):430; doi: 10.1007/s00381-025-07094-8

39.

Lonstein

. Congenital spine deformities: Scoliosis, kyphosis, and lordosis. Orthop Clin North Am, 1999; 30(3):387–405, viii; doi: 10.1016/s0030-5898(05)70094-8

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