Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy
- Beth Israel Deaconess Medical Center and Harvard Medical School, United States
- Uppsala University, Sweden
- The Francis Crick Institute, United Kingdom
- Instituto de Medicina Molecular, Portugal
- Harvard Medical School, United States
- Harvard University, United States
- University of Idaho, United States
- Universidade de Lisboa, Portugal
- The Francis Crick Institute / Kings College London, United Kingdom
Abstract
As the general population ages, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the mouse eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM); 2) flat-mounting retinas for confocal microscopy significantly distorts tissue morphology, confirmed by quantitative correlative LSFM-Confocal imaging of vessels; 3) LSFM readily reveals new features of even well-studied eye disease mouse models, such as the oxygen-induced retinopathy (OIR) model, including a previously unappreciated 'knotted' morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics when live-imaged. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of the eye, in particular pathological, neuro-vascular, degenerative processes.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Data has been provided for Figures 3d, e, Figure 4c, Figures 5b,c,d,e, Figures 7d,e,f, Supp. Figures 2c,d and Supp. Figures 5h, i
Article and author information
Author details
Lena Claesson-Welsh
Funding
National Eye Institute (1R21EY027067-01)
- Claudia Prahst
- Katie Bentley
European Research Council (starting grant (679368))
- Claudio A Franco
Fundação para a Ciência e a Tecnologia (grants: IF/00412/2012)
- Claudio A Franco
Fondation Leducq (17CVD03)
- Claudio A Franco
National Eye Institute (EY027067)
- Kin-Sang Cho
Knut och Alice Wallenbergs Stiftelse (KAW 2015.0030)
- Lena Claesson-Welsh
- Katie Bentley
Francis Crick Institute
- Thomas Mead
- Katie Bentley
Fundação para a Ciência e a Tecnologia (PRECISE-LISBOA-01-0145-FEDER-016394)
- Claudio A Franco
Harvard Catalyst (UL1 TR001102)
- Claudia Prahst
- Katie Bentley
Beth Israel Deaconess Medical Center (startup funds)
- Claudia Prahst
- Lakshmi Venkaraman
- Katie Bentley
Kjell och Märta Beijers Stiftelse
- Parham Ashrafzadeh
- Katie Bentley
Marfan Foundation (Victor A McKusick fellowship)
- Lakshmi Venkaraman
European Molecular Biology Organization (ALTF 2016-923 fellowship)
- Mark Richards
National Heart, Lung, and Blood Institute (T32 HL07893)
- Kyle Harrington
National Eye Institute (EY025259)
- Dong Feng Chen
National Eye Institute (P30 EY03790)
- Dong Feng Chen
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice used in experiments at Beth Israel Deaconess Medical Center were held in accordance with Beth Israel Deaconess Medical Center institutional animal care and use committee (IACUC) guidelines. Animal work performed at Uppsala University was approved by the Uppsala University board of animal experimentation. Transgenic mice were maintained at the Instituto de Medicina Molecular (iMM) under standard husbandry conditions and under national regulations.(ethics approval reference C134/14 and C116/15).
Reviewing Editor
- Anna Akhmanova, Utrecht University, Netherlands
Publication history
- Received: June 29, 2019
- Accepted: February 11, 2020
- Accepted Manuscript published: February 19, 2020 (version 1)
- Version of Record published: April 16, 2020 (version 2)
Copyright
© 2020, Prahst et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 6,081
- Page views
-
- 880
- Downloads
-
- 13
- Citations
Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy
eLife 9:e49779.
https://doi.org/10.7554/eLife.49779
Further reading
-
- Cell Biology
- Structural Biology and Molecular Biophysics
Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αβ-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.
-
- Cell Biology
- Developmental Biology
Heterozygous, missense mutations in α- or β-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation’s impact at the molecular and cellular levels scale with the severity of brain malformation. Here, we focus on two mutations at the valine 409 residue of TUBA1A, V409I, and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG (tumor overexpressed gene) domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.
