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First published online 6 December 2006
doi: 10.1242/dev.02721

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Transcriptome and phenotypic analysis reveals Gata3-dependent signalling pathways in murine hair follicles

¹ Department of Cell Biology, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
² Department of Genetics, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
³ Department of Obstetrics and Gynecology, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.

View larger version (70K): [in this window] [in a new window]   Fig. 1. (A) Generation of floxed/deleted Gata3 alleles. LoxP sites were introduced in the start codon (in exon 2) and in the second intron (blue flags) of Gata3. A Puro cassette, flanked by FRT sites (pink flags) and coupled to an nlsLacZ gene lacking a polyA signal was introduced in intron 2. Arrows indicate lengths of NotI-SalI restriction enzyme fragments that hybridize to the external 5' probe. A Polyoma thymidine kinase gene (PyTK) was added 5' to the construct to counter select against random integration. Flp recombinase mice (Buchholz et al., 1998) were used to remove the Puro cassette. After recombination by Cre recombinase, resulting in excision of the part of exon 2 that is 3' to the start ATG of Gata3 and the part of intron 2 that is 5' to the EcoRI site, the nlsLacZ reporter replaces the 3' end of the truncated Gata3 gene. Arrows below genes indicate direction of transcription. Black boxes correspond to exons. B, BamHI; C, ClaI; H, HindIII; N, NcoI; R, EcoRI; S, SacI. (B-T). Phenotypes caused by homozygous deletion of Gata3 in the skin (ko) versus wt. (B) Delayed hair growth in K14-Gata3-/- at P10. (C) Short stubby hairs instead of normal coat of fur. (D) Weight difference in the first months of life. (E,F) Eyes are still closed, whiskers are present but rudimentary, most facial hairs have disappeared and remaining hairs are still very short. (G) After 3 months, most K14-Gata3-/- mice are completely bald; eyes are still closed. (H,I) The absence of visible nipples in female K14-Gata3-/- mice. (J,K) The complete loss of hairs from the abdominal area at 2 months. (L,M) Baldness starts at the head and progress from anterior to posterior. (N-P) Coat of fur at P26. d, dorsal; v, ventral. (N) wt back hair; (O) K14-Gata3-/- back hair; (P) K14-Gata3-/- belly hair (and flakes; note the encircled, thick rounded tip of the hair instead of the normal fine hair tips). (Q,R) Scanning electron microscopy of dorsal wt (Q) and K14-Gata3-/- (R) hairs of 12-week-old mice. Note thickness of K14-Gata3-/- hair as well as absence of regular cuticle cells and presence of irregular flakes. (S,T) Around P24, K14-Gata3-/- skin starts flaking. Scale bar in Q: 50 µm for Q,R.

View larger version (101K): [in this window] [in a new window]   Fig. 2. Haematoxylin-Eosin staining of fresh-frozen dorsal skin sections of control and K14-Gata3-/- mice. (A,B) P0: skin of K14-Gata3-/- is indiscernible from control skin. (C,D) P7: in K14-Gata3-/- HFs are disorganized and do not produce hairs. Epidermis is thickened. (E,F) P11: in the mutant, (abnormal) hairs are still not penetrating the epidermis. (G,H) P13: some irregularly formed hairs penetrate the skin; both dermis and subcutaneous fat layer (white cells) are much thinner in K14-Gata3-/- than in control skin. Scale bar in A: 50 µm for A,B; 200 µm for C-H.

View larger version (129K): [in this window] [in a new window]   Fig. 3. Expression pattern of Gata3 during the cell cycle. Gata3 expression is visualized by in situ hybridization in the control (A,B,F,J), or by X-Gal staining in the Gata3 LacZ knockin skin (C-E,G-I,K,L). At E15.5 there is strong expression of Gata3 in the epidermis (A). During anagen (A, P3), Gata3 is highly expressed in the IRS, ORS, sebaceous glands (black arrowhead), epidermis and infundibulum (arrows) (B-F,I-L,), and closer inspection (L) reveals that some cells in the germinative layer around the dermal papilla are also Gata3 positive (white arrowhead). During catagen, Gata3 expression was restricted to the IRS (G) and was absent in HF during telogen but present in the epidermis and sebaceous gland (H). Scale bar in A: 200 µm for D,K; 150 µm for A-C,F,G-J,L; 50 µm for E.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Transcriptome analysis in K14-Gata3-/- HF. (A-F) Schematic of HF (A) and Gata3 expression in innermost layers of IRS at P11 (B). Fresh-frozen skin sections of P11 wt (C,D) and K14-Gata3-/- (E,F) before (C,E) and after (D,F) collecting HF tissue by laser-capture. RNA from the captured tissue was used for microarray analysis. DP, dermal papilla; He, Henle's layer of IRS; HS, hair shaft; Hu+Ci, Huxley's layer and cuticle of IRS; Me, medulla; ORS, outer root sheath. (G,H) Full mouse genome transcriptome analysis of laser-captured, K14-Gata3-/- and wt HFs. (G) Pie chart of significantly over-represented biological processes in K14-Gata3-/- as compared with wt HFs. (H) Significant gene expression changes in the K14-Gata3-/- as compared with wt mouse HFs (for an extensive overview, see Table S1 in the supplementary material). FC, fold change; P=P-value for two-tail analysis of variance.

View larger version (85K): [in this window] [in a new window]   Fig. 5. Altered distribution of dividing cells, T-cell number and apoptosis in K14-Gata3-/- skin. (A-F) BrdU labeling of dividing cells of wt (left) and K14-Gata3-/- (right) skin. In wt animals, BrdU labeling (brown-stained nuclei) is mostly present in the bulb of the HFs (D). The cells of the basal layer of the K14-Gata3-/- epidermis are dividing at a much higher rate (compare A with B). There is no obvious difference in the number of BrdU-labelled HF cells, but in their distribution (compare regions a and b in D-F). (G-I) TUNEL assay shows altered apoptosis in K14-Gata3-/- skin as compared with control skin at P11. Arrows indicate positive cells (J-L): K14-Gata3-/- skin contains less CD3 positive T-cells (red dots, arrows) than wt skin. White asterisks mark background staining of an artifact of the knots of highly keratinized material (G,H) and sebaceous glands (J,K). Data are given as mean±s.e.m.; *, P0.05; Student's t-test. Scale bar in A: 150 µm for A,B,D,E,J,K; 100 µm for G,H.

View larger version (97K): [in this window] [in a new window]   Fig. 6. Hair follicle in the absence of Gata3. (A,B) In situ mRNA hybridization of Krtap16-7 at P11; insets show normal cortex expression in the wt (A), whereas expression spreads into the medulla in K14-Gata3-/- hairs (B). (C,D) Cortical cells recognized by AE13 antibody were no longer separate from the pigmented cells in K14-Gata3-/- HFs and those HFs exhibited expanded precortex and cortex as compared with wt HFs. Arrowheads indicate lack of separation of cortical cells from the pigmented cells. DAPI was used to mark the nuclei. (E,F) AE15 antibody recognizes trychohyalin IRS and medulla of control HFs (E) and only wisps of cells in K14-Gata3-/- IRS (F). There is a lack of separation of AE15-positive cells from K14-positive cells in K14-Gata3-/- HFs as compared with control (E,F, insets). (G,H) Expression of Crym at P11 in control (G) and K14-Gata3-/- skin (H); arrows in G point to expression in IRS. Scale bar in G: 200 µm for A,B,G,H; 100 µm for C,D; 50 µm for E,F.

View larger version (74K): [in this window] [in a new window]   Fig. 7. Skin localization of differentiation and proliferation markers in control and K14-Gata3-/- skin. (A-D) Normal staining of the late-stage differentiation marker loricrin at P1 (A,B) and 5 months (C,D). (E,F) Normal staining of the differentiation marker K10 at P4. (G,H) K10 expression at P26; note the cyst-like HFs in K14-Gata3-/-. (I-M) Double-labelling of K10 (red) and K6 (green) at P11 (I-K) and at 5 months (L,M). There are clearly separated expression domains in the wt (I), but in K14-Gata3-/- K6 expression extends more distally (J); the arrow in K indicates a double-labelled (yellow) cell present in the K14-Gata3-/-. Scale bar in A: 200 µm for A-H; 150 µm for I,J,L,M; 10 µm for K.

View larger version (84K): [in this window] [in a new window]   Fig. 8. Expression pattern of signalling molecules in HFs in the absence of Gata3. (A,B) Reduced expression levels of Wnt5a in K14-Gata3-/- HFs (B) as compared with wt littermate controls (A). (C,D) Increased Wnt11 expression in the K14-Gata3-/- outer HF layer (D) as compared with wt controls (C) at P11. (E,F) Stronger nuclear ß-catenin staining in K14-Gata3-/- HFs in the matrix and in the distal part of the HF (F) as compared with control (E) at P11. (G,H) Decrease in Gli1 expression levels, particularly in the region of the dermal papilla in P7 K14-Gata3-/- HFs (H) as compared with controls (G). (I,J) Higher expression levels of Notch1 at P11 in the K14-Gata3-/- HF (J) as compared with wt (I). Scale bar in A: 150 µm for A-D,I,J; 100 µm for E,F; 200 µm for G,H.

View larger version (37K): [in this window] [in a new window]   Fig. 9. K6 expression pattern in the ear. Expression of K6 (green) versus MTS24 (red), which marks specific progenitor cells in the HF (Nijhof et al., 2006), at 5 months in wt (A) and K14-Gata3-/- (B) ears. The epidermis on the outside of the ear is towards the top of the image; the epidermis on the inside of the ear is towards the bottom of the image. Scale bar: 150 µm.