Meis2 is expressed in subclasses of DRG cutaneous mechanoreceptive neurons in mouse embryos (A-B).

A) ISH for Meis2 mRNA showed expression in a subpopulation of DRG sensory neurons at embryonic stages E11.5, E14.5 and E18.5, at P0 and at adult stages. Dashed lines delineate the DRG. Scale bar=50µm. B) IF for Meis2 (red) and c-Maf, Ntrk2 or Ntrk3 (blue) at P7 following injection of cholera toxin B (CTB in green) in the skin of new-born pups. Note that Meis2+/CTB+ retro-traced sensory neurons co-expressed c-Maf, Ntrk2 or Ntrk3 (arrows). Scale bar=50µm. We estimated that 30.5 ± 3.5% (mean ± SEM; n = 3) of Meis2-positive neurons co-expressed Ntrk2, and that 39.5 ± 5.4% co-expressed Ntrk3. Conversely, Meis2 was co-expressed in 53.6 ± 9.4% of Ntrk2-positive neurons, and in 78.5 ± 5.0% of Ntrk3-positive neurons. Meis2 expression depends on target-derived signals (C-D). C) Representative images of Meis2 mRNA expression (blue or pseudo-colored in red) and islet1 (green) in DRGs of Hamburger-Hamilton stage (HH) 36 chick embryos on the ablated and contralateral sides. Box plots showing the number of Islet1+/Meis2+ DRG neurons per section at stage HH36 following limb bud ablation. For Islet1-positive neurons, the contralateral side was taken as 100%. For Meis2, values represent the percentage of Meis2+ over Islet1 + neurons. D) Representative images of Meis2 mRNA expression (blue or pseudo-colored in red) and islet1 (green) in DRGs of HH29 chick embryos on the ablated and contralateral sides. Box plots showing the quantification of Islet1 +/Meis2+ neurons number per section at stage HH29 on the contralateral and ablated sides. Arrow heads point at remaining Meis2-positive VL neurons. Dashed lines encircle the DRGs. ** p≤0.005 *** p≤0.0005 ; ns= not significant following Student t-test. n=3 chick embryos. Scale bar=100µm. Alteredtouchperceptionin Meis2 mutant mice (E-H). E) Box plots showing the responses following application of Von Frey filaments of different forces. Isl1 +/Cre::Meis2LoxP/LoxP mice exhibited a significantly reduced sensitivity to the 0.16, 0.4 and 0.6 g Von Frey filaments but not to higher forces filaments compared to WT and Isl1 +/CRE littermates. * p≤0.05; ** p≤0.005; *** p≤0.001 following Kruskal-Wallis statistical analysis. F) Box plots showing the dynamic touch responses when the hind paw palms of individual mice were stroked with a tapered cotton-swab. Analysis showed that Isl1 +/Cre::Meis2LoxP/LoxP mice were less responsive to the stimulus than WT and Isl1 +/Cre littermates. *** p≤0.0001 following a one-way Anova statistical analysis. G) Box plots indicating that the latency to the first signs of aversive behavior in the hot plate test is similar in all groups of mice. WT, n=19; Isl1 +/Cre, n=16; Isl1+/Cre::Meis2LoxP/LoxP, n=9. H) Box plots showing the number of bouts when a sticky paper tape was applied on the back skin of mice. Analysis indicated a significant decrease in the number of bouts in Isl1 +/Cre::Meis2LoxP/LoxP mice compared to WT and Isl1+/Cre littermates. * p≤0.05 following a one-way Anova statistical analysis.

Meis2 is necessary for LTM neurons peripheral projections but dispensable for their survival and specification.

A) Box plots showing that the DRGs volumes along the rostro-caudal axis are similar in E16.5 WT and Isl1 Cre/+::Meis2LoxP/LoxP embryos. IF for Ntrk2 or Ntrk3 (red) and Islet1 (blue) and box plots analysis indicating that the percentage of Ntrk2 + and Ntrk3 + neurons are not affected in E16.5 Isl1 Cre/+::Meis2LoxP/LoxP. Dashed lines encircle the DRGs. n=4; n.s. = not significant. Scale bar = 20µm. B) Representatives images showing IF for Ntrk2 or Ntrk3 (green) with Pvalb or Maf (red) in P0 WT and Wnt1 Cre::Meis2LoxP/LoxP DRGs. Box plots showing that the number of Ntrk2 + and Ntrk3+ neurons are unchanged in P0 WT and Wnt1 Cre::Meis2LoxP/LoxP DRGs. n=3, n.s. = not significant. Scale bar = 20µm. C) Representative images showing IF for Ntrk1 (red), Ret, Ntrk2, Ntrk3 and Maf in E18.5 WT and Isl1 Cre/+::Meis2LoxP/LoxP DRGs. Box plots showing that the number of Ret +, Ntrk2+, Ntrk3+ and Maf + LTM neurons and of Ntrk1 + nociceptive neurons are similar in E18.5 WT and Isl1Cre/+::Meis2LoxP/LoxP DRGs. n=3; n.s. = not significant. Scale bar = 100µm.

Meis2 inactivation dysregulates genes linked to neuronal projections and synaptogenesis.

A- Venn diagram comparing the number of DEGs between each genotype (n=3; p<0.05). This comparison identified 51 DEGs genes that were differentially expressed compared to both control genotypes (WT or Isl1+/CRE embryos), and a total of 488 genes that were differentially expressed in Meis2 mutant compared to either WT or Isl1+/CRE embryos. B-Heat maps for the 488 DEGs compared to both control genotypes showed that about half of the DEGs were either up-(257) or down-(231) regulated in Meis2 mutant. Names of genes that are shown in F are labeled in red (up-regulated) or blue (down-regulated). Members of the protocadherin family are indicated. C-Gene ontology analysis for the 488 DEGs using David and the full RNAseq gene list as background. Graphs showing the result of gene ontology analysis using David. The upper graph shows the -log10(p value) associated with selected significant (p<0.05) GO terms or KEGG_PATHWAY terms. The lower graph shows the fold enrichment for the same selected GO terms or KEGG_PATHWAY terms. Only terms associated with a minimum of 5 genes were considered. Bars in red indicated terms for which the heat maps are shown in D. D-Heat maps showing the DEGs related to previous GO terms (Red bars in C). E-Representative images showing a strong overall deficits in Nfeh + (red) sensory projections in the hairy and the glabrous skin of P0 Wnt1 Cre::Meis2LoxP/LoxP neonates forepaw compared to WT littermates. Dashed lines delineate the hair follicle and the epidermis. Scale bar = 50µm.

Meis2 gene inactivation compromised Merkel cells innervation in the glabrous skin and increased SAM vibration threshold.

A) Percentage of tap units among A β fibers in the hairy and glabrous skin detected by electrophysiological recording in the nerve-skin preparation. Note that Tap-units are only present in both the hairy and glabrous skin of adult Isl1 +/Cre::Meis2LoxP/LoxP mice but not in WT and Isl1 Cre/+ littermates. B) Confocal images of Nefh + innervation (green) of CK8 + Merkel cells (red) in the forepaw glabrous skin of WT and Isl1 Cre/+::Meis2LoxP/LoxP adult mice. Dotted white squares indicate the close-up on CK8 + Merkel cells. Note the lack of Nefh + fibers innervating Merkel cells in mutant mice. Scale bar = 10µm. C) Representative images of whole mount staining for CK8 in the hairy back skin of adult WT and Isl1 Cre/+::Meis2LoxP/LoxP E18.5 embryos showing no difference in the number of touch dome between genotypes. Box plots show the number of touch domes per surface area and the number of Merkel cells per guard hair. No significant differences were found between both genotype in Mann-Whitney test. n=5 (WT) and 4 (Isl1 +/Cre::Meis2LoxP/LoxP). D) Confocal images of Nefh+ innervation (green) and CK8 + Merkel cells (red) of guard hairs in the hairy back skin of WT and Isl1Cre/+::Meis2LoxP/LoxP adult mice. Dotted white squares indicate the close-up on CK8 + Merkel cells with apparently normal Nefh+ innervation. Scale bar = 10µm. E) Box plot indicating the percentage of Merkel cells in contact with Nfeh + fibers in the forepaw glabrous and in the back hairy skin of adult WT and Isl1 +/Cre::Meis2LoxP/LoxP mice. n=4; * p≤0.05 in Mann-Whitney test. F) In the hairy and glabrous skins, SAMs in Isl1 +/Cre::Meis2LoxP/LoxP mice (n = 22 from 6 mice) had significantly increased vibration threshold compared to WT mice (n = 29 from 6 mice), but normal firing activity to a 25-Hz vibration. Trace shows the stimulation and red square indicate the time frame during when the number of spikes was calculated. G) SAM response to a ramp of 50 Hz vibration with increasing amplitude are similar in WT, Isl1 +/Cre and Isl1 +/Cre::Meis2LoxP/LoxP mice. SAM responses to ramp stimuli and their static force responses were also identical in the different genotypes. Fibers from WT and Isl1 +/Cre mice (n=5) displayed similar responses.* p≤0.05; ** p≤0.005.

Meis2 gene inactivation affects Meissner corpuscles morphology but not RAM fibers electrophysiological responses in the glabrous skin.

A) Representative images showing S100β+ Meissner corpuscles (red) and their innervation by Nefh + fibers (green) in the glabrous skin of WT and Isl1 +/Cre::Meis2LoxP/LoxP adult mice. Scale bar=10µm. The box plot shows the average number of time that Nefh+ fibers crossed the midline of the Meissner corpuscles. Dashed blue line indicate the Meissner corpuscle midline. Blue arrow heads indicate sites where Nefh + fibers cross this midline. B) RAMs of the glabrous skin exhibited similar vibration threshold and firing activity to a 25-Hz vibration in WT (n = 16 from 4 mice) and Isl1 +/Cre::Meis2LoxP/LoxP mice (n = 21 from 6 mice). Glabrous RAMs showed a non-significant decrease in firing activity to a ramp of 50 Hz vibration with increasing amplitude in Isl1 +/Cre::Meis2LoxP/LoxP compared to WT littermates, but their response to ramp stimuli was similar in both genotypes. Traces indicate the type of stimulation and red squares the time frame during which the number of spikes was calculated. *** p≤0.001.

Meis2 gene inactivation affects hair follicle innervation and RAM fibers electrophysiological responses in the hairy skin.

A) Representative images of whole mount immunostaining for Nfeh+ sensory projections in the hairy skin of adult WT and Isl1 +/Cre::Meis2LoxP/LoxP embryos. Scale bar=100µm. B) Box plots showing the quantification for the number of branch points in the innervation network and for the number of innervated hair follicles. n=3; * p≤0.05. C) RAMs in the hairy skin of Isl1 Cre::Meis2LoxP/LoxP mice (n = 24 from 3 mice) exhibited significantly increased vibration threshold and reduced firing activity to a 25-Hz vibration compared to WT mice (n = 20 from 3 mice). Traces show the stimulation and red square indicate the time frame when the numbers of spikes were calculated. RAMs in the hairy skin of Isl1 +/Cre::Meis2LoxP/LoxP mice also showed a reduced firing activity in response to a ramp of 50 Hz vibration with increasing amplitude compared to WT and Isl1 +/Cre animals. Fibers recorded from Isl1 +/Cre mice (n=5) showed similar responses than those recorded from WT mice. * p≤0.05; ** p≤0.005.