Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

Summary

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

Abstract

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

Introduction

The heart forms early during embryogenesis to start pumping nutrients to the whole embryo, while it continues developing¹. In mouse embryos, one and a half days after the initiation of gastrulation, a rudimentary heart organ assembles at the anterior pole^2,3. By Early Streak (ES) stage, cardiac progenitors in the epiblast ingress through the primitive streak to the nascent mesodermal layer^4,5,6 and start migrating to the anterior pole, where they differentiate to form the primitive heart tube. ....

Protocol

All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee, by the Community of Madrid (Reference PROEX 220/15) and conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.

This protocol includes the use of two males from the fluorescent transgenic mouse line reporting NOTCH activity Tg(CBF:H2BVenus,+)

Discussion

Early heart progenitors organize in a primitive heart tube that starts beating while it is still forming. Understanding how this process takes place is key to pinpoint the wide spectrum of congenital heart defects to specific morphogenetic events. For that, live imaging offers an opportunity to study normal and defective embryonic development with increased temporal resolution. This is especially useful to study early cardiac progenitor cells as they transition quickly through multiple differentiation and migration behav.......

Materials

Name	Company	Catalog Number	Comments
#55 Forceps	Dumont	11295-51
35 mm Dish with glass coverslip bottom 14 mm Diameter	Mattek	P35G-1.5-14-C
35 mm vise table	Grandado	SKU 8798771617573
50 mL tubes	BD Falcon	352070
Distilled water
DMEM - Dulbecco's Modified Eagle Medium	Gibco	11966025	with L-Glutamine, without Glucose, without Na Pyruvate
Fetal Bovine Serum	Invitrogen	10438-026
Fluorescent reporter transgenic mice (Tg(CBF:H2BVenus,+)	JAX
Fluorobrite DMEM	ThermoFisher	A1896701	DMEM for live-cell imaging
High-vacuum silicone grease	Dow Corning	Z273554-1EA
Holder for wires	Perlen Pressen	pwb1
LSM 780 Upright microscope	Zeiss
MaiTai Deepsee far red pulsed-laser tuned at 980 nm	Spectra-Physics
Non Descanned Detectors equipped with the filter sets cyan-yellow (BP450-500/BP520-560), green-red (BP500-520/BP570-610) and yellow-red (BP520-560/BP645-710)	Zeiss
Obj: 20x water dipping 1.0 NA, long working distance	Zeiss
P1000 and P200 pipettes
Paraffin Oil	Nidacon	VNI0049
Penicillin-streptomycin	Invitrogen	15070-063	(the final concentration should be 50 μg/mL penicillin and 50 μg/mL streptomycin)
Petri dishes 35 mm x 10 mm	BD Falcon	351008
Pipette tips
Polymethyl methacrylate			Reused from old laboratory equipment
Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD	Janvier Labs	9979
Set of 160 mm fines	RS PRO	541-6933
Standard 1.0 mm glass capillaries	Anima Lab	1B100F-3
Sterile 0.22 μm syringe filter	Corning	431218
Sterile 5 mL syringe	Fisher Scientific	15809152
Tungsten needles
Ultrasonic homogeniser (sonicator)	Bandelin	BASO_17021