(A) An anti-α-Tubulin antibody (green) reveals MTs in the FCs, while a nod::LacZ transgene (magenta) shows that the MT minus ends point toward the apical surface. Dynein is known to transport cargo toward MT minus ends.
(B–G) Wild-type cells are marked with GFP (green) in mosaic egg chambers.
(B) Crb is virtually absent in Dhc clones.
(C) Sdt is largely missing from the apical surface in Dhc clones and is instead found throughout the cytoplasm. Mis-localization of Sdt in Dhc clones cannot be due solely to the loss of Crb, as crb clones consistently retain a punctate Sdt signal near the apical side of the cell (D). Mislocalization of Sdt in Dhc clones could account for the loss of Crb, however, as Crb is strongly disrupted in sdt clones (E).
(F) Baz is only slightly reduced at the apical surface in Dhc cells.
(G) Dlg localization is largely normal in Dhc clones.

Fluorescent in situ hybridization (green) reveals the localization of the sdt mRNA in the FCs (A–E) and embryonic surface ectoderm (F and G).
(A–E) Pink lines denote the estimated position of the apical surface, and white lines mark the basal surface.
(C), (F and G) Nuclear envelopes are stained with wheat germ agglutinin (WGA, blue).
(A) At stages 2–4, sdt mRNA forms a punctate ring at the apical FC surface. An arrowhead points to sdt mRNA localization in the germline, which is better shown in Figure S3.
(B) Over-expression of the sdt cMAGUK transgene in the FCs reveals strong apical transcript localization.
(C) At stage 10, sdt mRNA is uniformly distributed in the FC cytoplasm. The apical ring of sdt puncta normally observed in wild-type egg chambers (D) disappears when the entire epithelium is mutant for Dhc^D12−5 (E).
(F) At embryonic stage 11, sdt transcripts are strongly enriched in the apical cytoplasm, but appear to be uniformly distributed by stage 15 (G and G')

(A) The sdt^EH681 allele contains a stop codon within the alternatively spliced exon 3.
(B and C) Wild-type cells are marked with GFP (green). In sdt^EH681 mutant FCs clones, Sdt (B) and Crb (C) are both reduced at the apical surface.
(D) Reverse transcriptase-PCR with primers flanking exon 3, showing the relative distribution of sdt A and sdt B transcripts at different developmental time points in embryos. Asterisk marks a non-specific band. The fragmented cuticle of sdt^EH681 embryos (F) when compared to wild-type embryos (E) demonstrates a strong loss of apico–basal polarity in embryonic epithelia.
(G) A sdt^EH681 mutant embryo in which the UAS-sdt A transgene has rescued more than two-thirds of the embryonic cuticle.
(H) Graph demonstrating that a UAS-sdt A transgene is more efficient at rescuing the sdt^EH681 cuticle phenotype than a UAS-sdt B transgene or the negative control, UAS-GFP transgene.

(A–F) Wild-type cells are marked with green fluorescent protein (GFP).
(A–D) Phalloidin staining (red) reveals FC morphology.
(A) Wild-type ECs are surrounded by a uniform monolayer of cuboidal FCs.
(B and C) Loss of Dhc function in the FCs, through either the newly isolated D12–5 allele (B) or the previously characterized 4–19 allele (C), leads to disruptions of cell shape and gaps in the epithelium (arrowheads).
(D) K194, a newly isolated allele of the Dynactin component Glued, phenocopies the loss of Dhc in the FCs.
(E) Immunohistochemistry on mosaic egg chambers containing Dhc⁴⁻¹⁹ mutant clones shows that Dhc protein levels (grayscale) are strongly reduced.
(F) Dhc levels are similarly reduced in Dhc^D12−5 mutant clones.

(A) Graph quantifying the strong genetic interaction between sdt and Dhc64C, demonstrated by the cuticle phenotypes expressed by sdt embryos from three different genetic crosses. sdt^P10 is a hypomorphic allele, which typically displays two patches of cuticle in addition to small cuticle fragments (B). In the strong sdt^XP96 allele, the epidermis only produces small cuticle fragments (D). When Dhc levels are reduced in sdt^P10 mutants, the cuticle in many embryos is reduced to a single patch (C) or strongly reduced to fragments, like sdt^XP96 (D).

(A) Diagram showing the relationship between the exon structure of two sdt mRNA isoforms and the protein domains they encode. Exon 3, which is deleted from the sdt B isoform, encodes no conserved functional domains. The stretch of amino acids encoded by exon 3 is not conserved in other Sdt homologs such as human MPP5.
(B) Diagram summarizing the experimental results from the six UAS-transgenes used to map the sdt apical localization signal to exon 3. All transgenes were expressed under the control of the GR1 FC driver. (C–H) Fluorescent in situ hybridization (green), using probes against either sdt (C–E) or GFP (F–H) reveals the localization of transgenic mRNAs in the FCs at stages 8–9.
(C) Transcripts from a transgene representing the sdt A isoform localize apically.
(D) sdt A transcripts continue to localize apically without the sdt 3′UTR.
(E) Transcripts from a transgene representing the sdt B isoform, which lacks exon 3, are distributed uniformly in the cytoplasm.
(F) Adding the sdt 3′UTR to an eGFP transgene is insufficient to target these transcripts apically.
(G) Adding sdt exon 3 to an eGFP transgene is sufficient to send the mRNA apical.
(H) Neither the SV40 3′UTR nor the eGFP coding region can direct a transcript to the apical cytoplasm.
(I and J) Fluorescent in situ hybridization (green) reveals the localization of endogenous mRNAs at stage 10. A probe specific to sdt exons 4–6 stains the unlocalized transcripts at stage 10 (J), while a probe specific to exon 3 does not (I).
(C–J) The nuclear envelope is marked with WGA (blue). The apical surface is denoted with a dashed line and the basal surface with a solid line. (A) has been adapted with permission from a figure by S. Berger and E. Knust (unpublished).