Hayes JM , Kim SK , Abitua PB , Park TJ , Herrington ER , Kitayama A , Grow MW , Ueno N , Wallingford JB .

R01 GM074104-03 NIGMS NIH HHS , R01 GM074104-04 NIGMS NIH HHS , R01 GM074104-04S1 NIGMS NIH HHS , R01 GM074104 NIGMS NIH HHS

	Fig. 1. A screen for differentially expressed genes in a mucociliary epithelium. (A) The Xenopus epidermis is a salt-and-pepper mix of mucus-secreting goblet cells and ciliated cells. Ciliated cells are marked by red α-tubulin staining. Small secretory cells are labeled with an asterisk. All other cells are goblet cells. (B) At higher magnification, membrane-GFP (green) reveals numerous exocytic vesicles at the apical surface of goblet cells, and α-tubulin staining (red) reveals cilia. (C) The vesicles of small secretory cells are shown by phalloidin stain (green) and a neighboring ciliated cell is marked by alpha-tubulin stain (red). (c′) A high magnification view of a small secretory cell shows visible vesicles. (D) A diagram of cell types in the Xenopus epidermis. Goblet cells are the predominant cell type. Ciliated and small secretory cells (asterisks) are scattered throughout. (E) In situ hybridization for genes expressed in ciliated cells produces regularly spaced dark spots on a light background. (F) Genes expressed in goblet cells produce a reciprocal pattern. (G) Small secretory cells are visible as unevenly scattered dark spots. The full set of differentially expressed genes can be found in Supplemental Table 1.
	Fig. 3. Representative expression patterns from screen. Expression of coiled-coil domain containing 19, (A) and Doublecortin domain-containing 2 (B) in ciliated cells are evenly spaced and colocalize with α-tubulin marking cilia. (C) The transcription factor CP2 is expressed in the scattered secretory cells, and its expression does not colocalize with cilia. (D) Similar to Otogelin is expressed in mucus-secreting goblet cells covering much of the epidermis. High magnification view reveals absence of expression in scattered secretory and ciliated cells.
	Fig. 5. TEX15, a ciliated cell marker is expressed in presumptive ciliated cells and other ciliated tissues and is negatively regulated early on by Notch. TEX15 is expressed in ciliated tissues such as the midline (A) and the future ear (B). Expression in presumptive ciliated cells in early development (C) is severely reduced by Notch-icd (D).
	Fig. 6. Small, scattered cells are negative regulated by Notch. In situ hybridization shows that genes expressed in scattered cells do not colocalize with cilia (A–A′). Foxa1and CP2 are expressed throughout the embryo at tadpole stages (B, D). The uneven distribution of the cells is seen at higher magnification (B′, D′). Injection of Notchicd reduces expression of scattered cell markers in Foxa1(C, C′) and CP2 (E, E′). Slc16a3 marks scattered cells early in development (F) and its expression is also reduced by Notchicd (G).
	Fig. 8. TTC25 is required for ciliogenesis. (A) TTC25 is expressed in the evenly distributed pattern of ciliated cells, and colocalizes with cilia (B–b′). (C) Ciliated cells in normal embryos develop dozens of long, thin cilia (red, anti-α-tubulin staining). (c′) Side view of cell shown in panel A. (D, d′) Ciliated cells in embryos injected with TTC25 MO develop only short, fat cilia. Such diminutive cilia have also been observed in embryos mutant for IFT proteins (Huangfu and Anderson, 2005).
	Fig. 9. Mig12 is required for ciliogenesis. (A–a′) Mig12 in situ shows expression in the evenly distributed pattern of ciliated cells and colocalizes with cilia. (B–D) Mosaic epidermis was generated by targeted co-injection of Mig12-MO and membrane-GFP. GFP-positive cells containing the MO fail to develop cilia (arrowheads). Neighboring GFP-negative cells do not contain MO and exhibit prominent tufts of large, normal cilia.
	Fig. 11. Novel ciliogenesis factors are required in the midline for neural tube closure. (A and D) Dorsal view of TTC25 and Mig12 in situ shows expression in the ventral midline of the neural plate during neural tube closure. Dorsal view of an embryo injected with TTC25-MO (B) or Mig12-MO (D) both displaying a severe neural tube closure defect.
	ubp1 (upstream binding protein 1 (LBP-1a)) gene expression in Xenopus laevis, NF stage 25, assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	mid1ip1 (MID1 interacting protein 1) gene expression in Xenopus laevis, NF stage 18, assayed by in situ hybridization, dorsal view, anterior up.
	slc16a3 ( solute carrier family 16 (monocarboxylate transporter), member 3) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 16, lateral view, anterior left, dorsal up.
	odad4 (outer dynein arm docking complex subunit 4) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 18, dorsal view, anterior up.
	odad4 (outer dynein arm docking complex subunit 4) gene expression in a Xenopus laevis embryo, assayed by in situ hybridization, NF stage 25, lateral view (trunk region only), anterior left, dorsal up.
	tex15 (testis expressed 15) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 17/18, lateral view, anterior left, dorsal up.
	tex15 (testis expressed 15) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 17/18, dorsal view, anterior left.
	tex15 (testis expressed 15) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 21, dorsal view, anterior left.
	Fig. 2. Ultrastructure of the Xenopus epidermis. (A) View of mucociliary epithelium including a ciliated cell, small secretory cell (marked by ∗) and several large goblet cells. (B) Goblet cells contain empty vesicles, noted with arrowheads, and vesicles with secretory granules being exocytosed. (C) Lateral view of a ciliated cell, showing the many motile, apical cilia.
	Fig. 4. Goblet cells in the Xenopus epidermis secrete Intelectin-2. (A) A Xenopus epidermis goblet cell; membrane-GFP (green) reveals the extensive exocytic vesicles; antibody staining demonstrates the presence of intelectin-2 (red) in these vesicles. Arrows mark vesicles absent of intelectin-2. (B–B′) High magnification of exocytic vesicles shown in panel A. (C–E) Transmission electron microscopy of a Xenopus epidermis goblet cell. (C) Three apically located vesicles at different stages of exocytosis are visible in an epidermal goblet cell. A mucus granule can be seen in the process of being secreted. (D) Membrane fusion intermediate during exocytosis. (E) Exocytic release.
	Fig. 7. Mig12 and TTC25 localize to basal bodies and ciliary axoneme. Inturned-GFP, shown here as a reference, is localized at the apical surface of the ciliated cell, but is not in the ciliary axoneme (A–a3). TTC25-GFP localizes to apical foci, presumably basal bodies, and also to the ciliary axoneme (B–b3). Mig12-GFP localizes to both basal bodies and the ciliary axoneme (C–c3). At normal gain levels, Mig12-GFP localizes very specifically to the basal bodies (C), clearly marking basal body orientation and ciliary polarity (c6). At high gain levels, the presence of Mig12-GFP in the ciliary axoneme is more apparent (c4–c5).
	Fig. 10. SEM confirms suppression of ciliogenesis in Mig12 and TTC25 morphants. (A) High magnification view of a control cell shows multiple, long cilia on the apical cell surface. (B, C) TTC25 morphants and Mig12 morphants both exhibit fewer, shortened apical cilia. (D–F) Five-base-pair mismatch morpholinos for TTC25 and Mig12 exhibit no ciliogenesis defects.
	Tuba4a AB2 (anti-α-tubulin), in red, showing ciliated cells in normal embryos which develop dozens of long, thin cilia.
	itln2 Ab1 (anti-intelectin2 ) in Xenopus laevis embryo, showing goblet cells of epidermis in red, NF stage 28.

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