A: The phenotypic analysis in this study was performed with Cad74A^38A, which has the entire coding region of Cad74A (on chromosome 3L) deleted. B: Protein domain structure of Cad74A. Based on electronic annotation, Cad74A is a conserved putative cell adhesion molecule with 13 cadherin domains, an N-terminal signal peptide, a transmembrane domain, and a short uncharacterized cytoplasmic domain. (; ). Putative cadherin domains were determined using the SMART tool (http://smart.embl-heidelberg.de) (). (C–F): Images of egg chambers after mRNA in situ hybridization. Cartoons highlighting the expression pattern are included below the images of egg chambers. C: Cad74A transcript is not detected during early oogenesis. D: Dorsal view of a stage 10 egg chamber; Cad74A is also expressed in the border cells (arrow) at the end of border cell migration. The following are marked on the cartoon: nurse cells (NCs), main body follicle cells (MBFCs), and roof cells. E: Dorsal view of a stage 12 egg chamber. F: Stage 13 egg chamber; Cad74A is strongly expressed in the apical region of the cells forming the respiratory appendages (dorsal appendages, DAs) and in the operculum domain. (G–J): In situ hybridization images of stage 10–11 egg chambers for fs(1)K10 and CY2>mae genetic backgrounds and D. pseudoobscura (D. pse.) and D. virilis (D. vir.). G: A late stage 10 fs(1)K10 mutant background shows repression of Cad74A expression along an anterior lateral ring that extends to the ventral side. Cad74A is expressed along the dorsal midline and in the anteriormost follicle cells. H: Cad74A expression in a stage 10 CY2>mae mutant background egg chamber. I: Cad74A expression in stage 10 of D. pse. using the D. mel. Cad74A antisense probe. J: Cad74A expression in D. vir. The patches of reduced Cad74A expression corresponds to the patches of BR protein reported for D. virilis. (). (K–N): ESEM images of the ventral appendage sheath of a fs(1)K10 egg (K), CY2>mae egg (L), a D. pse. egg (M), and the dorsal-anterior egg structures for D. vir. with four DAs (N).

(A, B): Immunostaining for Cad74A in a stage 11 egg chamber confirms the localization and expression of Cad74A along the apical surfaces and in the same expression pattern as the mRNA expression pattern with repression of protein in the roof cells. Cad74A is more punctate in dorsal midline and floor cells (marked by arrow) in a stage 11/12 egg chamber, cells expressing rho-LacZ (marked by green in A) provide a reference point for the location of the floor cells (). (C, D): Cad74A localization in a stage 12 egg chamber, lateral view. Punctate Cad74A is visible near the apices of the floor cells. (E): Localization of Cad74A in a magnified view of a single MBFC during mid-oogenesis (stage 10B). Cad74A is found in the microvilli and apical surface of the follicle cells (apical-basal axis indicated by arrow). (F): Cad74A remains localized to the apical surface at later stages in the MBFCs. The microvilli length is shorter in stage 12 MBFCs (a single magnified cell is shown). (G-I): At stage 12, Cad99C (G) and Cad74A (H) are at elevated levels in different cells. Phalloidin staining is brighter in cells with high levels of Cad99C (I). (J–M): No immunoreactivity to the Cad74A antibody is detected in Cad74A^38A/38A egg chambers (J, K), in contrast to CY2>Cad74A-GFP egg chambers (L, M), which show strong Cad74A and GFP (not shown) staining in stage 11 MBFCs. (N, O): In E4>Cad74ARNAi, egg chambers, Cad74A staining is lost in the poster MBFCs, demonstrating the efficacy of the UAS-Cad74ARNAi line and the specificity of the Cad74A antibody. (P–R): Ectopic Cad74A-GFP expression, driven by the GR1-GAL4 driver in an early stage 10B egg chamber (P), is recognized by the Cad74A antibody (Q).

A: In CY2>λtop (up-regulation of EGFR signaling in all MBFCs), Cad74A expression is uniform in the follicle cells overlaying the oocyte except for the anteriormost row of follicle cells. B: At later stages, the repression seen in the anterior region is replaced with a dark band of expression, followed by a band of repression (arrowhead), and a second band of dark expression. The band of repression likely corresponds to the boundary of operculum material. C: BR protein staining with slightly brighter staining in a band (arrowhead) that may correspond to Cad74A repression in (B) (arrowhead). D: In CY2>λtop, The eggshell morphology is completely dorsalized so no dorsal appendages, which are normally dorsolateral, form. E: In CY2>dnEGFR (down regulation of EGFR), Cad74A is expressed uniformly as well. The anteriormost row of follicle cells is stained more darkly. F: Uniform expression continues in later egg chambers. G: BR staining in stage 12 CY2>dnEGFR. H: In CY2>dnEGFR, the eggshell is ventralized. I: In CY2>tkv^* (ectopic BMP signaling), the dorsal-anterior expression of Cad74A is repressed, which corresponds to the loss of dorsal appendages and increased operculum seen in the final eggshell morphology. J: At later stages, a band of Cad74A repression is clearly seen, corresponding to the operculum boundary shown in (L). K: BR staining in stage 12 CY2>tkv^* egg chamber. L: In CY2>tkv^*, the dorsal appendages are absent and the operculum is enlarged. M: In CY2>Dad (downregulation of BMP signaling), the patches of Cad74A repression are larger at earlier stages. N: In a stage 12 egg chamber the patches of repression are smaller and shifted in the anterior direction. O: BR staining in a different stage 12 egg chamber. P: An anterior shift in the dorsal appendages is seen in a CY2>Dad egg. (Q–S): Complete loss of EGFR signaling in the roof cells homozygous for the null Ras allele (Ras^ΔC40b) reduces BR to a basal level equivalent to the level in the posterior MBFCs. Cad74A is ectopically expressed in the roof cells (marked by an arrow) (T, U): An earlier Ras clone located in the roof cells in a stage 10B egg chamber (T) does not show significant ectopic Cad74A (U). (V): In the hypomorph Ras background Ras^0503/E62K, Cad74A transcript is lost in the midline due to the loss of PNT and ectopic BR (not shown) in the dorsal in the dorsal anterior bridge. Here, and in other figures, the dotted line marks the dorsal midline. W: Summary of Cad74A expression and BR levels in three different regions of the follicular epithelium which is maintained across the various examined perturbations in EGFR and BMP signaling: Region I (dorsal midline): No BR staining is detected, and Cad74A expression is slightly delayed and at lower levels. This effect is most apparent in CY2>tkv^* egg chambers, but is also found in WT (, ). Region II: High BR levels in the roof cells and no Cad74A. Region III: Basal levels of BR and strong Cad74A expression.

(A–C): The high level of BR in the roof cells (A), which correlates with Cad74A repression in those cells (B), as seen in the merged image (C), suggests the possibility that BR represses Cad74A. (D–F): Overexpression of br-Z1 in CY2>br-Z1 (D) often leads to a loss of Cad74A in the dorsal anterior bridge (E) and more patchy expression in the posterior and ventral regions of the egg chamber (F). (G–I): BR-Z1 is more highly expressed in the anterior, even when driven by the driver CY2-GAL4, which drives patchy expression throughout the MBFCs (G, I), resulting in a loss of Cad74A in the dorsal anterior. (J–O): In mosaic egg chambers, clones homozygous for the null allele br^npr-3, marked by BR (J), result in relieved repression of Cad74A in the roof cells (L, N). Panels K, M, O are magnified images of the roof cells shown in panels J, L, N.

A: Ectopic expression of the transcription factor pntP1 (CY2>pntP1) results in ectopic and early expression of Cad74A (B) which overcomes repression of Cad74A in the roof cells (C). (D–F): Loss of PNT activity in the CY2>Mae background results in ectopic BR in the dorsal midline (D) accompanied by a loss of Cad74A (E). (G–I) Examination of Cad74A expression in numerous ct^db7 clones, marked by the loss of nuclear GFP (G), suggests that Cad74A is not regulated by the transcription factor Cut (H, I).

(A, B): In situ hybridization of Cad74A in control egg chambers (A) is completely absent in honmozygous Cad74A^38A egg chambers (B). (C–F): Severe DA phenotypes in homozygous Cad74A^38A egg chambers range from a complete loss of DAs (C) to DAs of varying lengths not seen in the control parental line (D–F).

A: DAs of br>GFP eggs project out of the main body of the egg. B: Shortened, flattened DAs in a br>Cad74A-GFP53A egg. C: The br>Cad74A-GFP53A flies lay eggs with a high penetrance of flattened, short DAs (285/308 eggs scored, 93%) compared to the sister flies (br-GAL4/TM6C, 42/188, 22%) or br>GFP (2/51, 4%) flies. (D–F): br-GAL4 strongly drives UAS-Cad74A-GFP53A in the roof cells, resulting in large aggregates of Cad74A in the apical half of the roof follicle cells. (G-N): Compared to control sister flies (G), br>Cad74A-GFP53A egg chambers at stage 11/12 show defects in apical constriction, accompanied by the formation of large aggregate bodies (H–J). The slanted lines mark the dorsal midline. (K–N) are magnified images of the roof cells, the disruption in apical constriction is indicated by an arrow in (M). (O-R): Compared to control sister flies (O), migration of the roof cells is severely affected in late stage 13/14 br>Cad74A-GFP53A eggs (P–R). Arrows in (P–R) mark the anterior extent of roof cell migration, as marked by BR in (R).

A: In model I, Cad74A is activated by a uniform signal starting at stage 10 and repressed by BR in the roof cells. B: In an alternate model, Cad74A expression requires a uniform signal, repression by an unknown dorsal TF, and activation by a midline activator (such as PNT). C: Regulation of Cad74A by the EGFR and BMP pathways based on the results of BR regulating Cad74A (this work) and previously reported research (). Based on ectopic expression of Cad74A in the roof cells, we propose that Cad74A mediates adhesion to the oocyte, but do not rule out other functions such as secretion of the chorion. D: Expression patterns for several effector genes and proteins at stage 10B, including: Cad74A (this work), Echinoid (ED) (), basal E-CAD (), FC-α-actinin (), 18w () and FAS3 () possibly are regulated by BR, either directly or indirectly, in a synergistic and coordinated manner to ensure robust eggshell morphogenesis and the formation of the DAs.