Renal tubule cells are polarised throughout development. (A-D) Stage 11; (E-H) stage 13; (I-L) stage 16. (A,E,I) Embryos stained for the transcription factor Cut (Ct), to mark renal tubule cells. The renal tubules bud out from the hindgut primordium during stage 11 (A, arrow) and undergo several rounds of cell division. These are completed by stage 13, when the tubules appear as short fat tubes (E, arrow). During stages 13 to 16 they elongate dramatically and narrow to a two-cell circumference (I, arrow). (B,F,J) Crb (red) and Baz (green) are localised to the apical membranes of tubule cells during stages 11-16, with Baz overlapping with, but slightly apical to Crb during stages 11-13. (C,G,K) Dlg (red) localises to the lateral membranes and shows no overlap with Baz (green). (D,H,L) ZAs, marked by Armadillo (Arm, red), are maintained between tubule cells (nuclear Ct, green) throughout cell rearrangements. Dashed white lines outline tubules.

Tubule cells do not require Crb for polarity during early development. (A,B) Stage 11 embryos that are wild type (A) or mutant for the amorphic allele crb^8F105 (B) stained for Crb (brown). In wild-type embryos Crb localises apically in both the epidermis (A, arrowhead) and in the renal tubules (A, arrow). In crb mutants the truncated Crb protein is distributed diffusely in the epidermis (B, arrowhead), yet is clearly restricted to the apical membrane of the renal tubules (B, arrow). (C-D″) Lateral view of wild-type (C) and dorsal view of crb (D) stage 11 renal tubules stained for Baz (green) and Dlg (red). crb embryos show a normal localisation of apical and basolateral proteins. (E,F) Transmission electron microscopy images of wild-type (E) and crb (F) tubule cells from stage 11 embryos. ZAs are observed at the apical ends of the lateral membranes of crb mutant tubule cells (F, arrows) as in wild type (E, arrows). Scale bars: 200 nm. (G-I) Overexpressed Crb localises to the apical domain of renal tubules during stages 11-13. (J-K″) Confocal optical sections of stage 13 renal tubules reveal that even when levels of Crb (green) are highly upregulated, Baz (red) localises correctly to the apical domain (K, compare with wild-type embryos in J).

Crb is required to maintain polarity during late tubule development as they undergo cell rearrangements. (A,A′) In early stage 13 crb mutant embryos, Baz localises normally to the apical membrane of the tubules and the tubule lumen is clearly visible (arrows). (B,B′) In late stage 13 crb mutant embryos there are regions of tubule cells in which Baz has become delocalised and the lumen of the tubule is disrupted (arrow). (C,C′) By stage 14, polarity is disrupted throughout the tubules and there is no apparent lumen (arrow). (D-E″) When Crb is overexpressed in the tubules, there is no effect on polarity at the beginning of stage 13; Baz remains tightly localised to the apical domain (D′). In late stage 13, Crb spreads down the basolateral membranes of the cells and Baz is no longer restricted to the apical domain (E). (F,G) In wild-type tubules Fas II (green) localises along the lateral membranes of tubule cells (arrow) and does not overlap with apical Baz (red) (F). Stellate cells (Tsh, green) integrate into the epithelium during tubule elongation (G). (H-J) In ct mutant embryos the tubules do not elongate but remain as blisters at the junction between the hindgut and midgut (H,I arrows). (I) Tsh-expressing stellate cells are absent from the tubules of ct mutant embryos. (J) Fas II (green) and Baz (red) localisation is not affected in ct mutant tubule cells. (K-L′) ct;crb double mutants have a tubule phenotype similar to single ct mutants; the cells form epithelial blisters of columnar cells, with Baz (red) and Fas II (green) localised to distinct domains.

Renal tubule polarity becomes dependent on the exocyst subunit Exo84 during late stages of development. (A-D) stage 11 wild-type (A,C) and exo84 mutant (B,D) embryos. (A,C) In wild-type embryos Crb (green, A), Baz (red, A) and Sas (red, C) localise apically in the renal tubules (arrows) and epidermis (arrowheads) and Fas II (green, C) localises to the lateral membrane of tubule cells. (B,D) In exo84 mutants the apical localisation of Crb, Baz and Sas is lost in epidermal cells (arrowheads), but is unaffected in the renal tubules and Fas II localises correctly to lateral tubule cell membranes (arrows). (E-H) Stage 16 wild-type (E,G) and exo84 mutant (F,H) tubules. (E,G) In wild-type tubules, Crb (E, green), Baz (E, red) and Sas (G, red) localise to apical cell surfaces and Fas II (G, green) concentrates laterally. (F,H) Stage 16 exo84 mutant tubules remain short and fat and the cells are irregular in shape (compare H with wild type in G). Apical proteins have spread into the lateral membranes (F, arrow) or are partially lost from the apical domains (compare H, with wild type in G, arrowheads). Although basolateral proteins localise as in wild type, the cells appear irregular and more rounded in shape (compare H with wild type in G).

Tubule cell polarity is lost in both baz and scrib mutant embryos from the earliest stages of development. Confocal microscopy images of stage 11 baz maternal/zygotic (M/Z) (A,B) and scrib M/Z (C,D) embryos stained for Ct (red) to visualise the renal tubules. (A,B) In baz M/Z mutant tubules Crb levels are strongly reduced and any remaining protein is mis-localised (A,A′, arrow). Dlg has spread around tubule cell membranes (B,B′, arrow). (C,D) In scrib M/Z mutant embryos, both Crb (C,C′, green) and Baz (D,D′, green) are delocalised in tubule cells and can be found all around the cell cortex (arrows).

Tubule cells require Crb to maintain polarity during late stages of development. (A,B) Stage 16 wild-type (A) and crb mutant (B) embryos stained for Ct (brown). The renal tubules (arrows) in crb mutant embryos do not elongate fully and lose their normal morphology. (C-F″) Stage 16 renal tubules stained for Dlg (red) and Baz (green) in C and D, Ct (red) and Par-6 (green) in E, and Ct (red) and aPKC (green) in F. The localisation of the apical proteins Baz (D, green), Par-6 (E, green) and aPKC (F, green) is lost in stage 16 crb tubules. Although Dlg (D, red) remains localised to the lateral membranes, cells are irregular in shape (compare D with the wild type in C). (G,H) Transmission electron microscope images of wild-type (G) and crb (H) tubule cells from stage 16 embryos. (G) ZAs are visible in the apical region of the lateral border between all wild-type tubule cells (arrows). (H) ZAs are no longer present between crb mutant tubule cells (arrows indicate the apical region of lateral membranes). Scale bars: 500 nm. (I-N″) Crb overexpression leads to severe polarity defects in stage 16 embryos; Baz (red; I and J) is delocalised (compare J, where two tubules lie next to each other, with the wild type in I), Dlg (red; K and L) spreads around the cell membranes (compare L with the wild type in K) and Arm (red; M and N) is fragmented and reduced in levels (compare N with wild type in M). Dashed white lines outline the tubules.

Stalling cell movements in the epidermis removes the requirement for Crb to maintain cell polarity. (A-C) As the stomodeum invaginates Baz localisation is progressively lost in the neighbouring epidermal cells (arrows). (D-K) Stage 11 wild-type (D), Kr (E), crb (F,H,J) and Kr;crb (G,I,K) mutant embryos stained for Baz (green) and Neurotactin (Nrt, red; D-G), Sas (green) and Dlg (red; H,I), and E-cad (J,K). Boxed areas in D-G are enlarged in D′-G′ and D″-G″. (D) Baz localises to the apical surfaces of epidermal cells and Nrt to the lateral membranes. (E) The germ band does not extend fully in Kr mutant embryos and cell polarity is unaffected. (F,H,J) In crb mutant embryos, epidermal polarity is lost by stage 11; apical proteins such as Baz (F, green) and Sas (H, green) are delocalised throughout the cell, basolateral proteins such as Nrt (F, red) and Dlg (H, red) spread around the cell membrane, and junctional proteins such as Ecad are fragmented (J). (G,I,K) In Kr;crb double mutants there is a marked rescue of epidermal cell polarity. Baz and Sas localise to the apical membrane (G,I), and Nrt and Dlg are restricted basolaterally (G,I). ZAs are largely rescued, as seen by circumferential labelling of Ecad (compare K with crb mutant J). (L) In crb mutants, most of the cuticle is absent with only fragments of cuticle remaining. (M) Embryos mutant for Kr and crb show a marked rescue of the crb phenotype, as a continuous cuticle is formed. (H, I) Images taken of the epidermis, just posterior to the tip of the germband. (J,K) Enface views of the dorsal epidermis.

Model outlining interactions between cell polarity proteins before (left) and during (right) morphogenetic cell rearrangements. The Crb protein group is not required for normal cell polarity in the absence of cell rearrangements. dPATJ, Drosophila PATJ.