Digits and fin rays share common developmental histories

Journal name:
Nature
Volume:
537,
Pages:
225–228
Date published:
DOI:
doi:10.1038/nature19322
Received
Accepted
Published online

Understanding the evolutionary transformation of fish fins into tetrapod limbs is a fundamental problem in biology1. The search for antecedents of tetrapod digits in fish has remained controversial because the distal skeletons of limbs and fins differ structurally, developmentally, and histologically2, 3. Moreover, comparisons of fins with limbs have been limited by a relative paucity of data on the cellular and molecular processes underlying the development of the fin skeleton. Here, we provide a functional analysis, using CRISPR/Cas9 and fate mapping, of 5′ hox genes and enhancers in zebrafish that are indispensable for the development of the wrists and digits of tetrapods4, 5. We show that cells marked by the activity of an autopodial hoxa13 enhancer exclusively form elements of the fin fold, including the osteoblasts of the dermal rays. In hox13 knockout fish, we find that a marked reduction and loss of fin rays is associated with an increased number of endochondral distal radials. These discoveries reveal a cellular and genetic connection between the fin rays of fish and the digits of tetrapods and suggest that digits originated via the transition of distal cellular fates.

At a glance

Figures

  1. Expression patterns of hox13 genes at 48–120 hpf.
    Figure 1: Expression patterns of hox13 genes at 48–120 hpf.

    a, hoxa13a. b, hoxa13b. c, hoxd13a. Hoxa13a is expressed in distal mesenchyme at 48 hpf, but expression continues in the proximal fin fold from 72 to 96 hpf (a). Hoxa13b is expressed in distal mesenchyme and expression can be observed at the distal part of the endochondral disk until 96 hpf (b). Hoxd13a is expressed in the posterior half of the mesenchyme at 48 hpf and expression continues in the posterior endochondral disk through 96 hpf. After 96 hpf, expression becomes weak (c). Scale bars are 100 μm. n = 20 embryos for each in situ hybridization at 48 hpf. n = 10 embryos after 72 hpf.

  2. Fate mapping of cells marked by the activity of hox enhancers.
    Figure 2: Fate mapping of cells marked by the activity of hox enhancers.

    a, In situ hybridization of Cre in Dr-CNS65x3–Cre and Lo-e16x4–Cre exhibits expression dynamics of early and late phase enhancers used for fate mapping. Cre regulated by early phase hox enhancer CNS65 is expressed throughout the fin from 31 to 38 hpf, whereas late phase expression (driven by e16) begins weakly in the distal fin at 38 hpf and ceases at ~55 hpf. Inset shows zoom in of the pectoral fin, black arrows point to the distal border of the endochondral disk. b, Lineage tracing of Dr-CNS65x3–Cre at 6 dpf and 20 dpf. Red: mCherry IF; blue: DAPI. Cells that experienced early phase expression (red) contribute to fin fold and endochondral disk. c, Lineage tracing of Lo-e16x4–Cre at 6 dpf and 20 dpf. Cells that underwent late phase expression are present mostly in the fin fold, though some cells are at the distal edge of the disk. Red cells at 6 dpf protrude filopodia in the distal direction, indicating that these cells are actively moving out into the fin fold. d, Lineage tracing of late phase hox cells in adult zebrafish fin (~120 dpf). mCherry cells are present only in the derivatives of the fin fold, and not in the endochondral disk. Inset: magnification of distal edge of fin rays. Green: Zns-5 osteoblast marker; red: Hox-positive; yellow: overlap of Zns5 and Cre. White dotted lines outline the fin (b, c) or endochondral bones in (d). n = 5 for stable lines. All scale bars are 100 μm except for the total fin in d, which is 500 μm.

  3. Adult fin phenotypes of hox13 deletion series.
    Figure 3: Adult fin phenotypes of hox13 deletion series.

    a–c, wild type. d–f, hoxd13a−/−. g–i, hoxa13b−/−. j–l, hoxa13a−/−. m–o, hoxa13a−/−, a13b−/−. p–r hoxa13a0% a13b0% and d13a0% (mosaic triple knockout; Methods and Extended Data Tables 3, 4). Each mutant hox sequence is found in Extended Data Tables 3, 4. a, d, g, j, m, p, Alzarin Red and Alcian Blue staining of pectoral fin. b, e, h, k, n, q, CT scanning of pectoral fins. Black: radials (endochondral bones); grey: fin rays (dermal bones). Note that hoxa13 single (g, h, j, k), double (m, n), and mosaic triple (p, q) mutant fins show shorter fin rays than wild type (a, b). Fins were scaled according to the bone staining pictures. c, f, i, l, o, r, Enlarged images of CT scanning without fin rays to reveal endochondral patterns. Dark grey; proximal radials, red; distal radials. Upper left side is the anterior and bottom right is the posterior side in each picture. Double and triple knockout mutants have 10–13 distal radials (o and r; Extended Data Fig. 4, Supplementary Information). Third and fourth proximal radials started to fuse into one bone in hoxa13a−/−, a13b−/− (o). Note that posterior distal radials are stacked along proximodistal axis (o). Posterior proximal radials are broken down into small parts in mosaic triple knockout (r). Scale bars are 2 mm. The size of specimens are not scaled in c, f, i, l, o and r to display the detail of distal radials. n = 3 fish for single and double mutants and n = 5 fish for mosaic triple mutant.

  4. Shared developmental histories in fin rays and digits.
    Figure 4: Shared developmental histories in fin rays and digits.

    In mice (top row), late phase Hox expression (red) marks the distal cells of the limb bud that result in bones of the autopod (wrists and digits). Double knockout of Hoxa13 and Hoxd13 results in the loss of the autopod. In zebrafish wild-type fins (middle row), cells marked by late phase hox expression (red) end up in the fin fold and within osteoblasts of the dermal rays. Hoxa13 double knockout fish (hoxa13a−/−, a13b−/−) and the triple knockout (mosaic for hoxa13b and hoxd13a) have extremely reduced fin rays with increased distal endochondral radials. Note that distal radials are stacked along the proximodistal axis in the posterior of the fins. The results lead to the the hypothesis (bottom row) that the knockout phenotype results from a deficit in migration of mesenchymal cells with more cells left in the distal fin bud (increased number of cells in the endochondral disk of mutants fins, Extended Data Fig. 4) and fewer migrating to the fold, thereby resulting in a larger number of endochondral bones and reduced dermal ones. Red cells: cells that experienced late phase hox expression. Mouse limbs consist of only endochondral bones, but fish fins contain endochondral (black) and dermal (transparent; fin rays) bones.

  5. Cre in situ hybridization of lineage tracing fish.
    Extended Data Fig. 1: Cre in situ hybridization of lineage tracing fish.

    a, Cre is expressed only from 31 hpf to 38 hpf in Dr-CNS65x3–Cre, whereas it is expressed from 38 hpf to 55 hpf in Lo-e16x4–Cre. These temporal expression patterns of Cre indicate that our transgenic lineage tracing labelled the cells which experienced only early or late phase hox. Scale bars are 100 μm. b, Cre expression pattern from 48–120 hpf in independent Lo-e16x4–Cre lines (different founders from a). The fin is outlined by a dashed white line. The expression patterns from different founders were investigated and all expression ceases before 72 hpf. Our in situ results indicate that Lo-e16x4–Cre marks only the cells that experienced late phase hox expression from 38-55 hpf. n = 5 embryos for all stages. Scale bars are 100 μm. c, The expression pattern of and1 and hox13 genes in wild type (10 dpf) and also Cre in Lo-e16x4–Cre line (10 dpf and 3 months, n = 10). Whereas and1 expression can be observed in fin fold (positive control, black arrow), hox13 genes are not expressed at 10 dpf in the wild type. Cre is not expressed at 10 dpf and at 3 months in the fin, indicating that Lo-e16x4–Cre activity is limited to only early embryonic development (38–55 hpf). Three month fins were dissected from the body of Lo-e16x4–Cre lines and subjected to in situ hybridization (n = 3). Scale bars are 500 μm at 10 dpf and 3 months.

  6. T7E1 assay of F0 CRISPR/Cas9 adult fish.
    Extended Data Fig. 2: T7E1 assay of F0 CRISPR/Cas9 adult fish.

    PCR products of hoxa13a, hoxa13b or hoxd13a were subjected to a T7E1 assay (Methods) and confirmed by gel electrophoresis. a, The result of the hoxa13a, hoxa13b or hoxd13a T7E1 assay for ten adult fish. ‘M.’ is a 100 bp DNA ladder marker (NEB). In the hoxa13a gel picture, 810 bp (black arrowhead) is the wild-type band as observed in cont. lane (wild type without gRNA injection). All ten fish showed smaller and bottom shifted products (red arrowheads) compared to negative control fish, indicating that all fish have mutations in the target region of hoxa13a. In the hoxa13b gel picture, 1,089 bp is the wild-type band. All ten fish into which hoxa13b gRNAs were injected showed smaller and bottom shifted products compared to negative control fish, indicating that all fish have mutations in the target region of hoxa13b. In the hoxd13a gel picture, 823 bp is the wild-type band. Eight of ten fish showed smaller and bottom shifted products, indicating that 80% of fish have mutations in the target region of hoxd13a. b, The efficiency of CRISPR/Cas9 deletion for hox13 in zebrafish. Almost all adult fish into which gRNAs and Cas9 mRNA were injected have mutations at the target positions. c, The efficiency of germline transmission of CRISPR/Cas9 mutant fish. Identified mutant fish were outcrossed to wild-type fish to obtain embryos and confirmed germline transmission. Obtained embryos were lysed individually at 48 hpf, genotyped by T7E1 assay and sequenced. Because of CRISPR/Cas9 mosaicism, some different mutation patterns, which result in a non-frameshift or frameshift mutation, were observed.

  7. Embryonic phenotypes of hox13 deletion mutants.
    Extended Data Fig. 3: Embryonic phenotypes of hox13 deletion mutants.

    a, e, i, m, q, Whole body pictures at 72 hpf. a, Wild type, e, hoxa13a−/− (4 bp del./4 bp del.), i, hoxa13b−/− (4 bp del./14 bp ins.), m, hoxd13a−/− (5 bp ins./17 bp del.), and q, hoxa13a−/−, hoxa13b−/− double homozygous embryo (8 bp del./29 bp del., 14 bp ins./14 bp ins.). The details of mutant sequences are summarized in Extended Data Table 3. Wild-type and single homozygous fish for hoxa13a or hoxa13b were treated by PTU to inhibit pigmentation. The body size and length of mutant embryos are relatively normal at 72 hpf. n = 5 embryos for all genotypes. b, f, j, n, r, Bright field images of pectoral fins. Pectoral fins were detached from the body and photographed (Methods). Hoxa13a−/−, a13b−/− double homozygous embryo shows 30% shorter pectoral fin fold compared to wild type (r, see also Extended Data Fig. 5). n = 5 embryos for all genotypes. c, g, k, o, s, and1 in situ hybridization at 72 hpf. Hox13 mutants show normal expression patterns, which indicates that fin fold development is similar to wild type in these mutants. n = 3 embryos for all genotypes. d, h, l, p, t, shha in situ hybridization at 48 hpf. Hox13 mutants show a normal expression pattern that is related to relatively normal anteroposterior asymmetry of adult fin (Fig. 3, Extended Data Fig. 4 and Supplementary Information). n = 3 embryos for all genotypes. Scale bars are 1 mm (a), 200 μm (b, c) and 100 μm (d).

  8. Phenotype of adult hox13 mutant fish.
    Extended Data Fig. 4: Phenotype of adult hox13 mutant fish.

    a, c, e, g, i, k, m, Whole body morphology of hox13 deletion mutants were photographed at 4 months old; hoxa13a−/− (8 bp del./29 bp del.), hoxa13b−/− (4 bp del./14 bp ins.), hoxd13a−/− (5 bp ins./10 bp ins.), hoxa13a−/−, hoxa13b−/− double homozygous fish (8 bp del./29 bp del., 14bp ins./14 bp ins.) and triple knockout (k, m, mosaic for hoxa13a hoxa13b and hoxd13a) fish (Methods). n = 3 fish for wild type, single and double mutants and n = 5 fish for triple mosaic mutants (same specimens were used as in Fig. 3). The details of mutant sequences are summarized in Extended Data Table 3. Each homozygous mutant fish shows normal morphology at 4 months old except for slightly short pectoral fin rays of hoxa13a−/− or a13b−/− single mutants. Hoxa13a−/−, hoxa13b−/− double homozygous fish shows a severe reduction of fin rays in pectoral, pelvic, dorsal and anal fins compared with wild type. The triple knockout (mosaic for hoxa13a, hoxa13b and hoxd13a) fish also showed a reduction in fin rays. Scale bar is 5 mm. Owing to the size of the adult fish, three different pictures for anterior, centre and posterior of the body were merged to make whole-body pictures. b, d, f, h, j, l, n, Bone staining pictures of mutant fish. The endochondral bones of pectoral fins are shown. Whereas single homozygous fish show relatively normal proximal radials (b, d, f, h and Fig. 3), double homozygous mutants show fused third and fourth proximal radials (j). One triple knockout (mosaic for hoxa13a, hoxa13b and hoxd13a, 0, 25, 50%) fish had fused third and fourth proximal radials (i), but another triple knockout (0, 0, 0%) had more broken down proximal radials (n). n = 3 fish for wild type, single and double mutants and n = 5 fish for triple mosaic mutants (same specimens were used as in Fig. 3). The scale bar is 500 μm. o, p, Examples of counting distal radials in wild-type and hoxa13a−/−, hoxa13b−/− double homozygous fish. First distal radials are not shown in CT segmentation because of a fusion with first fin ray. q, The number variation of distal radials in mutant fish. Multiple fins were investigated in wild type (25 fish/50 fins), hoxa13a−/− (4 bp del./4 bp del., 3 fish/6 fins), hoxa13b−/− (4 bp del./14 bp ins., 3 fish/6 fins), hoxd13a−/− (5 bp ins./17 bp del., 3 fish/6 fins), hoxa13a−/−, hoxa13b−/− double homozygous (8 bp del./29 bp del., 14 bp ins./14 bp ins., 3 fish/6 fins) and triple knockout (mosaic for hoxa13a, hoxa13b and hoxd13a) fish (five fish/10 fins). The number of distal radials increased to 10 and 13 in double and triple mutants, respectively. The difference in distal radial number between wild-type and double homozygous or wild-type and triple knockout fish (mosaic for hoxa13a, hoxa13b and hoxd13a) is statistically significant (P = 0.0014 or P = 0.00001, respectively, t-test comparing the means, two-tailed distribution).

  9. Analysis of embryonic fin fold and endochondral disk in hoxa13a−/−, hoxa13b−/− embryos.
    Extended Data Fig. 5: Analysis of embryonic fin fold and endochondral disk in hoxa13a−/−, hoxa13b−/− embryos.

    a, A bright field image of wild-type and hoxa13a−/−, hoxa13b−/− pectoral fins at 72 hpf. Pectoral fins were detached from the body and photographed (Methods). Scale bar is 150 μm. b, The difference in fin fold length between wild-type and hoxa13a−/−, hoxa13b−/− embryos. The length of the fin fold was measured in wild-type (n = 8) and hoxa13a−/−, hoxa13b−/− double homozygous (n = 5) embryos at 72 hpf and 96 hpf (Methods). The length of the fin folds was decreased to about 70% of wild type in double homozygous embryos (72 hpf; P = 0.006, 96 hpf; P = 0.004, t-test comparing the means, one-tailed distribution, see Source Data). The error bars indicate s.e.m. c, d, Images of DAPI staining of wild-type (c) and hoxa13a−/−, hoxa13b−/− mutant (d) pectoral fins captured by confocal microscopy. White circles indicate nuclei in the endochondral disks. Scale bar is 200 μm. e, The average number of cells in the endochondral disk of wild-type and hoxa13a−/−, hoxa13b−/− mutant fins (see Methods and Source Data). The difference is statistically significant (P = 0.041 by Student’s t-test, one-tailed distribution). The error bars indicate s.e.m.

Tables

  1. Primers and oligos sequence for lineage tracing
    Extended Data Table 1: Primers and oligos sequence for lineage tracing
  2. PCR primers for CRISPR/Cas9 deletion, T7E1 assay, genotypes and gene cloning
    Extended Data Table 2: PCR primers for CRISPR/Cas9 deletion, T7E1 assay, genotypes and gene cloning
  3. List of hox13 mutant sequences
    Extended Data Table 3: List of hox13 mutant sequences
  4. Genotyping of progeny from mutant crosses
    Extended Data Table 4: Genotyping of progeny from mutant crosses

References

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Author information

  1. These authors contributed equally to this work.

    • Tetsuya Nakamura &
    • Andrew R. Gehrke

Affiliations

  1. Department of Organismal Biology and Anatomy, University of Chicago, Chicago, Illinois 60637, USA

    • Tetsuya Nakamura,
    • Andrew R. Gehrke,
    • Justin Lemberg,
    • Julie Szymaszek &
    • Neil H. Shubin

Contributions

T.N., A.R.G. and N.H.S. designed research; T.N. and J.S. performed in situ hybridization and CRISPR experiments; A.R.G. did fate mapping of the hox enhancers; T.N. and J.L. obtained CT scanning data; T.N., A.R.G., J.L. and N.H.S. analyzed data; and T.N., A.R.G., J.L. and N.H.S. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks S. Burgess and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Cre in situ hybridization of lineage tracing fish. (637 KB)

    a, Cre is expressed only from 31 hpf to 38 hpf in Dr-CNS65x3–Cre, whereas it is expressed from 38 hpf to 55 hpf in Lo-e16x4–Cre. These temporal expression patterns of Cre indicate that our transgenic lineage tracing labelled the cells which experienced only early or late phase hox. Scale bars are 100 μm. b, Cre expression pattern from 48–120 hpf in independent Lo-e16x4–Cre lines (different founders from a). The fin is outlined by a dashed white line. The expression patterns from different founders were investigated and all expression ceases before 72 hpf. Our in situ results indicate that Lo-e16x4–Cre marks only the cells that experienced late phase hox expression from 38-55 hpf. n = 5 embryos for all stages. Scale bars are 100 μm. c, The expression pattern of and1 and hox13 genes in wild type (10 dpf) and also Cre in Lo-e16x4–Cre line (10 dpf and 3 months, n = 10). Whereas and1 expression can be observed in fin fold (positive control, black arrow), hox13 genes are not expressed at 10 dpf in the wild type. Cre is not expressed at 10 dpf and at 3 months in the fin, indicating that Lo-e16x4–Cre activity is limited to only early embryonic development (38–55 hpf). Three month fins were dissected from the body of Lo-e16x4–Cre lines and subjected to in situ hybridization (n = 3). Scale bars are 500 μm at 10 dpf and 3 months.

  2. Extended Data Figure 2: T7E1 assay of F0 CRISPR/Cas9 adult fish. (208 KB)

    PCR products of hoxa13a, hoxa13b or hoxd13a were subjected to a T7E1 assay (Methods) and confirmed by gel electrophoresis. a, The result of the hoxa13a, hoxa13b or hoxd13a T7E1 assay for ten adult fish. ‘M.’ is a 100 bp DNA ladder marker (NEB). In the hoxa13a gel picture, 810 bp (black arrowhead) is the wild-type band as observed in cont. lane (wild type without gRNA injection). All ten fish showed smaller and bottom shifted products (red arrowheads) compared to negative control fish, indicating that all fish have mutations in the target region of hoxa13a. In the hoxa13b gel picture, 1,089 bp is the wild-type band. All ten fish into which hoxa13b gRNAs were injected showed smaller and bottom shifted products compared to negative control fish, indicating that all fish have mutations in the target region of hoxa13b. In the hoxd13a gel picture, 823 bp is the wild-type band. Eight of ten fish showed smaller and bottom shifted products, indicating that 80% of fish have mutations in the target region of hoxd13a. b, The efficiency of CRISPR/Cas9 deletion for hox13 in zebrafish. Almost all adult fish into which gRNAs and Cas9 mRNA were injected have mutations at the target positions. c, The efficiency of germline transmission of CRISPR/Cas9 mutant fish. Identified mutant fish were outcrossed to wild-type fish to obtain embryos and confirmed germline transmission. Obtained embryos were lysed individually at 48 hpf, genotyped by T7E1 assay and sequenced. Because of CRISPR/Cas9 mosaicism, some different mutation patterns, which result in a non-frameshift or frameshift mutation, were observed.

  3. Extended Data Figure 3: Embryonic phenotypes of hox13 deletion mutants. (640 KB)

    a, e, i, m, q, Whole body pictures at 72 hpf. a, Wild type, e, hoxa13a−/− (4 bp del./4 bp del.), i, hoxa13b−/− (4 bp del./14 bp ins.), m, hoxd13a−/− (5 bp ins./17 bp del.), and q, hoxa13a−/−, hoxa13b−/− double homozygous embryo (8 bp del./29 bp del., 14 bp ins./14 bp ins.). The details of mutant sequences are summarized in Extended Data Table 3. Wild-type and single homozygous fish for hoxa13a or hoxa13b were treated by PTU to inhibit pigmentation. The body size and length of mutant embryos are relatively normal at 72 hpf. n = 5 embryos for all genotypes. b, f, j, n, r, Bright field images of pectoral fins. Pectoral fins were detached from the body and photographed (Methods). Hoxa13a−/−, a13b−/− double homozygous embryo shows 30% shorter pectoral fin fold compared to wild type (r, see also Extended Data Fig. 5). n = 5 embryos for all genotypes. c, g, k, o, s, and1 in situ hybridization at 72 hpf. Hox13 mutants show normal expression patterns, which indicates that fin fold development is similar to wild type in these mutants. n = 3 embryos for all genotypes. d, h, l, p, t, shha in situ hybridization at 48 hpf. Hox13 mutants show a normal expression pattern that is related to relatively normal anteroposterior asymmetry of adult fin (Fig. 3, Extended Data Fig. 4 and Supplementary Information). n = 3 embryos for all genotypes. Scale bars are 1 mm (a), 200 μm (b, c) and 100 μm (d).

  4. Extended Data Figure 4: Phenotype of adult hox13 mutant fish. (499 KB)

    a, c, e, g, i, k, m, Whole body morphology of hox13 deletion mutants were photographed at 4 months old; hoxa13a−/− (8 bp del./29 bp del.), hoxa13b−/− (4 bp del./14 bp ins.), hoxd13a−/− (5 bp ins./10 bp ins.), hoxa13a−/−, hoxa13b−/− double homozygous fish (8 bp del./29 bp del., 14bp ins./14 bp ins.) and triple knockout (k, m, mosaic for hoxa13a hoxa13b and hoxd13a) fish (Methods). n = 3 fish for wild type, single and double mutants and n = 5 fish for triple mosaic mutants (same specimens were used as in Fig. 3). The details of mutant sequences are summarized in Extended Data Table 3. Each homozygous mutant fish shows normal morphology at 4 months old except for slightly short pectoral fin rays of hoxa13a−/− or a13b−/− single mutants. Hoxa13a−/−, hoxa13b−/− double homozygous fish shows a severe reduction of fin rays in pectoral, pelvic, dorsal and anal fins compared with wild type. The triple knockout (mosaic for hoxa13a, hoxa13b and hoxd13a) fish also showed a reduction in fin rays. Scale bar is 5 mm. Owing to the size of the adult fish, three different pictures for anterior, centre and posterior of the body were merged to make whole-body pictures. b, d, f, h, j, l, n, Bone staining pictures of mutant fish. The endochondral bones of pectoral fins are shown. Whereas single homozygous fish show relatively normal proximal radials (b, d, f, h and Fig. 3), double homozygous mutants show fused third and fourth proximal radials (j). One triple knockout (mosaic for hoxa13a, hoxa13b and hoxd13a, 0, 25, 50%) fish had fused third and fourth proximal radials (i), but another triple knockout (0, 0, 0%) had more broken down proximal radials (n). n = 3 fish for wild type, single and double mutants and n = 5 fish for triple mosaic mutants (same specimens were used as in Fig. 3). The scale bar is 500 μm. o, p, Examples of counting distal radials in wild-type and hoxa13a−/−, hoxa13b−/− double homozygous fish. First distal radials are not shown in CT segmentation because of a fusion with first fin ray. q, The number variation of distal radials in mutant fish. Multiple fins were investigated in wild type (25 fish/50 fins), hoxa13a−/− (4 bp del./4 bp del., 3 fish/6 fins), hoxa13b−/− (4 bp del./14 bp ins., 3 fish/6 fins), hoxd13a−/− (5 bp ins./17 bp del., 3 fish/6 fins), hoxa13a−/−, hoxa13b−/− double homozygous (8 bp del./29 bp del., 14 bp ins./14 bp ins., 3 fish/6 fins) and triple knockout (mosaic for hoxa13a, hoxa13b and hoxd13a) fish (five fish/10 fins). The number of distal radials increased to 10 and 13 in double and triple mutants, respectively. The difference in distal radial number between wild-type and double homozygous or wild-type and triple knockout fish (mosaic for hoxa13a, hoxa13b and hoxd13a) is statistically significant (P = 0.0014 or P = 0.00001, respectively, t-test comparing the means, two-tailed distribution).

  5. Extended Data Figure 5: Analysis of embryonic fin fold and endochondral disk in hoxa13a−/−, hoxa13b−/− embryos. (436 KB)

    a, A bright field image of wild-type and hoxa13a−/−, hoxa13b−/− pectoral fins at 72 hpf. Pectoral fins were detached from the body and photographed (Methods). Scale bar is 150 μm. b, The difference in fin fold length between wild-type and hoxa13a−/−, hoxa13b−/− embryos. The length of the fin fold was measured in wild-type (n = 8) and hoxa13a−/−, hoxa13b−/− double homozygous (n = 5) embryos at 72 hpf and 96 hpf (Methods). The length of the fin folds was decreased to about 70% of wild type in double homozygous embryos (72 hpf; P = 0.006, 96 hpf; P = 0.004, t-test comparing the means, one-tailed distribution, see Source Data). The error bars indicate s.e.m. c, d, Images of DAPI staining of wild-type (c) and hoxa13a−/−, hoxa13b−/− mutant (d) pectoral fins captured by confocal microscopy. White circles indicate nuclei in the endochondral disks. Scale bar is 200 μm. e, The average number of cells in the endochondral disk of wild-type and hoxa13a−/−, hoxa13b−/− mutant fins (see Methods and Source Data). The difference is statistically significant (P = 0.041 by Student’s t-test, one-tailed distribution). The error bars indicate s.e.m.

Extended Data Tables

  1. Extended Data Table 1: Primers and oligos sequence for lineage tracing (443 KB)
  2. Extended Data Table 2: PCR primers for CRISPR/Cas9 deletion, T7E1 assay, genotypes and gene cloning (250 KB)
  3. Extended Data Table 3: List of hox13 mutant sequences (546 KB)
  4. Extended Data Table 4: Genotyping of progeny from mutant crosses (173 KB)

Supplementary information

PDF files

  1. Supplementary Figure (5.4 MB)

    This file contains a 3D pdf of wild-type and double mutant fin.

Additional data