(A–C) Transverse sections of embryos pulsed for 2 hours with EdU (green), counterstained with anti-laminin (red) and Hoechst stain (blue) showing that the region that will undergo dorsolateral expansion (boxed, HH 16), or was undergoing dorsolateral expansion at the time the embryo was collected (boxed, HH 18 and 20). The dorsolateral otocyst contains few EdU-positive (i.e., mitotic cells in the S-phase of the cell cycle) cells, as compared to other regions of the developing otocyst, indicating that cell proliferation is not increased in the dorsolateral otocyst during its rapid expansion. D, dorsal; L, lateral; no, notochord; nt, neural tube; ed, endolymphatic duct. (A’-C’) Enlargements of the boxes in A–C showing epithelial thinning in the dorsolateral otocyst during its expansion. (D–G) Confocal imaging showing that E-cadherin (red; cell nuclei labeled with Hoechst staining, blue) is broadly distributed along the entire apicobasal (a–b) axis of the dorsolateral epithelium at HH 16, and then fragments as dorsolateral epithelial cells undergo thinning during HH 18–24. 1, panel showing the entire otocyst. 2, boxed area in panel 1 enlarged. 3, area in panel 2 containing two cells marked with asterisks enlarged. 4, panel showing a lateral view of the epithelium at the level of the green line in panel 3. D, dorsal; A, anterior.

(A) Over expression of Bmp4 results in formation of an enlarged (hyperexpanded) otocyst (ot) (right side of figure), as compared to the control otocyst (left side of figure), as shown in whole mounts at HH 21. The wall of the otocyst is outlined, along with the endolymphatic duct (ed). A, anterior; D, dorsal; ed, endolymphatic duct. (B, C) Transverse sections (HH 20; B, labeled with pSMAD1/5/8 antibody [red] and Hoechst stain [blue]; C, labeled with E-cadherin antibody [green] and Hoechst stain [blue]) show that Bmp4 over expression results in an increase in the extent of the dorsolateral otocyst that expresses pSMAD1/5/8 (B; asterisks) as well as an enlargement of the area undergoing rapid thinning and expansion (C; asterisks). D, dorsal; L, lateral; ed, endolymphatic duct. (B’) Graph showing the mean number of pSMAD1/5/8-positive cells (plus/minus standard error of the mean) in the dorsolateral (thinned) region in control (Cont) otocysts, and in otocysts subjected to Bmp4 over expression (4–5 sections through the mid-anteroposterior extent of the otocyst in 3 control and 3 experimental embryos were counted). *P < 0.05 (Student’s T-test). (C’) Graph showing the mean length (in the mediolateral plane) (plus/minus standard standard error of the mean) of the dorsolateral (thinned) region in control (Cont) otocysts, and in otocysts subjected to Bmp4 over expression (3 control and 3 experimental embryos were counted, and the thinned region from 4–5 sections of individual embryos were measured). *P < 0.05 (Student’s T-test).

(A) Treatment of embryos with Dorsomorphin results in the formation of slightly smaller otocysts, as compared to those of the controls, as viewed in confocal reconstructions of otocysts labeled with E-cadherin at HH 20. D, dorsal; A, anterior. The asterisks mark the dorsolateral otocyst, which undergoes normal expansion in the control otocyst, but not in the experimental. (B) Transverse sections (HH 20; labeled with pSMAD1/5/8 antibody [red] and Hoechst stain [blue]) after Dorsomorphin treatment show that pSMAD1/5/8 expression occurs in the control dorsolateral otocyst but not in the experimental dorsolateral otocyst (these regions are marked with asterisks). D, dorsal; A, anterior; nt, neural tube; ed, endolymphatic duct. (C) Confocal imaging showing that the dorsolateral otocysts of Dorsomorphin-treated embryos display an apically-basally broad E-cadherin (red; Hoechst staining of nuclei, blue) distribution similar to that of the normal dorsolateral otocyst cells at stages prior to epithelial thinning and expansion (i.e., at HH 16), whereas control otocysts show fragmented E-cadherin. D, dorsal; A, anterior; a, apical; b, basal. 1, panel showing the entire otocyst. 2, boxed area in panel 1 enlarged. 3, area in panel 2 containing two cells marked with asterisks enlarged. 4, panel showing a lateral view of the epithelium at the level of the green line in panel 3.

(A) Confocal reconstructions of anti-E-cadherin-labeled otocysts at HH 16, 18, and 20. Asterisks mark the dorsolateral wall of the otocyst that undergoes rapid thinning and expansion. D, dorsal; V, ventral; A, anterior; P, posterior; ed, endolymphatic duct. (B–D) Transverse H&E-stained sections of otocysts at HH 16–20. (B’-D’) Enlargements of the boxes in B–D showing epithelial thinning in the dorsolateral otocyst wall during its expansion. D, dorsal; L, lateral; nt, neural tube; no, notochord; ed, endolymphatic duct. (E) Graph showing measurements of cells (maximal mean cell heights, red line, measured in the apicobasal dimension of the epithelium; maximal mean cell widths, blue line, measured at the widest part of the cell, which coincided with the mid apicobasal level of the nucleus; bars indicate standard error of the mean) and confocal reconstruction of cell shapes (red, Hoechst staining of nuclei; green, cytoplasmic GFP transfected into the dorsolateral otocyst at HH 15) over about 2.5 days of development showing that the pseudostratified columnar epithelial cells of the dorsolateral otocyst at HH 16 transform into cuboidal epithelial cells by HH 18, and then into squamous epithelial cells by HH 20 (HH 24 is pictured and measured). a, apical side of cell; b, basal side of cell.

(A–C) As shown by whole mount in situ hybridization, Smad6, an inhibitor of BMP signaling, is expressed in the otocyst (but not in its dorsolateral wall; asterisks) during HH 16–20 when rapid thinning and expansion occurs (HH 16: n = 6; HH 18: n = 18; HH 20: n = 16). The line in (B) marks the level of the section shown in (D). Sections (D) reveal more clearly that expression occurs only in the ventrolateral wall of the otocyst, not in its dorsolateral wall (asterisks). D, dorsal; A, anterior; L, lateral; nt, neural tube. (E–G) Transverse sections (labeled with pSMAD1/5/8 antibody [red] and Hoechst stain [blue]) showing that pSMAD1/5/8 is expressed in the dorsolateral otocyst (asterisks; HH 16: n = 9; HH 18: n = 10; HH 20: n = 7), in contrast to Smad6, during HH 16–20. D, dorsal; L, lateral; nt, neural tube; ed, endolymphatic duct.

(A, A’) Whole mount (A) and transverse section (A’; unstained) showing GFP-expressing cells (green) in the mesenchyme posterior to the otocyst 12 hours after sonoporation. The otocyst (ot) and endolymphatic duct (ed) are outlined in A. D, dorsal; A, anterior; L, lateral. (B) Reconstruction showing that at HH 30 (10 days of incubation) the semicircular canals (ssc, lsc, psc), but not the cochlear duct (cd), endolymphatic duct (ed), and endolymphatic sac (es; present but truncated), are absent on the side of the embryo (left side of figure) in which Noggin was over expressed (inner ear structures are shown in purple on the Noggin-over expression side; and in green on the control side). ca, cristae ampullaris; la, ampulla of the lateral semicircular canal, pa, ampulla of the posterior semicircular canal. (C) Confocal reconstructions of HH 24 otocysts labeled with E-cadherin showing that expansion of the dorsolateral otocyst is severely attenuated 3 days after sonoporation of the Noggin vector (bottom image), as compared to control otocysts (top image). Asterisks mark the dorsolateral otocyst, which has undergone normal expansion in the control otocyst but not in the experimental otocyst, whereas outgrowth of the ventral otocyst and endolymphatic duct (ed) is not affected by Noggin over expression. D, dorsal; A, anterior. (D) Confocal imaging showing that in otocysts subjected to Noggin over expression, dorsolateral cells remain as elongated pseudostratified columnar epithelial cells, and E-cadherin (red; Hoechst staining of cell nuclei, blue) remains broadly expressed along the apicobasal (a, b) surface of these cells, in contrast to control embryos in which dorsolateral cells become cuboidal to squamous and E-cadherin expression becomes fragmented. D, dorsal; A, anterior. 1, panel showing the entire otocyst. 2, boxed area in panel 1 enlarged. 3, area in panel 2 containing two cells marked with asterisks enlarged. 4, panel showing a lateral view of the epithelium at the level of the green line in panel 3.

(A, A’) Whole mount and transverse section of the otocyst after its electroporation with both GFP- and LacZ-expressing vectors (GFP: 10–12 hours after electroporation; LacZ: 24 hours after electroporation). The section (HH 20; counterstained with eosin) shows that vector-derived gene expression is restricted to the dorsolateral otocyst (blue staining). D, dorsal; A, anterior; L, lateral; en, endolymphatic duct. (B) Transverse sections (HH 20; labeled with pSMAD1/5/8 and Hoechst stain) showing that the number of pSMAD1/5/8-positive cells is decreased in the dorsolateral otocyst (asterisks) by 24 hours after Smad6 over expression, but not in the control. D, dorsal; L, lateral; nt, neural tube. (B’) Enlargement of the boxed region of the otocyst in which Smad6 was over expressed, showing that pSMAD1/5/8 labeling (red) is abolished only in transfected cells (green). (C) Confocal reconstructions of otocysts labeled with E-cadherin at HH 20 showing that Smad6 over expression results in hypoplastic otocysts that fail to undergo expansion of their dorsolateral region (asterisks), as compared to control otocysts. D, dorsal; A, anterior; ed, endolymphatic duct. (D) Confocal imaging showing that E-cadherin (red; cell nuclei stained with Hoechst, blue; GFP, green, indicating transfected cells) is broadly expressed along the apicobasal (a–b) axis of the dorsolateral epithelium of the otocyst in which Smad6 was expressed, rather than being expressed in a fragmented pattern as in control otocysts in which GFP was ectopically expressed. D, dorsal; A, anterior, a, apical; b, basal. 1, panel showing the entire otocyst. 2, boxed area in panel 1 enlarged. 2a shows only the red (E-cadherin) channel; 2b shows the green (GFP, indicating transfected cells) and blue (Hoechst stained nuclei) as well. 3, area in panel 2 containing two cells marked with asterisks enlarged. 4, panel showing a lateral view of the epithelium at the level of the green line in panel 3.