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First published online 2 February 2005
doi: 10.1242/dev.01645

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aPKC, Crumbs3 and Lgl2 control apicobasal polarity in early vertebrate development

¹ Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QR, UK
² Department of Anatomy, University of Cambridge, Downing Site, Cambridge CB2 3DY, UK
³ Division of Developmental Biology, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA

View larger version (80K): [in a new window]   Fig. 1. aPKC, Crumbs3 and Lgl2 show specific localisation in early epithelial cells. (A) GFP-Lgl2 localised exclusively to the basolateral membrane at stage 8. (B) Crumbs3-GFP localised to the apical (arrowhead) and basolateral membrane at stage 8, and to unknown internal structures (arrow). (C) GFP control. The examples shown are after injecting 1 ng of RNA. (D) RLDX control. GFP localised nonspecifically in the cytoplasm, nucleus and points of cell contact, as did the lineage label RLDX. Because of the high yolk content of early Xenopus cells, cytoplasmic fluorescence of the controls has a latticed appearance. This is very distinct from the localisation of the fusion proteins shown. (E) Antibody staining showing that aPKC localises to the apical membrane.

View larger version (61K): [in a new window]   Fig. 2. aPKC overexpression produces rounded, protruding, hyper-pigmented cells. (A,B) Mouse and Xenopus aPKC overexpression produced embryos with protruding superficial cells and extended pigmented (apical) surface when compared with controls (C,D). (E) Overexpression of a truncated version of the Xenopus tropicalis protein, PKC NT, which lacks the entire kinase domain, failed to produce this phenotype. (F) Crumbs3 overexpression caused cell protrusion and over-apicalisation, similar to that of aPKC, but was less effective in that the percentage of affected embryos was lower. Quantification was carried out blind, by counting the number of embryos with protruding cells. Right panels show the percentage of affected embryos at each concentration of injected RNA. Each experiment was carried out at least three times and the average is shown.

View larger version (76K): [in a new window]   Fig. 7. aPKC and Lgl act by a process of mutual inhibition. (A,B) GFP-Lgl was injected on its own (A) or with aPKC (B). Addition of aPKC inhibited the basolateral localisation of GFP-Lgl2. GFP was visualised by using an anti-GFP antibody. (C,D) Overexpression of Lgl2 inhibited the apical localisation of aPKC but overexpression of GFP did not. (E-G) Lgl2, but not GFP injections, can rescue the apicalisation caused by injecting aPKC. There are more rounded cells in aPKC plus GFP-injected embryos than in aPKC plus Lgl2-injected embryos. The graph shows the average percent of embryos with apicalised cells from three experiments. The experiment was scored blind as for Fig. 2.

View larger version (96K): [in a new window]   Fig. 3. aPKC is sufficient to promote apical and inhibit basal lateral membrane identity without disrupting tight junctions. (A,B) aPKC overexpression (B) caused expansion of the apical marker keratin compared with GFP control (A). (C-F) aPKC caused reduction in the basolateral markers, occludin (D) and ß1-integrin (F) compared with controls (C,E). (G,H) aPKC caused tight junctions (as marked by cingulin) to be maintained but relocated to the new apicobasolateral border. The borders of the markers used in each panel are delineated with arrows. (I,J) aPKC staining in GFP-injected controls (I) and aPKC staining in aPKC-injected (J) embryos. The apicalised cells have inherited overexpressed aPKC. (K) Diagrammatic representation of the result; aPKC causes protruding hyper-apical cells, which still have tight junction markers. Apical, red; basolateral, black; tight junctions, green. Albino embryos were injected with aPKC RNA and stained for antibody markers of cell polarity. Each experiment was carried out three times.

View larger version (79K): [in a new window]   Fig. 4. Loss of aPKC function expands basolateral membrane domain into the apical side and disrupts the apical domain. (A) The aPKC NT construct has the Par6-binding site but no kinase domain and so acts as a dominant-negative fragment. (B) The effect of this construct can be rescued by overexpressing full-length aPKC. Injections of 4.5 ng aPKC NT + 0.5 ng GFP, 4.5 ng aPKC NT + 0.5 ng aPKC, or 5 ng GFP were carried out. The average of four experiments scored blind is shown. (C,D) aPKC NT dominant-negative fragment caused pigment defects (D) compared with control (C) (5 ng of each). (E,H) aPKC NT was co-injected with GFP showing that the pigment defects occurred in the injected region. The arrows in C,E and D,H highlight the same cells. (F-J) aPKC NT (I,J) caused ectopic localisation of the basolateral markers ß1-integrin and occludin to the apical side (arrow) and tight junctions were also lost (J, arrowhead) when compared with GFP control (F,G). (K) Diagram of the observed phenotype. Colours are as above. Pigmented embryos were injected as this allowed the affected area to be easily identified, they were then fixed and stained for markers of cell polarity.

View larger version (90K): [in a new window]   Fig. 5. Lgl2 promotes basolateral and inhibits apical identity. (A) Injection of 5 ng GFP did not affect the cells. (B,C) Injection of Xenopus Lgl2 caused loss of pigment and also a block in cytokinesis at high doses (B, 5 ng; C, 0.5 ng). (D-K) Injection of GFP (D-G) or Lgl2 (H-K) and immunostaining with the markers shown in each panel. GFP-injected embryos were entirely normal. (I) Injection of Lgl2 caused a reduction in keratin to the levels normally seen in the basolateral region (arrow) and loss of tight junctions (cingulin, arrowhead). (J,K) Injection of Lgl2 caused ectopic localisation of ß1-integrin (J) and occludin (K) to the apical side (arrow) and loss of tight junctions (arrowhead). (L) diagrammatic representation of phenotype, colours as above. Experiments were carried out three times in both albino and pigmented embryos (except for the keratin where the staining is obscured by the pigment and therefore was carried out only in albinos), and the same result was obtained in both.

View larger version (72K): [in a new window]   Fig. 6. Time-lapse images showing the gradual but direct depigmentation of the apical side by Lgl2. A pigmented embryo was injected animally with Lgl2 RNA at the two-cell stage and filmed. A small site of cytoplasmic leakage helps to verify the site of injection. Evidence of apical membrane disruption starts as a concentration of pigment spots (arrow) that appear quite suddenly and spread quickly. The even distribution of pigmentation is lost and the pigment is gradually cleared from the apical side. Interestingly, pigment becomes concentrated to the periphery of the apical domain. There is no evidence of inner cells coming to the surface of the embryo or outer cells falling in.

View larger version (12K): [in a new window]   Fig. 8. A model showing the antagonistic action of aPKC and Lgl2 in maintaining the apical and basolateral domain. Increased aPKC causes expansion of the apical domain (red), while reduced aPKC or increased Lgl2 causes expansion of the basolateral domain (black). Tight junctions are shown in green.