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Figures
- Fig. 1.
Tg(col2a1aR2:KalTA4) enables stable and transient oncogene expression in the developing notochord. (A,B) Lateral view (A) and transverse histology section (stained with H&E) (B) of 5 dpf zebrafish embryos transgenic for the transgene col2a1aR2:KalTA4 and crossed with stable UAS:Kaede (visible as green fluorescence in A). Note expression in the notochord, craniofacial cartilage, otic vesicle and pectoral fins; the prominent green heart (white arrowhead, A) indicates the myl7:EGFP transgenesis marker associated with col2a1aR2:KalTA4. The developing notochord shows that the large vacuolated cells take up the vast majority of the notochord volume and are rimmed by a thin layer of sheath cells (B). (C,D) Expression of stable UAS:EGFP-HRASV12 (detectable by green fluorescence of the fusion protein, C) by col2a1aR2:KalTA4 causes invasive and widespread notochord hyperplasia (black arrowheads, D) and overgrowth of other cartilage tissue (i.e. otic vesicle, black asterisk in C); the white arrowhead indicates the myl7:EGFP transgenesis marker (A). (E-G) col2a1aR2 provides a potent driver for transient notochord expression. (E) Injection of UAS:EGFP into the one-cell-stage embryos with either twhh:Gal4 (F) or col2a1aR2:KalTA4 (G) to visualize the notochord mosaicism resulting from random integration of UAS:EGFP by Tol2 transposase. While injections into twhh:Gal4 result in highly patchy EGFP expression (F, green fluorescence; n=33/56), col2a1aR2:KalTA4 more consistently drives EGFP expression throughout the notochord (G, green fluorescence; n=25/47); n indicates representative EGFP-expressing embryos in an injected representative clutch. Scale bars: 500 μm in A,C,F,G; 200 μm in B,D. See also Fig. S1.
- Fig. 2.
Overexpression of brachyury genes in the zebrafish notochord is insufficient to initiate chordoma. (A) Workflow of injection-based notochord hyperplasia assessment: at the one-cell stage, Tg(col2a1aR2:KalTA4) embryos are injected with Tol2 transposase mRNA and a plasmid containing a fluorescently labeled candidate gene under UAS control; injected embryos are raised up to 5 dpf and candidate gene expression is monitored through notochord fluorescence. Embryos with consistent reporter expression are fixed, sectioned and stained with H&E to assess the notochord phenotype using light microscopy. (B-O) Close-up lateral view of embryo notochords at 5 dpf, for brightfield and fluorescence (left column) and H&E histology (right column; different embryos per condition); numbers indicate observed versus total from an individual representative experiment. (B,C) The col2a1aR2:KalTA4 control reference at 5 dpf, expressing UAS:Kaede to fluorescently label the notochord. (D,E) Transient injection of UAS:EGFP-HRASV12 causes localized notochord hyperplasia (arrowheads). (F-I) Forced expression of human TBXT (brachyury) (F,G) or of the zebrafish gene tbxta (H,I) does not affect notochord development or cell proliferation. Minor lesions caused by collapsed vacuolated cells developed in a few UAS:tbxta-expressing notochords (arrowheads, I). (J,K) Overexpression of the second zebrafish brachyury gene tbxtb does not affect notochord development or proliferation. (L,M) Combined overexpression of both zebrafish brachyury genes tbxta and tbxtb has no effect on proliferation and the notochord develops normally. (N,O) Notochord-driven expression of tbxta-VP16, encoding a dominant-active transcriptional activator, leads to non-autonomous defects in the trunk with a shortened and/or severely curled trunk (32% of analyzed embryos), whereas the notochord forms normally. Scale bars: 200 μm. See also Fig. S2.
- Fig. 3.
Transient overexpression of RTK genes drives notochord hyperplasia. (A-L) Close-up lateral views of embryo notochords at 5 dpf, with brightfield/fluorescence (left panels) and H&E histology (right panels; different embryos per condition); numbers in D,F,H and J indicate embryos with observed lesions versus phenotypically normal embryos observed in an individual representative experiment, and numbers in L indicate wild-type-looking embryos (n=3 experiments). (A,B) The col2a1aR2:KalTA4 control reference at 5 dpf, expressing UAS:Kaede to fluorescently label the notochord. (C,D) Transient injection of UAS:EGFP-HRASV12 causes localized hyperplasia (arrowheads, C,D) in the notochord. (E,F) Overexpression of human EGFR consistently causes local hyperplasia in the developing notochord (arrowheads, F). (G,H) Overexpression of zebrafish kdr (encoding the VEGFR2 ortholog) causes strong hyperplasia (arrowheads in G,H, compare with D,F). (I,J) Zebrafish rheb overexpression leads to enlarged vacuoles and no detectable hyperplasia. (K,L) Zebrafish stat3 overexpression leads to no detectable hyperplasia and allows for normal notochord development within 5 dpf. Scale bars: 200 μm. See also Fig. S3.
- Fig. 4.
Expression of chordoma markers in RTK-transformed zebrafish notochords. (A-O) Immunohistochemistry on sagittal sections through the notochord of 5 dpf zebrafish embryos of the indicated genotypes, expressing either stable or mosaic transgenes. (A-E) MAPK pathway activation through HRASV12, EGFR and kdr overexpression results in nuclear pERK staining in the notochord, whereas controls and tbxta,tbxtb-injected embryos are negative for pERK staining. (F-J) HRASV12, EGFR and kdr overexpression results in staining for pan-Cytokeratin, whereas tbxta,tbxtb-injected embryos are negative for pan-Cytokeratin staining akin to wild-type controls. (K-O) Whereas control notochords show faint to no Tbxta signal, owing to low cell density of the sheath layer (K), HRASV12-overexpressing notochords (L) as well as EGFR-overexpressing (M) and kdr-overexpressing (N) notochords show prominent nuclear Tbxta staining. The tbxta,tbxtb-overexpressing notochords also stain positive (O), confirming transgenic tbxta expression. Scale bars: 50 µm. See also Fig. S4.
- Fig. 5.
HRASV12-induced zebrafish chordomas have a deregulated UPR and suppress apoptosis. (A) Workflow of control and transformed notochord isolation. Notochords were dissected from twhh:Gal4 (wild-type morphology and transgenic base line) and twhh:Gal4;UAS:EGFP-HRASV12-expressing larvae at 8 dpf; see Materials and Methods for details. (B) Volcano plot depicting overall distribution of deregulated genes: gray, genes for which P<0.05; blue, genes with significant deregulation between control versus transformed notochords. Note that the zebrafish brachyury genes tbxta and tbxtb are unchanged but expressed. (C) Expression of human chordoma-associated genes in HRASV12-transformed zebrafish notochords; see text for details. Zebrafish orthologs of human chordoma genes are shown with all their orthologs: orthologs with changes considered to be not significant (n.s., green) are marked with asterisks, genes with no significantly altered ortholog are shown in gray boxes at the bottom. (D) Significantly deregulated UPR genes as per IPA pathway analysis of hyperplastic versus control zebrafish notochords. (E) Gene ontology (GO) enrichment in hyperplastic zebrafish notochords also highlights deregulation of the UPR, ER stress, ECM dynamics and cell death. The vertical red bars represent the expected number of genes per GO term under random selection based on the reference list, Homo sapiens REFLIST (21,042 genes in total). See also Fig. S5.
- Fig. 6.
Aberrant ECM and ER accumulation in HRASV12-induced zebrafish chordomas. (A-F) Transverse sections through wild-type (A,C,E) and HRASV12-transformed (B,D,F) zebrafish notochord at 8 dpf imaged using TEM. (A,B) Wild-type cells have a pill-shaped, regular nucleus (red outline, A) and form regular ECM layers (red letter ‘E’) secreted by the sheath cells at the outside of the notochord. Nuclei in transformed cells expand and develop lobed and distorted nuclear shapes (red outline, B); transformed cells further accumulate extensive ER lumen (white arrowheads, B). (C,D) In wild-type notochords, vacuolated cells (red letter ‘V’) take up the majority of the space inside the ECM-lined notochord (red letter ‘E’) to provide mechanical stability; transformed notochords become filled with non-vacuolated cells and secrete aberrant amounts of ECM that leads in extreme cases to entombed cells trapped in ECM layers (white asterisk, D). (E,F) Membrane details of wild-type versus transformed notochord sheath cells. Wild-type notochords show budding of vesicles that transport collagen for stereotypically layered ECM build up (red arrowheads); in transformed notochords, the secretion process appears to be overactive (ER accumulation shown by white arrowheads, F) and results in membrane inclusions within the ECM (red arrowheads). Scale bars: 1 μm (A,B); 2 μm (C,D); 0.5 μm (E,F). See also Fig. S6.
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