Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

Abstract

Background: Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported.

Results: We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle.

Conclusions: All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics.

Fig. 1

Generation and validation of Wash, WASp, SCAR, and Whamy monoclonal antibodies. A: Diagrams of Drosophila WASP family proteins indicating their characteristic domains. WHD1: Wash homology domain 1; WHD2: Wash homology domain 2; PPP: proline rich domain; VCA: actin and Arp2/3 binding; WH1: WASp homology 1; B: basic domain; GBD: GTPase binding domain; CC: coiled coil; SHD: SCAR homology domain; WJD: WHAMM and JMY homology domain. Amino acids are indicated (aa). B-C: Specificity of the mouse polyclonal antibodies generated against WASP family proteins by western blot analysis and cell staining. (B) Autoradiograph of ³⁵S-labeled in vitro translated (IVT) full-length WASP family proteins showing the relative levels of the translated proteins (upper panel). Western blots of IVT WASP family proteins probed with mouse polyclonal antibodies generated against Wash, WASp, SCAR, and Whamy as indicated. (C) Wildtype (top row) and WASP family RNAi treated (middle and bottom rows) Drosophila S2 cells stained with the indicated WASP family mouse polyclonal antisera (middle row) or with phalloidin (to visualize actin) and DAPI (to visualize nuclei; bottom row). Scale bars: 10µm. D-E: The specificity of the monoclonal antibodies used in this study was validated by western blot analysis. (D) Western blots of in vitro translated WAS family proteins probed with monoclonal antibodies generated against Wash (P3H3 and P4C9), WASp (P5E1 and P3B1), SCAR (P1C1 and P1C8) and Whamy (P4A8 and P1D1). (E) Western blots of Drosophila 0–2hours embryo whole cell extract performed with WASP family monoclonal antibodies or with WASP family polyclonal (poly) antibodies as indicated. Endogenous WASP family protein is indicated with an asterisk.

Fig. 2

WASP family protein expression during Drosophila oogenesis. A: Schematic of Drosophila oogenesis. ooc: oocyte; NC: nurse cells; SC: germline stem cell; FSC: follicle cell stem cell; FC: follicle cell; cysts: developing cysts; BC: border cells; mp: micropyle; DA: dorsal appendages. B-E: Expression patterns of Wash (B), WASp (C), SCAR (D) and Whamy (E) throughout oogenesis. The germarium (G) and individual egg chambers are shown from different stages (stg.2–stg.14) of oogenesis as indicated. (B) Wash becomes enriched at the oocyte cortex at stg.7 (arrows in stg.7, stg.9), at the nurse cell cortex at stg.10A-10B (arrows), and is highly expressed in dorsal appendages (stg.13–14) (arrows). (C) During stg.6–7 WASp becomes enriched in the posterior follicle cells overlying the oocyte (arrows in stg.6). WASp is also highly expressed in the border cells (arrow in stg.10A and stg.11), nurse cell cortices (arrow in stg.10B), and the lumen of the dorsal appendages (stg.13–14) (arrow in stg.13). (D) SCAR expression can be detected in the germarium, where it accumulates highlighting developing cysts (arrows in stg.G). SCAR also accumulates at the periphery of the oocyte (arrows in stg.6–7) and remains at the oocyte cortex through stg.10A. Beginning at stage 10B SCAR exhibits cortical accumulation within the nurse cells (arrow in stg.11). SCAR is highly expressed in dorsal appendages (stg.12–14) (arrows in stg.14). (E) Whamy exhibits asymmetric localization in nurse cells from stg.G-9, where it accumulates on the apical (outermost) side of the cell (arrows in stg.4 and stg.8). This asymmetric distribution is also observed in the follicle cells in stages 7–8 (arrow). Similar to Wash and SCAR, Whamy is enriched at the dorsal appendages (stg.14). Scale bars: stg.G-stg.6, 10µm; stg.7–stg.9, 20µm; stg.10–14, 100µm. Egg chamber stage (stg.) is indicated and in all panels single slice (2µm thickness) confocal micrographs are shown.

Fig. 3

WASP family proteins show distinct patterns of expression in follicle cells, as well as in the oocyte. A-Aiii: Surface Z-projection showing the asymmetric localization of Whamy in follicles cells (α-Whamy, green; DAPI, blue). (Ai-Aiii) Higher magnification views of regions indicated in A. B-E”: Surface Z-projections of egg chambers stained with α-Whamy (green or grayscale) and alpha-tubulin to visualize microtubules (MTs) (B-B”, red or grayscale), the golgi protein Lava lamp (C-D”, red or grayscale), or the mitochondria marker MitoTracker (E-E”, red or grayscale). DAPI (blue) is included to visualize nuclei in D”. F-I: Surface Z-projections of stage 8, 10, 12 and 14 egg chambers stained with α-Wash (F), α-WASp (G), α-SCAR (H), or α-Whamy (I). Scale bars: A-Aiii, B-B”, E-E”: 10µm; C-D”: 100µm, F-I, 20µm.

Fig. 4

WASP family protein co-localization with F-actin during Drosophila oogenesis. A-D: Confocal micrograph projections showing WASP family proteins (green or grayscale) co-localizing with F-actin (red or grayscale) and co-stained with DAPI (blue) as indicated. Wash (A), WASp (B), SCAR (C) and Whamy (D) expression patterns in the germarium (upper panels) and individual egg chambers from stages 7 (middle panels) or stages 10A and 10B (lower panels) showing co-localization with actin rich structures. Higher magnification views of germarium region 1 (yellow dashed box) and regions 2b-3 (red dashed box), stage 7 ring canals (red dashed box) and oocyte cortex (yellow dashed box), stage 10A border cells (red dashed box) and nurse cell actin bundles in stage 10B (yellow dashed box). Scale bars: Germarium: 10µm, stages 7, 10A and 10B: 100µm.

Fig. 5

Wash expression throughout Drosophila embryogenesis. A-R: Confocal micrograph projections showing Wash expression in successively older wildtype embryos (α-Wash (P3H3); grayscale or green; DAPI, blue). Embryonic stage (stg) is indicated. (A’-R”) Higher magnification views of region from A-R, respectively, without (A’-R’) and with (A”-R”) DAPI staining to visualize nuclei. Wash is expressed during embryogenesis throughout the whole embryo. Note increased Wash accumulation in energids at stages 1–2 (arrows in A). Wash exhibits dynamic subcellular localization in mitotic domains (arrows in F’). During gastrulation (stg.6–8), Wash is enriched in cephalic furrow (arrows in F), ventral furrow (arrows in G), and the posterior midgut invagination (arrow in H). In later staged embryos, Wash accumulates in spiracles (stg.13; arrow in N’) and malpighian tubules (stg.15–17; arrows in Q). Lateral views are shown throughout with the exception of G-G” (ventral view). Scale bars: A-R, 50µm; A’-R”, 20µm.

Fig. 6

WASp expression throughout Drosophila embryogenesis. A-R: Confocal micrograph projections showing WASp expression in successively older wildtype embryos (α-WASp (P5E1); grayscale or green; DAPI, blue). Embryonic stage (stg) is indicated. (A’-R”) Higher magnification views of region from A-R, respectively, without (A’-R’) and with (A”-R”) DAPI staining to visualize nuclei. WASp is expressed during embryogenesis throughout the whole embryo. Note the enrichment of WASp in ventral furrow (arrows in G) and the posterior midgut invagination (arrows in F and I). Beginning with germband retraction (stg.12) WASp accumulates in the amnioserosa (arrows in N) and remains until dorsal closure is finished (stg.15). In later staged embryos, WASp is enriched in the midgut (arrow in Q), malpighian tubules (arrows in R), and ventral denticles (arrows in S). Lateral views of embryos are shown in all cases, with the exception of G-G” (ventral view). (S-T) WASp polyclonal antibody (S; 1:100) and a second WASp monoclonal antibody (T; WASp (P3B1); 1:10) were used to show WASp accumulation in the ventral denticles. Scale bars: A-T, 50µm; A’-R”, 20µm; S’-T”, 10µm.

Fig. 7

SCAR expression throughout Drosophila embryogenesis. A-R: Confocal micrograph projections showing SCAR expression in successively older wildtype embryos (α-SCAR (P1C1); grayscale or green; DAPI, blue). Embryonic stage (stg) is indicated. (A’-R”) Higher magnification views of region from A-R, respectively, without (A’-R’) and with (A”-R”) DAPI staining to visualize nuclei. SCAR is expressed during embryogenesis throughout the whole embryo. SCAR accumulates in energids at stages 1–2 (arrows in B’) and in pseudocleavage furrows at stg.3 (arrows in C’). During gastrulation (stg.6–8), SCAR is enriched in cephalic furrow (arrows in G), ventral furrow (arrows in F’), and the posterior midgut invagination (arrow in H’). Scar exhibits altered subcellular localization in mitotic domains (arrows in I’). In later staged embryos, SCAR is enriched in the central nervous system (stg.13–17; arrows in O, Q) and brain (stg.17; arrow in R). Lateral views are shown throughout with the exception of F-F” (ventral view), G-G” (dorsal view), and Q-Q” (ventral view). Scale bars: A-R, 50µm; A’-R”, 20µm.

Fig. 8

Whamy expression throughout Drosophila embryogenesis. A-T: Confocal micrograph projections showing Whamy expression in successively older wildtype embryos (α-Whamy (P1D1/P4A8 mix); grayscale or green; DAPI, blue). Embryonic stage (stg) is indicated. (A’-T”) Higher magnification views of region from A-T, respectively, without (A’-T’) and with (A”-T”) DAPI staining to visualize nuclei. Whamy is expressed during embryogenesis throughout the whole embryo. Note increased Whamy accumulation in energids at stages 2–3 (arrows in B). During gastrulation (stg.6–8), Whamy is enriched in cephalic furrow (arrows in F), ventral furrow (arrows in G), dorsal transverse fold (arrow in H), and the posterior midgut invagination (arrow in H’). In later staged embryos, Whamy is enriched in the hindgut (stg.13–14; arrow in N), midgut (stg.15, dashed box in P represents higher magnification views in P’ and P”), and muscle (arrows in S). Lateral views are shown throughout with the exception of G-G”(ventral view) and Q-Q” (ventral view). Scale bars: A-T, 50µm; A’-M”, 20µm; N’-T”, 10µm.

Fig. 9

WASP family expression during cellularization. Expression pattern of WASP family proteins (green or grayscale) in confocal cross-sections of nuclear cycle (nc) 8–14B embryos expressing an actin reporter (sGMCA; red) and co-stained with DAPI (blue). A: Schematic representation of embryonic nuclear divisions (nc8–14B) and pole cell formation. B-E: Wash (B), WASp (C), SCAR (D), or Whamy (E) expression throughout the cellularization process. Note that SCAR is expressed in actin caps (arrows in nc10–11), in newly formed furrows (arrow in nc14A–14B), and at the cell cortex in forming pole cells (Pole Cells). All images are the same magnification. Scale bar in B (nc8): 10µm.

Fig. 10

WASP family proteins exhibit dynamic subcellular localization during gastrulation and CNS development. A-D””: Single section (1µm thickness) confocal micrographs of stg.7 wildtype embryos showing the expression pattern of Wash (A-A”’), WASp (B-B”’), SCAR (C-C”’), or Whamy (D-D”’) in mitotic domains, with (A-D, A’”-D’”) and without (A’-D”) DAPI staining to visualize nuclei (a-WASP family member, green or grayscale; DAPI, blue). Note that all WASP family proteins exhibit dynamic subcellular localization with exclusive cytoplasmic (cf. arrowheads in C”) or both nuclear and cytoplasmic (cf. arrows in C”) localization. (A”-D””) Higher magnification views of the regions outlined with dashed boxes in A’-D’, respectively. Scale bars: A-D’, 20µm; A”-D”’, 7µm. E: Western blots of Drosophila 0–12hour embryo nuclear extracts performed with WASP family monoclonal antibodies as indicated. F-F’”: WASp accumulates in the nuclei of CNS precursors. Ventral surface projections of early stg.9 wildtype embryos stained with WASp (green or gray). Neuroblasts are identified by Hunchback (Hb) nuclear staining (red; F, F”). (F”-F”) Higher magnification views of the region outlined by dashed box in F, with (F”) and without (F’”) α-Hb co-staining. Scale bars: F-F’, 20µm; F”-F”’, 10µm.