On the role of PDZ domain-encoding genes in Drosophila border cell migration

Abstract

Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in vivo RNAi knockdown. The PDZ domain is one of the largest families of protein-protein interaction domains found in eukaryotes. Proteins that contain PDZ domains participate in a variety of biological processes, including signal transduction and establishment of epithelial apical-basal polarity. Targeting PDZ proteins effectively assesses a larger number of genes via the protein complexes and pathways through which these proteins function. par-6, a known regulator of border cell migration, was a positive hit and thus validated the approach. Knockdown of 14 PDZ domain genes disrupted migration with multiple RNAi lines. The candidate genes have diverse predicted cellular functions and are anticipated to provide new insights into the mechanisms that control border cell movement. As a test of this concept, two genes that disrupted migration were characterized in more detail: big bang and the Dlg5 homolog CG6509. We present evidence that Big bang regulates JAK/STAT signaling, whereas Dlg5/CG6509 maintains cluster cohesion. Moreover, these results demonstrate that targeting a selected class of genes by RNAi can uncover novel regulators of collective cell migration.

Keywords: Drosophila; JAK/STAT; PSD95/Dlg/ZO-1 (PDZ) domains; border cells; collective migration.

Figure 1

In vivo RNAi knockdown to identify PDZ domain-encoding genes required for border cell migration. (A) Control border cells (arrowheads) migrate between the nurse cells (nc) from stage 9 to 10 of oogenesis to reach the oocyte (o). Border cell clusters that have migrated past the dashed line (stage 10 control) are considered to have completed their migration. Border cells and follicle cells (brackets) express UAS-mCD8:GFP (green) driven by c306-GAL4 in egg chambers at the indicated stages; genotype is c306-GAL4/+; UAS-mCD8:GFP/+. Egg chambers were co-stained for actin (red) and DAPI (blue) to label cell membranes and nuclei, respectively. (Lower right panel) A stage 10 c306-GAL4/+; UAS-mCD8:GFP/UAS-baz RNAi v2914 (baz RNAi) egg chamber in which border cells did not migrate. Scale bar is 20 μm. (B and C) Knockdown of baz in follicle cells (bottom panels) using the follicle cell driver T155-GAL4 disrupts the epithelium compared with control (top panels) at stage 9 (B) and stage 10 (C). Genotypes are T155-GAL4/+ (control) and UAS-baz RNAi/+; +/T155-GAL4. Scale bar is 20 μm. (B) baz RNAi follicle cell layer is thin (dashed line), and some nuclei are misaligned (bracket) compared with control. (C) baz RNAi follicle cells are multilayered (arrow) and fail to retract over the oocyte (square bracket) as in control. (D and E) Ovarioles showing GAL4 expression patterns in border cells (arrowheads) and follicle cells (brackets) as visualized by UAS-mCD8:GFP (green); stages are indicated. Egg chambers were co-stained for actin (red) and DAPI (blue). Scale bar is 50 μm. (D) slbo-GAL4 expression pattern (slbo-GAL4, UAS-mCD8:GFP/+). (E) c306-GAL4 expression pattern (c306-GAL4/+; UAS-mCD8:GFP/+). c306-GAL4 is also expressed in stalk cells, which connect egg chambers within the ovariole. (F) Quantification of migration in stage 10 egg chambers of the indicated genotypes, shown as the percentage with complete (green) or incomplete (red) border cell migration. Error bars represent SEM; n ≥ 50 egg chambers in each of three trials (**P < 0.01; ***P < 0.001; two-tailed unpaired t-test). (G) Outline of the scheme used to survey the role of PDZ genes in border cell migration. Anterior is to the left in this and all subsequent figures.

Figure 2

Confirmation of positive and negative hit genes. (A and C) Quantification of border cell migration at stage 10, shown as the percentage of border cells with complete (green) or incomplete (red) migration in egg chambers expressing RNAi to GFP (control) or the indicated RNAi transgenes driven by c306-GAL4. Error bars represent SEM; n ≥ 50 egg chambers in each of at least three trials (*P < 0.05; **P < 0.01; two-tailed unpaired t-test). (A) Knockdown of CG6498 using multiple transgenes disrupts border cell migration. (B) Representative example of an egg chamber with a border cell migration defect caused by CG6498 RNAi. Genotype is c306-GAL4/+; UAS-mCD8:GFP/UAS-CG6498 RNAi v35100. Border cells (green; arrowhead) stopped a little more than halfway to the oocyte (outlined). DAPI marks nuclei. Scale bar is 20 µm. (C) Border cell migration defects by RNAi knockdown of veli (v43094) and CASK (v34185). Normal border cell migration with RNAi knockdown of X11L (v28652) and in a PsGEF mutant (PsGEF^Δ55/PsGEF^Δ21). RNAi for ERR (line v108349), the predicted off-target gene for veli RNAi line v43094, did not disrupt border cell migration. (D) Border cells stained with an antibody to Veli. Control border cells (c306-GAL4/+; UAS-mCD8:GFP/+) had detectable Veli (red), which was strongly reduced in veli RNAi border cells (c306-GAL4/+; UAS-mCD8:GFP/UAS-veli RNAi v43094). GFP (green) shows GAL4 expression and DAPI (blue) labels nuclei. Scale bar is 10 µm.

Figure 3

The multi-PDZ domain protein Big bang regulates nuclear STAT levels in border cells. (A) Schematic diagram of five bbg predicted transcripts (adapted from FlyBase); coding exons are in orange. RNAi target sequences and c96-GAL4 insertion site are indicated. RNAi lines v15975 and v101691 target sequences common to all isoforms. RNAi line v36111 is specific to RC and RK (not shown; differs from RC only in a non-coding exon; see FlyBase) transcripts. (B) Schematic diagram of the eight Bbg protein isoforms, which have either two or three PDZ domains. (C) Egg chambers showing c96-GAL4 expression pattern visualized by UAS-mCD8:GFP (green) at the indicated stages. Egg chambers were co-stained for E-cadherin (red) to mark cell membranes and DAPI (blue) to mark nuclei. Scale bar is 50 µm. (Top panel) c96-GAL4 expression in anterior and posterior polar cells (arrows) at early stages. Inset shows c96-GAL4–positive polar cells (green) co-stained for FasIII (red; scale bar, 5 µm). (Bottom panels) c96-GAL4-driven GFP in border cells (arrowheads) and surrounding follicle cell epithelium during stages 9 and 10. (D) Quantification of border cell migration at stage 10, shown as the percentage of border cells with complete (green) or incomplete (red) migration in egg chambers expressing RNAi to GFP (control) or the indicated RNAi transgenes driven by c306-GAL4. Knockdown of bbg with RNAi line v15975 disrupted border cell migration. bbg RNAi line v36111 had variable effects, and line v101691 did not disrupt migration. RNAi to the predicted off-target genes, CG42724 (line v30629) and Irbp (line JF03273), did not disrupt migration. Error bars represent SEM; n ≥ 50 egg chambers in each of at least three trials (**P = 0.0071; two-tailed unpaired t-test). (E) Reduction of Stat92E levels in border cell nuclei when bbg is knocked down. Stage 9 border cells stained for Stat92E (magenta) and DAPI (green). Stat92E is expressed at higher levels in control border cell nuclei (yellow outline) compared with cytoplasm (c306-GAL4/+; UAS-mCD8:GFP/+). Stat92E is expressed at low levels in bbg RNAi (c306-GAL4/+; UAS-mCD8:GFP/UAS-bbg RNAi v15975) border cell nuclei (outlined). Polar cells (asterisks) were excluded from analyses. Scale bar is 5 μm. (F) Quantification of the fluorescence intensity ratio of STAT nuclear staining to DAPI staining for control (n = 59) or bbg RNAi (n = 88) border cells; genotypes as in (E). At least 16 individual clusters were analyzed. Error bars represent SEM (***P < 0.001; two-tailed unpaired t-test).

Figure 4

Markers of cell fate, cell adhesion, polarity, and cytoskeleton in bbg RNAi and CG6509 RNAi border cells. Representative immunofluorescent images of stage 9 control (c306-GAL4/+; UAS-mCD8:GFP/+), bbg RNAi (c306-GAL4/+; UAS-mCD8:GFP/UAS-bbg RNAi v15975), and CG6509 RNAi (c306-GAL4/+; UAS-mCD8:GFP/UAS-CG6509 RNAi v22496) border cells. (A and B) Border cells stained for antibodies to the cell fate markers Singed (A) and Stat92E (B). (A) Singed is enriched in the cytoplasm. (B) Stat92E is enriched in border cell nuclei compared with the cytoplasm. The same cluster is presented with and without border cell nuclei outlined with a dotted line (taken from DAPI staining of nuclei, not shown). Polar cells are marked with an asterisk (*). (C) Border cells stained for the cell adhesion protein E-cadherin, which is high in central polar cells and at the membrane interface between border cells. (D and E) Border cells stained for the cell polarity proteins aPKC (D) and Dlg1 (E). (D) aPKC is an apical cell marker and localizes between border cells; an apical view is shown. (E) Dlg1 is a basolateral cell marker that is enriched in the central polar cells and at lower levels at border cell membranes. (F and G) Border cells stained for the cytoskeletal markers α-tubulin to mark microtubules (F) and phalloidin to label F-actin (G). N ≥ 10 border cell clusters assayed for each genotype. Scale bar is 10 µm.

Figure 5

The MAGUK family member CG6509 regulates border cell cluster morphology. (A) Schematic of the CG6509 transcripts, which differ only in the 5′ non-coding exons (adapted from FlyBase); coding exons in orange. RNAi target sequences are indicated. (B) Schematic of CG6509 protein showing the conserved domains. (C) Quantification of border cell migration at stage 10, shown as the percentage of border cells with complete (green) or incomplete (red) migration in egg chambers expressing multiple RNAi lines or overexpression of full-length UAS-CG6509 (different insertions of same transgene) driven by c306-GAL4. Error bars represent SEM; n ≥ 50 egg chambers in each of at least three trials (*P < 0.05; **P < 0.01; two-tailed unpaired t-test). (D) Stage 9 border cells stained for GFP (green) and Singed (red) to reveal border cell cluster morphology. Representative example of a control (c306-GAL4/+; UAS-mCD8:GFP/+) border cell cluster. Two examples of CG6509 RNAi (c306-GAL4/+; UAS-mCD8:GFP/UAS-CG6509 RNAi v22496) border cells in which the cluster is partially dissociated (middle panel) or elongated (right panel). Scale bar is 20 μm.