Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Interviews
    • Sign up for alerts
  • About us
    • About BiO
    • Editors and Board
    • Editor biographies
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contact
    • Contact BiO
    • Advertising
    • Feedback
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

User menu

  • Log in

Search

  • Advanced search
Biology Open
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

supporting biologistsinspiring biology

Biology Open

Advanced search

RSS   Twitter   Facebook   YouTube

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Interviews
    • Sign up for alerts
  • About us
    • About BiO
    • Editors and Board
    • Editor biographies
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contact
    • Contact BiO
    • Advertising
    • Feedback
Research Article
The Golgi matrix protein giantin is required for normal cilia function in zebrafish
Dylan J. M. Bergen, Nicola L. Stevenson, Roderick E. H. Skinner, David J. Stephens, Christina L. Hammond
Biology Open 2017 6: 1180-1189; doi: 10.1242/bio.025502
Dylan J. M. Bergen
1Cell Biology Laboratories, School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK
2School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nicola L. Stevenson
1Cell Biology Laboratories, School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Roderick E. H. Skinner
2School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David J. Stephens
1Cell Biology Laboratories, School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: chrissy.hammond@bristol.ac.uk david.stephens@bristol.ac.uk
Christina L. Hammond
2School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Christina L. Hammond
  • For correspondence: chrissy.hammond@bristol.ac.uk david.stephens@bristol.ac.uk
  • Article
  • Figures & tables
  • Supp info
  • Info & metrics
  • eLetters
  • PDF + SI
  • PDF
Loading

Article Figures & Tables

Figures

  • Fig. 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 1.

    Experimental knockdown and knockout of giantin in vivo. (A) Schematic representation of the zebrafish golgb1 transcript (ENSDART00000131402.2) showing the binding sites of the designed morpholinos (ATG and E14), the relative exonic location of the human annotated p115 binding site, transmembrane domain (TMD), the various mouse mutants (black asterisks) and the ocd rat allele (white asterisk) as described in Lan et al. (2016), and Katayama et al. (2011), respectively. Coloured bars show the interspecies conserved sequence regions as assigned in Ensembl (release 87). Red asterisks indicate the location of zebrafish giantin mutant alleles. (B) Alternative spliced PCR products (blue arrows) from 32 hpf RT-PCR cDNA of two amplicons in E14 MO binding region. Black arrows indicate the expected size of PCR product. (C) Sa11389 line carrying a point mutation from C to T (yellow highlight, red letters) resulting in a premature stop codon at Q2948 (golgb1Q2948X) from the EZRC. (D) TALEN site directed mutagenesis resulted in an 8 bp deletion (spacer sequence in yellow, red letters), resulting in golgb1X3078 mutant line. (E) Alignment of Golgb1 WT protein sequence (Ensembl release 87) with predicted Golgb1 X3078 protein sequence showing translated spacer sequence (red) and site of deletion T3028 and A3029 with a subsequent frameshift from E3027 (red asterisk) changing 51 amino acids to a predicted stop codon at position 3078. Part of cDNA exon 14 sequence. (F) Stereomicroscope images of 48 hpf control, ATG, and E14 morphants exhibiting defects in the eye, heart, cranium, and various axis orientations (blue, green, black, red arrow, respectively). (G) Percentile quantification of scored phenotypes. (H) Stereomicroscope images of heterozygote in-cross 5 dpf larvae from both mutant lines and (I) 7 wpf female adults. (J) Dot plot for body length at 7 wpf, and (K) 44-46 wpf for golgb1Q2948X and 41-43 wpf for golgb1X3078 alleles. Scale bars: (F) 100 µm, (H) 200 µm, (I) 500 µm. (J, K) One-way ANOVA with Tukey's multiple comparison test. All experiments of three replicates. Bars show means with standard deviation. *P<0.05, and *** P<0.005.

  • Fig. 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 2.

    Giantin knockdown leads to changed cilia length and morphology with a ciliopathy-like phenotype in morphant zebrafish. (A) Lateral view confocal images of 24 hpf neural tube ependymal cilia (red in merge, white arrow) were quantified in a two nuclei wide area (dotted line). Nuclei (DAPI-labelled) are shown in blue. Scale bar: 10 μm. (B) Quantification of cilia number per field of image within the two nuclei window (panel A, two images per individual). Scale bar: 50 μm. (C) Scatter dot plot and (D) cumulative frequency plot (0.25 µm bins) of all cilia measured. (E) Average cilium length per individual measured. (F) Representative confocal images of in vivo imaging control and E14 morphants showing Arl13b-GFP-positive cilia (white arrow) in the myotome (lateral view). Scale bars: 10 µm. (G) Arl13b-GFP ciliary membrane length displayed in a cumulative frequency plot of all measured cilia (Ctrl MO: n=115, E14 MO n=152). (H) Average cilium length per individual imaged. On scatter plots (B, C, E, H), bars represent mean with standard deviation and statistical testing was done by one-way ANOVA with a Dunn's multiple comparison test. Data from three independent experiments. *P<0.05, ** P<0.01, and ***P<0.001.

  • Fig. 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 3.

    Knockout of giantin results in defects in cilia length. (A,B) Lateral view confocal images of 24 hpf cilia in ventral neural tube of Q2948X mutant line (A) and X3078 mutant line (B). Scale bars: 10 μm. White arrows indicate ciliary axonemes. (C) Cilia number per field of image (two per individual) in a two nuclei wide area adjacent to the notochord. (D,E) Ciliary length of all cilia measured depicted in a scatter dot plot (D) and a cumulative frequency plot (E) showing shift (blue arrow Q2948X and green arrow X3078) in percentile abundance of binned (0.25 µm bins) ciliary length between WT and homozygous 24 hpf embryos. (F) Average ciliary length per individual imaged. Data shown from at least three independent in-crosses. Bars show mean with standard deviation. Data was normally distributed and a subsequent unpaired t-test was performed between wild-type and homozygous groups. *P<0.05, **P<0.01, and ***P<0.001.

  • Fig. 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 4.

    Scanning electron microscopy reveals structural defects in cilia following knockdown of giantin. (A) SEM images olfactory pit (3 dpf) indicating ciliary tips (red arrows). (B) SEM images of LLC-PK1 cells exhibit bulbous ciliary tips (red arrow) with visible ciliary extension (blue arrow) after Ss-Golgb1 knockdown.

  • Fig. 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 5.

    Left-right asymmetry and phenotypic rescue of giantin MO. (A) Cardiomyocyte Cmcl2::GFP transgenic reporter line outlining the heart position (arrows, blue: left; green: midline; white: right) at 32 hpf. (B) Representative confocal image of 48 hpf with ATG morpholino and Rn-Golgb1 mRNA showing Golgi localisation of Rn-Golgb1 in dermal cells stained for anti-flag (white arrows). White dotted line indicates cell boundaries. N=3 individuals. (C) Quantification of heart position (32 hpf), additionally showing Rn-Golgb1 mRNA co-injection rescue. (D) Stereomicroscope images of 3 dpf control and E14 morpholinos injected with same dose in WT and golgb1Q2948X/Q2948X individuals derived from same day WTxWT and golgb1Q2948X/Q2948X x golgb1Q2948X/Q2948X in-crosses with (E) quantification of phenotypes scored. (F) Quantitative RT-PCR of golgins showing relative mRNA expression levels on 5 dpf larvae. Normalised to beta-actin, mean and standard deviation. All data from three independent experiments.

Previous ArticleNext Article
Back to top
Previous ArticleNext Article

This Issue

RSSRSS

Keywords

  • Golgi
  • Golgi matrix
  • Cilia
  • Zebrafish

 Download PDF

Email

Thank you for your interest in spreading the word on Biology Open.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
The Golgi matrix protein giantin is required for normal cilia function in zebrafish
(Your Name) has sent you a message from Biology Open
(Your Name) thought you would like to see the Biology Open web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Research Article
The Golgi matrix protein giantin is required for normal cilia function in zebrafish
Dylan J. M. Bergen, Nicola L. Stevenson, Roderick E. H. Skinner, David J. Stephens, Christina L. Hammond
Biology Open 2017 6: 1180-1189; doi: 10.1242/bio.025502
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Research Article
The Golgi matrix protein giantin is required for normal cilia function in zebrafish
Dylan J. M. Bergen, Nicola L. Stevenson, Roderick E. H. Skinner, David J. Stephens, Christina L. Hammond
Biology Open 2017 6: 1180-1189; doi: 10.1242/bio.025502

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Alerts

Please log in to add an alert for this article.

Sign in to email alerts with your email address

Article Navigation

  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • Acknowledgements
    • Footnotes
    • References
  • Figures & tables
  • Supp info
  • Info & metrics
  • eLetters
  • PDF + SI
  • PDF

Related articles

Cited by...

More in this TOC section

  • Smoking flies: Testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster
  • Bisphenol A promotes stress granule assembly and modulates the integrated stress response
  • Variation in alarm calls during different breeding stages of the common kestrel (Falco tinnunculus)
Show more RESEARCH ARTICLE

Similar articles

Other journals from The Company of Biologists

Development

Journal of Cell Science

Journal of Experimental Biology

Disease Models & Mechanisms

Advertisement

Biology Open and COVID-19

We are aware that the COVID-19 pandemic is having an unprecedented impact on researchers worldwide. The Editors of all The Company of Biologists’ journals have been considering ways in which we can alleviate concerns that members of our community may have around publishing activities during this time. Read about the actions we are taking at this time.

Please don’t hesitate to contact the Editorial Office if you have any questions or concerns.


2020 at The Company of Biologists

Despite 2020’s challenges, we achieved a lot at The Company of Biologists. In the midst of the pandemic, we have seen long-term projects and new ventures come to fruition. Read our full lowdown of 2020.


Interview- Sebastian Markert

Sebastian Markert is first author of a paper in BiO using C. elegans to model amyotrophic lateral sclerosis. In an interview, he talks about the potential implications of his work and his future plans.


Three communities to support biologists to everywhere

Online communities have never been more important. If you’re looking for somewhere to meet fellow scientists, take part in topical discussions and find virtual events in your field, take a look at each of our community sites:

  • The Node: the community site for and by developmental biologists
  • preLights: the preprint highlights service run by the biological community
  • FocalPlane: the community site for microscopists and biologists alike

Articles

  • Accepted manuscripts
  • Issue in progress
  • Latest complete issue
  • Issue archive
  • Archive by article type
  • Interviews
  • Sign up for alerts

About us

  • About BiO
  • Editors and Board
  • Editor biographies
  • Grants and funding
  • Journal Meetings
  • Workshops
  • The Company of Biologists

For Authors

  • Submit a manuscript
  • Aims and scope
  • Presubmission enquiries
  • Article types
  • Manuscript preparation
  • Cover suggestions
  • Editorial process
  • Promoting your paper
  • Open Access

Journal Info

  • Journal policies
  • Rights and permissions
  • Media policies
  • Reviewer guide
  • Sign up for alerts

Contact

  • Contact BiO
  • Advertising
  • Feedback

Twitter   YouTube   LinkedIn

© 2021   The Company of Biologists Ltd   Registered Charity 277992