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First published online 31 January 2006
doi: 10.1242/jcs.02789

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Par-1 kinase establishes cell polarity and functions in Notch signaling in the Drosophila embryo

Jennifer Bayraktar, Deborah Zygmunt^* and Richard W. Carthew

Department of Biochemistry, Molecular Biology and Cell Biology, 2205 Tech Drive, Northwestern University, Evanston, IL 60208, USA

View larger version (116K): [in a new window]   Fig. 1. Localization of Par-1 in the Drosophila embryo and eye imaginal disc. (A-E) Embryos stained for PAR-1 protein (green) and nuclei (red). All views except B are mid-sagittal; B is of the blastoderm at the apicolateral plane. (A,B,C,D,E) Low magnification views of embryos at different stages. (A',B',C',D',E') High magnification views of the developing blastoderm and ectoderm, with the apical region oriented towards the top. (A,A') Early cellularization. (B,B') Mid-cellularization; PAR-1 is reduced apically and localized laterally, including the furrow canal (arrow). (C,C') Late cellularization. (D,D') Stage 8 gastrulating embryo showing apical and apicolateral PAR-1 in the ectoderm. Weak staining is also present in neuroblasts. (E,E') Stage 15 embryo, with higher levels present in the nervous system and midgut. (F-H) Localization of PAR-1 (green) in eye imaginal discs. (F) A low magnification view of the eye disc. The band of strong staining is the morphogenetic furrow. (G) A transverse view of three ommatidia with the apical region oriented toward the top. PAR-1 is concentrated in apical and apicolateral regions. E-cadherin (red) marks the AJ in projections of photoreceptor neurons. (H) A planar view at the apicolateral plane of disc cells. Par-1 is detected around cell membranes and in the photoreceptor AJs containing E-cadherin (red).

View larger version (64K): [in a new window]   Fig. 2. Par-1 is required to establish blastoderm polarity. (A,B) Stage 15 embryos stained for Par-1 protein (red). Embryos were injected with (A) buffer or (B) with par-1 dsRNA. (C-P) Embryos were injected prior to cellularization with buffer (C,E,G,I,K,M,O) or par-1 dsRNA (D,F,H,J,L,N,P) and stained with antibodies near the end of cellularization. Blastoderm nuclei are shown in blue in some panels and all panels are oriented apical side towards the top. (C,D) Blastoderm stained with anti-Par-1 (red) and anti-E-cadherin (green). (E,F) Anti-phosphotyrosine (green) staining. (G,H) Anti-Patj staining (green) is expanded along the lateral domains of the par-1(RNAi) blastoderm. (I,J) Anti-Crb staining (green) in the apical domain. (K,L) Anti-Dlg staining (green) is present along the lateral membranes. (M,N) Anti-Delta staining (green) is reduced basolaterally by par-1(RNAi). (O,P) Anti-Tau (green) stains the apical cytoplasm capping the nuclei and extends down past the nuclei.

View larger version (119K): [in a new window]   Fig. 3. Par-1 is required for gastrula ectoderm cell polarity. Embryos injected at the syncitial stage with buffer (A,C,E) or par-1 dsRNA (B,D,F) are shown at stage 9. (A,B) Anti-E-cadherin (red) shows expanded localization within ectoderm, mesoderm and neuroblasts in par-1(RNAi) embryos. (C-F) Embryos triple-labeled for Patj (cyan), Dlg (green) and E-cadherin (red). (C,D) Patj staining is not significantly expanded along lateral membranes in par-1(RNAi). (E,F) Dlg staining is similar in control and par-1(RNAi) embryos.

View larger version (160K): [in a new window]   Fig. 4. Par-1 overexpression disrupts eye disc cell polarity. All eye imaginal discs are oriented as anterior to the left. (A) A disc with par-1w3 mutant clones marked by the absence of myr-GFP (green) and stained for E-cadherin (red). (B-D) Confocal z-section series from apical to basal through a disc containing a par-1w3 clone. Markers visualized as in (A). (E) The projected stack of z-sections from B-D. (F,F',I,I',K,K',M,O,P) Low and high magnification of GMR-Gal4 UAS-PAR-1 eye discs. (G,G',J,J',L,L',N,Q,R) Low and high magnification of GMR-Gal4 UAS-PAR-1(AA) eye discs. (H,H') Low and high magnification of GMR-Gal4 UAS-PAR-1(KR) eye discs. (F-H) Anti-Par-1 (green) staining shows all transgenes are expressed at similar levels. (I,J) E-cadherin (green) is altered by kinase-active Par-1. (K,L) Staining of cell nuclei (red) and neuron nuclei with Elav (green) shows neural specification is inhibited by kinase-active Par-1. (M,N) BrdU labeling (green) of disc cells. Insets show magnified views of boxed areas in discs, in the region of the second mitotic wave. This is greatly expanded in (M). Arrows mark regions that are normally mitotically quiescent in wild-type discs. (P,Q) Acridine Orange staining shows apoptosis is enhanced by kinase-active Par-1. (O,R) Discs stained for Crb (red) and the septate junction protein Coracle (green) show disorganization with kinase-active Par-1. However, Crb does not overlap with Coracle (lack of orange signal).

View larger version (50K): [in a new window]   Fig. 5. Loss of Par-1 causes a neurogenic phenotype in late stage embryos. Embryos stained for neuron-specific Neuroglian at stage 15; whole embryos (left panels) and representative segments of peripheral nerves (right panels). (A,B) Wild-type embryos develop highly organized central and peripheral nervous systems with characteristic numbers of neurons. (C,D) par-1(RNAi) embryos and (E,F) N55e11 mutants display hyperplasia of central and peripheral nervous systems. (G,H) scrib(RNAi) embryos and (I,J) shg2, (K,L) bazXI106 and (M,N) crb11A2 mutants all show defects in nervous system development, but have negligible to minor neural hyperplasia.

View larger version (45K): [in a new window]   Fig. 6. Par-1 acts specifically in the Notch pathway. Embryos were injected with buffer (A,C,E) or par-1 dsRNA (B,D,F) before analysis. (A,B) Anti-Achaete staining of four segments at stage 9 shows one positive cell per proneural cluster in control, whereas many proneural cells are positive after par-1 RNAi treatment. (C,D) Stage 15 arm-GAL4 UAS-NECN embryos stained for Neuroglian show severe reduction in neural tissue. (E,F) Stage 10 embryos stained for Engrailed.

View larger version (51K): [in a new window]   Fig. 7. Loss of Par-1 disrupts Delta localization during lateral inhibition. Embryos were injected with buffer (A,E) or par-1 dsRNA (B-D,F-H) and immunostained at stage 9 for Notch (A-D) or Delta (E-H). All embryos were stained for E-cadherin. (A,B) Merged image of Notch (green) and E-cadherin (red), with individual channels in B shown in (C-D). (E,F) Merged image of Delta (green) and E-cadherin (red), with individual channels in F shown in (G-H).

View larger version (72K): [in a new window]   Fig. 8. Par-1 act upstream of Notch in eye disc cell determination. Antenna-eye disc complexes were stained to identify cell nuclei (red) and for neuron-specific Elav (green), which marks differentiating eye tissue but not the antenna disc. (A-C) ey-Gal4 UAS-Par-1 complexes have a smaller eye disc, no eye disc, or transformation of an eye disc into an antenna disc. (D) ey-Gal4 UAS-Par-1(AA) disc complex shows normal determination. (E) ey-Gal4 UAS-Nintra disc complex shows development of extra eye tissue. (F) ey-Gal4 UAS-Nintra UAS-Par-1 disc complex shows development of extra eye tissue. This hyperplasia is significantly enhanced compared to E.