A conserved Shh cis-regulatory module highlights a common developmental origin of unpaired and paired fins

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Despite their evolutionary, developmental and functional importance, the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka (Oryzias latipes) ZRS and enhancer assays identify the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolishes shh expression and completely truncates pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.

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Fig. 1: The regulatory landscape of Shh and the enhancer activity of ZRS in fin or limb ZPAs are conserved across gnathostomes.
Fig. 2: Medaka ZRS ablation results in mild pectoral fin defects.
Fig. 3: The Lmbr1 intron containing ZRS harbors shadow enhancers for ZRS (sZRS) in zebrafish, medaka and humans, and deleting both ZRS and sZRS completely truncates pectoral fin development.
Fig. 4: Dorsal fin does not form in ZRS medaka mutants.


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We thank A. Fernández-Miñan from the CABD aquatic vertebrate platform for providing the medaka 4C-seq samples, R.D. Acemel for helping with the design of the medaka 4C-seq primers, and all members of JLGSK laboratory and F. Casares for discussions. We thank J. Westlund for illustration assistance and F. Sugahara for providing the clone of lamprey HhA and the metamorphic ammocoete larva. This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 740041), the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement #658521, the Spanish Ministerio de Economía y Competitividad (grants BFU2016-74961-P, BFU2014-53765-P and BFU2016-81887-REDT), the Andalusian Government (grant BIO-396), CNPq Universal Program Grant 403248/2016-7 and CAPES/Alexander von Humboldt Foundation fellowship (to I.S.). J.L. was supported by Becas Chile.

Author information

J.L. generated and analyzed the medaka mutants. E.d.l.C.-M. carried out the 4C-seq experiments and the zebrafish transgenic assays with the help of S.N. and J.L. J.P. generated the mouse transgenic data. T.N. performed the µCT experiments. J.P.-A. carried out the lamprey in situ experiments. J.L.G.-S., J.R.M.-M., I.S. and N.H.S. conceived, designed and coordinated the project with the help of J.L. and I.M.  I.S., J.L.G.-S., J.R.M.-M., N.H.S., J.L. and I.M. wrote the manuscript.

Correspondence to Neil H. Shubin or Igor Schneider or Juan Ramón Martinez-Morales or José Luis Gómez-Skarmeta.

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The authors declare no competing interests.

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Supplementary Figure 1 Sequence conservation and enhancer activity of the ZRS from different species and the corresponding amphioxus Lmbr1 intron in zebrafish fins and mouse limbs.

a, the ZRS enhancer is located between Exon 5 and 6 of the Lmbr1 gene. b, vista plot showing the conservation degree of the ZRS enhancer in different species. c-f, transgenic mouse embryos showing the activity of the ZRS region from different species in the E10.5 developing limbs. Scale bars, 200μm. g-k, stable transgenic zebrafish larvae showing the activity of the ZRS region from different species at 72hpf pectoral fins. Arrows point to the ZPA domain. Scale bars, 50μm. For each construct shown in c-k, three or more independent transgenic lines were generated.

Supplementary Figure 2 Hh expression pattern in the dorsal fins of the lamprey ammocoete larva during metamorphosis.

a, b, first dorsal (a) and second dorsal and caudal fins (b) of an ammocoete larva of the lamprey L. reissneri. Scale bars, 5 mm. c-g, in situ hybridizations of L. reissneri first (c, d) and second (e-g) dorsal fins in sections at different anterior-posterior levels (as shown in a and b), assayed with an equimolecular mix of digoxigenin-labeled riboprobes of the lamprey L. camtschaticum HhA, HhB and HhD genes. No expression of Hh genes in the underlying mesenchyme of developing dorsal fins was detected. Scale bars, 100 μm. h-j, L. camtschaticum whole embryos at stage 27 hybridized with individual riboprobes of HhA (h), HhB (i) and HhD (j) used as positive controls for the Hh genes’ probes. HhD expression pattern has not been hitherto described (see Sugahara et al., 2016). Scale bars, 200μm. k, l, section in situ hybridizations of L. reissneri second dorsal fin, at the levels shown in b, with digoxigenin-labeled riboprobes of the L. camtschaticum MyHC1 (k) and ColA (l) genes (used as positive control). Expression in the skeletal muscle (MyHC1) and cartilage (ColA), was observed. Scale bars, 200 μm. Lamprey expression experiments were repeated two times independently with similar results.

Supplementary Figure 3 µCT scanning of adult pectoral fins in wild-type and ∆401 mutant fish.

a, b the architecture of fin rays and proximal radials was revealed after scanning for three-month-old wild type and medaka mutants. Note the reduced number of endoskeletal elements in the mutants. µCT scanning experiments were performed for three WT and five ∆401 ZRS adult fish. Scale bars, 500μm c, in contrast to the endoskeletal defects, measurement of fin ray length did not show any significant differences (p-value=0.122) between wild type (mean=0.185) and ∆948 mutant fish (mean=0.204). A t-test was used for analysis of pectoral fin length measurements. Each point in the graph represent pectoral fin length measurements from independent animals.

Supplementary Figure 4 Potential fin and limb enhancers in the introns of the zebrafish and human lmbr1 and LMBR1 genes.

Genome coordinates are shown in the x axis and reads counts in the y axis. Several potential fin/limb enhancers, as predicted by H3K27ac signature are found in different introns of the zebrafish and human lmbr1/LMBR1 genes. Although conserved with other mammals, these enhancers are not conserved in the mouse genome (red arrowheads). We search for putative ETS transcription factor binding sites in the sZRS of the three different species tested in the transgenesis assay. We found 3 ETS transcription-binding sites in the sZRS from zebrafish, 7 sites in medaka sZRS and 9 in human sZRS.

Supplementary Figure 5 Progression of pectoral fin development is truncated in ∆3.4-kb ZRS mutants.

a-c, temporal series showing normal development of pectoral fins in wild type embryos (black arrowheads). d-f, at 3dpf pectoral fin buds appears reduced (black arrowhead) in mutant embryos and are absent during later developmental stages (asterisks). We could not analyse in detail the mature histology of other fins in the double mutant (ZRS-sZRS) due to fish lethality before the onset of pelvic and anal fins maturation (from 3–4 weeks post fertilization on). Nevertheless, the lack of activity of the sZRS in the anal fin (Extended data Figure 7d) and our observations of mutant hatchlings indicate that the ZRS-sZRS deletion does not affect significantly the development of the anal fin. For each stage represented in a-f, five or more individuals were analyzed. Scale bars, 100μm.

Supplementary Figure 6 Detailed analyses of the dorsal fin phenotype in the ZRS ∆948 mutant.

Bone and cartilage staining showing the dorsal fin from wild type (a) or ∆948 (b-d) 4 month old fish. 74% (31/42) of the mutants show complete ablation (black arrow) of the dorsal fin (d) while in 19% (8/42) no fin rays are observed and endoskeletal elements are highly reduced (c). Only 7% (3/42) of the mutants analysed show fin rays although severely affected (b). Bone and cartilage staining protocol was performed in three independent experiments. Scale bars, 1mm.

Supplementary Figure 7 ZRS and sZRS enhancer expression and skeletal architecture for anal, dorsal and pelvic fins in wild type and ∆401 and ∆948 ZRS mutants.

a-c, whole mount ISH showing ZRS expression in the posterior region of the anal, dorsal (black arrows) and pelvic fin (white arrows) buds in zebrafish. d-f, whole mount ISH showing sZRS expression in pelvic fins (white arrows), but not in anal or dorsal zebrafish fin buds. ZRS and sZRS expression assessment in a-f was performed in three independent experiments with similar results. Scale bars, 100μm. g-o, Comparative analysis of skeletal elements, as revealed by alcian blue/alizarin red staining, for each of the fins in wild type and mutant adult fish. Ectopic elements are observed in pelvic mutant fins (black arrowheads). Bone staining was performed in three or more independent experiments for wildtype, ∆401 and ∆948 ZRS mutant animals with similar results. Scale bars, 1mm. p-q, Quantitative analysis of pelvic fins length illustrate the phenotypic defects observed in these appendages in the ∆948 mutants (WT mean= 0.088, ∆948 mean=0.041), p-value=1.433x10−5 (***) . In contrast, no significant defects in structure and length are observed for the anal fin of ∆948 mutants (WT mean=0.239, ∆948 mean=0.248, p-value=0.512). Each point in p and q graphs represents measurements from single adult fins. Differences in fin length in p and q were analysed using a t-test.

Supplementary information

Supplementary Figures

Supplementary Figures 1–7 and Supplementary Table 1

Life Sciences Reporting Summary

Supplementary Table 1

Oligonucleotides used for PCR amplification of the ZRS and sZRS enhancer from different species. Also listed are primers used for the 4C-seq analyses, genomic deletion target sites (PAM sequence in bold), genomic deletions screening and lamprey cloning.

Supplementary Video 1

ZRS-sZRS double mutants lack pectoral fins. Video showing a ∆3.4-kb larva (orange arrow) and its siblings freely swimming in a Petri dish recently after hatching (9 dpf).

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