Lee JY , Harland RM .

F32 HD052374-02 NICHD NIH HHS , F32HD052374-01 NICHD NIH HHS , GM42341 NIGMS NIH HHS , R01 GM042341-21 NIGMS NIH HHS , R01 GM042341-25 NIGMS NIH HHS , R01 GM042341-26 NIGMS NIH HHS , R01 GM042341 NIGMS NIH HHS , F32 HD052374 NICHD NIH HHS

	Fig. 1. previous termXenopusnext term laevis bottle cell formation. (A) Embryo orientation. Lateral illustration (left panel, Stage 8) and vegetal illustration (middle, Stage 10) from Nieuwkoop and Faber (1994). Dotted line through vegetal view shows the mid-sagittal plane. Abbreviations: DMZ, dorsal marginal zone; BC, blastocoel; Br.c, Brachet's cleft; BP, blastopore. (B) Time-lapse images of bottle cell formation in a whole embryo (top) and in a dorsalateral marginal zone explant. Time elapsed (in minutes) noted in the bottom left-hand corner of each panel. Small arrows point to sites of bottle cell formation. Movies of this embryo and explant can be found in the Supplementary materials. (C) Confocal midsagittal images of DMZs stained with α-tubulin antibody from late stage 9 (left) to stage 10.25 (right) showing progression of bottle cell formation (small arrows) and blastopore groove formation. In this and all DMZ midsagittal views, embryos are oriented with vegetal to the left and apical side down. (D) Measuring apical constriction. Left panel shows a confocal image of a midsagittal section of a DMZ stained with α-tubulin antibody. Asterisks indicate cells undergoing apical constriction, which are illustrated in middle and right panels. Blastopore depth (d), indicated by the length of the arrow in the middle panel. The right panel illustrates the apical index (AI), which is the cell length (l) divided by the apical width (aw). Scale bar = 50 μm.
	Fig. 3. F-actin is required for bottle cell formation. (A) Actin inhibitors latrunculin B (top) and cytochalasin D (bottom) reversibly prevent bottle cell formation. Control embryos were in 1% DMSO. Bar graph shows percent of embryos making bottle cells in the presence of DMSO control or inhibitor. n, number of embryos. Error bars = 2S.E. (B) F-actin distribution as indicated by Oregon green-conjugated phalloidin in control, latrunculin B, and cytochalasin D-treated embryos.
	Fig. 4. Myosin function is required for blastopore groove formation and efficient constriction. (A) Blebbistatin-treated whole embryos (top right) and explants (middle right) make bottle cells but exhibit weak apical constriction compared to control (left panels). Bottom row of pictures show control and blebbistatin-treated embryos immunostained with pMLC primary antibody and Texas Red secondary antibody. The Texas Red secondary antibody results in nonspecific staining, allowing visualization of cell outlines for quantitative analysis (compare with Alexa 488 secondary shown in [Fig. 2] and [Fig. 4], and Supplementary Figs. 2A, B; see Materials and methods). (B) Apical width, cell length, and apical index in control versus blebbistatin-treated bottle cells. Asterisks denote p ≤ 0.001. (C) ML-7 treatment results in shallower blastopore invagination (top) and reduced pMLC staining. (D) Apical width, cell length, and apical index in control versus ML-7-treated bottle cells. (E) Blebbistatin and ML-7 treatment both result in significantly shallower blastopore depths compared to controls. Error bars = 2S.E.; p ≤ 0.001 for both control vs. blebbistatin and control vs. ML-7. Scale bar = 50 μm.
	Fig. 5. Intact microtubules are required for efficient apical constriction, but not elongation, of bottle cells. (A) Nocodazole affects bottle cell formation by affecting apical constriction. Middle panels show morphological differences between the bottle cells forming in control versus in nocodazole-treated embryos. Nocodazole treatment disrupts α-tubulin staining. Bar graph shows quantitation of bottle cell morphology. Only apical width and apical index are significantly different in presence of nocodazole (p ≤ 0.0001). (B) Taxol stabilizes microtubules (see also Supplementary Fig. 6) without affecting blastopore formation or bottle cell morphology. Error bars = 2S.E.

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