We thank Dr. Jessica Habashi for critically reading of the manuscript. We thank Dr. Xianyong Sheng of the Imaging Center, College of life Sciences, Capital Normal University (Beijing, China), for performing the localization of DR5-GFP assay. This work was supported by grants from the Beijing Municipal Government Science Foundation (CIT&#38;TCD20150102) and from the National Natural Science Foundation of China (31600248).

NOTE: This protocol has three parts: 1) observing the pattern formation in wild-type Arabidopsis embryos using DIC microscopy; 2) characterizing the embryo pattern formation via the observation of marker protein expression using confocal laser scanning microscopy; and 3) determining the role of a specific gene in embryogenesis (using the naa10 mutant as an example).
1. Ovule Clearing
<ol>
	<li>Plant material preparation 
	NOTE: The protocol described here is an example on how to grow healthy plants; other ways to grow healthy plants also work.
	<ol>
		<li>Add 100 seeds to a 1.5 mL tube, and then add 1 mL of 1.5% sodium hypochlorite (mix 15 mL of 10% chlorine with 85 mL of water) to sterilize the seeds for 5 min. Invert the tube several times to ensure that the seeds and solution are well-mixed to sterilize the surface seeds completely.</li>
		<li>Force the seeds to the bottom of the tube by centrifugation for 30 s at 1,200 x g, remove the supernatant, and wash the seeds with sterile water four times at a bench to remove the residual sodium hypochlorite.</li>
		<li>Add 4.4 g of Murashige and Skoog (MS) basal salt mixture and 10 g of sucrose to 1 L of ultrapure water. Stir the mixture to dissolve the salts and sucrose. Adjust the solution to pH 5.8 with 1 M KOH, add 3 g of gelling agent, such as Phytagel, to the MS-sucrose solution, and sterilize the solution at 121 &#176;C for 15 min to make MS medium.</li>
		<li>Pour 30 mL of the MS medium into a 12-cm culture dish to generate MS medium plates.</li>
		<li>Place the sterilized seeds on the MS plate. Transfer the plate to a growth chamber and keep the plate under long-day conditions (22 &#176;C for 16 h in the light and 18 &#176;C for 8 h in the dark) after seed stratification (3 days at 4 &#176;C in the dark).</li>
		<li>After 8 days of growth, transfer the plants to soil (mix nutrient-rich soil and vermiculite at a 1:1 ratio) in a tray, cover the tray with a white, transparent lid for 5 days to avoid water loss, and leave a gap to avoid vitrification of the plants.</li>
		<li>Take the lid off the tray and cultivate the plants in a walk-in chamber under long-day conditions (see 1.1.5). Switch the light on at 6:00 am and off at 10:00 pm. After about 20 days of growth in the walk-in chamber, the plants will have flowered and produced siliques.</li>
		<li>Mark the flower buds with a thread at the position of the seed stalks in the inflorescence at 8:00 pm (do not use the first to the third flower buds; the siliques generated from these flower buds usually develop a few aborted seeds, and this may alter the proportion of normal to abnormal ovules). Define the beginning of pollination of the ovule as when the marked bud opens to flower at 8:00 am the next morning.</li>
	</ol>
	</li>
	<li>Preparation of Hoyer&#39;s solution
	<ol>
		<li>Add 24 g of chloral hydrate to a 50-mL tube and wrap the tube completely with aluminum foil (solutions of chloral hydrate are not stable under light).</li>
		<li>Next, add 9 mL of ultrapure water and 3 mL of glycerol to the above solution and shake the tube to dissolve the chloral hydrate. To make Hoyer&#39;s solution, let the tube stand overnight at room temperature to eliminate any bubbles.</li>
	</ol>
	</li>
	<li>Ovule separation and clearance
	<ol>
		<li>Attach a piece of double-sided adhesive tape to a microscope glass slide. Place a silique grown for the desired number of days after pollination (DAP) on the slide with the pseudoseptum perpendicular to the surface; this will make it easy to split the carpels (Figures 1A and 1B).</li>
		<li>Split the carpels on both sides of the pseudoseptum with a syringe needle and attach the two dissected carpels to the slide so that the ovules in the silique are visible under a stereoscope (Figures 1C and 1D).</li>
		<li>Dispense 70 &#181;L of Hoyer&#39;s solution onto the glass slide, and then moisten the tip of a pair of fine-tipped tweezers with Hoyer&#39;s solution by dipping. Using the stereoscope, move the ovules into the Hoyer&#39;s solution on the slide with the tweezers to clean the embryos. Apply a coverslip to the slide gently to avoid generating any bubbles.</li>
		<li>Put the finished slide into a box in a dark room for 2 - 12 h (the more mature an ovule is, the more time will be needed) at room temperature. Embryos at the 2/4-cell stage (1 - 2 DAP) can be observed microscopically after 2 h, while embryos between the octant and early globular stages (3 DAP) can be observed after 4 h. More mature embryos (4 - 8 DAP) can be observed after 12 h.</li>
		<li>Examine the cleared ovules using the DIC function of a microscope. Locate the embryos under a low-power field (10X) and then change to a high-power field (100X oil immersion lens) to observe the cellular pattern in the developed embryos.</li>
		<li>Examine the embryos at different developmental stages.</li>
	</ol>
	</li>
</ol>
2. Characterization of Embryo Patterning and Cell Identity via the Observation of Marker Protein Expression
<ol>
	<li>Preparation of plant materials
	<ol>
		<li>Clone the promoter and coding sequence of the marker gene or hormone response element and then fuse it with a fluorescent protein (such as GFP); next, insert the construct into the binary vector (such as pCambia1300). Here, the auxin response element DR5 is presented as an example10.</li>
		<li>Introduce the resulting DR5-GFP plasmid into the wild-type plants by Agrobacterium tumefaciens-mediated transformation11. Select the T1 plants using MS medium plates containing hygromycin (50 mg/mL of mother liquor, diluted 1:1,000). The transgenic plants will exhibit long roots and generate two rosette leaves after 8 days of growth under the conditions described in 1.1.5.</li>
		<li>Transfer the transgenic plants to soil and cultivate them as indicated in 1.1.6 and 1.1.7.</li>
		<li>Collect the T2 seeds from the independent T1 lines. Sow the seeds on MS-hygromycin plates, select the plants carrying one copy of the DR5-GFP insertion (the ratio of resistant plants to sensitive plants will be roughly 3:1), and transfer them to soil.</li>
		<li>Cultivate the T2 plants as indicated in 1.1.6 and 1.1.7.</li>
		<li>Collect the T3 seeds from the T2 plants. Sow the seeds on MS-hygromycin plates, select the homozygous transgenic plants (all of the plants from a single line will exhibit hygromycin resistance), and transfer them to soil.</li>
		<li>Mark the flower buds of the homozygous T3 plants as indicated in 1.1.8.</li>
	</ol>
	</li>
	<li>GFP signal observation
	<ol>
		<li>Mix 3 mL of glycerol with 47 mL of ultrapure water to generate a 6% glycerol solution.</li>
		<li>Attach a piece of double-sided adhesive tape to a glass microscope slide and fix a silique as indicated in 1.3.1.</li>
		<li>Split the carpels on both sides of the pseudoseptum with a syringe needle and attach the two dissected carpels to the slide under a stereoscope as indicated in 1.3.2.</li>
		<li>Dispense 30 &#181;L of 6% glycerol onto the glass slide and place all of the ovules from the dissected carpels into the solution using fine-tipped tweezers. Place a coverslip on the slide gently.</li>
		<li>As the seed coat and endosperm in each ovule can prevent the observation of GFP signals in the embryo, extract the embryo from the ovule. Tap the slide quickly and gently to extrude the embryo from the ovule (the more mature an ovule is, the less force should be applied to the slide).</li>
		<li>Examine the slide for GFP fluorescence using confocal laser scanning microscopy. Find the isolated embryos under a low-power field (10X) and record their location, then change to a high-power field (100X oil immersion lens) to observe DR5-GFP expression in the embryos by setting the excitation light source to 488 nm for detection of the GFP signal.</li>
		<li>Detect the GFP expression in the embryos to select the suitable T3 line for further analysis.</li>
	</ol>
	</li>
</ol>
3. Characterization of Embryo Pattern Formation in an Embryo-lethal Mutant via Ovule Clearance and Marker Protein Observation: Naa10 as an Example
<ol>
	<li>Embryo patterning in naa10 mutant plants
	<ol>
		<li>Prepare the plant materials as indicated in 1.1.1-1.1.7.</li>
		<li>Identify the naa10+/&#8722; plants by PCR using gene-specific primers5, and then mark the flower buds of the naa10+/&#8722; plants as indicated in 1.1.8.</li>
		<li>Perform ovule clearance and the microscopic examination of naa10+/&#8722; embryos as indicated in 1.2 and 1.3.</li>
	</ol>
	</li>
	<li>Marker protein analysis using naa10 embryos
	<ol>
		<li>Obtain suitable DR5-GFP transgenic lines using the procedure outlined in 2.1 - 2.2.</li>
		<li>Cross a homozygous DR5-GFP transgenic line with an naa10+/&#8722; plant to obtain naa10+/&#8722; plants that are homozygous for the GFP fluorescence marker at the F2 generation (identified by gene-specific PCR) to characterize naa10+/&#8722;5 and observe the GFP signal under a microscope using a homozygous DR5-GFP line.</li>
		<li>Mark the flower buds of naa10+/&#8722; plants that are homozygous for DR5-GFP as indicated in 2.1.7.</li>
		<li>Search for the GFP signal in naa10 embryos that are homozygous for DR5-GFP as indicated in 2.2.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Jenik, P. D., Gillmor, C. S., Lukowitz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+patterning+in+Arabidopsis+thaliana.">Embryonic patterning in Arabidopsis thaliana.</a> Annu Rev Cell Dev Biol. 23, 207-236 (2007).</li><li>Lau, S., Slane, D., Herud, O., Kong, J., Jurgens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryogenesis+in+flowering+plants:+setting+up+the+basic+body+pattern.">Early embryogenesis in flowering plants: setting up the basic body pattern.</a> Annu Rev Plant Biol. 63, 483-506 (2012).</li><li>Wendrich, J. R., Weijers, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+embryo+as+a+miniature+morphogenesis+model.">The Arabidopsis embryo as a miniature morphogenesis model.</a> New Phytol. 199 (1), 14-25 (2013).</li><li>ten Hove, C. A., Lu, K. J., Weijers, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+plant:+cell+fate+specification+in+the+early+Arabidopsis+embryo.">Building a plant: cell fate specification in the early Arabidopsis embryo.</a> Development. 142 (3), 420-430 (2015).</li><li>Feng, 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=Protein+N-terminal+acetylation+is+required+for+embryogenesis+in+Arabidopsis.">Protein N-terminal acetylation is required for embryogenesis in Arabidopsis.</a> J Exp Bot. 67 (15), 4779-4789 (2016).</li><li>Berleth, T., Jurgens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+monopteros+gene+in+organising+the+basal+body+region+of+the+Arabidopsis+embryo.">The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo.</a> Development. 118, 575-587 (1993).</li><li>Luo, Y., 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=D-myo-inositol-3-phosphate+affects+phosphatidylinositol-mediated+endomembrane+function+in+Arabidopsis+and+is+essential+for+auxin-regulated+embryogenesis.">D-myo-inositol-3-phosphate affects phosphatidylinositol-mediated endomembrane function in Arabidopsis and is essential for auxin-regulated embryogenesis.</a> Plant Cell. 23 (4), 1352-1372 (2011).</li><li>Nodine, M. D., Yadegari, R., Tax, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPK1+and+TOAD2+are+two+receptor-like+kinases+redundantly+required+for+Arabidopsis+embryonic+pattern+formation.">RPK1 and TOAD2 are two receptor-like kinases redundantly required for Arabidopsis embryonic pattern formation.</a> Dev Cell. 12 (6), 943-956 (2007).</li><li>Ulmasov, T., Murfett, J., Hagen, G., Guilfoyle, T. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (16), 735-743 (1998).</li><li>Fiume, E., Fletcher, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Arabidopsis+embryo+and+endosperm+development+by+the+polypeptide+signaling+molecule+CLE8.">Regulation of Arabidopsis embryo and endosperm development by the polypeptide signaling molecule CLE8.</a> Plant Cell. 24 (3), 1000-1012 (2012).</li><li>Jurkuta, R. J., Kaplinsky, N. J., Spindel, J. E., Barton, M. 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The authors have nothing to disclose.

Ovule clearance is a useful method for detecting cell division and assessing morphology during embryogenesis in Arabidopsis; using this technique, embryos can be observed directly under DIC microscopy5,12. The critical step for ovule clearance is step 1.3.4. The time required for ovule clearance in step 1.3.4 is variable. Embryos at the 2/4-cell stage can be clearly observed after 2 h in the Hoyer&#39;s solution, whereas they look indistinct after 12 h. ...

Embryogenesis is the earliest event in higher plant development. A mature embryo forms from a zygote through cell division and differentiation under strict genetic control1,2. Arabidopsis embryos are a useful model to study the control of morphogenesis because the sequence of cell divisions during embryogenesis follows an expected pattern3,4. However, an Arabidopsis embryo containing one to several cells is too small to be observed and analyzed. In addition, the mutation of certain genes can cause embryo lethality5, indicating the key role of those genes in embryogenesis. The characterization of pattern formation in the embryo-lethal mutants provides a basis for understanding the molecular mechanism whereby essential genes regulate embryo development.
A detailed method is described here for the characterization of embryo pattern formation in the model plant Arabidopsis by direct microscopic observation. The method involves ovule clearance followed by the microscopic observation of an embryo. The characterization of an embryo-lethal mutant is also described.
The morphology of embryos at different developmental stages can be determined directly by microscopy using the ovules that have been cleared with Hoyer&#39;s solution6,7. Such ovules exhibit a high refractive index; thus, this ovule clearing method is especially advantageous for specimens that must be observed by differential interference contrast (DIC) microscopy.
In addition, the genes that are expressed in a particular cell or a limited number of cells during embryogenesis can be used as markers to identify the cells in an embryo. Therefore, the cell identity specification during embryogenesis can be annotated by monitoring the expression of GFP-tagged marker proteins using confocal laser scanning microscopy. Conversely, observing the expression pattern of an unknown functional gene-GFP fusion during embryogenesis can provide a link between embryo patterning and the expression of that gene, and facilitate the identification of new genes that are required for embryogenesis in Arabidopsis.
The method described here can also be used to characterize the phenotype of an embryo-lethal mutant, and to determine the impact of the affected gene on embryogenesis at the cellular level. Furthermore, in combination with a comparison of the expression pattern of marker genes during embryogenesis between wild-type and mutant plants, the method can yield clues about the molecular mechanisms and signaling pathways involved in embryogenesis8,9. Indeed, the method was applied to naa10 plants, and it was found that the abnormal division of the hypophysis in the mutant may be caused by a change in the auxin distribution during embryogenesis5.

<table border="0" cellpadding="0" cellspacing="0" width="847">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Chloral Hydrate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">15307</td>
			<td style="width:293px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Murashige and Skoog Basal Medium</td>
			<td style="width:155px;">Sigma</td>
			<td>M5519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Phytagel</td>
			<td style="width:155px;">Sigma</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Hygromycin&#160;</td>
			<td>Roche</td>
			<td>10843555001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Growth chamber&#160;</td>
			<td>Percival</td>
			<td style="width:119px;">CU36L5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence microscope</td>
			<td>Zeiss Oberkochen</td>
			<td>Zeiss Image M2</td>
			<td>camera: AxioCam 506 color</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Laser Scanning Microscopy</td>
			<td>Zeiss Oberkochen</td>
			<td>Zeiss LSM 5</td>
			<td>filters: BP 495-555 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereoscope</td>
			<td>Zeiss Oberkochen</td>
			<td>Axio Zoom V16</td>
			<td>camera: AxioCam MRc5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nutrient-rich&#160;soil</td>
			<td colspan="2">KLASMANN, Germany</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50-ml tube</td>
			<td>Corning Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5-ml tube</td>
			<td>Corning Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">10% sodium hypochlorite solution</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic analytical reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sucrose</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic analytical reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KOH</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic analytical reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic analytical reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">culture dish</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vermiculite</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass slide</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">syringe needle</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fine-tipped tweezers</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">super clean bench</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;Domestic equipment</td>
		</tr>
	</tbody>
</table>

a method for characterizing embryogenesis in arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

The method described in this paper can be used to observe embryogenesis directly under a microscope, annotate the cell identity specification during embryogenesis with specific markers, and characterize the role of a particular gene during embryogenesis.
Representative results from the analysis of embryo patterning (from the elongated zygote stage to the mature walking-stick stage) in the wild-type Arabidopsis are shown...

Watch this Scientific Journal Video about A Method for Characterizing Embryogenesis in Arabidopsis at JoVE.com

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

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11.0K Views.  Capital Normal University. This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Video: A Method for Characterizing Embryogenesis in Arabidopsis

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 ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter&#39;s needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...