Urotensin II-related peptides, Urp1 and Urp2, control zebrafish spine morphology
- Institute of Molecular Biology, Department of Biology, University of Oregon, United States
Figures
Figure 1 with 3 supplements
Urp1 and Urp2 are dispensable for axial straightening.
(A) urp1 and urp2 are 5-exon genes (gray boxes). The final exon codes for the 10-amino acid peptides produced after cleavage from the prodomain at a dibasic site (KR). Pairs of gRNAs were used to …
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Figure 1—source data 1
Raw data for qRT-PCR and crispant body angle measurements.
- https://cdn.elifesciences.org/articles/83883/elife-83883-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Urotensin family peptides.
(A) Successive rounds of genome duplications (*) and divergence converted an ancient urotensin protein (U) into the urotensin II (UII) and urotensin II-related (URP) proteins, some of which have …
Figure 1—figure supplement 2
Generation of urp1∆P and urp2∆P mutants.
(A–B) Pairs of guide RNAs (gRNAs) were used to delete genomic regions coding for the Urp1 and Urp2 peptides. The urp1b1420 (urp1∆P) allele encodes a 279 bp deletion and 1 base pair insertion that …
Figure 1—figure supplement 3
Generation of sspob1446 mutants.
(A) Schematic of zebrafish SCOspondin protein showing domain architecture based on Troutwine et al., 2020. VWD – von Willebrand factor type D domain; C8 – C8 domain; TIL – trypsin inhibitor like …
Figure 2 with 11 supplements
Urp1 and Urp2 are required for proper adult spine morphology.
(A–C) Lateral views of microcomputed tomography reconstitutions of wild-type (A), urp1∆P;urp2∆P (B) and uts2r3b1436 (C) mutants at 3 mpf. (D) Cobb angle measurements for individual fish in the …
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Figure 2—source data 1
Raw data from spinal curve phenotypic measurements.
- https://cdn.elifesciences.org/articles/83883/elife-83883-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Phenotyping spinal curves.
(A) Lateral views of adult zebrafish. (B) Cobb angles were measured from lateral views of microcomputed tomography reconstitutions. Right angles are assigned parallel to the rostral and caudal face …
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Figure 2—figure supplement 1—source data 1
Raw data from spinal curve phenotypic measurements.
- https://cdn.elifesciences.org/articles/83883/elife-83883-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Spinal curves in urp1∆P, urp2∆P, urp1∆P;urp2∆P, and pkd2l1icm02 mutants degenerate with age.
(A–B) Lateral views of microcomputed tomography reconstituted skeletons at 3 mpf (A) and 12 mpf (B) show curves worsen with age. All fish shown are female. Scale bar: 10 mm.
Figure 2—figure supplement 3
Generation of uts2r3b1436 mutants.
The uts2r3b1436 allele was generated with a single-guide RNA which induced a deletion of 534 base pairs, resulting in an in-frame 178 amino acid deletion that removes around half the protein …
Figure 2—video 1
Three-dimensional reconstitution of a 3-mpf wild-type male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary. The adult zebrafish spine …
Figure 2—video 2
Three-dimensional reconstitution of a 3-mpf urp1∆P male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary.
Figure 2—video 3
Three-dimensional reconstitution of a 3-mpf urp2∆P male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary. Mutants demonstrate a slight …
Figure 2—video 4
Three-dimensional reconstitution of a 3-mpf urp1∆P;urp2∆P male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary. Mutants exhibited planar …
Figure 2—video 5
Three-dimensional reconstitution of a 12-mpf wild-type male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary.
Figure 2—video 6
Three-dimensional reconstitution of a 12-mpf urp1∆P male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary.
Figure 2—video 7
Three-dimensional reconstitution of a 12-mpf urp2∆P male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary.
Figure 2—video 8
Three-dimensional reconstitution of a 3-mpf uts2r3b1436 male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary. Mutants exhibited planar …
Figure 3 with 3 supplements
urp1∆P;urp2∆P mutants exhibit adolescent-onset spinal curves without significant structural vertebral defects.
(A) Lateral views of control fish and age-matched urp1∆P;urp2∆P mutants. Arrows point to forming body curves. (B) Traces of body shape every 2 days for 5 fish per time point from 3 to 17 dpf. (C) …
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Figure 3—source data 1
Raw data from larval growth measurements and vertebral quantitation.
- https://cdn.elifesciences.org/articles/83883/elife-83883-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Spinal curves are variable in 1 mpf urp1∆P;urp2∆P mutants.
(Ai-Bi) Dorsal and lateral views of microcomputed tomography (µCT) reconstitutions of wild type and urp1∆P;urp2∆P mutants at 1 mpf. (Aii-Bii) Lateral views of additional examples of µCT …
Figure 3—video 1
Time course of juvenile development in wild-type and urp1∆P;urp2∆P siblings.
Fish were imaged every other day over a 2.5-week period.
Figure 3—video 2
Vertebral reconstitutions from wild-type, urp1∆P;urp2∆P and uts2r3b1436 adults.
Alternating vertebrae are shown at 25% transparency to highlight subtle vertebral shape defects, including a reduced length and slight changes to length aspect ratios.
Figure 4 with 5 supplements
urp1∆P;urp2∆P mutants and cfap298tm304 mutants are phenotypically distinct.
(A–D) Lateral views of microcomputed tomography (µCT) reconstitutions of wild-type (A), cfap298tm304 mutants (B), urp1∆P;urp2∆P double mutants (C), and cfap298tm304;urp1∆P;urp2∆P triple mutants (D). …
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Figure 4—source data 1
Raw data for quantition of spinal phenotypes.
- https://cdn.elifesciences.org/articles/83883/elife-83883-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Analysis of lateral curvature.
Dorsal views of microcomputed tomography reconstituted spines from wild type and mutants at 3 mpf. Asterisks refer to spines shown in Figure 4Fi. Scale bar: 5 mm.
Figure 4—video 1
Three-dimensional reconstitution of a 3-mpf cfap298tm304 male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary. Mutants develop severe …
Figure 4—video 2
Three-dimensional reconstitution of a 3-mpf cfap298tm304;urp1∆P;urp2∆P male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary.
Figure 4—video 3
Three-dimensional reconstitution of a 3-mpf pkd2l1icm02 male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary.
Figure 4—video 4
Three-dimensional reconstitution of a 12-mpf pkd2l1icm02 male zebrafish.
Scans were performed using an 18-µm voxel resolution. Surface reconstructions were performed using a threshold of 3200, scales were digitally removed where necessary.
Figure 5
Reissner fiber (RF) breakdown in cfap298tm304 mutants but not urotensin-deficient mutants.
(A–F) Grayscale maximal intensity projection of Sspo-GFP localization in the central canal in 28 hpf embryos (A–C) and 12 dpf adolescents (D–F). RF is denoted by arrow heads in D and F. Arrows point …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Commercial assay, kit | DNA Clean and Concentrator Kit | Zymo Research | Cat no: D4013 | |
| Commercial assay, kit | RNA Clean and Concentrator Kit | Zymo Research | Cat no: R1016 | |
| Commercial assay, kit | Direct-zol RNA MiniPrep Kit | Zymo Research | Cat no: R2050 | |
| Commercial assay, kit | GeneJET Gel Extraction Kit | Thermo Fisher Scientific | Cat no: K0691 | |
| Commercial assay, kit | High Capacity RNA-to-cDNA Kit | Thermo Fisher Scientific | Cat no: 4387406 | |
| Commercial assay, kit | MEGAshortscript T7 Transcription Kit | Thermo Fisher Scientific | Cat no: AM1354 | |
| Commercial assay, kit | HiScribe T7 High Yield RNA Synthesis Kit | New England Biolabs | Cat no: E2040 | |
| Commercial assay, kit | FS DNA Library Prep Kit for Illumina | New England Biolabs | Cat no: E7805 | |
| Commercial assay, kit | HCR-RNA FISH Hybridization Buffer | Molecular Instruments | ||
| Commercial assay, kit | T7 Endonuclease I | New England Biolabs | Cat no: E3321 | |
| Commercial assay, kit | HCR-RNA FISH Amplification Buffer | Molecular Instruments | ||
| Commercial assay, kit | AlexaFluor-647 Hairpins | Molecular Instruments | ||
| Commercial assay, kit | AlexaFluor-546 Hairpins | Molecular Instruments | ||
| Commercial assay, kit | AlexaFluor-488 Hairpins | Molecular Instruments | ||
| Commercial assay, kit | urp1 HCR-RNA FISH probe | Molecular Instruments | Project-specific design | |
| Commercial assay, kit | urp2 HCR-RNA FISH probe | Molecular Instruments | Project-specific design | |
| Commercial assay, kit | pkd2l1 HCR-RNA FISH probe | Molecular Instruments | Project-specific design | |
| Commercial assay, kit | Taq Polymerase | New England Biolabs | Cat no: M0273 | |
| Commercial assay, kit | TURBO DNase | Thermo Fisher Scientific | Cat no: AM2238 | |
| Commercial assay, kit | Phusion High-Fidelity DNA Polymerase | New England Biolabs | Cat no: M0530 | |
| Commercial assay, kit | Phusion High-Fidelity PCR Master Mix | New England Biolabs | Cat no: M0531 | |
| Commercial assay, kit | SYBR Green PCR Master Mix | Thermo Fisher Scientific | Cat no: 4309155 | |
| Strain, strain background (Danio rerio) | AB strain | University of Oregon | ||
| Strain, strain background (D. rerio) | WIK strain | University of Oregon | ||
| Strain, strain background (D. rerio) | TU strain | University of Oregon | ||
| Strain, strain background (D. rerio) | cfap298tm304 line | Jaffe et al., 2016 | ZDB-FISH-150901–23024 | |
| Strain, strain background (D. rerio) | pkd2l1icm02 line | Sternberg et al., 2018 | ZDB-FISH-160811–9 | |
| Strain, strain background (D. rerio) | sspob1446 line | This study | Figure 1—figure supplement 3 | |
| Strain, strain background (D. rerio) | sspo-GFPut24 line | Troutwine et al., 2020 | ZDB-FISH-190313–20 | |
| Strain, strain background (D. rerio) | urp1b1420 line | This study | Figure 1—figure supplement 2 | |
| Strain, strain background (D. rerio) | urp2b1421 line | This study | Figure 1—figure supplement 2 | |
| Strain, strain background (D. rerio) | uts2r3b1436 line | This study | Figure 2—figure supplement 3 | |
| Software, algorithm | 3D Slicer | Fedorov et al., 2012 | ||
| Software, algorithm | IMARIS 9.9 | Oxford Instruments | ||
| Software, algorithm | NIS-Elements | Nikon Instruments Inc | ||
| Software, algorithm | ZEN Software | Carl Zeiss AG | ||
| Software, algorithm | QuantStudio Design and Analysis Software | Applied Biosystems | ||
| Software, algorithm | Integrated Genome Viewer (version 2.13.1) | Robinson et al., 2011 | ||
| Sequence-based reagent | Bottom strand ultramer_1 | This study | Tail ultramer for generating singe gRNA oligos | AAAAGCACCGACTCG GTGCCACTTTTTC AAGTTGATAACGGACT AGCCTTATTTT AACTTGCTAT |
| Sequence-based reagent | urp1_gRNA_1 | This study | gRNA_1 oligo for generating urp1b1420 line | taatacgactcactataGGCGT TGGTCAGCCTGACAT gttttagagctagaa |
| Sequence-based reagent | urp1_gRNA_2 | This study | gRNA_2 oligo for generating urp1b1420 line | taatacgactcactataGGG TCCTCTGTCCATCTCCG gttttagagctagaa |
| Sequence-based reagent | urp2_gRNA_1 | This study | gRNA_1 oligo for generating urp2b1421 line | taatacgactcactataGGCA GATGGAGAAAGATTGA gttttagagctagaa |
| Sequence-based reagent | urp2_gRNA_2 | This study | gRNA_2 oligo for generating urp2b1421 line | taatacgactcactataGGC GTTTGCAGAAATCAGCG gttttagagctagaa |
| Sequence-based reagent | uts2r3_gRNA | This study | gRNA oligo for generating uts2r3b1436 line | taatacgactcactataGGG TGAAGGGGAAGAGAAGA gttttagagctagaa |
| Sequence-based reagent | sspo_gRNA_1 | This study | gRNA_1 oligo for generating sspob1446 line | taatacgactcactataGGT CCCCAGTGGTCCGCG GTgttttagagctagaa |
| Sequence-based reagent | sspo_gRNA_2 | This study | gRNA_2 oligo for generating sspob1446 line | taatacgactcactataGGCAC AGTGTGTGAGACCAG gttttagagctagaa |
| Sequence-based reagent | Bottom strand ultramer_2 | This study | Tail ultramer for generating multiplexed gRNA oligos | AAAAGCACCGACTCGG TGCCACTTTTTC AAGTTGATAA CGGACTAGCCTTATT TTAACTTGCTATTTC TAGCTCTAAAAC |
| Sequence-based reagent | urp1_F0_gRNA_1 | Wu et al., 2018 | gRNA_1 oligo for generating urp1 F0 embryos | TAATACGACTCACTAT AGGAAAGTGAAGAT CGCGGCCGTTTTAG AGCTAGAAATAGC |
| Sequence-based reagent | urp1_F0_gRNA_2 | Wu et al., 2018 | gRNA_2 oligo for generating urp1 F0 embryos | TAATACGACTCACT ATAGGACACGG CTCTGCC ACAACGTTTTAGA GCTAGAAATAGC |
| Sequence-based reagent | urp1_F0_gRNA_3 | Wu et al., 2018 | gRNA_3 oligo for generating urp1 F0 embryos | TAATACGACTCACTA TAGGTTCAGAAGC TGGTAGCAGGTTTTA GAGCTAGAAATAGC |
| Sequence-based reagent | urp1_F0_gRNA_4 | Wu et al., 2018 | gRNA_4 oligo for generating urp1 F0 embryos | TAATACGACTCACT ATAGGGAAAATAAAT AACATGGTGTTTTA GAGCTAGAAATAGC |
| Sequence-based reagent | urp2_F0_gRNA_1 | Wu et al., 2018 | gRNA_1 oligo for generating urp2 F0 embryos | TAATACGACTCACTA TAGGTGACTGTCGC TTCAATCGGTTTTAG AGCTAGAAATAGC |
| Sequence-based reagent | urp2_F0_gRNA_2 | Wu et al., 2018 | gRNA_2 oligo for generating urp2 F0 embryos | TAATACGACTCACT ATAGGGACATTTCCT GACGGAGAGTTTTA GAGCTAGAAATAGC |
| Sequence-based reagent | urp2_F0_gRNA_3 | Wu et al., 2018 | gRNA_3 oligo for generating urp2 F0 embryos | TAATACGACTCACTA TAGGTGGACACGA GGAGACCGAGTTTT AGAGCTAGAAATAGC |
| Sequence-based reagent | urp2_F0_gRNA_4 | Wu et al., 2018 | gRNA_4 oligo for generating urp2 F0 embryos | TAATACGACTCACT ATAGGTCACCAGGTAG TGACGGAGTTTTAG AGCTAGAAATAGC |
| Sequence-based reagent | sspo_F0_gRNA_1 | Wu et al., 2018 | gRNA_1 oligo for generating sspo F0 embryos | TAATACGACTCACT ATAGGTTCGTCCCC AGTGGTCCGGTTTT AGAGCTAGAAATAGC |
| Sequence-based reagent | sspo_F0_gRNA_2 | Wu et al., 2018 | gRNA_2 oligo for generating sspo F0 embryos | TAATACGACTCACT ATAGGAAACGG CCGTCAGTGTCGGT TTTAGAGCTAGAAATAGC |
| Sequence-based reagent | sspo_F0_gRNA_3 | Wu et al., 2018 | gRNA_3 oligo for generating sspo F0 embryos | TAATACGACTCAC TATAGGTGTTGC AACACCAACCGGGT TTTAGAGCTAGAAATAGC |
| Sequence-based reagent | sspo_F0_gRNA_4 | Wu et al., 2018 | gRNA_4 oligo for generating sspo F0 embryos | TAATACGACTCACT ATAGGAGCCTAGACC TGCTCACGGTTTTA GAGCTAGAAATAGC |
| Sequence-based reagent | cfap298_F0_gRNA_1 | Wu et al., 2018 | gRNA_1 oligo for generating cfap298 F0 embryos | TAATACGACTCAC TATAGGTTCTCTT CAACACTACGGGT TTTAGAGCTAGAAATAGC |
| Sequence-based reagent | cfap298_F0_gRNA_2 | Wu et al., 2018 | gRNA_2 oligo for generating cfap298 F0 embryos | TAATACGACTCAC TATAGGGCTCC ACAATCTGATCATG TTTTAGAGCTA GAAATAGC |
| Sequence-based reagent | cfap298_F0_gRNA_3 | Wu et al., 2018 | gRNA_3 oligo for generating cfap298 F0 embryos | TAATACGACTCAC TATAGGCATTC TTATTGGATCATGG TTTTAGAGCTA GAAATAGC |
| Sequence-based reagent | cfap298_F0_gRNA_4 | Wu et al., 2018 | gRNA_4 oligo for generating cfap298 F0 embryos | TAATACGACTCACT ATAGGTCTCTGG CAGGTGCGCCCGTT TTAGAGCTAGAAATAGC |
| Sequence-based reagent | pkd2l1_geno_1 | Sternberg et al., 2018 | pkd2l1icm02 genotyping oligo 1 | TGTGTGCTAGG ACTGTGGGG |
| Sequence-based reagent | pkd2l1_geno_2 | Sternberg et al., 2018 | pkd2l1icm02 genotyping oligo 2 | AGGGCAAGAGAA TGGCAAGACG |
| Sequence-based reagent | urp1_geno_1 | This Study | urp1b1420 genotyping oligo 1 | GCACCCAAAAT CCAACGACT |
| Sequence-based reagent | urp1_geno_2 | This Study | urp1b1420 genotyping oligo 2 | TGTATGGGGAA AACAAAGGCA |
| Sequence-based reagent | urp2_geno_1 | This Study | urp2b1421 genotyping oligo 1 | TTGGGGTTGT AACAGGTAGTG |
| Sequence-based reagent | urp2_geno_2 | This Study | urp2b1421 genotyping oligo 2 | AACAAGGAAGA CGCTGCAAG |
| Sequence-based reagent | uts2r3_geno_1 | This Study | uts2r3b1436 genotyping oligo 1 | ATGGATCCCC TGATGTCCTG |
| Sequence-based reagent | uts2r3_geno_2 | This Study | uts2r3b1436 genotyping oligo 2 | TCGAACTCTGC TCATCCCAG |
| Sequence-based reagent | sspo_geno_1 | This Study | sspob1446 genotyping oligo 1 | CGCAAACACTT CCACTTCCA |
| Sequence-based reagent | sspo_geno_2 | This Study | sspob1446 genotyping oligo 2 | TTGAAGCCAGATGT AAAGGATGAGTGT |
| Sequence-based reagent | urp1_gRNA1+2_T7E1_F | This Study | Forward primer to amplify genomic DNA for T7E1 assay in crispants | GACAGCGCAC CCTTAATTGT |
| Sequence-based reagent | urp1_gRNA1+2_T7E1_R | This Study | Reverse primer to amplify genomic DNA for T7E1 assay in crispants | ACATTTAGCCTT AACAAGCACAA |
| Sequence-based reagent | urp1_gRNA3+4_T7E1_F | This Study | Forward primer to amplify genomic DNA for T7E1 assay in crispants | CAGACAAGGG AACAGAGAGGA |
| Sequence-based reagent | urp1_gRNA3+4_T7E1_R | This Study | Reverse primer to amplify genomic DNA for T7E1 assay in crispants | CCACTGCTTTTA AATCATCCACC |
| Sequence-based reagent | urp2_gRNA1_T7E1_F | This Study | Forward primer to amplify genomic DNA for T7E1 assay in crispants | ATCTTAGAGG CGCATTGGTG |
| Sequence-based reagent | urp2_gRNA1_T7E1_R | This Study | Reverse primer to amplify genomic DNA for T7E1 assay in crispants | GCATGAGGCG GTTTGTTTTG |
| Sequence-based reagent | urp2_gRNA2+3_T7E1_F | This Study | Forward primer to amplify genomic DNA for T7E1 assay in crispants | TGAAGCAACT GAGGAGCAAA |
| Sequence-based reagent | urp2_gRNA2+3_T7E1_R | This Study | Reverse primer to amplify genomic DNA for T7E1 assay in crispants | ACAGTACAGTT CAGCACACCT |
| Sequence-based reagent | urp2_gRNA4_T7E1_F | This Study | Forward primer to amplify genomic DNA for T7E1 assay in crispants | TGACCTATACATC AAAGCCAAGG |
| Sequence-based reagent | urp2_gRNA4_T7E1_R | This Study | Reverse primer to amplify genomic DNA for T7E1 assay in crispants | CCTGGGCTGA TCATACCTCT |
| Sequence-based reagent | rpl13_qPCR_F | This Study | Forward primer for quantitative RT-PCR | TAAGGACGGAG TGAACAACCA |
| Sequence-based reagent | rpl13_qPCR_R | This Study | Reverse primer for quantitative RT-PCR | CTTACGTCTGC GGATCTTTCTG |
| Sequence-based reagent | urp1_qPCR_F | This Study | Forward primer for quantitative RT-PCR | ACATTCTGG CTGTGGTTTG |
| Sequence-based reagent | urp1_qPCR_R | This Study | Reverse primer for quantitative RT-PCR | GTCCGTCTTCA ACCTCTGCTAC |
| Sequence-based reagent | urp2_qPCR_F | This Study | Forward primer for quantitative RT-PCR | AGAGGAAACA GCAATGGACG |
| Sequence-based reagent | urp2_qPCR_R | This Study | Reverse primer for quantitative RT-PCR | TGTTGGTTTTG GTTGACG |
Author response table 1
| 3-months | 12-months | Position of curve | |
|---|---|---|---|
| urp1∆P | no curves | mild D/V curves | Mostly caudal |
| urp2∆P | mild D/V curves | intermediate D/V curves | Mostly caudal |
| urp1∆P;urp2∆P | severe D/V curves | severe D/V curves | Mostly caudal |
| uts2r3b1436 | severe D/V curves | severe D/V curves | Mostly caudal |
| cfap298tm304 | severe 3D curves | severe 3D curves | Caudal and pre-caudal |
| pkd2l1icm02 | no curves | very mild D/V curves | Mostly pre-caudal |
