The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.


FIGURE S1. Vav function regulates SSC numbers.
(A) Representative images of the colonies (stained with Giemsa) formed by epidermal cells from mice of indicated genotypes and ages. The right panels show magnifications of representative colonies obtained in these assays (boundaries of the colony are indicated with a dashed line). Scale bar, 50 µm (n = 4).
(B) Quantification of the colony-forming efficiency of epidermal cells isolated from mice of indicated genotypes and ages according to the data obtained in A. **, P < 0.01 (two-way ANOVA and Sidak's multiple comparisons test, n = 4 per genotype and time point).
(E) Quantification of the colony-forming efficiency of epidermal cells isolated from mice of indicated genotypes and ages. **, P < 0.01 (two-way ANOVA and Sidak's multiple comparisons test, n = 3).
(F) Representative whole mount immunofluorescence images showing the presence of BrdU (green color) and the expression of Ki67 (red color) and keratin 15 (blue color) in the tail epidermis from mice of indicated genotypes. Scale bar, 200 µm (n = 6).
(G) Quantification of the number of label-retaining (left) and Ki67 + (right) cells in the hair follicle bulge of mice of the indicated genotypes upon the indicated treatments according to the data gathered from the experiment shown in panel F. *, P < 0.05; ***, P < 0.001 (ANOVA and Tukey's HSD tests, n = 6).
In B, C, D, E, G and H, data represent mean ± SEM. TABLE S1. Summary of the SSC-related phenotypes found in Vav2 Onc/Onc and Vav2 -/-;Vav3 -/mice. Upregulation and downregulation events are indicated by red and blue arrows, respectively. Grades of blue represent the relative intensity of the interrogated biological parameter. No effect is depicted as a "=" symbol. The figure number in which the experimental data are found in indicated on the right column (left, data from Vav2 Onc/Onc mice; right, data from Vav2 -/-;Vav3 -/mice. (C) Quantification of the skin area regenerated by indicated cell type combinations according to the data obtained in A. ***, P < 0.001 (ANOVA and Tukey's HSD tests, n = 4).

(D)
Representative histological sections of the skin area regenerated by the indicated cell mixes at the endpoint of the xenograft protocol. Scale bar, 100 µm (n = 4).

(E to H) Quantification of the thickness of the skin (E), epidermis (F) and dermis (G) as well as of the number of hair follicles (H) in the histological sections from the experiment shown in D. Differences were not significant (Student's t-test, n = 4).
(I) Representative image of immunodeficient mice at the endpoint of the skin xenograft protocol performed with the indicated cell type combinations. The regenerated skin area is indicated with an arrow. Scale bar, 2 cm (n = 4).
(J) Representative images of hair regeneration in immunodeficient mice at the endpoint of the skin xenograft protocol shown in I and performed with the indicated cell type combinations. n = 4 per cell mix.
(K) Quantification of the skin area regenerated by the indicated cell type combinations according to the data obtained in I. Differences were not significant (Student's t-test, n = 4). In C, E, F, G, H, K, M, N, O and P, data represent mean ± SEM. NS, not significant.

FIGURE S3. Vav proteins regulate transcriptional programs involved in stem cell homeostasis.
Protein-protein interaction network of genes involved in SSC homeostasis that are upregulated by Vav2 Onc . Genes associated with the extracellular matrix and the signaling through Wnt, Hippo/Yap, Hedgehog, cAMP and PI3K/Akt pathways are highlighted in color as indicated. ECM, extracellular matrix. (C) qRT-PCR showing the abundance of interrogated transcripts in SSCs of indicated genotypes. **P ≤ 0.01, ***P ≤ 0.001 (two-way ANOVA and Sidak's multiple comparisons test, n = 3). a.u., arbitrary units. Data represent mean ± SEM.