Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Summary

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Abstract

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

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....

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

1. 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:GFP) fish, kind gifts from Thomas Schilling and Mary Halloran, respectively, were crossed into the Casper (roy -/-, nacre -/-) b....

Results

Multi-photon fluorescence time-lapse imaging generated a series of videos that revealed the migration patterns of cranial neural crest cells that give rise to the craniofacial structures and anterior segment of the eye in the Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish lines. As an example, sox10-positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the craniofacial region (Video 1, F.......

Discussion

Multi-photon time-lapse imaging enables the in vivo tracking of transient and migratory cell populations. This powerful technique can be used to study embryonic processes in real time, and in the present study, the results of this method enhanced the current knowledge of neural crest cell migration and development. Previous time-lapse imaging studies typically utilize confocal laser scanning microscopy.^29,30,31,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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