Evolution of Hoxa11 regulation in vertebrates is linked to the pentadactyl state

Journal name:
Nature
Volume:
539,
Pages:
89–92
Date published:
DOI:
doi:10.1038/nature19813
Received
Accepted
Published online

The fin-to-limb transition represents one of the major vertebrate morphological innovations associated with the transition from aquatic to terrestrial life and is an attractive model for gaining insights into the mechanisms of morphological diversity between species1. One of the characteristic features of limbs is the presence of digits at their extremities. Although most tetrapods have limbs with five digits (pentadactyl limbs), palaeontological data indicate that digits emerged in lobed fins of early tetrapods, which were polydactylous2. How the transition to pentadactyl limbs occurred remains unclear. Here we show that the mutually exclusive expression of the mouse genes Hoxa11 and Hoxa13, which were previously proposed to be involved in the origin of the tetrapod limb1, 2, 3, 4, 5, 6, is required for the pentadactyl state. We further demonstrate that the exclusion of Hoxa11 from the Hoxa13 domain relies on an enhancer that drives antisense transcription at the Hoxa11 locus after activation by HOXA13 and HOXD13. Finally, we show that the enhancer that drives antisense transcription of the mouse Hoxa11 gene is absent in zebrafish, which, together with the largely overlapping expression of hoxa11 and hoxa13 genes reported in fish3, 4, 5, 6, 7, suggests that this enhancer emerged in the course of the fin-to-limb transition. On the basis of the polydactyly that we observed after expression of Hoxa11 in distal limbs, we propose that the evolution of Hoxa11 regulation contributed to the transition from polydactyl limbs in stem-group tetrapods to pentadactyl limbs in extant tetrapods.

At a glance

Figures

  1. The proximal restriction of Hoxa11 is linked to antisense transcription at the Hoxa11 locus.
    Figure 1: The proximal restriction of Hoxa11 is linked to antisense transcription at the Hoxa11 locus.

    ac, Expression of Hoxa10 (a), Hoxa11 (b) and Hoxa13 (c) in wild-type limb bud from embryonic day (E) 11.5 mouse. d, Replacement of the Hoxa11 gene with the PGK-neomycin cassette (Hoxa11Neo; scheme to the left), results in neomycin expression both in the proximal and distal domains. e, f, Expression of all antisense transcripts (e) and antisense transcripts overlapping with Hoxa11 exon 1 (f) in E11.5 wild-type limb. Schemes of the antisense transcripts and the probes used (blue boxes) are on the left. Note that the antisense transcripts overlapping with Hoxa11 exon 1 (Hoxa11as-b) are distally restricted (f), reminiscent of Hoxa13 expression (c) and mutually exclusive with the Hoxa11 pattern (b). g, Deletion of the antisense transcript start sites in Hoxa11 exon 1, via replacement of most of exon 1 with the eGFP coding sequence (Hoxa11eGFP; scheme to the left) and expression of gfp under the control of the Hoxa11 promoter (right). Original magnification, ×31.5 (for all images).

  2. Deletion of the distal enhancer in Hoxa11 intron results in impaired antisense transcription and gain of sense transcription in distal cells.
    Figure 2: Deletion of the distal enhancer in Hoxa11 intron results in impaired antisense transcription and gain of sense transcription in distal cells.

    a, Left, scheme of the Tg(m-Inta11-LacZ) transgene carrying the predicted distal enhancer (Int, red box). Right, X-gal staining of E12.5 transgenic embryos (n = 5). be, Expression of Hoxa11as-b (b, c) and Hoxa11 (d, e) in wild-type (WT; b, d) and Hoxa11ΔInt/ΔInt (c, e) mouse limbs at E12.5. Note that based on the observed gain of Hoxa11 expression, other regulatory input(s) could be implicated in Hoxa11 regulation in distal cells. Pr, minimal promoter. Original magnification, ×31.5 (for all images).

  3. Hox13 inactivation disrupts Hoxa11 antisense transcription in distal cells and distal Hoxa11 expression results in the formation of supernumerary digits.
    Figure 3: Hox13 inactivation disrupts Hoxa11 antisense transcription in distal cells and distal Hoxa11 expression results in the formation of supernumerary digits.

    af, Hoxa11as (probe A) (a, c, e) and Hoxa11 (b, d, f) expression in E11.5 limb buds from wild-type (a, b), Hoxa13−/− Hoxd13+/ (c, d) and Hoxa13−/− Hoxd13−/− (e, f) mouse embryos. Arrows in c and d show the group of cells still expressing Hoxa11as in Hoxa13−/−Hoxd13+/− limbs (c), which corresponds to distal cells in which Hoxa11 expression is not gained (d). g, h, Skeleton of RosaHoxa11/Hoxa11 (g) and Prx1Cre; RosaHoxa11/Hoxa11 (h) distal forelimb at postnatal day 0 (P0). Anterior is up. Original magnification, ×31.5 (af) and ×20 (g, h).

  4. The mouse Hoxa11 antisense enhancer is functional in distal fins.
    Figure 4: The mouse Hoxa11 antisense enhancer is functional in distal fins.

    a, mVISTA sequence conservation plot of the mouse Hoxa11 intron (red) with tetrapod (rat, human, chicken and frog) and fish representatives (coelacanth and zebrafish). Ex1, exon 1; Ex2, exon 2. Note that zebrafish has two hoxa11 genes expressed in developing fins, hoxa11a and hoxa11b. b, c, GFP expression in fin buds of Tg(m-Inta11-eGFP) transgenic zebrafish embryos at 60 hpf (b) and 72 hpf (c), revealing the enhancer activity of the mouse Hoxa11 intron in fish. Note the filopodia-like protrusions in GFP+ mesenchymal cells suggestive of a migration towards the fin fold. d, e, hoxa13a expression in developing fins at 60 hpf (d) and 72 hpf (e). Original magnification, ×400.

  5. Absence of antisense transcription 3′ to the Hoxa11 promoter in the Hoxa11eGFP/eGFP limb and evidence that Hoxa11as-b transcripts produced in trans have no effect on Hoxa11 expression.
    Extended Data Fig. 1: Absence of antisense transcription 3′ to the Hoxa11 promoter in the Hoxa11eGFP/eGFP limb and evidence that Hoxa11as-b transcripts produced in trans have no effect on Hoxa11 expression.

    a, b, Detection of Hoxa11as-b transcripts in wild-type limb buds at E12.5 (a), and whole-mount in situ hybridization to detect gfp antisense transcripts in Hoxa11eGFP/eGFP limb buds at E12.5 (b). ce, Hoxa11 expression in wild-type limb buds (c), and Hoxa11as-b (d) and Hoxa11 (e) expression in Prx1-Hoxa11as limb buds. Original magnification, ×31.5.

  6. Deletion of the distal enhancer in Hoxa11 intron using CRISPR-Cas9.
    Extended Data Fig. 2: Deletion of the distal enhancer in Hoxa11 intron using CRISPR-Cas9.

    a, Scheme of the wild-type and targeted (Hoxa11ΔInt) loci. Sites targeted by the single-guide RNAs (sgRNA_1 and sgRNA_2) for the CRISPR-Cas9-mediated deletion of the distal enhancer. The blue rectangles indicate the position of the DNA probe used to confirm the deletion by Southern blot in b. b, Lane 1 shows the 6-kb KpnI band resulting from the CRISPR-Cas9-mediated deletion. Lane 2 was loaded with wild-type DNA. c, PCR reaction using a forward primer located upstream of sgRNA_1 and a reverse primer located downstream sgRNA_2 shows the presence of a 300 bp (ΔInt 300 bp) fragment expected for the Hoxa11ΔInt allele. d, The sequence of the 300-bp PCR fragment confirms the CRISPR-Cas9-mediated deletion of the Hoxa11 intronic region containing the distal enhancer (only the sequence encompassing the deletion breakpoints is shown).

  7. The distal enhancer located in the Hoxa11 intron is bound by HOXA13 and HOXD13 in distal limb cells and its activity is increased by HOXA13 in 293T cells.
    Extended Data Fig. 3: The distal enhancer located in the Hoxa11 intron is bound by HOXA13 and HOXD13 in distal limb cells and its activity is increased by HOXA13 in 293T cells.

    a, Integrative genomics viewer (IGV) screenshot showing HOXA13 and HOXD13 ChIP–seq data at the Hoxa11 locus. These ChIP–seq data were obtained using chromatin from distal forelimb buds of wild-type E11.5 mouse embryos (R. Sheth et al., manuscript submitted). b, Transfection assay shows HOXA13 dependent activation of Hoxa11 intron driving reporter gene expression. Two-tailed Tukey’s multiple comparisons test was performed. Error bars indicate s.d (n = 3). RQ, relative quantification.

  8. Individual inactivation of Hoxa13 or Hoxd13 is not sufficient to fully abrogate antisense transcription in distal limbs.
    Extended Data Fig. 4: Individual inactivation of Hoxa13 or Hoxd13 is not sufficient to fully abrogate antisense transcription in distal limbs.

    a, b, Whole-mount in situ hybridization, using probe A (see Fig. 1) to detect all antisense transcripts, on Hoxd13−/− (a) and Hoxa13−/− (b) mouse limb buds at E11.5. Antisense transcription in distal limbs remains robust in both mutants but a clear reduction is seen in the distal Hoxa13−/− limbs. Original magnification, ×31.5.

  9. Inactivation of both Hoxa13 and Hoxd13 disrupts antisense transcription overlapping with the Hoxa11 exon 1.
    Extended Data Fig. 5: Inactivation of both Hoxa13 and Hoxd13 disrupts antisense transcription overlapping with the Hoxa11 exon 1.

    ad, Hoxa11as-b expression (probe B in Fig. 1) in limb buds (a, b) and tail buds (c, d) from wild-type (a, c) and Hoxa13−/− Hoxd13−/− (b, d) E12.5 mouse embryos. Whole-mount in situ hybridization shows that Hoxa11as-b expression in tail buds (internal control) is similar in both the wild-type (c) and double-mutant (d) embryos, whereas there is almost no expression remaining in Hoxa13−/− Hoxd13−/− limb buds (b). Original magnification, ×31.5.

  10. Generation of the RosaHoxa11 knock-in mouse line.
    Extended Data Fig. 6: Generation of the RosaHoxa11 knock-in mouse line.

    a, Targeting of the endogenous Rosa26 locus (top three lines). The wild-type Rosa26 locus is shown below (middle). Regions used as homologous arms for the recombination in ES cells are indicated by brown rectangles labelled 5′ and 3′, respectively. Scheme of the targeted locus after homologous recombination in ES cells and after Cre-mediated recombination is shown at the bottom. The position of the internal (IP) and external (EP) probes and restriction sites used for Southern blot analysis are indicated on both the wild-type and targeted locus. b, c, Southern blots of ES cells clones using the internal probe (b) and external probe (c) to detect the targeted allele (lane 1). d, Southern blot of wild-type (lane 2) and heterozygous (lane 1) mice. A, AscI; E, EcoRV; P, PacI; S, SwaI.

  11. The conditional gain of Hoxa11 using the Hoxa13Cre allele results in the formation of supernumerary digits.
    Extended Data Fig. 7: The conditional gain of Hoxa11 using the Hoxa13Cre allele results in the formation of supernumerary digits.

    a, b, Autopod of RosaHoxa11/+ (a) and RosaHoxa11 Hoxa13Cre (b) at E15.5. Anterior is up. The Rosa26 locus and Hoxa13Cre allele being on the same chromosome (Chr6), the gain-of-function phenotype was assessed with only one copy of the RosaHoxa11 allele. ce, Autopod skeletons of Prx1Cre; RosaHoxa11/Hoxa11 mice at P0 from four distinct mutants (anterior is up). The number of digits varies from 6 to 7, with often a small post-axial extra-digit (posterior). The extra-digit phenotype is fully penetrant upon Cre-activation of two copies of the RosaHoxa11 allele (n = 10). Original magnification, ×20.d, Quantification of Hoxa11 expression level by quantitative reverse transcriptase PCR (RT–qPCR) on RNA extracted from E11.5 forelimb, relative to both Gapdh and Tbp mRNA of Prx1Cre; RosaHoxa11/Hoxa11 embryos. Two-tailed t-test was performed. Error bars indicate s.d (n = 4).

  12. hoxa11 and hoxa13 are expressed in overlapping domains in zebrafish fins.
    Extended Data Fig. 8: hoxa11 and hoxa13 are expressed in overlapping domains in zebrafish fins.

    ad, Expression of hoxa13b (a, b) and hoxa11b (c, d) in zebrafish fins at 60 hpf (a, c) and 72 hpf (b, d). Dotted lines indicate the boundary between the endochondral disc and the fin fold. Original magnification, ×400.

  13. Absence of antisense transcription at the hoxa11a and hoxa11b loci in zebrafish fins.
    Extended Data Fig. 9: Absence of antisense transcription at the hoxa11a and hoxa11b loci in zebrafish fins.

    a, b, Whole-mount in situ hybridization with probes designed to detect putative antisense transcription at hoxa11a (a) and hoxa11b (b). cf, No antisense transcription is detected, whereas expression of hoxa11a (c), hoxa11b (d), hoxa13a (e) and hoxa13b (f) is observed in zebrafish fins at the same stage. Asterisks correspond to the staining from the fin on the other side of the embryo. Original magnification, ×63.

Tables

  1. Summary of transient transgenic embryos analysed
    Extended Data Table 1: Summary of transient transgenic embryos analysed

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Author information

  1. Present address: Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada.

    • Gemma de Martino

Affiliations

  1. Genetics and Development Research Unit, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada

    • Yacine Kherdjemil,
    • Rushikesh Sheth,
    • Annie Dumouchel,
    • Gemma de Martino &
    • Marie Kmita
  2. Département de Médecine (Programme de Biologie Moléculaire), Université de Montréal, Montréal, Québec H3T 1J4, Canada

    • Yacine Kherdjemil &
    • Marie Kmita
  3. Department of Biology and CAREG, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada

    • Robert L. Lalonde &
    • Marie-Andrée Akimenko
  4. Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan, Ann Arbor, Michigan 48109-2200, USA

    • Kyriel M. Pineault &
    • Deneen M. Wellik
  5. Shriners Hospital for Children, Portland, Oregon 97239, USA

    • H. Scott Stadler
  6. Department of Experimental Medicine, McGill University, Montreal, Quebec H3A 1A3, Canada

    • Marie Kmita

Contributions

Y.K. and M.K. conceived the study and analysed the data. Y.K. designed and conducted all mouse experiments with the help of R.S. for the generation of the mouse lines. All fish experiments were performed by R.L.L. under the supervision of M.-A.A. R.S. performed the ChIP–seq experiments. A.D. provided technical help for the mouse experiments. G.M. performed preliminary experiments related to Figs 2a and 3c, e. D.M.W. and K.M.P provided Hoxa11eGFP/eGFP embryos. H.S.S. provided the HOXA13 and HOXD13 antibodies. M.K. wrote the paper. All authors commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks R. Freitas, J. L. Gomez-Skarmeta and S. Mackem 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: Absence of antisense transcription 3′ to the Hoxa11 promoter in the Hoxa11eGFP/eGFP limb and evidence that Hoxa11as-b transcripts produced in trans have no effect on Hoxa11 expression. (168 KB)

    a, b, Detection of Hoxa11as-b transcripts in wild-type limb buds at E12.5 (a), and whole-mount in situ hybridization to detect gfp antisense transcripts in Hoxa11eGFP/eGFP limb buds at E12.5 (b). ce, Hoxa11 expression in wild-type limb buds (c), and Hoxa11as-b (d) and Hoxa11 (e) expression in Prx1-Hoxa11as limb buds. Original magnification, ×31.5.

  2. Extended Data Figure 2: Deletion of the distal enhancer in Hoxa11 intron using CRISPR-Cas9. (150 KB)

    a, Scheme of the wild-type and targeted (Hoxa11ΔInt) loci. Sites targeted by the single-guide RNAs (sgRNA_1 and sgRNA_2) for the CRISPR-Cas9-mediated deletion of the distal enhancer. The blue rectangles indicate the position of the DNA probe used to confirm the deletion by Southern blot in b. b, Lane 1 shows the 6-kb KpnI band resulting from the CRISPR-Cas9-mediated deletion. Lane 2 was loaded with wild-type DNA. c, PCR reaction using a forward primer located upstream of sgRNA_1 and a reverse primer located downstream sgRNA_2 shows the presence of a 300 bp (ΔInt 300 bp) fragment expected for the Hoxa11ΔInt allele. d, The sequence of the 300-bp PCR fragment confirms the CRISPR-Cas9-mediated deletion of the Hoxa11 intronic region containing the distal enhancer (only the sequence encompassing the deletion breakpoints is shown).

  3. Extended Data Figure 3: The distal enhancer located in the Hoxa11 intron is bound by HOXA13 and HOXD13 in distal limb cells and its activity is increased by HOXA13 in 293T cells. (147 KB)

    a, Integrative genomics viewer (IGV) screenshot showing HOXA13 and HOXD13 ChIP–seq data at the Hoxa11 locus. These ChIP–seq data were obtained using chromatin from distal forelimb buds of wild-type E11.5 mouse embryos (R. Sheth et al., manuscript submitted). b, Transfection assay shows HOXA13 dependent activation of Hoxa11 intron driving reporter gene expression. Two-tailed Tukey’s multiple comparisons test was performed. Error bars indicate s.d (n = 3). RQ, relative quantification.

  4. Extended Data Figure 4: Individual inactivation of Hoxa13 or Hoxd13 is not sufficient to fully abrogate antisense transcription in distal limbs. (327 KB)

    a, b, Whole-mount in situ hybridization, using probe A (see Fig. 1) to detect all antisense transcripts, on Hoxd13−/− (a) and Hoxa13−/− (b) mouse limb buds at E11.5. Antisense transcription in distal limbs remains robust in both mutants but a clear reduction is seen in the distal Hoxa13−/− limbs. Original magnification, ×31.5.

  5. Extended Data Figure 5: Inactivation of both Hoxa13 and Hoxd13 disrupts antisense transcription overlapping with the Hoxa11 exon 1. (765 KB)

    ad, Hoxa11as-b expression (probe B in Fig. 1) in limb buds (a, b) and tail buds (c, d) from wild-type (a, c) and Hoxa13−/− Hoxd13−/− (b, d) E12.5 mouse embryos. Whole-mount in situ hybridization shows that Hoxa11as-b expression in tail buds (internal control) is similar in both the wild-type (c) and double-mutant (d) embryos, whereas there is almost no expression remaining in Hoxa13−/− Hoxd13−/− limb buds (b). Original magnification, ×31.5.

  6. Extended Data Figure 6: Generation of the RosaHoxa11 knock-in mouse line. (126 KB)

    a, Targeting of the endogenous Rosa26 locus (top three lines). The wild-type Rosa26 locus is shown below (middle). Regions used as homologous arms for the recombination in ES cells are indicated by brown rectangles labelled 5′ and 3′, respectively. Scheme of the targeted locus after homologous recombination in ES cells and after Cre-mediated recombination is shown at the bottom. The position of the internal (IP) and external (EP) probes and restriction sites used for Southern blot analysis are indicated on both the wild-type and targeted locus. b, c, Southern blots of ES cells clones using the internal probe (b) and external probe (c) to detect the targeted allele (lane 1). d, Southern blot of wild-type (lane 2) and heterozygous (lane 1) mice. A, AscI; E, EcoRV; P, PacI; S, SwaI.

  7. Extended Data Figure 7: The conditional gain of Hoxa11 using the Hoxa13Cre allele results in the formation of supernumerary digits. (1,211 KB)

    a, b, Autopod of RosaHoxa11/+ (a) and RosaHoxa11 Hoxa13Cre (b) at E15.5. Anterior is up. The Rosa26 locus and Hoxa13Cre allele being on the same chromosome (Chr6), the gain-of-function phenotype was assessed with only one copy of the RosaHoxa11 allele. ce, Autopod skeletons of Prx1Cre; RosaHoxa11/Hoxa11 mice at P0 from four distinct mutants (anterior is up). The number of digits varies from 6 to 7, with often a small post-axial extra-digit (posterior). The extra-digit phenotype is fully penetrant upon Cre-activation of two copies of the RosaHoxa11 allele (n = 10). Original magnification, ×20.d, Quantification of Hoxa11 expression level by quantitative reverse transcriptase PCR (RT–qPCR) on RNA extracted from E11.5 forelimb, relative to both Gapdh and Tbp mRNA of Prx1Cre; RosaHoxa11/Hoxa11 embryos. Two-tailed t-test was performed. Error bars indicate s.d (n = 4).

  8. Extended Data Figure 8: hoxa11 and hoxa13 are expressed in overlapping domains in zebrafish fins. (774 KB)

    ad, Expression of hoxa13b (a, b) and hoxa11b (c, d) in zebrafish fins at 60 hpf (a, c) and 72 hpf (b, d). Dotted lines indicate the boundary between the endochondral disc and the fin fold. Original magnification, ×400.

  9. Extended Data Figure 9: Absence of antisense transcription at the hoxa11a and hoxa11b loci in zebrafish fins. (686 KB)

    a, b, Whole-mount in situ hybridization with probes designed to detect putative antisense transcription at hoxa11a (a) and hoxa11b (b). cf, No antisense transcription is detected, whereas expression of hoxa11a (c), hoxa11b (d), hoxa13a (e) and hoxa13b (f) is observed in zebrafish fins at the same stage. Asterisks correspond to the staining from the fin on the other side of the embryo. Original magnification, ×63.

Extended Data Tables

  1. Extended Data Table 1: Summary of transient transgenic embryos analysed (177 KB)

Additional data