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First published online 16 November 2005
doi: 10.1242/dev.02159

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The RNA-binding protein Squid is required for the establishment of anteroposterior polarity in the Drosophila oocyte

Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027, USA

View larger version (78K): [in a new window]   Fig. 1. PKA mutant phenotypes and attempted rescue by constitutive PKA activity or LKB1. (A-D) PKA-C1 null germline clones do not prevent normal anterior migration of the oocyte nucleus (A, arrow), marked by orb-PZ expression in the germline nuclei (blue), normal expression of the dorsal follicle cell marker mirror6D1 (B, purple), normal expression of the posterior follicle cell marker pnt998/12 (C, red; green shows GFP-Stau mislocalized to the center of the oocyte, arrow) or normal expression of the centripetal follicle cell marker BB127 (D, blue). (E,F) Mislocalization of kin-ß-gal (green) and fusion of nurse cells, revealed by rhodamine-phalloidin staining of actin filaments (red), in PKA-C1 null germline clones (E). Both phenotypes are rescued by expression of the constitutively active mouse PKA catalytic subunit mC* from an actin promoter (F). (G,H) GFP-Stau (green), like kin-ß-gal, is mislocalized toward the center of the oocyte in PKA-C1 null germline clones (G, arrow) and is restored to a normal posterior localization by expression of mC* (H, arrow). Rhodamine-phalloidin (red) marks cell outlines. (I,J) Expression of the GFP-LKB1S535E transgene (green) carrying a mutation that mimics phosphorylation at the putative PKA site does not perturb kin-ß-gal localization (red) in wild-type oocytes (I, arrow) but fails to rescue either kin-ß-gal mislocalization (red, arrow) or nurse cell fusions (outlined by GFP-LKB1S535E in green) in PKA-C1 null germline clones (J).

View larger version (74K): [in a new window]   Fig. 2. sqd mutants display AP patterning defects at mid-oogenesis. (A,C,E) In sqdj4B4 germline clones, kin-ß-gal (A, blue), GFP-Stau (C, green) and osk RNA (E, purple) are mislocalized to an ectopic spot in the center of the oocyte at almost complete penetrance. (B,D,F) Weaker mislocalization phenotypes are observed for kin-ß-gal (B, blue), GFP-Stau (D, green) and osk RNA (F, purple) in sqdj4B4/sqd1 (B,F) and sqd1/sqd1 (D) egg chambers. Arrows point to ectopic GFP-Stau (C,D) and osk RNA (E,F). Rhodamine-phalloidin (red) marks cell outlines in C and D. (G,H) Dynein heavy chain (green) localizes to the posterior in wild-type stage 9-10 oocytes (G) but not in sqdj4B4 germline clone egg chambers (H). (I,J) GFP-Stau (green) appears as diffuse particles in the nurse cell cytoplasm of wild-type egg chambers (I), while in sqdj4B4 germline clones, it aggregates around the nurse cell nuclei (J). Nuclei are labeled with propidium iodide (red). Note that sqdj4B4 germline clone oocytes are often smaller than wild-type oocytes in mid- to late oogenesis.

View larger version (84K): [in a new window]   Fig. 3. MT distribution is altered in sqdj4B4 germline clones at mid-oogenesis, but bcd localization is normal. (A,B) FITC-conjugated -tubulin antibody staining of stage 9 wild-type oocytes (A) shows a gradient of MTs extending from the anterior cortex to the posterior, with the posterior being relatively free of tubulin staining. By contrast, stage 9 sqdj4B4 germline clone oocytes (B) display an even distribution of tubulin staining along the entire oocyte cortex. (C-F) -tubulin staining following partial MT depolymerization reveals short MT stubs emanating from the anterior cortex of stage 9 wild-type oocytes (C), whereas in sqdj4B4 germline clone oocytes, such stubs are visible along the entire oocyte cortex (D,F). The oocyte in F is slightly older than that shown in D. In PKA-C1H2 germline clone oocytes, MT stubs are visible along the entire oocyte cortex, similarly to sqdj4B4 mutants, but an additional strong posterior focus is also sometimes visible (E). (G,H) bcd RNA localization (purple) to the anterior cortex of stage 8-10 oocytes is not detectably altered in sqdj4B4 germline clones (H), in contrast to another oocyte polarity mutant, mago1/Df(2R)F36, which shows ectopic posterior bcd RNA (G). sqd mutant oocytes are smaller than wild-type oocytes at stages 8-10.

View larger version (55K): [in a new window]   Fig. 4. sqd mutant oocytes display polarity defects in early oogenesis. In early wild-type egg chambers, Grk protein (A, red), Orb protein (C, green) and -tubulin (E) form a cap at the oocyte posterior, while in sqdj4B4 germline clones, Grk (B, red), Orb (D, green) and -tubulin (F) are more evenly distributed throughout the cytoplasm of stage 2-6 oocytes.

View larger version (68K): [in a new window]   Fig. 5. Both sqd and PKA-C1 mutants display ectopic Osk protein localization. (A,B) Osk protein (green) is detectable only at the posterior of wild-type stage 9 oocytes (A). By contrast, Osk protein is seen in some sqdj4B4 germline clone oocytes mislocalized away from the posterior (B). (C,D) Expression of a lacZ reporter for osk translation, m1414lacwt (blue), is limited to the posterior pole of stage 9 and older wild-type oocytes (C, arrow); it is not expressed in younger wild-type oocytes (arrowhead). In sqdj4B4 germline clones, expression of m1414lacwt is detected in the middle of stage 9 oocytes (D, arrow), although it is never expressed earlier (arrowhead). (E,F) Ectopic Osk protein (green) is seen in the middle of some oocytes that are homozygous for a PKA-C1 null allele (E) or for both sqdj4B4 and a PKA-C1 null (F). Lack of GFP (red) in the germline marks the PKA-C1 homozygote in (F).

View larger version (85K): [in a new window]   Fig. 6. Loss of Sqd in the germline does not affect posterior follicle cell specification, and loss of Sqd in somatic cells does not affect their polarity. (A) Expression of the posterior follicle cell marker pntrm254 (blue) is normal in sqdj4B4 germline clones, while kin-ß-gal is mislocalized (arrow). (B) The centripetal follicle cell marker BB127 (blue) is expressed normally at the anterior of sqdj4B4 germline clones (arrow). (C) Baz protein (red) localizes to the apical membrane of follicle cells. The absence of nuclear GFP (green) marks sqdj4B4 mutant clones. Note that this egg chamber is also mutant for sqd in the germline. Baz localization is normal in sqdj4B4 follicle cell clones. (D) The wing blade trichome bristles display normal planar cell polarity in sqdj4B4/Df(3R)urd escaper flies.

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