Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia

Abstract

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes.

Figure 1.

Characterization of the aPKC^k06403 allele. (A) Confirmation of the aPKC ^k06403 allele. A pair of PCR primers, one in the P element and one in the aPKC gene, was used to detect the P element insertion in single-mutant aPKC lines (lanes 3 and 4), lgl, aPKC lines (lanes 2 and 5), and an FRTG13 aPKC line (lane 6), but not when template genomic DNA was generated from a line containing a different transposon insertion (lane 7). Mol wt markers from bottom to top: 0.5, 1, 1.5, 2, 3, and 4 kb. (B) Northern blot to detect aPKC transcript in wild-type, heterozygous aPKC ^k06403, and homozygous aPKC ^k06403 second larval instar brains. Total RNA was visualized with ethidium bromide (left). The major band is likely rRNA. The mol wt markers from bottom to top: 1,350, 2,370, 4,400, 7,460, and 9,490 nucleotides. The major component of total RNA bound some aPKC probes, presumably nonspecifically (star). Higher mol wt products detected likely represent different aPKC transcripts (arrowhead), and are greatly reduced in the homozygous mutant brains. (C) Antibody staining reveals no full-length aPKC protein in aPKC ^k06403 second larval instar brain neuroblasts. Staining shows aPKC in main panels (green in inset) and tubulin (red in inset).

Figure 2.

aPKC zygotic null mutant larvae have defects in neuroblast cell polarity. Second instar larval neuroblasts; apical cortex to the top; cell cycle stage indicated; insets show double labels for the indicated protein (green) and α-tubulin (red) to document cell cycle stage and spindle orientation. Wild-type larval neuroblasts (first and third rows): aPKC, Pins, Baz/Par3, Lgl, and Par6 are localized to the apical cortex, whereas Miranda is localized to the basal cortex. aPKC mutant neuroblasts (second and fourth rows): aPKC is undetectable; Baz/Par3, Pins, and Dlg are normally localized to the apical cortex; Lgl and Par6 show abnormal cytoplasmic localization (Par6 can form cortical patches at the telophase bud neck); Miranda shows abnormal uniform cortical localization.

Figure 3.

Positively marked aPKC null mutant clones reveal defects in neuroblast cell polarity. A MARCM strategy was used to generate aPKC mutant clones positively marked by mCD8::GFP (indicated by dashed surrounding lines) in brains of aPKC/+ heterozygous animals. First column shows GFP marking the mutant clones (levels are low in neuroblasts, but can be readily detected when viewed at higher gain; not depicted); second column shows a DNA marker (phosphohistone H3) and/or apical polarity proteins; third column shows Miranda staining to identify neuroblasts and to assay basal protein targeting. (A) Neuroblast within an aPKC mutant clone lacks detectable aPKC protein (arrowhead); wild-type neuroblast outside the clone has high levels of aPKC protein (arrow). Neuroblasts are at interphase and aPKC is cytoplasmic. (B) Wild-type anaphase neuroblast shows normal apical Baz/Par3 (arrow) and basal Miranda. (C) aPKC mutant anaphase neuroblast shows normal apical Baz/Par3 (arrow), but Miranda is abnormally localized uniformly around the cortex. (D and E) Both wild-type and mutant prophase neuroblasts show normal apical Insc (arrows); Miranda is not strongly localized at this stage of the cell cycle.

Figure 4.

aPKC zygotic null mutants have defects in larval epithelial cell polarity. (A and B) aPKC mutant epithelia show decreased levels of apical/basal cell polarity markers. Wild-type (A) and aPKC/aPKC mutant (B) eye imaginal discs 48 h after larval hatching (second instar larvae). Apical/basal polarity is assayed using the apical marker aPKC (green), the apical adherens junctions marker E-cadherin (Ecad; blue), and the septate junction and basolateral membrane marker Dlg (red). Wild-type imaginal disc epithelia show well defined apical/basal polarity, normal disc morphology, and form a smooth monolayer; aPKC mutant imaginal discs show no detectable aPKC and reduced Dlg and Ecad levels. Discs in A and B were stained in parallel and imaged at the same confocal settings to allow protein levels to be compared. (C and D) aPKC mutant epithelia with extremely small disc size, defects in apical/basal polarity, and loss of epithelial monolayer morphology. Discs are imaged at high gain to visualize the subcellular protein distribution and disc morphology. Dlg and Ecad are delocalized around the cell cortex, although occasional clusters of Ecad were observed. Eye discs (arrowhead) are from late second instar larvae and were identified based on the presence of the optic nerve (brackets) connecting to the brain (arrow). (E and F) Clones of wild-type and aPKC mutant mushroom body neurons. aPKC mutant clones have fewer neurons (cell bodies, arrowheads) than wild-type clones. The calyx (dendritic projections) of each clone is marked by arrows.

Figure 5.

Reduced aPKC levels suppress the lgl neuroblast polarity and brain tumor phenotypes. (A–C) Reduced aPKC levels suppress the lgl neuroblast polarity phenotype in embryonic stage 15 neuroblasts. (A) In the wild type, Miranda is localized to the basal cortex. (B) In lgl ⁴/lgl⁴ mutants, Miranda is predominantly delocalized into the cytoplasm and onto the mitotic spindle and centrosomes. (C) In lgl ⁴ aPKC/lgl ⁴ aPKC double mutants, there is noticeably more basal Miranda localization than in lgl ⁴/lgl⁴ single mutants, although some cytoplasmic/spindle association remains. See Table II for quantification. (D–F) Reduced aPKC levels suppress the lgl brain tumor phenotype. (D) Wild type, (E) lgl ³³⁴ /lgl ³³⁴, and (F) lgl ³³⁴+/lgl ³³⁴ aPKC. Third instar larval brains 74 h after hatching were stained for the Scrib membrane marker (green) to identify neuroblasts (large cells, arrowheads) and GMCs (smaller cells), and with Elav (red) to mark neurons. In each panel, the optical cross section was taken at the same level, just apical to the mushroom body. See Table II for quantitation. Bar, 25 μm.

Figure 6.

aPKC suppresses the lgl epithelial polarity phenotype. (A–C) Low magnification view of a third instar larval nervous system (brain lobes outlined) and leg discs (arrowheads), fixed 74 h after hatching at 25°C. (A) In the wild type, imaginal discs are highly ordered. (B) In lgl ³³⁴/lgl ³³⁴ larvae, the discs are large, disorganized, and fused with surrounding tissue. (C) In lgl ³³⁴ aPKC/lgl ³³⁴+ larvae, the discs recover nearly normal morphology. (D–F) Confocal optical section of similarly staged third instar wing discs stained for the apical marker aPKC (green), the apical adherens junction marker Ecad (blue), and the septate junction and basolateral marker Dlg (red). Higher magnification views are shown below. Individual cells are outlined in D′–F′. (D and D′) Wild-type epithelia. (E and E′) lgl ³³⁴ discs lose their gently folded monolayer morphology, but individual cells still show normal topology of the apical/basal markers. However, the size of the apical domain appears enlarged at the expense of the basolateral domain. (F and F′) In lgl ³³⁴ aPKC/lgl ³³⁴+, the imaginal discs have nearly normal morphology and relative size of the apical and basolateral membrane domains. (G–I) Confocal optical section of second instar wing discs stained for Scrib (green) to visualize cell outlines and tissue morphology. (G) Wild-type discs are unfolded epithelial monolayer. (H) In lgl ³³⁴ discs, the cells are rounder and form a multilayer aggregate in the center of the disc. (I) In lgl ³³⁴ aPKC/lgl ³³⁴+, the imaginal discs recover nearly normal morphology. Bars: 100 μm (A–C); 50 μm (D–I).