Name: isolation and time-lapse imaging of primary mouse embryonic palatal mesenchyme cells to analyze collective movement attributes
Uploaded: 2021-02-13T14:30:00
Description: Watch this Scientific Journal Video about Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes at JoVE.com

This project was supported in part by the National Institutes of Health grants DE026172 (I.S.), and GM102801 (A.C.). I.S. was also supported in part by the Center of Biomedical Research Excellence (COBRE) grant (National Institute of General Medical Sciences P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence grant (National Institute of General Medical Sciences P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (KIDDRC) grant (U54 Eunice Kennedy Shriver National Institute of Child Health and Human Development, HD090216).

All experiments involving animals were carried out with a protocol approved by the KUMC Institutional Animal Care and Use Committee, in accordance with their guidelines and regulations (Protocol Number: 2018-2447).
1. Harvest E13.5 embryos
<ol>
	<li>Euthanize pregnant female mice using a CO2 inhalation chamber or by a procedure approved by the Institutional Animal Care and Use Committee. Immediately proceed to dissection.</li>
	<li>Expose the inferior half of the abdominal cavity ...

Palatal shelf elevation constitutes a vertical to horizontal remodeling event1,3,4,9,11. It is postulated that this remodeling process requires palatal shelf mesenchymal cells to behave coordinately. The analyses with wildtype MEPM cells show that this cell behavior is intrinsic and can be quantitated21. Thus, these assays can be used t...

Palate development has been studied extensively as defects in palatogenesis lead to cleft palate-a common birth defect that occurs in isolated cases or as part of hundreds of syndromes1,2. The development of the embryonic palate is a dynamic process that involves movement and fusion of embryonic tissue. This process can be divided into four major steps: 1) induction of palatal shelves, 2) vertical growth of the palatal shelves next to the tongue, 3) elevation of the palatal shelves above the tongue, and 4) fusion of the palatal shelves at the midline1,3,4. Over the past several decades, many mouse mutants have been identified that manifest cleft palate5,6,7,8. Characterization of these models has&#160;indicated defects in palatal shelf induction, proliferation, and fusion steps; however, palatal shelf elevation defects have been rare. Thus, understanding the dynamics of palatal shelf elevation is an intriguing area of research.
Careful analysis of some mouse mutants with palatal shelf elevation defects has led to the current model showing&#160;that the very anterior region of the palatal shelf appears to flip up, while a vertical to horizontal movement or &#34;remodeling&#34; of the palatal shelves occurs in the middle to posterior regions of the palate1,3,4,9,10,11. The medial edge epithelium of the palatal shelf likely initiates the signaling required for this remodeling, which is then driven by the palatal shelf mesenchyme. Recently, many researchers have identified palatal shelf elevation delay in mouse models that showed transient oral adhesions involving palatal shelves12,13. The mesenchymal remodeling involves reorganization of the cells to create a bulge in the horizontal direction, while simultaneously retracting the palatal shelf in the vertical direction9,10,14. Among the several mechanisms proposed to affect palatal shelf elevation and the underlying mesenchymal remodeling are cell proliferation15,16,17, chemotactic gradients18, and extracellular matrix components19,20. An important question arose: is the palatal shelf elevation delay observed in Specc1l-deficient mice also partly due to a defect in the palatal shelf remodeling, and could this remodeling defect manifest in an intrinsic defect in behavior of primary MEPM cells21?
Primary MEPM cells have been used in the craniofacial field for many studies involving gene expression22,23,24,25,26,27,28,29, and a few involving proliferation30,31 and migration25,31,32, but none for collective cell behavior analysis. Time-lapse imaging of MEPM cells was performed in 2D culture and wound-repair assays to show that MEPM cells displayed directional movement and formed density-dependent cell streams-attributes of collective movement21. Furthermore, Specc1l mutant cells formed narrower cell streams and showed highly variable cell migration trajectories. This lack of coordinated motility is considered to contribute to the palate elevation delay in Specc1l mutant embryos13,21. Thus, these relatively simple assays using primary MEPM cells may serve as a proxy for studying mesenchymal remodeling during palatal shelf elevation. This paper describes the isolation and culture of primary MEPM cells, as well as the time-lapse imaging and analysis, for the 2D and wound-repair assays.

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	<tbody>
		<tr>
			<td>Beaker, 250 mL (x2)</td>
			<td>Fisher Scientific</td>
			<td>FB-100-250</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2</td>
			<td>Matheson Gas</td>
			<td>UN1013</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes, 15 mL (x1)</td>
			<td>Midwest Scientific</td>
			<td>C15B</td>
			<td></td>
		</tr>
		<tr>
			<td>Debian operating system</td>
			<td></td>
			<td></td>
			<td>computational analysis of time-lapse images</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium/High Glucose with 4 mM L-Glutamine and Sodium Pyruvate</td>
			<td>Cytiva Life Sciences</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH, 100%</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Auto</td>
			<td>ThermoFisher Scientific</td>
			<td>AMAFD1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS Onstage Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>AMC1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS Onstage Vessel Holder, Multi-Well Plates</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEPVH028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine point #5 Stainless Steel Forceps (x2)</td>
			<td>Fine Science Tools</td>
			<td>11295-10</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Instrument sterilizer bead bath</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcetrifuge tubes, 1.5mL</td>
			<td>Avant</td>
			<td>2925</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Dissecting Stainless Steel Scissors, Straight</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>NucBlue (Hoechst) Live Ready Probes</td>
			<td>ThermoFisher Scientific</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Solution, 100x</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Insert, 2-well</td>
			<td>Ibidi</td>
			<td>80209</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Perforated Stainless Steel Spoon</td>
			<td>Fine Science Tools</td>
			<td>MC17C</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Spring Scissors, 4 mm</td>
			<td>Fine Science Tools</td>
			<td>15018-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 10 cm dishe(s)</td>
			<td>Corning</td>
			<td>430293</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 12-well plate(s)</td>
			<td>PR1MA</td>
			<td>667512</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 6-well plate(s)</td>
			<td>Thermo Fisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic bulb transfer pipette</td>
			<td>ThermoFisher Scientific</td>
			<td>202-1S</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, 0.25%</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and time-lapse imaging of primary mouse embryonic palatal mesenchyme cells to analyze collective movement attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

The dissection of palatal shelves is illustrated in Figure 1. The sequence of incisions is designed to minimize slippage of the tissue. Following the removal of the head (Figure 1A,B), the lower jaw is removed (Figure 1B,C). The incision of the upper part of the head (Figure 1C,D) is done to stabilize the tissue when placed upside down (<strong class="x...

Watch this Scientific Journal Video about Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes at JoVE.com

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

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