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: Live imaging media was freshly made right before the imaging. This media contains phenol-red which may lead to phototoxicity over long time courses of live imaging. Substituting for media without phenol-red may improve long-term imaging.</li>
	</ol>
	</li>
	<li>Preparation of low melting agarose solution
	<ol>
		<li>Dissolve 350 mg of low-melting agarose in 10 mL of autoclaved distilled water to make a 3.5% stock solution.</li>
		<li>Mix the agarose solution well and incubate at 70 &#176;C in a water bath to dissolve the agarose completely.</li>
		<li>Cool down the agarose solution in a 37 &#176;C water bath before adding it to the live imaging media.</li>
		<li>Add 1 mL of the agarose stock solution to 5 mL of the liquid live imaging media to make a final concentration (0.6%).</li>
		<li>Keep the media with agarose in a 37 &#176;C water bath to prevent premature solidification.</li>
	</ol>
	</li>
</ol>
2. Preparation of Palate Explant Culture for Live Imaging
<ol>
	<li>Dissection of recently-adhered E14.5 secondary palatal shelves 
	NOTE: In our experience imaging fusion requires starting with a slightly adhered pair of palatal shelves; this adherence prevents explant movements that prevent fusion from occurring during imaging.
	<ol>
		<li>Dissect E14.5 stage embryos in 1x phosphate-buffered saline (PBS) and choose green fluorescent protein (GFP)-expressing (from K14-cre; ROSA26-mTmGflox) or (Red fluorescent protein) RFP-expressing (from Lifeact-mRFPruby) positive embryos using a dissecting microscope equipped with fluorescent lamps. Transfer tissue to pre-warmed live imaging culture media without agarose.</li>
		<li>Cut the embryo head and remove upper brain area by using two fine No. 5 forceps (Figure 1A-C). Use one No. 5 forceps to hold the embryo while the other No. 5 forceps to cut away extra tissue outside the palatal shelves. 
		NOTE: Fine scissors can be used to cut embryo head (the first step) instead of one No. 5 forceps. However, two No. 5 fine forceps will give better control to make precise cuts.</li>
		<li>Remove mandible carefully without damaging palatal shelves as shown in Figure 1D and 1E. To reduce the chance of damaging palates, keep the tongue through dissection of the mandible, followed by careful removal of the tongue.</li>
		<li>After removing the mandible and tongue, examine the oral side of the secondary palate with a stereomicroscope with fluorescence to confirm strong reporter signal in the MES (Figure 1M).</li>
		<li>Dissect out posterior parts including hindbrain and brain stem after removing mandible and tongue (Figure 1F).</li>
		<li>Remove both maxillae with adhered palatal shelves (Figure 1G).</li>
		<li>Cut the anterior upper jaw tissue keeping both primary and secondary palate intact (Figure 1H and I).</li>
		<li>Remove nasal septum on nasal side of explant exposing the MES. 
		NOTE: These later steps of dissections need careful attention not to disrupt the contacts between two secondary palatal shelves. Removal of the nasal septum helps to reduce background signal from other areas and also reduce vertical tissue movement, allowing stable imaging (Figure 1J-L).</li>
		<li>After dissecting the palatal shelves, examine the midline reporter signal again to make sure that there is no damage to the epithelial layer between the tissues.</li>
	</ol>
	</li>
	<li>Setting up palate explants in culture dish with live imaging media
	<ol>
		<li>Put live imaging media into 35-mm glass bottom dish.</li>
		<li>Put dissected palatal shelves oral side facing down as close to the glass bottom as possible for imaging with an inverted confocal microscope (Figure 1N).</li>
		<li>Place dry ice pellets on a conductive surface nearby to the culture dish to expedite agarose solidification by decreasing temperature fast or put the culture dish at 4 &#176;C. A cold plate, such as one used for paraffin embedding could also be used at this stage. 
		NOTE: When dry ice is used, agarose media change needs to be monitored carefully to prevent freezing media.</li>
	</ol>
	</li>
</ol>
3. Confocal Time-lapse Imaging of Palate Explant
<ol>
	<li>Imaging and Data acquisition
	<ol>
		<li>After agarose was semi-solidified, mount the culture dish in an inverted confocal microscope equipped with 37 &#176;C incubation chamber. Use a white light confocal microscope and cell observer spinning disk confocal microscope. 
		NOTE: We used a modified media containing 15 mM HEPES without CO2 gas.</li>
		<li>Put petroleum jelly between the culture dish and cover using a syringe to prevent evaporation of media (Figure 1N). 
		NOTE: Some condensation on top of the culture dish cover after overnight (O/N) culture occurs but the volume of media does not change suggesting that the petroleum jelly prevents media from evaporating.</li>
		<li>Locate the region of interest with low magnification objectives (10x) and use higher magnification objectives (20x objective with 3x digital zoom or a 40x objective with Immersol W) for time-lapse live imaging. 
		NOTE: Dissected secondary palatal shelves are not flat. The secondary palate is a curved structure and this makes it difficult to image the entire MES in the same plane. Low magnification (10x) imaging can allow whole palate imaging but 40x objectives will provide better image resolution to examine detailed cellular movements in deeper Z positions. The curvature is high in the middle palate region. Anterior and posterior palate are relatively flat compared to the middle palate. We often image toward the mid-anterior of the secondary palate to avoid regions of greatest curvature.</li>
		<li>Scan multiple Z stacks with 5 &#181;m each step for 16-24 h using proper laser excitation (488 nm for GFP and 532/561 nm for RFP) (Figure 2). 
		NOTE: To reduce potential phototoxicity, use minimum laser power and exposure time. This is important especially in case of multi-color imaging. Use 22% of 552 nm laser power for membraneTomato (mT), 30% of 495 nm laser power for membraneGFP (mG) and 25% of 561 nm laser power for membraneRFP. Average exposure time was 550 ms.</li>
		<li>Use image analysis software to perform cell tracking and quantitative analysis.</li>
	</ol>
	</li>
</ol>
4. Data Image Analysis
NOTE: Here, Imaris was used. Similar data analyses may also be performed with ImageJ or Volocity software packages.
<ol>
	<li>Rendering 3-dimensional (3D) surfaces from image sequences. 
	NOTE: This section enables the generation of a surface from a movie generated with a membrane GFP signal.
	<ol>
		<li>Click Edit | Show Display Adjustment and use the histogram slider to adjust the signal intensity so that the membrane signal is clear throughout the length of the movie (Figure 3A).</li>
		<li>Correct bleached signal by using Image Processing | Attenuation Correction. Adjust the Intensity Front and Intensity Back values so that the membrane signal is clear throughout the length of the movie. Some trial and error might be required in setting these values. 
		NOTE: The Intensity Back (Deeper Z position) value will be less than the Intensity Front (Surface Z position) value due to limited laser penetration. These values can therefore also be changed to normalize signal intensity in the Z series. Both values may be adjusted to achieve bleaching correction. Attenuation correction is also called Emission attenuation correction19.</li>
		<li>In order to generate a surface from the membrane signal, select Surpass | Surfaces, choose a source channel and Threshold type (Absolute Intensity) and under the Algorithm Settings select Segment only a Region of Interest and Process of entire image finally, followed by clicking on the forward arrow; continue to push the forward arrow (use default settings, or specify Surface Area Detail Levels and Thresholding method by trial and error), checking whether the generated surface reflects the fluorescence signal. To specify a Region of Interest, select region and time(s) to be rendered by cropping X, Y, Z positions. 
		NOTE: Select Process of entire image finally so that adjustments and thresholds will be applied across the entire movie.</li>
		<li>Generate a surface by clicking the forward arrow button again. Then adjust the threshold by changing the histogram in Filters to correspond to the membrane signal.</li>
		<li>After the surface is generated, choose surface in Surfaces/Settings to visualize the surface signal and finish the surface generation process by pushing the double forward arrow button (Figure 3B).</li>
		<li>Go to Surface | Edit and click Mask all in the Mask Properties to generate a masked channel image.</li>
		<li>After clicking ok, the Mask Channel window will automatically open; select the GFP channel, set voxels outside the surface to the maximum signal intensity value (found in Edit | Display adjustment) and voxels inside the surface to the minimum GFP intensity value (also found in Edit | Display adjustment), in Mask Settings to generate a mask of the membrane signal.</li>
		<li>As the mask of the membrane signal will be shown in Volume view, generate inverted images by using Image Processing | Contrast Change | Invert (Figure 3C).</li>
	</ol>
	</li>
	<li>Detecting spots from inverted surface images. 
	NOTE: This section allows the center of each cell to be marked with a unique spot and tracked over space and time.
	<ol>
		<li>Select Surpass Scene | Spots | Create; in Algorithm Settings choose Segment only a Region of Interest, Process of entire image finally and Track Spots (over time).</li>
		<li>Click on the forward arrow and specify a Region of Interest; select the region and time(s) to track spots; continue to push the forward arrow, choose the masked channel as a source channel and determine estimated XY diameter of spots.</li>
		<li>As clicking the forward arrow button will automatically open a filter window; adjust the spot detection threshold by changing values in the histogram to generate single spots at the center of each cell (Figure 3D).</li>
		<li>As clicking the forward arrow button again will open the Algorithm window., choose Autoregressive motion tracking model20 and specify the maximum tracking distance and maximum gap size to connect tracks that become discontinuous for a short period of time.</li>
		<li>As clicking the forward arrow button again will open the Filters window, set the track duration threshold by adjusting the histogram so that every cell is tracked. Clicking the double forward arrow button will finalize spot detection.</li>
		<li>In order to correct tissue drift, click Spots | Track Editor and select Correct Drift. An Algorithm window will open automatically; select the select Translational drift, Include entire result, and Correct objects positions.</li>
	</ol>
	</li>
	<li>Generating vantage plots for quantitative analysis. 
	NOTE: The program&#39;s Scatterplot is a tool to generate multi-dimensional (X, Y, Z, Color and Scale) graphs with multiple objects. These plots are useful for viewing trends of many cell movements over time.
	<ol>
		<li>Make 2D or 3D plots of cell tracking data using the Vantage-Add New Vantage plot menu (Figure 3E and 3F).</li>
		<li>Select Volume or Spots and Choose Create/ Category and Plot Type</li>
		<li>Adjust Plot Values to generate different Vantage graphs.</li>
		<li>Export graphs using the Snapshot function. 
		NOTE: Statistical data can also be exported as a spreadsheet for further quantitative analysis by clicking Plot Numbers Area | Save.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Ray, H. J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+tissue+fusion+during+development.">Mechanisms of tissue fusion during development.</a> Dev. Camb. Engl. 139, 1701-1711 (2012).</li><li>Dudas, M., Li, W. -. Y., Kim, J., Yang, A., Kaartinen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palatal+fusion+-+where+do+the+midline+cells+go?+A+review+on+cleft+palate,+a+major+human+birth+defect.">Palatal fusion - where do the midline cells go? A review on cleft palate, a major human birth defect.</a> Acta Histochem. 109, 1-14 (2007).</li><li>Bush, J. O., Jiang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palatogenesis:+morphogenetic+and+molecular+mechanisms+of+secondary+palate+development.">Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development.</a> Development. 139, 231-243 (2012).</li><li>Lan, Y., Xu, J., Jiang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+Molecular+Mechanisms+of+Palatogenesis.">Cellular and Molecular Mechanisms of Palatogenesis.</a> Curr Top Dev Biol. 115, 59-84 (2015).</li><li>Taya, Y., O'Kane, S., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+cleft+palate+in+TGF-b3+knockout+mice.">Pathogenesis of cleft palate in TGF-b3 knockout mice.</a> Development. 126, 3869-3879 (1999).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergence+and+Extrusion+Are+Required+for+Normal+Fusion+of+the+Mammalian+Secondary+Palate.">Convergence and Extrusion Are Required for Normal Fusion of the Mammalian Secondary Palate.</a> PLOS Biol. 13, 100122 (2015).</li><li>Yoshida, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periderm+cells+covering+palatal+shelves+have+tight+junctions+and+their+desquamation+reduces+the+polarity+of+palatal+shelf+epithelial+cells+in+palatogenesis.">Periderm cells covering palatal shelves have tight junctions and their desquamation reduces the polarity of palatal shelf epithelial cells in palatogenesis.</a> Genes Cells. 17, 455-472 (2012).</li><li>Fitchett, J., Hay, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medial+edge+epithelium+transforms+to+mesenchyme+after+embryonic+palatal+shelves+fuse.">Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse.</a> Dev. Biol. 131, 455-474 (1989).</li><li>Akira, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+TGF-beta3+for+epithelial-mesenchyme+transdifferentiated+MEE+in+palatogenesis.">The expression of TGF-beta3 for epithelial-mesenchyme transdifferentiated MEE in palatogenesis.</a> J Mol Hist. 41, 343-355 (2010).</li><li>Sani, F. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate-mapping+of+the+epithelial+seam+during+palatal+fusion+rules+out+epithelial-mesenchymal+transformation.">Fate-mapping of the epithelial seam during palatal fusion rules out epithelial-mesenchymal transformation.</a> Dev. Biol. 285, 490-495 (2005).</li><li>Jin, J. -. Z., Ding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+migration,+transdifferentiation+and+apoptosis+during+mouse+secondary+palate+fusion.">Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion.</a> Development. 133, 3341-3347 (2006).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+autonomous+requirement+for+Tgfbr2+in+the+disappearance+of+medial+edge+epithelium+during+palatal+fusion.">Cell autonomous requirement for Tgfbr2 in the disappearance of medial edge epithelium during palatal fusion.</a> Dev. Biol. 297, 238-248 (2006).</li><li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro:+an+analysis+by+Dil+labelling+and+confocal+microscopy.">The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by Dil labelling and confocal microscopy.</a> Development. 114, 379-388 (1992).</li><li>Muzumdar, M., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li><li>Dassule, H., Lewis, P., Bei, M., Mass, R., McMahon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+growth+and+morphogenesis+of+the+tooth.">Sonic hedgehog regulates growth and morphogenesis of the tooth.</a> Development. 127, 4775-4785 (2000).</li><li>Eisenhoffer, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crowding+induces+live+cell+extrusion+to+maintain+homeostatic+cell+numbers+in+epithelia.">Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.</a> Nature. 484, 501-546 (2012).</li><li>Riedl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5, 605-607 (2008).</li><li>Riedl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact+mice+for+studying+F-actin+dynamics.">Lifeact mice for studying F-actin dynamics.</a> Nat Methods. 7, 168-169 (2010).</li><li>Can, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+correction+in+confocal+laser+microscopes:+a+novel+two-view+approach.">Attenuation correction in confocal laser microscopes: a novel two-view approach.</a> J Microsc. 211, (2003).</li><li>Veenman, C., Reinders, M., Backer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+motion+correspondence+for+densely+moving+points.">Resolving motion correspondence for densely moving points.</a> IEEE Trans Pattern Anal Mach Intell. 23, 54-72 (2001).</li><li>Udan, R., Piazza, V., Hsu, C., Hadjantonakis, A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+cell+dynamics+in+mouse+embryos+using+light-sheet+microscopy.">Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy.</a> Development. 141, 4406-4414 (2014).</li><li>Stoller, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+mouse+expressing+a+nuclear+localized+fusion+of+GFP+and+beta-galactosidase+reveals+new+derivatives+of+Pax3-expressing+precursors.">Cre reporter mouse expressing a nuclear localized fusion of GFP and beta-galactosidase reveals new derivatives of Pax3-expressing precursors.</a> Genesis. 46, 200-204 (2008).</li></ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12</td><td>Life technology</td><td>11330-032</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life technology</td><td>16000-044</td><td></td></tr><tr><td>L-glutamine</td><td>Life technology</td><td>25030-081</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sigma</td><td>A4544-100G</td><td></td></tr><tr><td>Pennicillin/Streptomycin</td><td>Life technology</td><td>15140-122</td><td></td></tr><tr><td>Low melting agarose</td><td>BioExpress</td><td>E-3111-125</td><td></td></tr><tr><td>35 mm glass bottom dish</td><td>MatTek</td><td>P35G-1.5-10-C</td><td></td></tr><tr><td>Petrolieum Jelly (Vaseline)</td><td>Sigma</td><td>16415-1Kg</td><td></td></tr><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Keratin14-cre&lt;/em&gt;</td><td></td><td>MGI: J:65294</td><td>Allele = Tg(KRT14-cre)1Amc</td></tr><tr><td>&lt;em&gt;ROSA26&lt;sup&gt;mTmG&lt;/sup&gt;&lt;/em&gt;</td><td></td><td>MGI: J:124702</td><td>Allele = Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo</td></tr><tr><td>&lt;em&gt;Lifeact-mRFPruby&lt;/em&gt;</td><td></td><td>MGI: J:164274</td><td>Allele = Tg(CAG-mRuby)#Rows</td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>White Light SP5 confocal microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>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 ...

live-imaging-of-mouse-secondary-palate-fusion

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

7.5K Views.  University of California San Francisco. 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.

Video: Live Imaging of Mouse Secondary Palate Fusion

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Bioengineering

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

Cancer Research

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Neuroscience

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

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.

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 ...

Live Tracking of Dental Cells via Two-Photon Microscopy

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

Author Spotlight: Understanding Dynamic Cellular Behaviors in Adult Mouse Dental Tissue Renewal and Repairment 

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...

Live Imaging of Mouse Secondary Palate Fusion

Summary

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Chapters in this video

Method of Studying Palatal Fusion using Static Organ Culture

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Longitudinal Intravital Imaging Through Clear Silicone Windows

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

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

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging