Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Summary

Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

Abstract

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis is becoming increasingly recognized. In particular, the embryonic chick heart and brain tube, which undergo drastic morphological changes as they develop, are among the prime candidates to study the role of physical forces in morphogenesis. Progressive ventral bending and rightward torsion of the tubular embryonic chick brain happen at the earliest stage of organ-level left-right asymmetry in chick embryonic development. The vitelline membrane (VM) constrains the dorsal side of the embryo and has been implicated in providing the force necessary to induce torsion of the developing brain. Here we present a combination of new ex-ovo experiments and physical modeling to identify the mechanics of brain torsion. At Hamburger-Hamilton stage 11, embryos are harvested and cultured ex ovo (in media). The VM is subsequently removed using a pulled capillary tube. By controlling the level of the fluid and subjecting the embryo to a fluid-air interface, the fluid surface tension of the media can be used to replace the mechanical role of the VM. Microsurgery experiments were also performed to alter the position of the heart to find the resultant change in the chirality of brain torsion. Results from this protocol illustrate the fundamental roles of mechanics in driving morphogenesis.

Introduction

Modern developmental biology research largely focuses on understanding development from the perspective of molecular genetics^{1,2,3,4,5,6,7,8,9,10,11,12,13}. It is known that physical phenomena play a central role in morphogenesis, or....

Protocol

1. Preparation of Tissue Culture Media

1. Use a 0.5 L bottle of Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/L glucose, sodium bicarbonate, and L-glutamine as the base for the culture media.
2. In a sterile laminar flow hood, add 10 mL of antibiotics to the 0.5 L of DMEM.
3. Using a sterile pipette, transfer 50 mL of the DMEM antibiotics solution to a sterile 50 mL conical tube.
4. Add 50 mL of chick serum to the remaining DMEM antibiotics solution in the 0.5 L bottle in the sterile hood.
5. Store the final solution (referred to hereafter as chick culture media [CCM]) in 50 mL conical tube aliquots at -20 °C.

Results

In this study, the VM of the embryo at HH11 was removed from the anterior end to the thoracic flexure. The embryos were imaged by an OCT system. At this stage, the torsion of brain tube has not started (Figure 1A). After being incubated to HH15-16, embryos with their VM removed exhibited reduced brain tube torsion, approximately 35 degrees (Figure 1B) compared to control embryos, which exhibit torsion of around 90 degrees. When t.......

Discussion

While physical phenomena play an integral role in morphogenesis^{26,27,28,29,30}, the specific mechanical mechanisms, along with the coordination of mechanical and molecular mechanisms, remain largely unexplored. It is known that the ventral flexure and rightward torsion of the primitive brain are two central processes that contribute to early embryonic morphogen.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fertilized Specific pathogen-free White Leghorn chicken eggs	Charles River
Optical Coherent Tomography Microscope	Thorlabs	GAN220C1
Silicone elastomer	Smooth-On, Inc.	EcoFlex 00-50
Dissecting microscope	Leica	MZ8
Dulbecco’s Modified Eagle’s Medium (DMEM)	Lonza	12-604F
Antibiotics	Sigma	P4083
Chick serum	Sigma	C5405
Micropipette puller	Sutter Instrument	Model P-30
Filter paper	Whatman	5202-110
Phosphate buffered saline (PBS)	Corning	21-040-CV
Comsol MultiPhysics	Comsol
3D computer graphics software	Rhino 5
Microscope attached with OCT	Nikon	FN1
Digital single-lens reflex camera	EOS	Rebel T3i