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. 2008 Jan;4(1):e8.
doi: 10.1371/journal.pgen.0040008.

Dynein regulates epithelial polarity and the apical localization of stardust A mRNA

Sally Horne-Badovinac  1 , David Bilder
Affiliations

Dynein regulates epithelial polarity and the apical localization of stardust A mRNA

Sally Horne-Badovinac et al. PLoS Genet. 2008 Jan.

Abstract

Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico-basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt) to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico-basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cytoplasmic Dynein Function Is Required for Epithelial Cell Shape and Integrity
(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 DhcD12 −5 mutant clones.
Figure 2
Figure 2. Loss of Dhc Disrupts the Apical Localization of Sdt and Crb
(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.
Figure 3
Figure 3. sdt Genetically Interacts with Dhc64C
(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. sdtP10 is a hypomorphic allele, which typically displays two patches of cuticle in addition to small cuticle fragments (B). In the strong sdtXP96 allele, the epidermis only produces small cuticle fragments (D). When Dhc levels are reduced in sdtP10 mutants, the cuticle in many embryos is reduced to a single patch (C) or strongly reduced to fragments, like sdtXP96 (D).
Figure 4
Figure 4. The Apical Localization of sdt mRNA Is Dynein-dependent and Developmentally Regulated
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 DhcD12 −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')
Figure 5
Figure 5. Exon 3 Contains the sdt Apical Localization Signal
(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).
Figure 6
Figure 6. sdt A Is Specifically Required for the Early Formation of Embryonic Epithelia
(A) The sdtEH681 allele contains a stop codon within the alternatively spliced exon 3. (B and C) Wild-type cells are marked with GFP (green). In sdtEH681 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 sdtEH681 embryos (F) when compared to wild-type embryos (E) demonstrates a strong loss of apico–basal polarity in embryonic epithelia. (G) A sdtEH681 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 sdtEH681 cuticle phenotype than a UAS-sdt B transgene or the negative control, UAS-GFP transgene.
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