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First published online September 28, 2005
doi: 10.1242/10.1242/dev.02019

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Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation

¹ Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS, INSERM, ULP, BP 10142-67404, Illkirch, C.U. de Strasbourg, France
² Institut Clinique de la Souris (ICS), BP 10142, CU de Strasbourg, 67404 Illkirch, France
³ Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France
⁴ Unité Expression Génétique et Maladies, CNRS URA 1644, Département de Biologie du Développement, Institut Pasteur, 75724 Paris, France

View larger version (58K): [in a new window]   Fig. 1. Generation of mice harboring floxed Brg1 alleles and characterisation of the Cre activity of K14-Cre mice during embryogenesis. (A) Targeting strategy of the mouse Brg1 locus. Partial structure of the Brg1 locus [Brg1 (+) allele]. Exons are indicated by black boxes. The Brg1 L3 allele containing the three loxP sites (lox) and the hygromycin resistance marker (Hygro) is presented. The expected genomic maps after excision of the floxed marker (Brg1 L2 allele) and excision of both the marker and exons 2 and 3 (Brg1 L- allele) are shown. The arrows indicate the PCR primer location (P1-P3). The location of the Southern blot 5'-probe (Sumi-Ichinose et al., 1997) is shown. The size of DNA segments obtained after BamHI restriction digest and detection with the 5'-probe are indicated. B, BamHI; N, NheI; X, XbaI; E, EcoRV. (B) Southern blot analysis of genomic DNA isolated from Brg1+/+, Brg1L3/+ and Brg1L2/+ ES cells, digested by BamHI and hybridized with the 5' probe. (C) Whole-mount X-Gal stained E9.5 (a,b) and E10.0 (c) control ROSAfl/+ (a) and K14-Cretg/0/ROSAfl/+ (b,c) embryos. fl, forelimb; hl, hindlimb. (D) X-Gal-stained dorsal skin sections from E12.5 (a,b), E14.5 (c,d) and E18.5 (e,f) control ROSAfl/+ (a,c,e) and K14-Cretg/0/ROSAfl/+ (b,d,f) fetuses. Sections were counterstained with safranin. E, epidermis; D, dermis. Scale bar in a: 24 µm.

View larger version (61K): [in a new window]   Fig. 2. Selective ablation of BRG1 in the surface ectoderm and epidermal keratinocytes of K14-Cretg/0/Brg1L2/L2 fetuses. (A) Immunohistochemical (IHC) detection (green fluorescence) of Brg1 (a-h) on E11 forelimb (a,b,e,f) and hindlimb (c,d,g,h) bud tranverse sections from control (a,c,e,g) and Brg1ep-/-(c) (b,f,d,h) fetuses. Superimposition of Brg1 (green fluorescence) and DAPI staining of nuclei (blue fluorescence; e-h). Scale bar in a: 24 µm. (B) IHC detection of Brg1 on transverse sections through the dorsal ectoderm of E12.5 Brg1L2/L2 (control; a,c) and Brg1ep-/-(c) (b,d) fetuses. Superimposition of Brg1 (green fluorescence) and DAPI staining of nuclei (blue fluorescence; c,d). Scale bar in a: 24 µm. (C) IHC detection of Brg1 on dorsal skin sections from (a,b) control Brg1L2/L2 and (c,d) Brg1ep-/-(c) E18.5 fetuses. Red staining corresponds to BRG1 antibody and blue staining to DAPI. E, epidermis; D, dermis. The dotted line indicates the dermal-epidermal junction. Scale bar in a: 10 µm. (D) PCR detection of the Brg1 L2 and L- alleles in the epidermis and dermis of E18.5 fetuses. DNA was extracted from tail epidermis (E; lanes 1,3,5,7,9) or dermis (D; lanes 2,4,6,8,10) of fetuses with the indicated genotypes, isolated from females that were Tam treated from either E9.5-E13.5 (lanes 3,4) or E12.5-E16.5 (lanes 5-8), or oil (vehicle) treated from E12.5-E16.5 (lanes 9,10).

View larger version (57K): [in a new window]   Fig. 3. Morphological and functional analysis of Brg1ep-/-(c) mutant fetuses. (A) Gross morphology of Brg1L2/L2 (control; a) and Brg1ep-/-(c) (b) E18.5 fetuses. fl, forelimb. (B) Hindlimb skeletal preparation from Brg1L2/L2 (a) and Brg1ep-/-(c) (b,c) E18.5 fetuses, stained for cartilage (Alcian Blue) and bone (Alizarin Red). Hindlimbs of two different mutants illustrate a mild (b) and the most severe (c) observed malformations. Two (b) and four (c) digits are missing in the autopods (au). The zeugopod (z) exhibits mild deformations of the tibia (ti) and fibula (fi) in b, and lacks the tibia in c. Note that the stylopod (st) and pelvic girdle (pg) are normal. fe, femur. (C) Whole-mount Fgf8 RNA in situ hybridization in control (a,c) and Brg1ep-/-(c) (b,d) fetuses collected at E10.5 (a,b) and E11.5 (c,d). The arrowhead in b points to a mild abnormal domain of Fgf8 expression at E10.5. fl, forelimb; hl, hindlimb. (D) Anteroposterior transversal semi-thin sections of E11.5 (a) control (CT) and (b) Brg1ep-/-(c) mutant hindlimb buds. The AER in control limb bud is indicated by an arrow. **, the altered AER region in the Brg1ep-/-(c) mutant limb bud. Scale bar in b: 50 µm. (E) In vivo Lucifer yellow diffusion in (a) Brg1L2/L2 and (b) Brg1ep-/-(c) E18.5 fetuses. Dorsal skin sections were counterstained with DAPI (blue) and analysed by fluorescent microscopy. E, epidermis; D, dermis. Scale bar in a: 33 µm. (F) Barrier-dependent X-Gal diffusion assay on Brg1L2/L2 (a) and Brg1ep-/-(c) (b) E18.5 fetuses. (G) TEWL on dorsal and ventral skin of Brg1L2/L2 and Brg1ep-/-(c) E18.5 fetuses. ***P<0.001.

View larger version (109K): [in a new window]   Fig. 4. Characterisation of skin defects in Brg1ep-/-(c) fetuses. (A) Histological analysis of Toluidine Blue-stained semithin sections of dorsal skin biopsies from E18.5 Brg1L2/L2 (a,c) and Brg1ep-/-(c) (b,d) fetuses. E, epidermis; D, dermis. Epidermal cell layers are indicated: C, cornified; G, granular; S, spinous; B, basal. Scale bars: in a, 60 µm for a,b; in c, 18 µm for c,d. (B) Scanning electron microscopy of the dorsal skin surface from E18.5 Brg1L2/L2 (a) and Brg1ep-/-(c) (b) fetuses. Scale bar: 10 µm. (C) Transmission electron microscopy of dorsal skin from E18.5 Brg1L2/L2 (a,c,e,g) and Brg1ep-/-(c) (b,d,f,h) fetuses. (a,b) Stratum granulosum (SG). D, desmosomes; KF, keratin filaments; KG, keratohyalin granules. Arrows indicate lamellar granules (LG). Insets show LGs. (c,d,e,f) Junction between stratum granulosum (SG) and stratum corneum (SC). Large black arrows in c and e point towards the LG(s) in the SG-SC interface, and arrowheads in e point towards aligned extracellular lipid discs at the junction between the granular and the cornified layer. The white arrow in c,d indicates desmosomes (D); small black arrows in d,f indicate vesicles at the interface of SG and SC. (g,h) Consecutive C1, C2, C3 and C4 cornified cell layers. Black arrows in h point towards vesicles. Black and white brackets in g and h indicate lipid lamellae (LL). CD, corneodesmosomes. Scale bar in a: 0.2 µm for a,b,c,d; 0.1 µm for e,f; and 0.25 µm for g,h.

View larger version (65K): [in a new window]   Fig. 5. Staining for neutral lipids and analysis of genes involved in epidermal barrier formation in skin from Brg1ep-/-(c) fetuses. (A) Nile Red staining for neutral lipids in the skin of E18.5 control (a) and Brg1ep-/-(c) fetuses (b). Arrowheads in b indicate an absence of neutral lipids. Scale bar in a: 16 µm. C, cornified layer; SB, suprabasal layer; B, basal layer. (B) Quantitative RT-PCR analysis of skin RNA from E18.5 Brg1L2/L2 (lanes 1 and 3) and Brg1ep-/-(c) (lanes 2 and 4) fetuses. (C) Real-time RT-PCR analysis of corneodesmosin on skin RNA from E18.5 Brg1L2/L2 and Brg1ep-/-(c) fetuses. **P<0.001.

View larger version (55K): [in a new window]   Fig. 6. Characterization of Tam-induced Cre recombinase activity in the surface ectoderm and forming epidermis of K14-Cre-ERT2 transgenic mice. (A) Whole-mount X-Gal-stained E10.5 K14-Cre-ERT2(tg/0)/ROSAfl/+ embryo after (a) oil and (b) 0.1 mg Tam injection to a female at E9.0. fl, forelimb; hl, hindlimb; p, first pharyngeal arch. (B) X-Gal-stained dorsal skin sections of E12.5 (a,b), E14.5 (c,d) and E18.5 (e,f) K14-Cre-ERT2(tg/0)/ROSAfl/+ fetuses after daily injections of oil (a,c,e) or Tam (b,d,f) to females from E9.5-E11.5 (a,b), E 9.5-E13.5 (c,d) and E12.5-E16.5 (e,f) days gestation. Sections were counterstained with safranin. E, epidermis; D, dermis; hb, hair bulb. Scale bar in a: 16 µm.

View larger version (73K): [in a new window]   Fig. 7. Characterisation of fetuses after temporally controlled Brg1 ablation in the forming epidermis. (A) Gross morphology of (a,c) control Brg1L2/L2, (b) Brg1ep-/-(ia) and (d) Brg1ep-/-(ib) E18.5 fetuses after daily Tam administration to pregnant females from E9.5-E13.5 (a,b) and E12.5-E16.5 (c,d). (B) Histological analysis of Toluidine Blue-stained semithin sections of dorsal skin biopsies from (a) control Brg1L2/L2 and (b) Brg1ep-/-(ib) E18.5 fetuses. C, cornified layer; G, granular layer; S, suprabasal layer; B, basal layer; D, dermis. Scale bar in a: 18 µm. (C) Transmission electron microscopy of stratum corneum (SC) (C1,C2 and C3 consecutive cornified cell layers) from dorsal skin of (a) Brg1L2/L2 and (b) Brg1ep-/-(ib) E18.5 fetuses. White arrows and white arrowheads point towards corneodesmosomes (CD) and lipid lamellae (LL), respectively. Scale bar in a: 0.25 µm. (D) X-Gal diffusion assay on (a) control Brg1L2/L2 and (b) Brg1ep-/-(ib) mutant E18.5 fetuses. (E) TEWL measured on E18.5 control Brg1L2/L2 and Brg1ep-/-(ib) fetuses. ***P<0.001.

View larger version (73K): [in a new window]   Fig. 8. Morphological and functional analysis of Brm-/-/Brg1ep-/-(ib) mutant fetuses. (A) Gross morphology of (a) Brm+/-/Brg1L2/L2 and (b) Brm-/-/Brg1ep-/-(ib) E18.5 fetuses. (B) Histological analysis of Toluidine Blue-stained semithin sections of dorsal skin biopsies from E18.5 Brm+/-/Brg1L2/L2 (a) and Brm-/-/Brg1ep-/-(ib) (b) fetuses. Epidermal cell layers are indicated: C, cornified; G, granular; S, spinous; B, basal; D, dermis. Scale bar in a: 12 µm. (C) Immunohistochemical detection of proliferation marker KI67 (pink colour) on dorsal skin sections from E18.5 control (a), Brg1ep-/-(c) (b) and Brm-/-/Brg1ep-/-(ib) (c) fetuses. Nuclei are stained with DAPI (blue). B, basal; SB, suprabasal; D, dermis. Scale bar in a: 25 µm. The dotted line indicates the dermal-epidermal junction. (D) Percentage of KI67-positive basal cells in the dorsal skin of E18.5 control, Brg1ep-/-(c) and Brm-/-/Brg1ep-/-(ib) fetuses. The number of KI67-positive cells out of 3000 DAPI-stained interfollicular basal cells were determined on four 10-µm thick frozen skin sections from three controls and three mutants. Values expressed as a percentage are the mean values±s.e.m. (n=12). (E) Transmission electron microscopy of dorsal skin from E18.5 Brm+/-/Brg1L2/L2 (a) and Brm-/-/Brg1ep-/-(ib) (b) fetuses. SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum. Arrows point to swollen cells in the spinous and granular layers. Scale bar: 4 µm. (F) Barrier-dependent X-Gal diffusion assay on E18.5 Brm+/-/Brg1L2/L2 (a) and Brm-/-/Brg1ep-/-(ib) (b) fetuses. (G) TEWL on dorsal and ventral skin of E18.5 Brg1L2/L2, Brg1ep-/-(ib), Brm+/-/Brg1L2/L2 and Brm-/-/Brg1ep-/-(ib) fetuses. *P<0.05.