Figure 2.
Figure 2. Notch signaling is associated with ciliogenesis in differentiating embryonic epidermis but is diminished upon localization of ARL13b and Presenilin-2 to cellular junctions. (A) E17.5 epidermis immunolabeled with an antibody against acetylated tubulin (green) or ARL13b (red) to visualize primary cilia in either basal or suprabasal layers. (B) Image of suprabasal ciliated cells (magnified from boxed region in A), colabeled for the Notch reporter GFP-TNR. Note that in the suprabasal layers, a good correlation exists between GFP-TNR expression and ciliation. In contrast, nonciliated cells within the same suprabasal plane display low GFP-TNR expression (indicated by an asterisk). (C) Quantification of the number of cilia in GFP (+) vs. GFP (−) E17.5 TNR epidermis. Histogram shows percentage of ciliated cells per confocal plane and is an mean of data from three embryos. Error bars are SEM. *, P < 0.05, Student’s t test. (D) Transmission electron microscopy of E17.5 WT epidermis, showing an example of a primary cilium from one cell appearing to “poke into” an adjacent cell. Suprabasal cells are false-colored pink to highlight their position relative to ciliated cell below. Kf, keratin filaments. Image from boxed panel is magnified at right; note the plasma membrane of one cell juxtaposed with ciliary membrane from neighboring cells. (E) Single confocal planes either basal or suprabasal layers (as indicated) of E16.5–E17.5 WM epidermis immunolabeled with an antibody against ARL13b. Note junctional localization in upper suprabasal layers. (F) Sagittal section of E17.5 epidermis immunolabeled with antibody to Presenilin-2 (green) and DAPI (blue) to mark chromatin. Note junctional localization of Presenilin-2 in the upper suprabasal layers. (G) Single confocal plane of E16.5 WM epidermis immunolabeled with an antibody against Presenilin-2. Boxed region is shown magnified in right panel; arrowhead points to basal body. (H) Quantification of the percentage of cells in the three different suprabasal layers of developing skin that show either nuclear HES1 or junctional Presenilin-2 and ARL13b immunolabeling. Data in histogram were analyzed from E17.5 epidermis; n = 2 embryos per condition. Error bars are SEM. Bars: (A–C, E, and G) 15 µm; (D) 0.5 µm; (F) 30 µm.

Notch signaling is associated with ciliogenesis in differentiating embryonic epidermis but is diminished upon localization of ARL13b and Presenilin-2 to cellular junctions. (A) E17.5 epidermis immunolabeled with an antibody against acetylated tubulin (green) or ARL13b (red) to visualize primary cilia in either basal or suprabasal layers. (B) Image of suprabasal ciliated cells (magnified from boxed region in A), colabeled for the Notch reporter GFP-TNR. Note that in the suprabasal layers, a good correlation exists between GFP-TNR expression and ciliation. In contrast, nonciliated cells within the same suprabasal plane display low GFP-TNR expression (indicated by an asterisk). (C) Quantification of the number of cilia in GFP (+) vs. GFP (−) E17.5 TNR epidermis. Histogram shows percentage of ciliated cells per confocal plane and is an mean of data from three embryos. Error bars are SEM. *, P < 0.05, Student’s t test. (D) Transmission electron microscopy of E17.5 WT epidermis, showing an example of a primary cilium from one cell appearing to “poke into” an adjacent cell. Suprabasal cells are false-colored pink to highlight their position relative to ciliated cell below. Kf, keratin filaments. Image from boxed panel is magnified at right; note the plasma membrane of one cell juxtaposed with ciliary membrane from neighboring cells. (E) Single confocal planes either basal or suprabasal layers (as indicated) of E16.5–E17.5 WM epidermis immunolabeled with an antibody against ARL13b. Note junctional localization in upper suprabasal layers. (F) Sagittal section of E17.5 epidermis immunolabeled with antibody to Presenilin-2 (green) and DAPI (blue) to mark chromatin. Note junctional localization of Presenilin-2 in the upper suprabasal layers. (G) Single confocal plane of E16.5 WM epidermis immunolabeled with an antibody against Presenilin-2. Boxed region is shown magnified in right panel; arrowhead points to basal body. (H) Quantification of the percentage of cells in the three different suprabasal layers of developing skin that show either nuclear HES1 or junctional Presenilin-2 and ARL13b immunolabeling. Data in histogram were analyzed from E17.5 epidermis; n = 2 embryos per condition. Error bars are SEM. Bars: (A–C, E, and G) 15 µm; (D) 0.5 µm; (F) 30 µm.

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