Research ArticleGENETICS

Proximo-distal positional information encoded by an Fgf-regulated gradient of homeodomain transcription factors in the vertebrate limb

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Science Advances  03 Jun 2020:
Vol. 6, no. 23, eaaz0742
DOI: 10.1126/sciadv.aaz0742
  • Fig. 1 Elimination of Meis1 and Meis2 produces proximal skeletal element specification defects resulting in phocomelia.

    Recombination pattern of Dll1Cre (A to Ab) revealed by Rosa26RLacZ activation in whole-mount embryonic day 9.5 (E9.5) embryos (A), forelimbs (FL) (Aa), and hindlimbs (HL) (Ab). Black arrowheads point to the anterior border in the FLs and posterior in the HLs. (Ac) Schematic showing the recombination pattern of the Dll1Cre driver in limb bud precursors as they are recruited to the primordium. (B) Skeletal preparations of Meis mutants and WT embryos stained with Alcian Blue/Alizarin Red at E18.5 or Victorian Blue at E14.5. At E18.5, M1M2DKO FLs showed minor phenotypical defects (n = 3/3), while HLs showed severe phocomelia, with rudimentary skeletal elements in all limb segments except the autopod, which was completely normal (n = 3/3 at birth + n = 3/5 at E14.5). In Dll1Cre;Meis1+/f;Meis2f/f (M1HT;M2KO) and Dll1Cre;Meis1f/f;Meis2+/f (not shown) fetuses, FLs showed minor alterations, while HLs display smaller pelvis and severe specific stylopod reduction (n = 7/7). An extra anterior digit is observed in one specimen (n = 1/7). At E14.5, a proportion of FLs in M1M2DKO fetuses showed strong reductions or absence of all skeletal except the autopod (n = 3/5). (C) Sox9 mRNA whole-mount in situ hybridization in E11.5 WT and M1M2DKO HL buds, showing alterations of the chondrogenic precursor pattern in the presumptive stylopod and zeugopod (n = 2/2). Black arrowheads point to the proximal-most appendicular pre-condensations and to the prospective zeugopod-autopod boundary.

  • Fig. 2 Meis protein forms a PD gradient that changes dynamically in correlation with the progressive distalization of the limb bud.

    (A) Meis immunofluorescence in a WT FL bud from a 32-somite (so) embryo (~E10.5) and (Aa) its quantification represented in three dimensions (3D). (B and Ba) As a quantification control, the same analysis is shown in a HoxB6CreERT;M1KO;M2KO FL bud, in which only the posterior region lacks Meis proteins according to the reported HoxB6CreER recombination pattern after tamoxifen induction at E8.5 (27). (C to Fa) Temporal sequence of Meis distribution and corresponding 3D quantification plots showing individual representative examples of 20-21so to 34so stages (~E9.5 to E10.5). (Cb to Fb) PD cross sections of Meis abundance along the indicated rectangles in (Ca) to (Fa), with exclusion of the ectodermal signal. The rectangles align with the line of maximal Meis variation along the limb bud PD axis (see Materials and Methods). (Cc to Fc) Graphical representation of Meis gradient quantification at each stage. Black lines represent the individual replicates (see Materials and Methods and fig. S4 for a detailed explanation), and blue lines represent the loess curve fitted from the replicates. The gray shadow represents the 95% confidence interval of the loess curve. A, anterior; Po, posterior; P, proximal; D, distal; Do, dorsal; V, ventral. Scale bars, 50 μm (C) and 100 μm (D to F).

  • Fig. 3 Meis expression does not adjust to limb PD segmental borders.

    (A) Lineage tracing of Meis1CreERT2-labeled cells by tamoxifen (TM) injection at different stages (TM.E8.5, n = 11; TM.E10.5, n = 3). (B) Lineage tracing of Hoxa13CreERT2-labeled cells by tamoxifen injection at different stages (TM.E9.5, n = 7; TM.E10.5, n = 4; TM.E11, n = 4). (C and D) Schemes showing the boundaries of the regions colonized by Meis-expressing cells (C) and Hoxa13-expressing cells (D) at different labeling time points. Besides minor leakiness observed following injections at E9.5 (C), the lineage of Hoxa13-expressing cells respects the zeugopod-autopod boundary.

  • Fig. 4 Correlation between the Meis gradient and Hoxa11 and Hoxa13 activation schedule in FLs.

    (A to Ba) Meis detection by immunofluorescence and (Ab and Bb) 3D representation of Meis level quantification in representative specimens at the stages in which Hoxa11 (so23) or Hoxa13 (so32) expression is first detected. (Ac and Bc) Graphical representation of Meis gradient quantification at so23 and so32. (C to F) Hoxa11 and Hoxa13 expression by whole-mount mRNA in situ hybridization at so23 and so32. At so23, when Hoxa11 mRNA is first detected (C), the Meis gradient is incipiently detected along the limb bud PD axis (A to Ac). At 32 somites, when Hoxa13 mRNA is first detected (F) and Hoxa11 has expanded proximally (D), the Meis gradient is mature (B to Bc), and Meis levels indistinguishable from background are detected in the distal limb bud (B to Ba). In (Ac) and (Bc), black lines represent the individual replicates (see Materials and Methods and fig. S4 for a detailed explanation), and blue lines represent the loess curve fitted from the replicates. The gray shadow represents the 95% confidence interval of the loess curve. Scale bars, 100 μm (A and Ba), 50 μm (B), and 20 μm (Aa).

  • Fig. 5 Meis gradient controls the activation timing and positioning of HoxA expression domains along the limb bud PD axis.

    (A) Hoxa11 mRNA expression detected by whole-mount in situ hybridization in FLs (E10.5) and HLs (E10.5 and E11.5) of WT and M1M2DKO embryos. Black lines indicate the proximal border of Hoxa11 expression, and red lines indicate the point of limb insertion in the flank. Note the proximalization of the expression domain in the anterior limb bud (n = 3/4). (B) Hoxa13 expression in FLs (E10.5) and HLs (E10.5 and E11.5) of WT and M1M2DKO embryos reveals premature Hoxa13 activation and expression domain expansion in mutant limbs (n = 4/5). At E11.5, Hoxa13 proximal expression border (black lines) in mutant HLs is closer to the flank (red lines) than in controls. White lines on FL specimens indicate the maximal PD extension of the expression domains. Black arrowheads on E10.5 HLs mark the absence of expression in controls and the robust activation in mutants. (C) Chromatin immunoprecipitation sequencing (ChIPseq) profile of Meis binding to the HoxA complex in FL and HL buds at E10.5. Dark blue arrowheads, shared peaks between FL and HL; light blue arrowheads, peaks specific for either FL or HL. A detailed view of the HoxA cluster region for the FL together with ChIPseq profiles for chromatin marks (32) shows that there is no correlation between enhancer/promoter chromatin marks and Meis binding sites.

  • Fig. 6 Transcriptional regulation of Meis gradient formation.

    Detection of Meis protein distribution by immunofluorescence (A) and Meis1 mRNA by in situ hybridization (B) in adjacent sections of FL buds. (Aa and Ba) 3D representation of the signals detected in (A) and (B), respectively. (Ab and Bb) PD cross sections of Meis abundance along the indicated rectangles in (Aa) and (Ba). The rectangles align with the line of maximal Meis variation along the limb bud PD axis (see Materials and Methods). Detection of Meis protein (C) and CFP (D) detection by immunofluorescence in limb buds of mice heterozygous for a Meis1 knock-in allele in which the CFP reporter is inserted at the endogenous Meis ATG. Limb cells express CFP from this allele and endogenous Meis from the unaltered allele. (Ca and Da) 3D representation of the signals detected in (C) and (D), respectively. (Cb to Db) PD cross sections of Meis abundance along the indicated rectangles in (Ca) and (Da), with exclusion of the ectodermal signal. The rectangles align with the line of maximal Meis variation along the limb bud PD axis (see Materials and Methods). (E to G) Graphical representation of Meis protein, Meis mRNA, and CFP quantifications. Black lines represent the individual replicates (see Materials and Methods and fig. S4 for a detailed explanation), and colored lines represent the loess curve fitted from the replicates. The gray shadow represents the 95% confidence interval of the loess curve. (E and F) Graphs showing the quantification of MEIS protein and Meis mRNA, respectively, in adjacent sections. (G) Graph showing the quantification of Meis protein and CFP in adjacent sections.

  • Fig. 7 Regulation of Meis gradient formation by AER-FGFs.

    (A to Da) Meis distribution and corresponding 3D quantification plots showing individual representative examples of WT limbs and limbs with different combinations of AER-FGF mutant alleles. (Ab to Db) PD cross sections of Meis abundance along the indicated rectangles in (Aa) to (Da), with exclusion of the ectodermal signal. The rectangles align with the line of maximal Meis variation along the limb bud PD axis (see Materials and Methods). While the Meis PD gradient is detectable in controls (A to Ab and C to Cb), the gradient is swallower or completely undetectable in different combinations of AER-FGF mutants (B to Bb and D to Db) (n = 5/5). (E) Graphical representation of Meis expression profile against size in individual examples of WT limbs and limbs with different combinations of AER-FGF mutant alleles. While mutant limbs show no signs of Meis gradient formation, WT limbs of a similar or smaller size show a detectable PD Meis gradient. F4, FGF4; F8, FGF8; F9, FGF9. Scale bars, 100 μm.

Supplementary Materials

  • Supplementary Materials

    Proximo-distal positional information encoded by an Fgf-regulated gradient of homeodomain transcription factors in the vertebrate limb

    Irene Delgado, Alejandra C. López-Delgado, Alberto Roselló-Díez, Giovanna Giovinazzo, Vanessa Cadenas, Laura Fernández-de-Manuel, Fátima Sánchez-Cabo, Matthew J. Anderson, Mark Lewandoski, Miguel Torres

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