Z.C. acknowledges the support from Dartmouth startup fund and the Branco Weiss- Society for Science fellowship, administered by ETH Zurich. The authors thank Drs. Larry A. Taber, Benjamen A. Filas, Qiaohang Guo, and Yunfei Shi for helpful discussions, as well as the anonymous reviewers for comments.&#160;This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1313911. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.&#160;

1. Preparation of Tissue Culture Media
<ol>
	<li>Use a 0.5 L bottle of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 4.5 g/L glucose, sodium bicarbonate, and L-glutamine as the base for the culture media.</li>
	<li>In a sterile laminar flow hood, add 10 mL of antibiotics to the 0.5 L of DMEM.</li>
	<li>Using a sterile pipette, transfer 50 mL of the DMEM antibiotics solution to a sterile 50 mL conical tube.</li>
	<li>Add 50 mL of chick serum to the remaining DMEM antibiotics solution in the 0.5 L bottle in the ...

<ol>
	<li>Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+Growth,+Remodeling,+and+Morphogenesis.">Biomechanics of Growth, Remodeling, and Morphogenesis.</a> Appl. Mech. Rev. 48, 487-545 (1995).</li><li>Wyczalkowski, M. A., Chen, Z., Filas, B. A., Varner, V. D., Taber, L. 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The authors declare no conflicts of interest.

While physical phenomena play an integral role in morphogenesis26,27,28,29,30, 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...

Modern developmental biology research largely focuses on understanding development from the perspective of molecular genetics1,2,3,4,5,6,7,8,9,10,11,12,13. It is known that physical phenomena play a central role in morphogenesis, or the generation of biological form14,15,16,17; however, specific mechanical mechanisms of development remain largely unstudied. Ventral flexure and rightward torsion of the primitive brain tube after Hamburger-Hamilton stage 11 (HH 11)18 are the two main processes that contribute to embryonic shape change19,20. In particular, the physical mechanism underlying the torsional development in the embryonic brain remains incompletely understood.
The embryonic torsion in chick embryo is among the earliest morphogenetic events of left-right (L-R) asymmetry in development. When the process of L-R asymmetry is perturbed, birth defects such as situs inversus, isomerism, or heterotaxia will occur21. 
Here we present a protocol which combines ex-ovo experiments22,23 with physical modeling to characterize mechanical forces during early embryonic brain development. The goal of the method presented is to identify the mechanical forces responsible for brain torsion and the factors that affect the degree of torsion during early development12. Based on the experimental observation that the vitelline membrane (VM) constrains the dorsal side of the embryo, we hypothesized that the VM provides the force necessary to induce torsion of the developing brain. Therefore, in this method, we removed the part of the VM that covers the brain area to find out the effects on brain torsion. Furthermore, the method of applying fluid surface tension was used to confirm the mechanical role of the VM and provide an estimate of the force needed for brain torsion, which had not been done previously. Measuring the forces during embryonic morphogenesis is a challenging task. Notably, in a pioneering study, Camp&#224;s and co-workers24 developed a novel method to quantify the cellular stresses using injected microdroplets. Nevertheless, this method was limited to measure forces at the cellular level, hence not applicable to probe forces at tissue- or organism-level. The protocol presented in this paper was developed to partially fill this gap.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Fertilized Specific pathogen-free White Leghorn chicken eggs</td>
			<td style="width:177px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:288px;">Optical Coherent Tomography Microscope</td>
			<td style="width:177px;">Thorlabs</td>
			<td style="width:119px;">GAN220C1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:288px;">Silicone elastomer</td>
			<td style="width:177px;">Smooth-On, Inc.</td>
			<td style="width:119px;">EcoFlex 00-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dissecting microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:119px;">MZ8</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:288px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Lonza</td>
			<td style="width:119px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Antibiotics</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">P4083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Chick serum</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">C5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Micropipette puller</td>
			<td style="width:177px;">Sutter Instrument</td>
			<td style="width:119px;">Model P-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Filter paper</td>
			<td style="width:177px;">Whatman</td>
			<td style="width:119px;">5202-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Phosphate buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:288px;">Comsol MultiPhysics</td>
			<td style="width:177px;">Comsol</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">3D computer graphics software</td>
			<td style="width:177px;">Rhino 5</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Microscope attached with OCT</td>
			<td style="width:177px;">Nikon&#160;</td>
			<td style="width:119px;">FN1</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:288px;">Digital single-lens reflex camera</td>
			<td style="width:177px;">EOS&#160;</td>
			<td style="width:119px;">Rebel T3i</td>
			<td></td>
		</tr>
	</tbody>
</table>

probing the roles of physical forces in early chick embryonic morphogenesis

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&#160;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.

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...

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Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

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.

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis ...

The overall goal of this experimental protocol is to study the role of mechanical forces in early embryonic brain development through ex ovo experiments. This method can help answer key questions in developmental biology and biomechanics, such as the roles of mechanical cues in morphogenesis, which is the generation of biological form. The main advantage of this technique is that it enables the characterization of the role of mechanical forces in morphogenesis at the tissue and organism level.To begin this procedure, use delicate wipes with 70%ethanol to clean the fertilized specific-pathogen-free White Leghorn chicken eggs. Then, arrange the eggs in a longitudinal orientation on a holder. After that, set the egg incubator at a target temperature of 37.5 degrees Celsius, and maintain the humidity at 48 to 55%The humidity is controlled by adding an appropriate amount of water to the incubator.Incubate the eggs for approximately 40 to 44 hours to HH11 to 13. Afterward, let the eggs cool down at room temperature for approximately 15 to 30 minutes prior to cleaning with 70%ethanol. In this step, crack the eggs from the bottom, pull apart the shell gently, and carefully deposit the contents into a 150 by 15 millimeter Petri dish.To ensure the embryo side is up, keep the eggs in the same orientation as they were incubated while cracking them. Remove the thin albumin using a disposable Pasteur pipette. Separate the thick albumin from the yolk using the blunt-ended forceps.Ensure that the thick albumin has been removed by lightly scraping the top of the yolk. Then, using fine-tipped forceps, place a filter paper ring over the embryo and center it, matching the long axis of the ring with the long axis of the embryo. Subsequently, cut the yolk surrounding the filter paper ring with scissors.Following that, pull the ring and embryo off the yolk in an oblique direction towards the site where the yolk was first cut. Then, rinse the embryo in two sequential 100-millimeter dishes with room temperature PBS. Next, place a filter paper ring into a 35-millimeter Petri dish.Place the embryo dorsal side up onto the other filter paper in the 35-millimeter Petri dish. After that, place a stainless-steel ring on top of the filter paper sandwich. Take care not to damage the embryo.Now, add three milliliters of the previously prepared CCM to each Petri dish. Remove the vitelline membrane of each embryo by lightly skimming the pulled capillary needle across the top of the embryo. Then, peel the vitelline membrane away, starting from the anterior end and proceeding to the notochord.Following that, place eight 35-millimeter Petri dishes into one 150-millimeter dish lined with water-saturated wipes to maintain humidity. Then, place the 150-millimeter Petri dish into a sealable plastic bag, and fill the bag with a gas mixture comprised of 95%oxygen and 5%CO2. Seal the bag, and place it in a 37.5 degree Celsius incubator.Now, remove the embryo from the incubator, and use an OCT system to image them and to determine the torsional angle of the neural tube. Transfer the embryos to a light microscope, and visualize at 10X magnification. Use a 200-microliter pipette to incrementally remove 0.2 milliliters of media from the Petri dish.Take bright-field images following each round of media removal to observe the effect of the media-air interface on the embryo. Continue removing media until the surface tension across the embryo induces torsion. Image the embryo using the OCT system once again to establish a final torsional angle for comparison to control embryos.To alter the direction of the heart loop, use a pair of forceps to flip the filter paper so that the embryo becomes ventral side up. Then, use the pulled capillary tube to cut open a slit in the splanchnopleure membrane and to exert a mechanical force to push the heart from the right-hand side to the left-hand side. Apply the fluid surface tension to keep the heart on the left-handed side.Then, incubate the embryo for another 20 hours to see the change in chirality of the torsion. Shown here is a harvested embryo with VM removed at HH11. The same embryo incubated for 27 hours post VM removal showing reduced torsion.Here is the same embryo that underwent brain torsion upon application of fluid surface tension, and here is the control embryo with normal brain torsion at a comparable stage. While attempting this procedure, it's important to be careful not to damage the embryo. Following this procedure, other methods like finite element analysis can be performed in order to computationally model how physical forces can influence morphology.Therefore, this technique developed here paved the way for future studies on mechanics of morphogenesis in chick embryos. After watching this video, you should understand how to harvest chick embryos for in vitro culture, micro-surgically remove the vitelline membrane, recapitulate the force provided by the vitelline membrane, and monitor embryonic morphology through OCT and bright-field imaging.

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Research

JoVE Journal

Bioengineering

7.2K Views.  Dartmouth College. The overall goal of this experimental protocol is to study the role of mechanical forces in early embryonic brain development through ex ovo experiments. This method can help answer key questions in developmental biology and biomechanics, such as the roles of mechanical cues in morphogenesis, which is the generation of biological form. The main advantage of this technique is that it enables the characterization of the role of mechanical forces in morphogenesis at the tissue and organism level...