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First published online 14 March 2007
doi: 10.1242/dev.001750

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The chromatin remodeler Mi-2ß is required for establishment of the basal epidermis and normal differentiation of its progeny

Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.

View larger version (91K): [in this window] [in a new window]   Fig. 1. Expression of Mi-2ß mRNA during epidermal development and conditional inactivation of Mi-2ß in the skin. (A) In situ hybridization studies reveal Mi-2ß mRNA expression at E10.5, E14.5, E18.5 and P1. Mi-2ß is uniformly expressed in the embryonic ectoderm (E10.5). Mi-2ß transcripts are also detected in E14.5 epidermis in the basal and stratum intermediate layers. In the differentiating hair follicle, increased levels of Mi-2ß mRNA are first detected in the placode (arrowhead) and then in the matrix (arrow). Scale bar: 50 µm. (B) Cre-dependent conversion of the floxed allele (LoxPF) to the mutant allele (F) in the P1 dorsal epidermis revealed by PCR of genomic DNA (lanes 2 and 4). (C) Wild-type (WT) and mutant littermates at P1. (D) Hematoxylin and Eosin-stained cross-sections of WT and mutant skin at P1. The dotted line demarcates the dorsal region and the unbroken line the ventral region. The mutant skin exhibits an exacerbation of phenotypes from the dorsal to the ventral side that includes thinning of the epidermis and reduction in hair follicles. Scale bar: 100 µm. A further magnification of the mutant skin is provided beneath to show the absence of the basal layer and the thinning of the suprabasal and cornified layers in the ventral region (right), and the more normal epidermal differentiation in the dorsal region (left). (E) The presence of Mi-2ß protein (green) in WT (left) and in the dorsal (middle) and ventral (right) mutant skin was evaluated by immunofluoresence at successive stages of development. DAPI-stained nuclei are shown in red, and the white dotted line demarcates the dermal-epidermal junction. Expression of Mi-2ß protein is indicated by the presence of yellow nuclei, depletion of Mi-2ß by red nuclei. Depletion of Mi-2ß protein occurs earlier (E10.5) in the ventral skin, and later (E13.5 and later) in the dorsal skin. Scale bar: 50 µm.

View larger version (112K): [in this window] [in a new window]   Fig. 2 . Early depletion of Mi-2ß in the ventral epidermis results in late depletion of the basal and suprabasal layers. (A) Hematoxylin and Eosin-stained sections of WT and mutant ventral skin isolated from E10.5-P1 stages of development. A reduced cellularity in the ventral epidermal layers was apparent from E16.5 to P1. (B-D) Expression of keratin 5 (K5) (basal epidermis), keratin 1 (K1) (suprabasal epidermis), and loricrin (granular epidermis) was examined by immunofluoresence. DAPI-stained nuclei are shown in blue. The timing of induction and expression of these epidermal differentiation markers is not initially affected. Their progressive reduction later in development reflects a progressive depletion of basal and suprabasal layers. Scale bars: 50 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 3 . Late effects on proliferation and apoptosis in the Mi-2ß-depleted ventral epidermis. (A,B) The percent of Pcna-positive cells within the basal layer (K5-positive) of WT and mutant skin was estimated during development. Sagittal sections were co-labeled with antibodies against the proliferation marker Pcna and K5. Nuclei were counterstained with DAPI (A). The data (B) represent the mean percentage of Pcna-positive cells within the basal layer (K5- and DAPI-positive) from five independent animals. From E14.5 through E16.5, a similar number of Pcna-positive basal cells were seen in both the mutant and WT skin. However, starting at E18.5 and through P1, a significant reduction of Pcna-positive cells was detected in the ventral, but not the dorsal, mutant regions. (C) Apoptotic cell nuclei (brown) were detected by TUNEL analysis on ventral skin at P1. In both the WT and mutant, TUNEL-positive cells were detected in the uppermost layer of the epidermis, but the dramatic increase in their number seen in the mutant suggests persistence of nucleated dead cells and a defect in terminal differentiation. Scale bars: 50 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 4. Effects of Mi-2ß depletion in the dorsal epidermis. (A-D) Development of the basal and suprabasal layers was examined by immunofluoresence using antibodies to K5, K1, loricrin and keratin 6 (K6). DAPI-stained nuclei are shown in blue. Expression of K5, K1 and loricrin was similar in WT and mutant throughout development (A-C). The K6-positive periderm detected at E16.5 was shed at E18.5 in both WT and mutant (D). From E18.5 through P1, K6 induction was observed in the suprabasal layers of the mutant but not WT skin (D). (E) Skin barrier function was analyzed by a barrier-dependent dye exclusion assay at E19.5. The WT and, for the most part, the mutant dorsal epidermis prevented dye penetration indicating intact barrier function. By contrast, the ventral part of the mutant epidermis was readily penetrated by the dye indicating lack of a barrier. (F) K6 expression (red) is confined to cells that lack Mi-2ß (green). Scale bar: 50 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 5 . Mi-2ß depletion causes severe effects on hair follicle morphogenesis. (A) Hematoxylin and Eosin-stained sagittal sections of dorsal skin from WT and mutant at E18.5 and P1. The number next to the follicles designates their developmental stage according to Hardy (Hardy, 1992). (B,C) The number of hair follicles per unit length (B) and the percentage of follicles at distinct developmental stages (C) were evaluated from E18.5 through P1. The number of hair follicles in the mutant was reduced by approximately 50% relative to WT from E18.5 through P1. At E18.5 in the WT, primary follicles have developed to stage 3 or 4, whereas secondary follicles have reached stage 1-2. In the mutant, a reduction was detected from stage 3a onwards. By P1 in the WT, primary, secondary and tertiary follicles have developed to stages 5-6, 3-4, and 1-2, respectively. However, by P1 in the mutant, follicles at stage 1 and after stage 3c were severely reduced relative to WT.

View larger version (124K): [in this window] [in a new window]   Fig. 6 . Effects of Mi-2ß depletion on the signaling network that controls hair follicle morphogenesis. (A-D) Immunofluorescence of E18.5 dorsal skin labeled with antibodies against Edar or Edar and E-cadherin (A), or against Mi-2ß and Edar (B,C), or against Mi-2ß and E-cadherin (D). (C,D) In situ hybridization of E18.5 dorsal skin with probes against Shh and Wnt5a. The depletion of Mi-2ß in follicles was confirmed by immunofluoresence staining of adjacent serial sections using antibodies to Mi-2ß and E-cadherin. DAPI-stained nuclei are blue. (A,B) In the WT, a local increase in Edar expression was detected among basal epithelial cells (asterisks) that give rise to the hair placode, as well as within the hair follicle (arrowhead). Edar upregulation was followed by a decrease in E-cadherin expression (A, WT). In the mutant, no Edar upregulation or E-cadherin downregulation was seen in areas of the mutant skin in which Mi-2ß was absent (A, mutant). By contrast, in areas with mosaic Mi-2ß depletion, follicular structures expressing Edar were detected in the Mi-2ß mosaic area in the mutant (arrowhead in B mutant, Cb). Shh expression was seen at the tip of stage-2 and stage-3a follicles in the WT (Cc and Da). In the mutant, Shh transcript was seen in stage-2 follicles with mosaic Mi-2ß depletion (Cd), but was significantly reduced in the Mi-2ß-null counterparts (Ce). By contrast, Shh was seen in Mi-2ß-null stage-3a follicles (Db). Expression of Wnt5a was observed in the dermal condensate of stage-3a follicles in the WT and in Mi-2ß-positive stage-3a follicles in the mutant (Dc and De). Wnt5a was, however, significantly reduced in the Mi-2ß-null counterparts (Dd).

View larger version (35K): [in this window] [in a new window]   Fig. 7. The role of Mi-2ß in skin development: a model for the development of the skin and its appendages and the role of Mi-2ß in this process. Successive stages in the development of wild-type skin are depicted on the upper time line. We propose that ectodermal cells (yellow) are first committed to an epidermal TA cell (blue) that can make epidermis but has limited proliferative potential and developmental plasticity. This cell type is then converted to an epidermal stem cell (green) with extensive proliferative capacity and plasticity to adopt alternative fates. Starting at E14.5, some of these cells are induced to become follicular progenitor cells (pink), and sometime thereafter other epidermal stem cells give rise to TA cells of the epidermis with more restricted proliferative and developmental potential. Follicular progenitors proliferate to make the hair peg, while epidermal stem and TA cells generate the stratified epidermis. Finally, a subset of follicular progenitors at the base of the follicle are specified as the matrix stem cells (orange) that give rise to the hair shaft and inner root sheath over the anagen phase of the hair cycle (right). These matrix stem cells are distinct from the follicular bulge stem cells (purple) that regenerate the lower follicle in the adult. The three phenotypes resulting from deletion of Mi-2ß at different stages of development are shown (1-3, indicated by a red cross) and interpretations of these phenotypes in the context of the model are shown beneath.

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