Generation of Shlp antibodies

To further analyze the function of Shlp during development of Drosophila, antibodies against different peptides of Shlp were generated. Fig. A.2-6 schematically represents the epitopes in Shlp against which three different antibodies have been generated (CG7739 EP023003, CG7739 EP023004 and CG7739 SAC115).

Figure A.2-13: Schematic representation of Shlp and the epitopes against which antibodies have been raised. Positions of epitopes are indicated by black bars. SAC115 was generated against aa 20-551, EP023004 was generated against aa 435-449 and EP023003 was generated against aa 580-596. Different domains are color coded in the legend. Numbers in the legend represent the length of the indicated domains in aa.

CG7739 EP023003 and CG7739 EP023004 are antibodies raised in rabbits against peptides with short amino acid sequences of Shlp. CG7739 EP023003 is directed against the most carboxy terminal region of Shlp (aa 580-596). Immunofluorescent antibody stainings with this antibody performed on wildtype embryos showed a ubiquitous staining.

On the subcellular level a staining of the mitotic spindle of mitotic cells of Drosophila as well as of the apical region of epithelial cells and a blurry apical crescent of interphase neuroblasts could be observed (data not shown). Antibody staining performed on embryos lacking Shlp revealed that this staining represents artifacts (see A.2.5 and data not shown).

When antibody staining was performed on wildtype embryos with the CG7739 EP023004 antibody no specific staining could be observed (data not shown). Therefore an antibody against a fusion between Glutathion-S-Transferase and the extracellular domain of Shlp termed CG7739 SAC115 was raised in guinea pig. When antibody staining with this antibody was performed on wildtype embryos, no difference could be observed between this staining and embryos stained with the according preimmune serum. The same results were observed when stainings were performed on brains of third instar larvae as well as on

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ovaries (data not shown). We conclude from these observations that either the sensitivity of the generated antibodies for the endogenous Shlp epitopes is low or that the Shlp protein levels are very low during oogenesis, embryonic and larval brain development.

To investigate the subcellular distribution of Shlp in the embryo, engrailed::GAL4 (en::GAL4) was used to drive expression of untagged Shlp under the control of a somatic promoter (pUASt-shlp) in the pattern of the segment polarity gene engrailed. Embryos were stained with CG7739 SAC115 and analyzed by confocal laser scanning microscopy.

We found Shlp protein enriched in the engrailed expression domain (Fig. A.2-7 A). On the subcellular level, ectopic Shlp in the engrailed domain localized to the plasma membrane and it could also be detected in small intracellular vesicular structures (A7 B, arrow).

Similar results could also be observed with the two above mentioned rabbit anti-Shlp antibodies (data not shown).

Figure A.2-14: Antibody staining with CG7739 SAC115 on embryos expressing pUASt-Shlp under engrailed::GAL4 control. (A) CG7739 SAC115 detects Shlp specifically in the engrailed expression domain. (B-C) Shlp localizes to the plasma membrane and to intracellular vesicular structures (B, arrow). Scale bar is 50 µm in A and 10 µm in B and C. Embryo is stage 11. Anterior is to the left, dorsal is up.

en::GAL4>pUASt::Shlp

A B

C

31 Next, we analyzed the subcellular localization of ectopically expressed Shlp in epithelia and neuroblasts of the Drosophila embryo. Therefore, pUASt-Shlp was expressed under the control of tubulin::GAL4 (tub::GAL4), a GAL4 line that expresses GAL4 in all cells.

Embryos were stained with CG7739 SAC115 and with an antibody directed against Bazooka as marker for the subapical region of epithelial cells and the apical membrane of dividing neuroblasts. As shown in Fig. A.2-8 Shlp localizes to the basolateral plasma membrane in neuroectodermal cells when overexpressed ubiquitously. In these epithelial cells it localizes basally to Bazooka (Fig. A.2-8 D). In neuroblasts it localizes to the plasma membrane, however not in a polarized way (see Fig. A.2-9 D, asterisk).

Figure A.2-15: Shlp localizes to basolateral membrane in the neuroectoderm. Embryos were stained with anti-Shlp SAC115 antibody (A), against Bazooka (B) and DAPI (C). (D) is a merge of all previous panels. Embryo is stage 11. Scale bar is 10 µm.

tub::GAL4>pUASt::Shlp

Shlp Bazooka

merge DAPI

A B

C D

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Figure A.2-16: Shlp localizes to the plasma membrane but not in a polarized way. Embryos were stained with anti-Shlp SAC115 antibody (A), against Bazooka (B) and DAPI (C). (D) is a merge of all previous panels. The white asterisk in (D) marks a metaphase neuroblast. Embryo is stage 11.

Scale bar is 10 µm.

As another approach to clarifiy the localization of Shlp in embryos we overexpressed a carboxy-terminally GFP-tagged Shlp protein with daughterless::GAL4 (da::GAL4), another GAL4 line that ubiquitously activates expression in the embryo and stained for GFP and Bazooka (Fig. A.2-10). While Bazooka localizes to the subapical region in the neuroectoderm, Shlp-GFP localizes to the basolateral membrane, similar to untagged Shlp protein. In neuroblasts Shlp-GFP localizes ubiquitously to the plasma membrane, again similar to untagged Shlp.

tub::GAL4>pUASt::Shlp

Shlp Bazooka

merge DAPI

A B

C D

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Figure A.2-17: Localization of Shlp-GFP in embryos. Embryos were stained with anti-GFP (A), anti-Bazooka (B) and DAPI (C). The asterisk marks a dividing neuroblast in metaphase. Embryo is stage 11. Scale bar is 10 µm.

TIP, the mammalian orthologue of Shlp, was identified as a secreted factor (Fiscella et al., 2003). Therefore, we also checked the localization of overexpressed untagged Shlp in a secretory epithelium, the salivary gland of the Drosophila embryo. In this experiment we overexpressed Shlp ubiquitously with da::GAL4 and stained for Shlp as well as for the apical marker Bazooka and for the septate junction marker Discs Large (Dlg). In contrast to the neuroectodermal epithelium where Shlp localized to the basolateral membrane, ectopic Shlp was strongly enriched apically in the secretory cells of the salivary gland (Fig.

A.2-11 A).

GFP Bazooka

merge DAPI

A B

C D

da::GAL4>pUASt::Shlp-GFP

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Figure A.2-18: Localization of overexpressed Shlp in the salivary glands of embryos. Embryos were stained with anti-Shlp EP023003, Discs Large (Dlg), a basolateral marker, Bazooka and DAPI. Note apical accumulation of Shlp close to the luminal space. Luminal space is marked with an asterisk. Embryo is stage 15. Scale bar is 20 µm