Targeted Downregulation of s36 Protein Unearths its Cardinal Role in Chorion Biogenesis and Architecture during Drosophila melanogaster Oogenesis

Abstract

Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion's formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In our study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion's regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion's fragility but also induces severe structural irregularities on chorion's surface and entirely impairs fly's fertility. Moreover, we herein unveil a novel function of s36 chorionic protein in the regulation of number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress.

Figure 1. Follicle cell-specific and RNAi-mediated targeting of s36 chorionic gene causes generation of s36 protein-depleted ovaries in Drosophila melanogaster.

(A) Confocal laser scanning microscopy images of representative stage-10 (left) and late stage-13 (right) follicles of c355 > eGFP transgenic control cross. (B) Western blotting analysis of ovaries derived from s36-targeted (c355 > s36_RNAi) and control (c355-GAL4/+ and s36_RNAi/+) flies via utilization of an s36-specific antiserum. Actin served as protein of reference. (C) Gene-expression profiling of the six major chorionic genes s36, s38, s18, s16, s19 and s15 in s36-depleted (c355 > s36_RNAi) and control (c355-GAL4/+) ovaries through employment of an RT-sqPCR protocol (also, see Table S1). (D) Graphical presentation of comparative measurements (in fold: “x”) of relative protein abundance, using a Mascot score-based evaluation process, in between s36-targeted (c355 > s36_RNAi) (also, see Table S2) and control (c355-GAL4/+) ovaries. Scale Bars: 50 μm.

Figure 2. The s36 chorionic protein controls the development of regionally specialized structures of Drosophila melanogaster eggshell.

(A,B) Light microscopy images of representative control (c355-GAL4/+) (A) and s36-targeted (c355 > s36_RNAi) (B) follicles. (C–H) Scanning electron microscopy images of representative control (C–E) and s36-depleted (F–H) follicles. (C,F) Far-off view of follicle’s general morphology. (D,G) Follicle’s anterior-end architecture. (E,H) Follicle’s posterior-end structure. DA: Dorsal Appendages, O: Operculum, M: Micropyle, C: Collar and A: Aeropyle. Scale Bars: 50 μm.

Figure 3. The indispensable contribution of s36 protein in biogenesis of chorion’s radial complexity.

(A,B) Transmission electron micrographs of representative control (c355-GAL4/+) follicles. (C–H) Transmission electron microscopy images of eggshell’s ultrastructural organization in s36-downregulated (c355 > s36_RNAi) follicles. Phenotypic variation (C–H) dictates the ability of s36 to modulate developmental randomness likely evolved during chorion’s assembly. VM: Vitelline Membrane, ICL: Inner Chorionic Layer, IE: Inner Endochorion (Floor), P: Pillar(s), OE: Outer Endochorion (Roof), EX: Exochorion, RN: Roof Network and FC: Follicle Cell(s). Scale Bars: 0.5 μm.

Figure 4. Targeted downregulation of s36 chorionic protein in the follicle-cell cluster of Drosophila melanogaster ovary does not seem to affect vitelline-membrane’s impermeability.

Light microscopy images of control (c355-GAL4/+) (A) and s36-downregulated (c355 > s36_RNAi) (B) laid de-chorionated follicles after their incubation with neutral red, demonstrating the impermeability of control and s36-targeted follicles to the dye. The arrow in (A) indicates a developing embryo. Scale Bars: 50 μm.

Figure 5. AFM-mediated imaging, and quantification of eggshell’s surface deformity and fragility in s36-targeted ovarian follicles.

Atomic force microscopy imaging of control (c355-GAL4/+) (A–C) and s36-depleted (c355 > s36_RNAi) (D–F) laid follicles. (A) Representative follicular imprint in a hexagonal form on control follicle’s surface. (B) Typical side (~1 μm width and ~400 nm height) of a follicular imprint that specifies control follicles. (C) Canonically distributed “hill and valley” structures within a representative hexagonal area of control follicle. The s36-depleted follicles lack the characteristic follicular imprints (D,E) and “hill-valley” typical assemblies (F). (Ga–Gd) Mean values of surface -geometrical- parameters, such as z-height (Ga), RMS roughness (Gb), skewness (Gc) and kurtosis (Gd) of control and s36-depleted follicles. (H–K) Lower (H,J) and upper (I,K) boundaries of force-distance curves of control (H,I) and s36-downregulated (J,K) -laid- follicles. (L) Young’s modulus average values of control and s36-targeted follicles.

Figure 6. Depletion of s36 protein in the follicle-cell cluster of Drosophila melanogaster ovary is associated with perturbed patterning of chorionic peroxidase activity and absence of post-zygotic mitotic divisions in early embryogenesis.

(A,B) Light microscopy images of a DAB-based assay for peroxidase activity in control (c355-GAL4/+) (A) and s36-downregulated (c355 > s36_RNAi) (B) follicles. (C,D) Confocal laser scanning microscopy images, after a PI nuclear-staining process, of two control (de-chorionated and de-vitellinized) embryos at different developmental stages (C) and a representative s36-downregulated (de-chorionated and de-vitellinized) laid follicle that appeared unable to proceed to early embryogenesis as dictated by the absence of PI-positive post-zygotic nuclei (D). Scale Bars: 50 μm.

Figure 7. Lack of s36 chorionic protein from regionally specialized follicle-cell subpopulations does not harm micropylar or polar integrity, but causes an age-dependent sporadic derailment of dorsal-appendages’ patterning.

(A,D) Confocal laser scanning microscopy (CLSM) images of representative stage-10 control follicles specifically expressing the eGFP protein either in the border-cell cluster (c522 > eGFP) (A) or in the (anterior and posterior) polar-cell clusters (109c1 > eGFP) (D). (B,C) Scanning electron microscopy (SEM) images of follicles being specifically targeted for the s36 protein in border cells (c522 > s36_RNAi). (E,F) SEM images of follicles being specifically targeted for the s36 protein in (anterior and posterior) polar cells (109c1 > s36_RNAi). (E) Anterior end. (F) Posterior end. (G) CLSM image of a representative stage-14 control follicle specifically expressing the eGFP protein in follicle cells of dorsal appendages (109-28 > eGFP). (H,I) SEM images of follicles being specifically targeted for the s36 protein in follicle cells of dorsal appendages (109-28 > s36_RNAi). (J) Bar-chart presenting the percentage (%) of follicles with a sporadically acquired and fly age-driven (0–29 and 30–60 days) pathology of dorsal appendages being produced by 109-28 > s36_RNAi but not 109-28-GAL4/+ (control) fly strains. (K,L) Light microscopy images of 109-28 > s36_RNAi follicles being sporadically generated by aged flies. Dysmorphic follicles are characterized by the development of four (K) or three (L), instead of two (e.g. Fig. 7H), dorsal appendages. Scale Bars: 50 μm in A,D,G,H,K,L; 10 μm in B,C,E,F,I.

Figure 8. Targeted depletion of s36 chorionic protein in the follicle-cell cluster of dorsal appendages sporadically induces a broad spectrum of follicular-pathology phenotypes in elderly flies.

(A–J) Scanning electron microscopy images of 109-28 > s36_RNAi transgenic -dysmorphic- follicles with pleiotropic dorsal-appendages’ pathology being sporadically produced by aged flies. (A,B) Four normally-sized dorsal appendages with almost typical paddle-shaped tips. (C) Appendages with abnormal tips. (D,F) Three almost typical dorsal appendages. (E) Two typically-shaped and -sized appendages together with an extra short and stubby one. (G,H) Dorsal appendages with bulky bifurcated tips (H: higher magnification of G). (I,J) Fused and antler-shaped dorsal appendages. Scale Bars: 50 μm.