Zebrafish homolog establishes and maintains cell adhesion and tissue integrity

Milad Adloo

NBP, a zebrafish homolog of human Kank3, is a novel Numb interactor essential for epidermal integrity and neurulation

Boggetti B., Jasik J., Takamiya M., Strähle U., Reugels A. M., and Campos-Ortega J. A.

Institut für Entwickl ungsbiologie, University of Cologne, 50923 Cologne, Germany
Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany
Institute of Cellular and Molecular Botany, University of Bonn, 53115 Bonn, Germany
Institute for Toxicology and Genetics, Forschungszentrum Karlsruhe, Postfach 3640, 76021 Karlsruhe, Germany

Developmental Biology 365 (2012) 164-174.

Introduction

Numb is an adaptor protein implicated in diverse basic cellular processes. Numb was originally identified as a gene required for normal development of the peripheral nervous system in Drosophila. In recent years, many studies in mammals have shown the importance of Numbs in different fields such as developmental neurobiology, cancer biology and degenerative diseases. Although the exact biochemical role of Numbs has yet to be discovered, increasing amounts of evidence show that Numbs may act as adaptors facilitating the formation of multi-protein complexes (1).

In this study, a new interactor of Numb is isolated in zebrafish that is an ortholog of human Kank3. Kank3 belongs to the kidney ankyrin repeat-containing protein family defined in 2009 by Kakinuma et. al. Kank1 was originally found as a tumor supressor of renal cell carcinoma and its deletion was connected with cerebral palsy (2). The authors reveal that zebrafish Numbs Binding Protein (NBP) interacts with Numbs through a novel motif well conserved in all vertebrates Kanks. They also demonstrate that NBP is a protein with nonredundant function during zebrafish embryogenesis as its deficiency causes serious embryo defects and lethality within 48 hours. They also discover a role of NBP in establishing and/or maintaining cell adhesion and polarity.

Methods

Yeast two-hybrid assay — Used to screen zebrafish cDNA library for either Numb1 or Numb3. Also used for quantitative analysis of protein binding and for identification of interaction domains

Domain and Protein Structure Analysis — NBP amino acid sequence was compared to the protein NCBI database(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Full-length protein sequences with structures resembling NBP were aligned by ClustalW XXL (http://www.ch.embnet.org/software/ClustalW-XXL.html).

Creation of knockdown embryos and EGTA treatment — NBP and Numbs knockdown embryo lines were generated using the antisense morpholino oligonucleotides (MO). NBP MO and Numb MO were injected into one-four-cell stage embryos to create single or double knockdown morphants. For EGTA treatment, embryos were kept with the chorion and briefly washed with calcium-magnesium free medium followed by 1 hour incubation in 20 mM EGTA.

Biomolecular fluorescence complementation analysis (BiFC), GFP technology and immunohystochemistry — Cappes mRNAs for BiFC and GFP technologies were amplified in vitro. RNA samples were injected into one-four-cell stage wild-type embryos. For immunohistochemistry, embryos from the BiFC assay were sectioned and probed with primary antibodies, washed, then treated for appropriate secondary antibodies.

Results

Key Points:

  • NBP interacts with Numbs through a well conserved region among Kank proteins
  • Numb proteins and NBP bind in vitro and in living embryos
  • The expression pattern of NBP is developmentally regulated
  • NBP is polarly localized
  • NBP is required for embryo developement and surviving
  • Numb and NBP interact on genetic level

NBP and Numbs morphants show defects in development.

Panels compare NBP morphants (NBP MO),NBP control morphants (NBP MOC) and Numbs morphants (NumbsMO) with wild-type embryos (WT). (A) 24 hpf embryos injected with either NBP MO or NumbsMO are delayed in growth and abnormally developed in comparison with wild-type embryos. Arrows point to dissociated cells in the dorsal region of the NBP morphants (lateral view, anterior towards the left). (B) Western blot analysis of protein extracts from 24hpf wild-type and NBP MO-injected embryos with the N1 polyclonal antibody against NBP. The wild-type extract gives a NBP-positive band of the expected size (99.57 kDa), whereas only a negligible amount of NBP is seen in extracts from the morphants. (C, E– H) Anomalous development of the epidermis. The epidermal cells of the NBP MO-injected embryos varied in size and shape, were separated by large intercellular spaces (C) and frequently were released from the surface (E , asterisk denotes the gap below the epidermal cells; arrowheads in F and H). Tumor-like structures emerged from the dorsal part of the embryo (as terisks in F, G, and H). They were composed of enlarged cells with round,β-catenin strongly positive nuclei (F) and with α-catenin (G), β-catenin (F) and E-cadherin (H) enriched in the cell – cell contacts. Stars point to inner tissue located just underneath the tumor-like structur es where cells lack β -catenin in both nuclei and membranes (F) and α-catenin in membranes (G). Arrows point to junctions between neighboring epidermal cells enriched in β -catenin (F), α-catenin (G) and E-cadherin (H) in WT and to mislocalized E-cadheri n in epidermal cells of NBP MO (H). Nuclei in H are stained by acridine orange. Scale bar: A, D = 100μm, C, F– H= 20μ m, and E = 1 μm.

NBP is required for neural tube development.

Panels compare NBP morphants (NBP MO) and double knockdown NBP; Numb mutants (NBP;Numb ) with wild-type embryos (WT). (A) Many cells in the neural tube of the 24 hpf morphants, particularly of the embryos injected with 1 mM NBP MO, gave positive reaction in the TUNEL assay when compared to embryos either injected with the mismatch NBP MOC (at 1 mM, not shown) or un-injected (flat preparations, anterior to the left). (B) Abnormal growth in the ventricle of the hindbrain in NBP MO embryos (flat preparations, anterior to the left). (C –F) Neural tube defects in the NBP morphants. In embryos depleted fromNBP (E, right picture) the cells of the neural tube frequently failed to form the usual epithelial structure compose d of elongated columnar cells (arrows point toβ -catenin membrane staining of baso-apically oriented polarized wild-type cells and of randomly oriented rounded cells in the morphant), the tissue appeared to be uno rganized and did not form the typical midline enriched in β -catenin and actin ring characteristic for wild-type (D; E, arrowheads in the left and middle pictures). The nuclei (visualized by acridine orange) we re not placed in their characteristic positions (C, left picture) but were scattered in the whole volume and their divisions were not restricted to the apical side of the neural tube (a rrow in C, right picture, points to an anaphase in a deep region of the neural tube) . For the morphant, E and C represent the same section probed by anti-β-catenin antibody (E) and acridine orange (C), respectively. (D) More than one neurocoel developed in the NBP morphants (middle panel), a phenotypic aberration enhanced in the NBP;Numb double morphants (arrowheads, neurocoel; arrows; HuC positive cells). (F) In the NBP morphant, Numb1 did not display the typical accumulation at the basal pole of cells (arrowhead, midline; arrow, basal pole of the neural cells). Scale bar: A, B = 100 μm, and C–F=5μm.

Conclusion

The authors demonstrate  that NBP, the zebrafish Kank 3, directly interacts with Numbs. The NBP expression experiments and embryo lethality of the NBP zebrafish morphants directly implies the importance of this non-redundant gene in embryo development. The authors also characterize another domain responsible for binding NBP to Numbs, which is conserved among all Kanks and may bind other proteins containing PTB domains.

Review/Strengths & Weaknesses:

Although the authors did an outstanding job with their experimental assay and displaying their data, they were not truly able to answer their initial question about what exactly does NBP do. Although they made some revelations and have promising theories, the precise biochemical function of NBP remains a mystery. The authors also did not develop any additional findings regarding Kanks, only mentioning that, in general, Kanks is rather poorly investigated and warrants more research as to its specific biological role.

References:

  1. Gulino, A., Di Marcotullio, L., Screpanti, I., 2010. “The multiple functions of Numb.” Exp. Cell Res. 316, 900–906.
  2. Sarkar, S., Roy, B.C., Hatano, N., Aoyagi, T., Gohji, K., Kiyama, R., 2002. “A novel ankyrin repeat-containing gene (Kank) located at 9p24 is a growth suppressor of renal cell carcinoma.” J. Biol. Chem. 277, 36585 –36591.

2 Responses to Zebrafish homolog establishes and maintains cell adhesion and tissue integrity

  1. camuzie says:

    Why are there references listed at the top?

    • madloo says:

      It is not really a reference, it is the title of the article, the authors, their affiliations and the journal the article was published in. It is more the source of the work

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