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. 2014 Jun 20;9(6):e99991.
doi: 10.1371/journal.pone.0099991. eCollection 2014.

Ablation of coactivator Med1 switches the cell fate of dental epithelia to that generating hair

Keigo Yoshizaki  1 , Lizhi Hu  2 , Thai Nguyen  2 , Kiyoshi Sakai  1 , Bing He  1 , Chak Fong  2 , Yoshihiko Yamada  1 , Daniel D Bikle  2 , Yuko Oda  2
Affiliations

Ablation of coactivator Med1 switches the cell fate of dental epithelia to that generating hair

Keigo Yoshizaki et al. PLoS One. .

Abstract

Cell fates are determined by specific transcriptional programs. Here we provide evidence that the transcriptional coactivator, Mediator 1 (Med1), is essential for the cell fate determination of ectodermal epithelia. Conditional deletion of Med1 in vivo converted dental epithelia into epidermal epithelia, causing defects in enamel organ development while promoting hair formation in the incisors. We identified multiple processes by which hairs are generated in Med1 deficient incisors: 1) dental epithelial stem cells lacking Med 1 fail to commit to the dental lineage, 2) Sox2-expressing stem cells extend into the differentiation zone and remain multi-potent due to reduced Notch1 signaling, and 3) epidermal fate is induced by calcium as demonstrated in dental epithelial cell cultures. These results demonstrate that Med1 is a master regulator in adult stem cells to govern epithelial cell fate.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Deletion of Med1 generated hairs on the labial side of incisors while disrupting enamel formation.
(A, B) Hairs were generated from the labial side of chalky-colored incisors lacking enamel in Med1 KO mice (12 wk) (A) and in dissected jaws (12 wk) (B). (C) Hairs (black triangle) were observed in the tissue between the bone and the dental epithelial layer (left panel) in Med1 KO. Hairs originated from abnormal tissues (red triangles) underlying the non-polarized ameloblasts and the papillary layer (enlarged image in right panel) (12 wk). (D, E) Micro CT analysis shows the enamel hypoplasia in the Med1 KO. The 3D-reconstructed µCT images showed the structure of the mandible in CON and KO mice (10 wk) and the location of hairs (red triangle) (D). Enlarged sections of the µCT images (squares) are shown (E). The high density mineralized layer (enamel) was present on the labial side of incisors in CON incisors but absent in Med1 KO (triangles) (10 wk) with changes starting at P17. Hair was observed consistently in over 20 litters of Med1 KO mice. The entire mandibles of two mice (CON and KO) from 2 litters (10 and 12 wk) were scanned using µCT, and reproducibility was confirmed. A mineralized layer was never detected in Med1 KO incisors.
Figure 2
Figure 2. Med1 ablation prevents dental fate but drives epidermal fate.
The IPA pathway analysis on 4(A) The expressions of dental genes were decreased in KO at the Mat stage. Instead, hair genes (B) and epidermal genes (C) increased in KO at the Sec and Mat stages. The full list of altered genes with fold changes and detailed information is shown in Tables S1, 2 and 3. Green letters indicate down-regulation and red letters indicate up-regulation. The mRNA levels of representative genes for dental (A, b Klk4), hair (B, b Krt71), and epidermal (C, b Lor) are confirmed by QPCR. The average and SD of relative expressions (% of GAPDH) in CL, Sec and Mat at 4 wk are shown. The numbers show the value of the relative expression, which are too low to represent using bars. The statistically significant increases/decreases in Med1 KO (closed bars) compared to KO (open bars) at each stage are shown by asterisks (n = 3, p<0.05). (D) Immuno-staining of epidermal keratin Krt1 (left) and hair keratin Krt71 (right) (Krt1 or Krt71 green, Notch1 red, DAPI blue) in the Med1 KO and CON at 4 wk. Equivalent sections stained by HE are presented on the right side of each image.
Figure 3
Figure 3. Med1 ablation resulted in defects in the DE-SC niche.
(A) A diagram to show the location of the labial CL in mouse mandible, where DE-SCs reside. The CL is composed of OEE, IEE, SI, and SR (left). (B) Histological analyses of CL in Med1 KO and CON at 4 wk. Med1 is abundantly expressed in OEE/IEE/SR in the CL but diminished in the KO (Med1, brown signals with blue counterstaining). Histological staining shows the morphological alterations of the CL in KO (HE, red triangles). Cell proliferation is increased in OEE/IEE and IDE equivalent to transient amplifying cells (TA) in KO (PCNA staining, brown signals with blue counterstaining). Vwa2 expression in the basement membrane of OEE and IEE decreased in Med1 KO (Vwa2 brown staining with blue counterstaining). The results were reproduced in two independent experiments using two Med1 KO mice from different litters.
Figure 4
Figure 4. Med1 ablation extended Sox2 and stem cell signatures.
(A) Stem cell marker Sox2 expression in dental epithelia at three different stages; the CL (left panels), the Sec (second left), and the Mat stage (third left) in Med1 KO and CON at 4 wk (green Sox2, red Notch1, blue DAPI). The location of the 3 stages is shown in the upper diagram. Enlarged images of the boxed area of the Mat stage are also shown (far right panels), and papillary structures are indicated by dotted lines. The lower diagram shows the location of Sox2 (green) in the papillary layer. (B) The mRNA expression of Sox2 and Vwa2 at three stages (CL, Sec, and Mat) in KO (closed bars) compared to CON (open bars) at 4 wk. The SD of relative expression (% of GAPDH) and the statistical significance (n = 3, * p<0.05) are shown. Red arrows indicate their retention in the Mat stage in KO.
Figure 5
Figure 5. Gene expression profiling predicted changes in Notch and calcium signaling upon Med1 deletion.
(A) Strategy of microarray analyses. CL and Mat tissues were dissected from Med1 KO and CON mice at 4 wk and 10 wk. Array 1 calculated fold changes in CL/Mat in control mice revealed genes that are enriched in CL compared to the Mat stage, which are DE-SC signature candidates. Array 2 calculated fold changes in KO/CON at CL shows genes up-or down-regulated in Med1 KO compared with CON. Arrays were performed on RNA samples from Med1 KO and CON. (B) These analyses revealed a list of DE-SC signatures affected in the CL of Med1 KO in comparison with gene pools identified through other stem cells of ESC or HF-SC. Green letters show down-regulation, and the red letters indicate up-regulation. Fold increases and other details are listed in Table S4 (10 wk). (C, D) The IPA pathway analysis predicted that Notch1 (C) and calcium (D) are potential upstream regulators to cause these changes in Med1 KO. The sub-cellular location and the up- and down-regulation of genes are shown by red and green, respectively. (E) The protein expressions of Notch1 at 4 wk are shown in CL of Med1 KO and CON.
Figure 6
Figure 6. Med1 regulates cell fate through calcium and Notch signaling.
(A) Cervical loop derived dental epithelial (CLDE) cells were established, and Med1 was silenced by siRNA (siMed1). (B) Sox2 was present in CLDE cells that were maintained in low calcium (upper panels), was decreased in high calcium (1.5 mM) (middle panels) (4 days culture), but sustained in siMed1 cells in high calcium (lower panels) (Sox2 green, DAPI blue). The numbers of Sox2 positive cells were counted (bar graph). (C) Control (open bars) and siMed1-treated cells (closed bars) were maintained for 1–13 days in 1.5 mM calcium, and the mRNA expression of Alpl (blue in A), Lor (red in A), Sox2, and Hes1 were measured. Data are shown as the mean±SD of triplicate measurements. The statistically significant increases or decreases in siMed1 compared with sicontrol at each stage are shown by asterisks (p<0.05). (D) Notch inhibitor DAPT suppressed the induction of Alpl, but induced the expressions of Lor (* p<0.05) when cells were maintained in a high calcium condition. The control mRNA level at Day 1 without DAPT for Alpl or Lor was set as 1. (E) The protein levels of Lor, cleaved Notch (c-Notch), total Notch, and the loading control β-actin (4 days in high calcium) are shown by the Western blot analysis. The results were reproduced in two different batches of CLDE cells.
Figure 7
Figure 7. Hair differentiation was induced adjacent to the blood vessels in which calcium is supplied.
(A) Hair keratin Krt71 was detected in the papillary layer adjacent to the blood vessels (right panel). Enlarged images are shown in the middle panel (Krt71 green, Notch1 red, DAPI blue). Yellow dotted circles and white arrows show the blood vessels in which erythrocytes are detected (insert). Sox2 was expressed in basal cells in the papillary layer (left panel, Sox2 green, Notch1 red, DAPI blue).
Figure 8
Figure 8. A proposed model in which Med1 ablation alters epithelial cell fate.
Med1 regulates Notch signaling by activating Notch1 target genes (upper diagram). Med1 maintains the niche architecture containing Sox2-expressing dental epithelial stem cells (DE-SC) (blue). Enamel is formed as dental epithelia differentiate in control incisors partly due to Notch signaling. In contrast, DE-SCs fail to commit to the dental lineage when Med1 is ablated (lower diagram). Instead, Sox2-expressing cells (blue) extend into the differentiating zones. Med1 deficient cells are exposed to extracellular calcium through the blood vessels (red-dotted circles) adjacent to the papillary layers (yellow). Med1 deficient cells are differentiated into epidermal cells (light green) and hair keratinocyte-like cells (green) and produce mature hair shafts (black) in the Med1 KO incisors (inserted picture).
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