Severe whisker follicle development in Meis2 cKO embryos.

(A) micro-CT images of control and Meis2 cKO embryos at E15.5 showing aberrant whisker phenotype in Meis2 cKO mice. (B) Light-sheet microscopy of Sox9 whole-mount immunostaining of WF. Arrow indicates example of an “escaper” whisker. (C) MEIS2 immunofluorescence on frontal frozen section of E13.5 snouts showing Meis2 expression in dermis, DC and epithelium including Pc. Arrowheads indicate epithelial expression of Meis2 in both control and Meis2 cKO mice. Arrow and asterisk show disappearing Meis2 expression in the DC and dermis, respectively, in the mutant. Scale bar: 150 μm. (D) Wnt1-Cre; mTmG embryos showing Cre recombination specificity in the craniofacial area, midbrain and dorsal spinal cord. Recombined cells are labeled by membrane-localized GFP in green and non-recombined cells labeled by membrane-localized tdTomato in red. (E) Frontal sections of Wnt1-Cre; mTmG snouts documenting Cre recombination in the neural crest-derive mesenchyme, cranial nerve projections without recombination in the overlying epithelium. (F) Whole-mount staining of Meis2 cKO heads at E13.5 with SOX9 and TRKA (Ntrk1) antibodies showing almost absence of WF in mutants and compromised branching of the trigeminal nerve. Scale bar: 500 μm.

Neurog1−/− (KO) embryos lack the trigeminal nerve but WF development is normal.

(A) Triple immunostaining of 100-μm sections shows absence of trigeminal nerve projections (Tuj1+, TrkA+) and normal WF (Sox9+) in Neurog1−/− mice. Arrows in black and white images show examples of invagination of whisker placodes labeled by Sox9 antibody. Scale bar: 500 μm. (B) Whole-mount immunostaining of WFs with SOX9 and EDAR antibodies showing normal WF morphology and patterning in mutants at E12.5 (top) and 13.5 (bottom). Scale bars: 300 μm. (C) EDAR staining of 10-μm sections at E12.5 showing normal initiation of placode formation in Neurog1 mutants. Scale bar: 150 μm. (D) micro-CT images reveal normally developed whiskers (top) at E17.5 in mutants while TGs are lacking (bottom, arrows).

Induction and progression of WF development is compromised in Meis2 cKO.

(A) Whole-mount in situ hybridization of Shh mRNA documenting loss of WFs in mutants. Arrow shows an escaper whisker. (B) Whole-mount immunostaining of WFs with SOX9 and EDAR antibodies showing absence of WF in Meis2 cKO at E12.5. Two examples for each genotype are shown. Scale bar: 300 μm. (C) EDAR staining of 10-μm sections showing placode formation arrest in mutants. Scale bar: 150 μm.

WNT signaling in the epithelium is affected by deletion of Meis2 in the dermal mesenchyme.

(A) LEF1 staining of 10-μm paraffin frontal sections of snouts showing abundance of LEF1 in the dermal mesenchyme while in the epithelium it is concentrated in placodes in regions of WF appearance. Similar to missing placodes, LEF1 is also lost in Meis2 cKO epithelium. Scale bars: 150 μm. (B) beta-galactosidase immunostaining of sections from WNT reporter BAT-gal controls and Meis2 cKO;BAT-gal mutants showing widespread galactosidase signal in the dermis and epithelium. Scale bars: 150 μm. (C) in situ hybridization HCR-FISH using probes for Axin2 and Wnt10b. In the epithelium, Wnt10b is detected in placodes and the signal is lost in Meis2 cKO. Similarly, upregulation of Axin2 is not observed in the expected WF loci. In the dermal mesenchyme, Axin2 mRNA is detected throughout dermis regardless of WFs. This pattern was not changed in Meis2 cKO. Scale bar: 50 μm.

Dermal condensation and proliferation are reduced in Meis2 cKO.

(A) Uniform Manifold Approximation and Projection (UMAP) diagram of single-cell RNA seq analysis of the dermal mesenchyme subset with 6-cluster resolution in which cluster 2 (green) represents dermal condensate (DC) of WFs. (B) UMAP representations of typical DC markers Sox2, Sox18, Tbx18, Bmp4, Lef1 or Cdkn1 shown by FeaturePlots. (C) Relative cell numbers in 6 clusters showing a higher cell count in cluster 1 (nonspecialized cells) at the expense of clusters 0 (dividing cells) and 2 (DC). (D) Analysis of cell proliferation by EdU incorporation after 2-hour pulse at E12.5 and 18-hour pulse at E13.5. (E) Overall proliferation rate was reduced in Meis2 cKO from 52.96 ± 2.05% to 42.38 ± 2.77% (mean ± sem, t-test p= 0.0057) in 2-hour pulse (top) and from 52.17 ± 0.81% to 45.76 ± 1.85% (mean ± sem, t-test p= 0.0052) in 18-hour pulse (bottom). Each data point indicates the average value for one section. n = 2 mice and at least 9 sections. (F) Increase of cell adhesion module score in mutants at E13.5 by scRNA seq analysis. Module scores are generated by indicated genes. (G) Increase of extracellular matrix (ECM) module score in mutants at E13.5 by scRNA seq analysis. Module scores are generated by indicated genes.

Expression of the earliest pre-DC and DC marker Foxd1 is lost in Meis2 cKO.

(A) UMAP representations of decline in Foxd1 (top) and Sox2 (bottom) expression in DC cluster (#2) shown by FeaturePlots. Expression of both genes is significantly reduced in mutants. (B) Immunofluorescence of FOXD1 and SOX2 at initial stages of WF showing FOXD1 in pre-DC stage while the expression shifts from DC to peri-DC regions during WF progression. SOX2 appears as a typical DC marker with no earlier expression. Scale bar: 50 μm. (C-D) Whole-mount immunostaining of FOXD1 and SOX9 of heads from controls and Meis2 cKO at E12.5 (C) and E13.5 (D). It shows absence of WF including pre-DC marker FOXD1 at E12.5 stage when proximal columns of WFs have already formed. A few WF escapers develop in mutants at E13.5 in proximal regions while at least 6 WF columns have formed in controls Scale bars: 400 μm (C) and 500 μm (D). Lower panels: FOXD1 staining in FFPE sections. Scale bars: 150 μm.

WFs normally develop in loss-of-function Foxd1 mutants.

(A) Whole-mount immunostaining of FOXD1, TUJ1 and SOX9 of heads from controls and Foxd1 null mutants at E13.5 showing normal formation of WFs in mutants in which FOXD1 signal disappears. Normal WF development is also reflected in normal WF innervation represented by TUJ1 staining. Scale bars: 500 μm. (B) Whole-mount immunostaining of EDAR confirmed normal Pc appearance in Foxd1 null mutants. Scale bars: 500 μm.