Fig. 5. Sponge body organization affects distribution of Stau protein and osk mRNA.

Panel A shows a portion of a stage 8 egg chamber with dispersed sponge bodies, with a higher magnification of the oocyte in panel B. Stau is red and Me31B∷GFP is green. Some of the sponge bodies in the oocyte contain Stau (the yellow puncta such as the one indicated by a long arrow), and others do not (green puncta such as that indicated by the arrowhead). Most of Stau, which is enriched in the oocyte relative to the nurse cells, appears in structures that do not contain high levels of Me31B∷GFP (short arrow). Panels C and D show stage 8 egg chambers with reticulated bodies labeled with Me31∷GFP (green) and stained for either Stau (C) or osk mRNA (D) in red (osk mRNA was detected by fluorescent in situ hybridization). Both Stau and osk mRNA are strongly concentrated in the reticulated bodies. The dashed lines in panels C-E, G-I, and O-Q trace the oocyte outline.

Panels E-J are of Stau∷GFP in live egg chambers (all other images in this figure are from fixed samples) in conditions with reticulated bodies (E-H) and dispersed bodies (I,J). Panels F and J are higher magnification views of E and I, respectively. As for endogenous Stau, Stau∷GFP is highly concentrated in reticulated bodies (arrows in E,F; early stage 8), is progressively enriched in bodies near the posterior of stage 8 oocytes (G), and is tightly localized to the posterior after stage 9 (H). In egg chambers with dispersed bodies (I,J), Stau∷GFP parallels the pattern of endogenous Stau shown in A and is cytoplasmic, but is also present in many small particles that are not seen for endogenous Stau in fixed tissue (arrows in I,J). J is a close up view of I and the arrows indicate the same cytoplasmic region in both images. Stau∷GFP localizes tightly to the posterior after stage 9 when dispersed bodies are present (data not shown) and its distribution is indistinguishable from that when reticular bodies are present (as in H). Examples of the artifactual class of Stau∷GFP particles, which do not correspond to endogenous Stau (see text), are indicated by arrowheads in E and I.

Panels K and L show the concentration of osk mRNA (red in K) and Stau (red in L) in subdomains of reticulated bodies labeled uniformly with Me31B∷GFP. Panel M and N are images from a 3D reconstruction of Stau (red in M, white in N) and Me31B∷GFP (green in M) in a stage 8 egg chamber. Reticulated bodies appear to form a highly interconnected network with Stau enriched at the posterior (M). N shows the highly interconnected nature of Stau protein distribution in a 3D reconstruction of Stau immunostaining in reticulated bodies in a stage 8 egg chamber. The perspective in this 3D reconstruction is of a viewer at the posterior looking towards the anterior.

Panels O-Q are of Stau (Q and red in O) and Me31B∷GFP (P and green in O) in an egg chamber with reticulated bodies. Endogenous Stau exits reticulated bodies in stage 9 egg chambers and compacts tightly against the posterior cortex where it still colocalizes with Me31B∷GFP. Scale bars are 2.5μm in K and L, 10μm in panels B,F,J,M,N and 20μm in panels A,C,D,E, G-I and O-Q.