Control of mouse limb initiation and antero-posterior patterning by Meis transcription factors

Abstract

Meis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis.

Fig. 1. Deletion of Meis genes in the limb-forming region.

a Tamoxifen injection and experimental timeline. b Whole-mount X-gal staining in E9.5 HoxB6CreER; R26RlacZ embryos (N = 6 embryos). Scale bar = 100 μm. c, d Meis immunofluorescence at E10.5 in wild-type and M1KO;M2KO forelimbs (FL) and hindlimbs (HL) (N = 4 limbs). Scale bar = 100 μm. e M1KO;M2KO limb phenotype (N = 9 embryos). Scale bar = 500μm. Arrowheads in b and d indicate the limits of Cre recombination. In e, the dashed line delineates the FL soft tissues and the arrowhead indicates the HL region. Source images are available at Mendeley Data [https://data.mendeley.com/datasets/r774bxyf8d/2].

Fig. 2. Skeletal defects in limb buds lacking three Meis1/2 alleles.

a–f Victoria blue cartilage staining of HLs at E14.5 in WT embryos, with homozygous deletion of Meis2 (M2KO), and M2KO embryos compound with a Meis1 knockout allele in heterozygosity (M1HT;M2KO). Scale bar = 1000 μm. g Percentage size change in skeletal elements of M2KO and M1HT;M2KO HLs. Limbs analyzed: WT N = 6; M2KO N = 4 and M1HM2KO N = 10. ns, non-significant; *p < 0.05, **p < 0.01, ***p < 0.001. Two-tailed, unpaired Mann–Whitney test. Red bars are mean values. Exact p-values from left to right: 0.7619; 0.0420; 0.4121; 0.0001; 0.6485; 0.9623; 0.3152; 0.3148. h Percentage occurrence of skeletal alterations in M1HT;M2KO HLs. Arrowheads in f indicate the absence of fibula and posterior digits in M1HT;M2KO specimens. N = 10 embryos. Source data are provided as a Source Data file. Source images are available at Mendeley Data [https://data.mendeley.com/datasets/r774bxyf8d/2].

Fig. 3. Molecular analysis of Meis function in limb buds.

a RNA-seq analysis comparing wild-type and M1KO;M2KO HLs, showing the clustering of WT and mutant samples by expression profile. b Volcano plot and heat map with the most relevant genes involved in AP patterning, PD patterning, Wnt and Fgf pathways highlighted in color code. p-values are obtained by a Benjamini–Hochberg procedure, which adjusts for multiple comparisons. The underlined highlighted genes show Meis-binding sites in their regulatory regions by ChIP-seq. c–f ChIP-seq analysis, showing the number of binding sites identified in E10.5 FLs and HLs (c), the distribution of Meis peaks with respect to transcriptional units (d) and de novo (e), and known (f) sequence motif identification. FC: fold change. See the “Data availability” section for source data.

Fig. 4. Expression of factors required for limb initiation in Meis mutants.

Whole-mount in situ hybridization analysis of the expression of Fgf8 (a–d), Fgf10 (e–h), and Lef1 (i–l) at E10.5 in WT and M1KO;M2KO limbs. For Fgf8, N = 6 FLs and N = 10 HLs. For Fgf10, N = 10 FLs and N = 6 HLs. For Lef1, N = 4 limbs for both, FLs and HLs. m, n Tbx5 expression in E9.5 FLs. o, p Tbx4 expression in E10.5 HLs. N = 13 limbs for Tbx5 and N = 8 limbs for Tbx4. Scale bars = 100μm. Arrowheads in a–d indicate the region of Fgf8 expression in control limb buds and its absence in mutants. Arrowheads in i, j indicate the expression of Lef1 in control limb buds and its absence on the posterior part of mutant limb buds. Source images are available at Mendeley Data [https://data.mendeley.com/datasets/r774bxyf8d/2].

Fig. 5. Meis, Tbx, and Hox transcription factors co-bind regulatory sequences of genes involved in limb initiation.

a Overlap (%) between ChIP-seq-binding sites for Tbx5, Hoxc10, and Meis. The size of the circles representing each class is proportional to the number of binding sites for each factor (Meis: 6811; Tbx5: 10273; Hoxc10: 1611). b Meis–Tbx5 summit distance distribution at different resolutions. c Binding sites shared by Meis, Tbx5, and Hoxc10 in the Fgf10 and Lef1 loci. See the “Data availability” section for source data.

Fig. 6. Regulatory elements bound by Meis, Tbx, and Hox factors control Fgf10 expression at limb initiation.

a Fgf10 expression at E9.5 and E10.5 in embryos with the CRISPR/Cas9-mediated-targeted Meis deletions indicated in c. Solid arrowheads indicate Fgf10 expression domain in FLs. Asterisks indicate non-limb Fgf10 expression domains. Empty arrowhead indicates the specific absence of Fgf10 expression in the anterior limb bud. Limbs analyzed: N = 12 for 5′−0.4∆, N = 10 for 3′−0.4∆, and N = 8 for 14.5∆. Scale bars = 100 μm in FLs and 1000 μm in whole embryos. b Quantitative RT-PCR showing a reduction of Fgf10 transcripts in E9.5 FL buds of embryos with the indicated regulatory region deletions. Limbs analyzed: For 5′−0.4∆, N = 7; For 3′−0.4∆, N = 8. For 14.5∆, N = 6. Two-tailed, one-sample t-test. Exact p-values from left to right: 0.0037; 0.0013; 0.0081 respectively. Red bars represent median values. c Examples of limb skeletons of genetic combinations between Meis mutants and Fgf10 regulatory region deletions. Scale bar = 1000 μm. d Comparison of pelvis and femur length in genetic combinations of Meis mutants and Fgf10 regulatory region deletions. Two-tailed, unpaired Mann–Whitney test. Limbs analyzed: M1HTM2KO N = 10; M1HTM2KO;5′−0.4∆, N = 6; M1HTM2KO;14.5∆, N = 4. Exact p-values from left to right: 0.3132; 0.0759; 0.0019; 0.0020. Red bars represent mean values. e Examples of limb skeletons of genetic combinations between Meis mutants and Fgf10 regulatory region deletions, showing the limb AP axis. Scale bar = 1000 μm. f Scoring of skeletal element loss in the genetic combinations of Meis mutants and Fgf10 regulatory region deletions shown in c. Loss of each skeletal element (fibula or digit) scores 1. Embryos analyzed: M1HTM2KO N = 10; M1HTM2KO;5′−0.4∆, N = 6; M1HTM2KO;14.5∆, N = 4; 14.5∆ N = 12; 5′−0.4∆, N = 8. Two-tailed, unpaired Mann–Whitney test. Exact p-values from top to down and left to right: 0.0357; 0.0245; 0.0360; 0.0005. Red bars represent mean values. ns, non-significant; *p < 0.05, **p < 0.01, ***p < 0.001. Statistics were not adjusted for multiple comparisons. Source data are provided as a Source Data file. Source images are available at Mendeley Data [https://data.mendeley.com/datasets/r774bxyf8d/2].

Fig. 7. Meis function in limb-bud AP prepatterning.

a Whole-mount in situ hybridization analysis of Shh and its targets Cdon and Patched1 (Ptch1) in WT and M1HT;M2KO HLs at E10. Limbs analyzed: N = 4, N = 4, and N = 2, respectively. Whole-mount in situ hybridization analysis of the expression of Hand2 (b) (N = 6 FLs and N = 6 HLs), Gli3 (c) (N = 2 FLs and N = 2 HLs), Hoxd13 (d) (N = 8 FLs and N = 8 HLs) and Hoxd9 (e, upper panels) (N = 4 FLs) at E10.5 in WT and M1KO;M2KO FLs and HLs. Scale bars = 100 μm. (e lower panels) HOXD9 immunofluorescence in E10.5 FLs (N = 3 FLs). Scale bar = 100 μm. f Map of the Hand2 locus with its regulatory regions and ChIP-seq signal for Meis and HOXD13. The position of the mm1689 enhancer (gray bar), the associated Meis and HOXD13-binding sites (blue peaks and green bar, respectively), and the 3 kb region deleted (orange bar) are shown in a zoomed region. g Whole-mount in situ hybridization analysis of Hand2 at E10 in embryos carrying the 3 kb regulatory region deletion (N = 4 homozygous FLs and HLs and N = 4 heterozygous FLs and HLs). Arrowheads point to protein or gene expression downregulation. Source images are available at Mendeley Data [https://data.mendeley.com/datasets/r774bxyf8d/2].

Fig. 8. Proposed role of Meis in limb initiation and AP prepatterning.

During limb initiation (a), Meis is required in parallel to Tbx factors for activation of the Wnt target Lef1 and Fgf10, both essential for limb-bud formation and progression. This function is similar in FLs and HLs. During AP patterning in the FL (b), Meis is required directly or indirectly for the activation of various prepatterning genes, like Hoxd9, Hoxd13, and Hand2, all involved in Shh activation.