Urotensin II-related peptides, Urp1 and Urp2, control zebrafish spine morphology

Figures

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  Figure 1.. 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 induce deletions of Urp1 and Urp2 peptide coding sequences, resulting in urp1∆P and urp2∆P mutants, respectively. SP – signal peptide. (B) Urp1 and Urp2 peptide sequences with identical hexacyclic regions. (C) Zebrafish posterior axial straightening, the morphogenetic process which straightens the embryonic body. (D) Fluorescence in situ hybridization based on hybridization chain reaction analysis of pkd2l1, urp1, and urp2 expression in the central canal at 28 hpf. pkd2l1 expression marks CSF-cNs. urp1 expression is restricted to ventral CSF-cNs while urp2 is expressed in all CSF-cNs. Both urp1 and urp2 are expressed in cfap298tm304 and sspob1446 mutants, though comparison of expression between samples was non-quantitative. (i) Shows the zebrafish trunk with the yolk stalk labeled (*). (ii) Shows zoomed regions taken at the rostro-caudal level at the end of the yolk stalk. Scale bars: 150 µm (i), 10 µm (ii). (E) Lateral views of…

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  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 subsequently been lost. (B) The UII and URP proteins in zebrafish and human are 8–12 amino acid peptides with a fully conserved hexacyclic region of sequence CFWKYC.

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  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 removes a portion of intron 4–5 and the coding part of exon 5 including the entire region coding for the Urp1 peptide. The urp2b1421 (urp2∆P) allele encodes a 61 base pair deletion which removes the region coding for the Urp2 peptide. Red arrows show the location of gRNA sites (1–4) used to generate crispants. Green arrows show the location of PCR primers used to amplify regions around gRNA sites. (C–D) T7E1 assays show significant mutagenesis at urp1 (C) and urp2 (D) loci in crispant embryos. Embryos were injected with Cas9 only (−) or Cas9 plus gRNAs 1–4 (+) then, at 1 dpf, DNA was extracted and regions amplified using the indicated primer pairs. PCR product from crispant embryos showed wider bands, indicating insertion-deletion (indel) mutations, as well as deletion bands (white arrow head), indicating large deletions between two gRNA sites. After PCR, product was purified and subjected to digestion with T7E1, which cleaves heteroduplex DNA.…

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  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 cysteine rich domain; LDLrA – low-density lipoprotein receptor class A domain; TSP1 – thrombospondin type 1 domain; FA58C – coagulation factor 5/8 C-terminal domain. In sspob1446 mutants, a genomic deletion results in a frame shift mutation at Valine 147 resulting in an early premature truncation codon. (B) The sspob1446 mutant line harbors a large deletion and a downstream small deletion which disrupt exons 4 and 5 causing the early truncation of Sspo.

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  Figure 2.. 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 sagittal plane for urp1∆P;urp2∆P and uts2r3b1436 mutants. Circles represent angles for individual curves. (E-E’) Total Cobb angles with each circle representing an individual fish. The mean ± s.d. is shown. (G’) is the data from G parsed for sex. p-Values are given from two-tailed unpaired student’s t tests. (F) The position of curve apex is plotted and shows that most curves are in caudal vertebrae. n=9 and 8 for urp1∆P;urp2∆P and uts2r3b1436 mutants, respectively. Figure 2—source data 1.Raw data from spinal curve phenotypic measurements.

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  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 of the first and last displaced vertebra, respectively, and the external angle is taken at their intersection. The angle was assigned to its most displaced apex vertebra (cyan asterisk) in heat map representations. Note that displaced vertebrae do not always demonstrate an easily identifiable wedge in intervertebral space, as is typical in human data. In these cases, the first or last vertebrae of the curve is designated as ‘least parallel’ to the local orientation of the spine. (C) The position of curve apex is plotted for cfap298tm304 mutants and cfap298tm304;urp1∆P;urp2∆P triple mutants (triple) alongside urp1∆P;urp2∆P and uts2r3b1436 mutants for comparison. See also Figure 2F. (D) Cobb angle measurements for individual fish in the sagittal plane for cfap298tm304 and cfap298tm304;urp1∆P;urp2∆P mutants. Circles represent angles for individual curves. (E) Total Cobb angles with each circle representing an individual fish. The mean ± s.d. is shown. p-Values…

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  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.

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  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 including transmembrane regions.

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  Figure 3.. 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) Growth curves for control and urp1∆P;urp2∆P mutants were indistinguishable. Arrow shows time of curve onset. Mean ± s.d. is plotted. (D) Microcomputed tomography (µCT) reconstitutions of spines at 1 mpf with heads, fins, and ribs digitally removed. Scale bar: 1 mm. (E) µCT reconstitutions of three pre-caudal and two caudal vertebrae including frontal and lateral views of the highlighted vertebra. No major structural defects such as fusions were observed in urp1∆P;urp2∆P mutants. (F) Vertebral body rostral-caudal length (L1/L2) and dorsal-ventral height (H1/H2) aspect ratios for six vertebrae of wild type (n=4) and urp1∆P;urp2∆P mutants (n=4). Length aspect ratios were significantly more variable in mutants, but height aspect ratios were unchanged (p=0.022 and 0.745, respectively, Bartlett’s test for equal variances). (G) Calcein staining revealed well-structured vertebrae…

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  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 reconstitutions. The position of curve apex is variable, with 5 of 7 urp1∆P;urp2∆P mutants exhibiting pre-caudal curves. Note that dorsal fins were digitally dissected for clarity. Scale bars: 1 mm.

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  Figure 4.. 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). All fish shown are female. Scale bar: 10 mm. (E) Total Cobb angles with each circle representing an individual fish. The mean ± s.d. is shown. p-Values are given from two-tailed unpaired student’s t tests. U — urp1∆P;urp2∆P double mutants; C — cfap298tm304 mutants; UC — cfap298tm304;urp1∆P;urp2∆P triple mutants. (Fi) Dorsal views of µCT reconstitutions with ribs and fins removed. Scale bar: 5 mm. (Fii) Quantitation of degree of lateral curvature for wild type (n=5) and urp1∆P;urp2∆P (n=8), uts2r3b1436 (n=3), cfap298tm304 (n=3), and cfap298tm304;urp1∆Purp2∆P (n=6) mutants. y-axis is the arbitrary units. Figure 4—source data 1.Raw data for quantition of spinal phenotypes.

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  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.