Summary

Multiphotonique Time Lapse imagerie de visualiser le développement en temps réel : visualisation de la migration des cellules neurales de crête dans des embryons de poisson-zèbre

Published: August 09, 2017
doi:

Summary

Une combinaison des techniques optiques avancées de lecture avec excitation de fluorescence multi-photons longue longueur d’onde de laser a été mis en œuvre pour capturer l’imagerie haute résolution, en trois dimensions, en temps réel de la migration de la crête neurale en Tg (sox10:EGFP) et les embryons de poissons zèbres Tg (foxd3:GFP).

Abstract

L’oeil congénitales et des anomalies cranio-faciales reflètent les perturbations dans la crête neurale, une population nomade de cellules souches migratrices qui donnent lieu à nombreux types de cellules dans tout le corps. Comprendre la biologie de la crête neurale a été limité, ce qui reflète un manque de modèles génétiquement tractable pouvant être étudié in vivo et en temps réel. Poisson zèbre est un modèle de développement particulièrement important pour l’étude des populations de cellules migratrices, comme la crête neurale. Afin d’examiner la migration de la crête neurale dans le œil en voie de développement, une combinaison des techniques optiques avancées de lecture avec excitation de fluorescence multi-photons de grande longueur d’onde de laser a été mis en place pour capturer des vidéos de haute résolution, en trois dimensions, en temps réel de le œil en développement chez les embryons de poisson-zèbre transgéniques, nommément Tg (sox10:EGFP) et Tg (foxd3:GFP), comme sox10 et foxd3 ont été présentés dans plusieurs modèles animaux de réglementer une différenciation précoce crête neurale et représentent probablement des marqueurs de cellules neurales de crête. Les Time-lapse imagerie multiphotonique servait à discerner le comportement et les habitudes migratoires des deux populations de cellules neurales de crête contribuant au développement d’oeil au début. Ce protocole fournit des informations pour générer des vidéos time-lapse pendant la migration de poisson-zèbre de crête neurale, à titre d’exemple et peut être davantage appliqué pour visualiser le début du développement de nombreuses structures dans le poisson-zèbre et autres organismes modèles.

Introduction

Congenital eye diseases can cause childhood blindness and are often due to abnormalities of the cranial neural crest. Neural crest cells are transient stem cells that arise from the neural tube and form numerous tissues throughout the body.1,2,3,4,5 Neural crest cells, derived from the prosencephalon and mesencephalon, give rise to the bone and cartilage of the midface and frontal regions, and the iris, cornea, trabecular meshwork, and sclera in the anterior segment of the eye.4,6,7,8 Neural crest cells from the rhombencephalon form the pharyngeal arches, jaw, and cardiac outflow tract.1,3,4,9,10 Studies have highlighted the contributions of the neural crest to ocular and periocular development, emphasizing the importance of these cells in vertebrate eye development. Indeed, disruption of neural crest cell migration and differentiation lead to craniofacial and ocular anomalies as observed in Axenfeld-Rieger Syndrome and Peters Plus Syndrome.11,12,13,14,15,16,17 Thus, a comprehensive understanding of the migration, proliferation and differentiation of these neural crest cells will provide insight into the complexities underlying congenital eye diseases.

The zebrafish is a powerful model organism for studying ocular development, as the structures of the zebrafish eye are similar to their mammalian counterparts, and many genes are evolutionarily conserved between zebrafish and mammals.18,19,20 In addition, zebrafish embryos are transparent and oviparous, facilitating the visualization of eye development in real-time.

Expanding on previously published work,6,7,20 the migratory pattern of neural crest cells was described using multi-photon fluorescence time-lapse imaging on transgenic zebrafish lines labeled with green fluorescent protein (GFP) under the transcriptional control of the SRY (sex-determining region Y)-box 10 (sox10) or Forkhead Box D3 (foxd3) gene regulatory regions.21,22,23,24. Multi-photon fluorescence time-lapse imaging is a powerful technique that combines the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with fluorophores.25,26,27 The use of the multi-photon laser has distinct advantages over standard confocal microscopy, including increased tissue penetration and decreased fluorophore bleaching.

Using this method, two distinct populations of neural crest cells varying in timing of migration and migratory pathways were discriminated, namely foxd3-positive neural crest cells in the periocular mesenchyme and developing eye and sox10-positive neural crest cells in the craniofacial mesenchyme. With this method, an approach to visualize the migration of ocular and craniofacial neural crest migration in zebrafish is introduced, making it easy to observe regulated neural crest migration in real time during development.

This protocol provides information for generating time-lapse videos during early eye development in Tg(sox10:EGFP) and Tg(foxd3:GFP) transgenic zebrafish, as an example. This protocol can be further applied for the high-resolution, three-dimensional, real-time visualization of the early development of any ocular and craniofacial structure derived from neural crest cells in zebrafish. Moreover, this method can further be applied for the visualization of the development of other tissues and organs in zebrafish and other animal models.

Protocol

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA). 1. Embryo Collection for Time-lapse Imaging Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating. NOTE: The Tg(sox10:EGFP) and Tg(foxd3:G…

Representative Results

L’imagerie de fluorescence multi-photons Time-lapse a généré une série de vidéos qui a révélé les patrons de migration des cellules de la crête neurale crânienne qui donnent lieu à des structures cranio-faciales et les segment antérieur de le œil dans le Tg (sox10:EGFP) et Tg (foxd3:GFP) lignes de poisson-zèbre. À titre d’exemple, sox10 -cellules neurales de crête positive entre 12 et 30 hpf migrent du bord du tube neural dans la région cran…

Discussion

Les Time-lapse imagerie multiphotonique permet le suivi in vivo des populations de cellules migratrices et transitoire. Cette technique puissante permet d’étudier les processus embryonnaires en temps réel, et dans la présente étude, les résultats de cette méthode a amélioré les connaissances actuelles de la migration des cellules neurales de crête et le développement. Les études d’imagerie Time-lapse antérieures utilisent généralement laser confocal, microscopie à balayage. 2…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Les auteurs remercient Thomas Schilling pour gifting gentiment le poisson Tg (sox10:eGFP) et Mary Halloran pour gentiment gifting le poisson(foxd3:GFP) de Tg.

Materials

Breeding Tanks with Dividers Aquaneering ZHCT100 Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert
M205 FA Combi-Scope Leica Microsystems CMS GmbH Stereofluorescence Microscope – FusionOptics and TripleBeam
Sodium Chloride Millipore (EMD) 7760-5KG Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3
Potassium Chloride Millipore (EMD) 1049380500 Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.
Calcium Chloride Dihydrate Fisher Scientific C79-500 Poly bottle; 500 g. CAS No. 10035-04-8
Magnesium Sulfate (Anhydrous) Millipore (EMD) MX0075-1 Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2
Methylene Blue Millipore (EMD) 284-12 Glass bottle; 25 g. Powder, Certified Biological Stain
Sodium Bicarbonate Millipore (EMD) SX0320-1 Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8
N-Phenylthiourea Sigma P7629-25G >98%. CAS Number 103-85-5, EC Number 203-151-2
Dimethylsulfoxide Sigma D8418-500ML Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3
Tricaine Methanesulfonate Western Chemical Inc. MS222 Tricaine-S
Low-Melt Agarose ISC Bioexpress E-3112-25 GeneMate Sieve GQA Low Melt Agarose, 25 g
Open Bath Chamber Warner Instruments RC-40HP High Profile
Glass Coverslips Fisher Scientific 12-545-102 Circle cover glass. 25 mm diameter
High Vacuum Grease Fisher Scientific 14-635-5C 2.0-lb. tube. DOW CORNING CORPORATION
1658832
Quick Exchange Platform Warner Instruments QE-1 35 mm
Stage Adapter Warner Instruments SA-20LZ-AL 16.5 x 10 cm
TC SP5 MP multi-photon microscope Leica Microsystems CMS GmbH
Mai Tai DeepSee Ti-Sapphire Laser SpectraPhysics
Laser Safety Box Leica Microsystems CMS GmbH
Leica Application Suite X (LAS X)  Software Leica Microsystems CMS GmbH
Photoshop CS 6 Version 13.0 x64 Software Adobe
iMovie Version 10.1.4 Software Apple

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Cite This Article
Williams, A. L., Bohnsack, B. L. Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos. J. Vis. Exp. (126), e56214, doi:10.3791/56214 (2017).

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