Name: live imaging of mouse secondary palate fusion
Uploaded: 2017-07-27T14:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

We thank M. Douglas Benson for initial conversations regarding secondary palate imaging. We also acknowledge David Castaneda-Castellanos (Leica) and Chris Rieken (Zeiss) for their help to adjust imaging conditions in confocal microscopy. We appreciate Lynsey Hamilton (Bitplane) for helpful suggestions for quantitative image analysis using Imaris software. This work was funded by NIH/NIDCR R01 DE025887.

All animal experiments were performed in accordance with the protocols of the University of California at San Francisco Institutional Animal Care and Use Committee.
1. Preparation of Live Imaging Media
<ol>
	<li>Preparation of liquid culture media
	<ol>
		<li>Add 20% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 200 &#181;g/mL L-ascorbic acid, 15 mM HEPES to Dulbecco&#39;s Modified Eagle Medium (DMEM)/F12 media. 
		NOTE: L...

<ol>
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Live imaging of tissue morphogenesis with 3D organ explant culture can provide detailed information regarding cellular processes that cannot be shown in conventional staining analysis of fixed tissue sections. Using in vitro explant culture of mouse embryonic secondary palate, we observed several interesting cellular behaviors leading us to propose a novel mechanism of palate fusion that involves epithelial convergence and cell extrusion.
One common challenge in studies of this type i...

Tissue fusion is an important step in the development of multiple organs. Major human birth defects such as cleft lip and palate, spina bifida and malformations of the heart can result from defects in tissue fusion1. Mouse secondary palate fusion has been extensively studied to identify the cellular and molecular mechanisms controlling tissue fusion in development2,3,4. In the mouse, secondary palate development starts at around E11.5 with the outgrowth of a secondary palatal shelf from each of the bilateral maxillary processes. Initial growth of the palatal shelves occurs vertically along the tongue, until approximately E14.0, at which time, the palatal shelves become elevated horizontally above the tongue. Medially-directed growth results in physical contact between the apposing epithelia of the two palatal shelves, forming the midline epithelial seam (MES) at E14.5. The intervening MES must be removed from between the secondary palatal shelves to allow mesenchymal confluence and the development of an intact, completely fused secondary palate by E15.53.
How a shared epithelial MES cell layer is formed between two separate palatal shelves, and then removed to achieve mesenchymal confluency, has been a central question in palate development. Based on mouse histological and electron microscopy (EM) studies, explant culture studies, and functional mouse genetics experiments, several fundamental cell behaviors have been implicated in this process. Filopodia-like projections from the medial edge epithelium MEE of each palatal shelf facilitates initial contact5,6, followed by intercalation of these epithelial cells to a shared single MES6,7. Removal of the resulting shared MES has been proposed to proceed by three non-exclusive mechanisms. Early studies employing histological observation and ex vivo lineage tracing with vital dyes indicated that the MES might be removed by epithelial to mesenchymal transition (EMT) of MES cells8,9, though more recently, genetic lineage tracing of epithelial cells has raised uncertainty as to the long-term contribution of epithelial cells to the palatal shelf mesenchyme10,11,12. Significant numbers of apoptotic cells, and a reduction in their number in some mutants that fail to undergo proper palate fusion has led to the idea that apoptosis may be a major driver of MES dissolution2,3. Finally, based initially on studies involving epithelial labeling and static observation at progressive timepoints, MES cells were proposed to migrate in the oronasal and anteroposterior dimensions11,13, but such dynamic cell behaviors were initially unconfirmed due to an inability to observe them in live palatal tissue. Recently, we were able to directly observe these behaviors by developing a new live imaging methodology that combines mouse genetic methods of fluorescent labeling with confocal live imaging of explanted palatal shelves.
First, to visualize dynamic cellular behaviors in palate epithelial cells during palate fusion, we generated an epithelial-specific reporter mouse by crossing ROSA26-mTmGflox mice with Keratin14-cre mice14,15. Confocal live imaging of palate explant culture of the resulting embryos confirmed some previously proposed cellular behaviors and identified novel events in the fusion process6. Epithelial cell membrane protrusions preceded initial cell-cell contact followed by epithelial convergence by cell intercalation and oronasal cell displacement. Notably, we also discovered that cell extrusion, a process reported to play important roles in epithelial homeostasis, was a major mechanism driving mouse secondary palate fusion6,16. This imaging method can be used with other reporter lines; we utilized Lifeact-mRFPruby transgenic mice17,18 to examine actin cytoskeletal dynamics during the fusion process. Other reporters can also be employed to observe other specific aspects of palate fusion and this method can be adapted either to laser scanning confocal microscopy or spinning disk confocal microscopy, depending on imaging needs and microscope availability. Live imaging is increasingly becoming a keystone approach in developmental biology. Particularly, craniofacial morphogenesis is complex and human birth defects that affect the face are common. This confocal live imaging method will help to enable an improved understanding of underlying basic developmental mechanisms as well as origins of human craniofacial abnormalities.

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	<tbody>
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			<td height="20" style="height:20px;width:348px;">Reagents</td>
			<td style="width:360px;"></td>
			<td style="width:225px;"></td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM/F12</td>
			<td>Life technology</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal Bovine Serum</td>
			<td>Life technology</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-glutamine</td>
			<td>Life technology</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544-100G</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pennicillin/Streptomycin</td>
			<td>Life technology</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Low melting agarose</td>
			<td>BioExpress</td>
			<td>E-3111-125</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">35mm glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Petrolieum Jelly (Vaseline)</td>
			<td>Sigma</td>
			<td>16415-1Kg</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Keratin14-cre</td>
			<td></td>
			<td>MGI: J:65294</td>
			<td>Allele = Tg(KRT14-cre)1Amc</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">ROSA26mTmG</td>
			<td></td>
			<td>MGI: J:124702</td>
			<td>Allele = Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Lifeact-mRFPruby</td>
			<td></td>
			<td>MGI: J:164274</td>
			<td>Allele = Tg(CAG-mRuby)#Rows</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">White Light SP5 confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cell Observer spinning disk confocal microscope</td>
			<td>Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of mouse secondary palate fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Live imaging of palate explant culture revealed multiple cellular processes mediating palate fusion6. Initial contacts between two epithelial cells are made by membrane protrusions (Figure 2B-2E). When two epithelial layers meet, they form a multi-cell layered MES followed by intercalation to make an integrated single cell-layer MES through epithelial convergence(Figure 2A-2E</stro...

Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

Live Imaging of Mouse Secondary Palate Fusion

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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