Journal: Genes & Development
Article Title: GATA-3: an unexpected regulator of cell lineage determination in skin
Figure Lengend Snippet: Expansion and aberrancies in expression patterns of cortical and IRS precursor populations in GATA-3 -null follicles. Skins from matched P35 wild-type and GATA-3 -null grafts were frozen, sectioned (10 μm), and subjected to either triple indirect immunofluorescence ( A – K ) or in situ hybridization ( L,M ). The genotype of each section is denoted at the upper right . Abs used are color-coded and indicated across the bottom of each frame. Asterisks in B and I denote autofluorescence, an artifact of the knots of highly keratinized material in some KO shafts. Boxed areas are shown at higher magnification in the insets . DAPI (blue) denotes nuclei. Abs were against GATA-3, present in the IRS lineage, and absent in the GATA-3 -null sections; β-galactosidase (Bgal), specific for GATA-3 promoter activity, which was indistinguishable from the anti-GATA IRS labeling in wild-type follicles, and still present in the expanded IRS precursor population of KO follicles; AE13, specific for the keratins of differentiating cortical cells; FOG1, a corepressor for GATA-3 protein, present in IRS precursor cells, where it marked the nuclei of wild-type but the cytoplasm of KO cells ( D and E , respectively; see inset to E as well); Lef-1, specific for cortical precursor cells, expanded in the KO; Ki67, specific for proliferating cell nuclei; and K5, specific for the ORS of the follicle. The cRNA probe is against Shh , Sonic hedgehog, which marks a small subset of matrix cells in wild-type follicles, unchanged in KO follicles. Note that in KO follicles, some AE13-positive cortical and Lef-1-positive precortical cells were also positive for GATA-3 promoter activity (representative cells indicated by arrows in C and G ). Note also that Ki67 labeling confirms the anagen state of both wild-type and KO follicles, and some Ki67-positive cells were also positive for GATA-3 promoter activity ( J,K ).
Article Snippet: The primary antibodies at the indicated concentrations were AE13 (mouse, 1:50–1:100; ), AE15 (mouse, 1: 10; ), K5 (gp, 1:250; Fuchs lab), GATA-3 (mouse, 1:100; Santa Cruz, HCG3-31), β-galactosidase (rabbit, 1:400; mouse, 1:100; Harlan; Sigma), K1 (rabbit, 1:250; Fuchs lab), loricrin (rabbit, 1:300; Fuchs lab), Lef-1 (rabbit, 1:250; Fuchs lab), filaggrin (rabbit, 1:1000; Covance, PRB-417P), K6 (rabbit, 1:500; Fuchs lab), involucrin (mouse, 1:200; Babco), Ki67 (rabbit, 1:1000; NovoCastra Laboratories Ltd.), and FOG1 (goat, 1:50; Santa Cruz, sc-9361).
Techniques: Expressing, Immunofluorescence, In Situ Hybridization, Activity Assay, Labeling