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First published online 15 July 2008
doi: 10.1242/jcs.033167

Journal of Cell Science 121, 2503-2510 (2008)
Published by The Company of Biologists 2008
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Divergent polarization mechanisms during vertebrate epithelial development mediated by the Crumbs complex protein Nagie oko

Nana Bit-Avragim1,2,*, Nicole Hellwig1,*, Franziska Rudolph1, Chantilly Munson3, Didier Y. S. Stainier3 and Salim Abdelilah-Seyfried1,{ddagger}

1 Max Delbrück Center (MDC) for Molecular Medicine, 13125 Berlin, Germany
2 Department of Cardiology, The Charité University Medical School of Berlin, Campus Buch, Campus Virchow Clinics, 13353 Berlin, Germany
3 Department of Biochemistry and Biophysics and Programs in Developmental Biology, Genetics, and Human Genetics, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-2711, USA


Figure 1
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  		Fig. 1. Structure of Nok deletion mutants and their expression. (A) Nok is a MAGUK protein which contains ECR1, a bipartite L27 domain, PDZ, SH3, 4.1 and GUK domains. Overview of different Nok deletions functionally tested in this study. Asterisk indicates the position of the nokm520 mutation. Expected interactions (black arrows) between Nok with different members of the Crb and the Par6-aPKC protein complexes are indicated. (B) Western blot of 32 hpf tissues with Myc-tagged Nokwt (asterisk; expected size of 82 kDa), Nok{Delta}Crb (asterisk; expected size of 74.5 kDa), Nok{Delta}C (dot; expected size of 50.6 kDa), Nok{Delta}Par6 (asterisk; expected size of 81.1 kDa), Nok{Delta}Patj (asterisk; expected size of 75.7 kDa), and Nok{Delta}Patj{Delta}Lin7 (asterisk; expected size of 69.4 kDa). Loading control is acetylated tubulin (bottom).

 

Figure 2
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  		Fig. 2. Functional assays for Nok deletions. (A-G) Phenotypes of (A) wt, (D) nokm520 mutant, or (B,C,E-G) nokm520 mutants that were injected with mRNA encoding different deletions of Nok. Insets are details of the corresponding RPE phenotype. Note that the cobblestone-like appearance of this tissue is severely disrupted in nokm520 (D), nok{Delta}Crb (F) and nok{Delta}C (G) mutants but rescued in the other mutants and WT. (H) Summary of rescue efficiencies of the different deletion constructs in the nokm520 mutant background for two aspects of the phenotype. RPE, retinal pigment epithelium.

 

Figure 3
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  		Fig. 3. Cardiac development in Nok deletion mutants. (A-F) Images represent reconstructions of confocal Z-stack sections of 32 hpf Tg[cmlc2:GFP] transgenic and nok morphant embryos (coinjected with MOp53 to suppress MOnok off-target effects) expressing different Nok deletion proteins. Cardiac morphology is assessed using the atrium-specific antibody S46 (red). Arrows indicate the extent of atrial expansion. (C) Whereas nokwt or (E) nok{Delta}Patj{Delta}Lin7 mRNA complements nok morphant heart defects, all other Nok deletions lack rescue function in this assay. The corresponding mutant cardiac phenotypes are characterized by an arrest at the heart cone stage or by a failure to extend the heart tube. (G) Summary of rescue efficiencies of the different Nok deletions expressed within the nok morphant background.

 

Figure 4
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  		Fig. 4. Neural retinal polarity in Nok deletion mutants. Optical cross sections through the neural retina at 32 hpf. Images represent reconstructions of confocal Z-stack sections. Tissues are stained for ZO-1 (red) and Nok (green). Small insets are magnifications of ZO-1 staining indicated by white arrowheads. (A) The noks305 mutant neural retina displays disruptions (white asterisks) and ectopic non-polarized clusters of junction-associated ZO-1 bundles throughout the entire tissue (white arrowhead). (B) Embryos expressing Nokwt protein within the noks305 mutant background display WT retinal polarity. The Nok domains thought to be required for interaction with (C) Patj, (D) Par6, or (F) Crb, as well as (E) the C-terminus of Nok are essential for retinal polarity. All of these deletion mutants display the characteristic noks305 mutant phenotype suggesting that Nok functions as an important scaffold for a highly conserved assembly of Crb and Par6-aPKC complex proteins within this tissue.

 

Figure 5
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  		Fig. 5. Localization of Nok deletion proteins within myocardial cells. Images represent reconstructions of confocal Z-stack sections imaged on 32 hpf Tg[cmlc2:GFP] transgenic myocardial cells of (A-F) noks305 mutant embryos expressing different Nok deletion proteins, (G) WT, or (H) omem289 mutant. ZO-1 (blue) and Nok (red) label apical cell junctions. (A) Apical junctions are lost and ZO-1-positive belts are missing in noks305 mutant embryos. (B) ZO-1-positive apical junctions are present and myocardial organization is restored in noks305 mutant embryos injected with mRNA encoding Nokwt protein which colocalizes with ZO-1 at apical junction belts (white arrowheads). (C) ZO-1-positive apical junctions are present and myocardial organization is restored in noks305 mutant embryos injected with mRNA encoding Nok{Delta}Patj{Delta}Lin7 which is consistent with the rescue of cardiac morphology by this deletion protein. The deletion protein colocalizes with ZO-1 at apical junctions (white arrowheads). The Nok domains required for interaction with (D) Par6, (E) Crb or (F) the C-terminus of the protein are functionally required for apical junction maintenance and myocardial organization. Although each deletion protein is diffusely expressed within the cytoplasm, ZO-1-positive apical junctions are missing. (A'-F') Nok protein localization is shown in gray. Whereas endogenous Nok protein colocalizes with ZO-1 to apical junctions in WT (G), it is mislocalized in ome/crb2am289 mutant myocardial tissue (H). Orientation: all images are apical views.

 

Figure 6
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  		Fig. 6. Apical localization of Pard6{gamma}b within the neural tube does not require the PDZ domain. (A-C) Live 24 hpf embryos injected with H2b-mRFP and pard6{gamma}bwt-GFP or pard6{gamma}b{Delta}KPLG-GFP mRNA as indicated (dorsal views of the neurocoel between the first and sixth somite). (A,B) In WT, both pard6{gamma}bwt-GFP and pard6{gamma}b{Delta}KPLG-GFP localize along the apical ventricular surface of the neural tube (white arrowheads). (C) Within the pard6{gamma}bs441 mutant background, pard6{gamma}b{Delta}KPLG-GFP localizes correctly to apical surfaces of the neural tube suggesting alternative modes of apical localization of pard6{gamma}b independent of the PDZ-domain-mediated association with Crb or Nok. (D-F) Localization of pard6{gamma}bwt-GFP within the neural tube is dependent on either (E) Nok or (F) Ome/Crb2a. (H) Apical localization of Nok requires Ome/Crb2a. Orientation: all images are apical views.

 

Figure 7
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  		Fig. 7. Alternative multi-protein assemblies of the Crb and Par6-aPKC complexes at the tight junction. Model indicating essential protein-protein interaction partners of Nok within different tissues. Protein-binding domains that are dispensable for Nok function are white. The C-terminal GUK and SH3 domains and the PDZ domain, which is thought to mediate the interaction with Crb proteins, are generally essential within each epithelial tissue. (A) Each of the Nok interaction domains thought to be required for association with Crb, Par6, Patj or Lin-7 is essential for correct polarity of the neural retina, indicating that a highly conserved assembly of Crb and Par6-aPKC complex proteins is present within this tissue. (B) Within the neural tube, interaction with Patj via the L27(N) domain is essential for epithelial integrity. The apical localization of Par6 is independent of the Nok ECR1 domain, which suggests that Par6 associates with Patj within this tissue (see text for further discussion). (C) Association of Nok with Patj or Lin-7 via the two L27 domains is not essential for maintenance of apical ZO-1 junction belts within myocardial cells and for correct myocardial morphogenesis. Therefore, an alternative multi-protein assembly that requires interaction with Par6 via the ECR1 domain may be present within myocardial cells (see text for further discussion). (D) Within RPE cells, Nok requires the PDZ-domain, which is thought to mediate the interaction with Crb, to confer correct polarity to this tissue. Integrity of the RPE is not dependent on the presence of the Nok ECR1 domain (association with Par6) or the two L27 domains (association with Patj and Lin-7). It remains to be investigated whether alternative binding sites are utilized to tether Par6, Lin-7 or Patj to Crb-Nok or whether these proteins are not essential for the maintenance of the Crb complex within the RPE.

 

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© The Company of Biologists Ltd 2008