Name: kinematic analysis of cell division and expansion: quantifying the cellular basis of growth and sampling developmental zones in zea mays leaves
Uploaded: 2016-12-02T11:00:00
Description: Watch this Scientific Journal Video about Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves at JoVE.com

This work was supported by a PhD fellowship from the University of Antwerp to V.A.; a PhD fellowship from the Flemish Science Foundation (FWO, 11ZI916N) to K.S.; project grants from the FWO (G0D0514N); a concerted research activity (GOA) research grant, &#34;A Systems Biology Approach of Leaf Morphogenesis&#34; from the research council of the University of Antwerp; and the Interuniversity Attraction Poles (IUAP VII/29, MARS), &#34;Maize and Arabidopsis Root and Shoot Growth&#34; from the Belgian Federal Science Policy Office (BELSPO) to G.T.S.B. Han Asard, Bulelani L. Sizani and Hamada AbdElgawad all contributed to the video.

NOTE: The following protocol for kinematic analysis is only valid for leaves during steady-state growth. This implies a stable leaf elongation rate and spatial patterns of cell length and expansion in a leaf during a period of several days6.
1. Plant Growth and Measurements of Leaf Elongation Rate (LER)
<ol>
	<li>Choose a leaf in steady-state growth and a developmental stage of interest. 
	NOTE: There is a difference between steady-state growth and repetitive growth,...

A full kinematic analysis on maize leaves enables the determination of the cellular basis of leaf growth and allows for the design of efficient sampling strategies. Although the protocol is relatively straightforward, some caution is recommended in the following critical steps: (1) It is important to detach the younger, enclosed leaves (step 2.3) without damaging the meristem, since meristem length determination (step 3) requires the complete meristem to be present. Some practice beforehand might be needed. (2) Meristema...

Growth analysis depends on a set of tools that are commonly used by plant scientists to describe genotype determined growth differences and/or phenotypic responses to environmental factors. They include size and weight measurements of the whole plant or an organ and calculations of growth rates to explore the underlying mechanisms of growth. Organ growth is determined by cell division and expansion at the cellular level. Therefore, including the quantification of these two processes in growth analyses is key to understanding differences in whole-organ growth1. Consequently, it is crucial to have an appropriate methodology to determine cellular growth parameters that is relatively easy to use by non-specialized laboratories.
Kinematic analysis has already been established as an approach providing a powerful framework for the development of organ growth models2. The technique has been optimized for linear systems, such as Arabidopsis thaliana roots and monocotyledonous leaves, but also for non-linear systems, such as dicotyledonous leaves3. Nowadays, this methodology is increasingly being used to study how genetic, hormonal, developmental, and environmental factors influence cell division and expansion in various organs (Table 1). Moreover, it also provides a framework to link cellular processes to their underlying biochemical, molecular, and physiological regulations (Table 2), although limitations can be imposed by organ size and spatial organization for techniques that require higher amounts of plant material (e.g., metabolite measurements, proteomics, etc.).
Monocotyledonous leaves, such as the maize (Zea mays) leaf, represent linear systems in which cells move from the base of the leaf towards the tip, sequentially passing through the meristem and elongation zone to reach the mature zone. This makes it an ideal model system for quantitative studies of the spatial patterns of growth4. Moreover, maize leaves have large growth zones (meristem and elongation zone spanning several centimeters5) and provide possibilities for studies at other organizational levels. This allows for the investigation of the (putative) regulatory mechanisms controlling cell division and expansion, quantified by kinematic analysis through a range of molecular techniques, physiological measurements, and cell biology approaches (Table 2).
Here, we provide a protocol for performing a kinematic analysis in monocot leaves. First, we explain how to conduct a proper analysis of both cell division and cell elongation as a function of position along the leaf axis and how to calculate kinematic parameters. Secondly, we also show how this can be used as a basis for sampling design. Here, we discuss two cases: high-resolution sampling and focused sampling, enabling improved data interpretation and the saving of time/money, respectively.
Table 1. Overview of kinematic analyses methods for quantification of cell division and expansion in various organs.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>organ</td>
			<td>reference</td>
		</tr>
		<tr>
			<td>monocotyledonous leaves</td>
			<td>16, 20, 21, 22</td>
		</tr>
		<tr>
			<td>root tips</td>
			<td>2, 23, 24, 25, 26, 27, 28, 29</td>
		</tr>
		<tr>
			<td>dicotyledonous leaves</td>
			<td>21, 30, 31</td>
		</tr>
		<tr>
			<td>shoot apical meristem</td>
			<td>32</td>
		</tr>
	</tbody>
</table>
Table 1. Overview of kinematic analyses methods for quantification of cell division and expansion in various organs.
<img alt="Figure 3" src="/files/ftp_upload/54887/54887table2.jpg" />
Table 2. Link between cellular processes quantified by the kinematic analysis to their regulation at the molecular level. References to various studies linking the quantification of cellular processes to results from biochemical and molecular assays in various species and organs. Xyloglucan endotransglucosylase (XET), malondialdehyde (MDA), cyclin-dependent kinases (CDK). <a href="http://cloudflare.jove.com/files/ftp_upload/54887/54887table2large.jpg" target="_blank">Please click here to view a larger version of this table.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1670">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Pots</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">We use pots with the following measueres, but can be different depending on the treatment/study : bottom diameter: 11cm, opening diameter: 15 cm, height: 12 cm. We grow one maize plant per pot.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Planting substrate</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td>We use potting medium (Jiffy, The Netherlands), but other substrates can be used, depending on treatment/study.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ruler</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td>An extension ruler that covers at least 1,5 meters is needed to measure the final leaf length of the plants.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Seeds&#160;</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">NA</td>
			<td>Seeds can be ordered from a breeder.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Scalpel</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">The scalpel is used during leaf harvesting to detach the leaf of interest from its surrounding leaves and right after harvesting to cut a proper sample for cell length and meristem length measurements.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">15 ml falcon tubes</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">The 15 ml falcon tubes are used for storing samples used for cell length measurements during sample clearing with absolute ethanol and lactic acid.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Eppendorf tubes</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">The eppendorf tubes are used for storing samples used for meristem length measurements in ethanol:acetic acid 3:1 (v:v) solution.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Gloves</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td>Latex gloves, which protect against corrosive reagents.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Acetic acid</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">CAUTION: Corrosive to metals, category 1 Skin corrosion, categories 1A,1B,1C Serious eye damage, category 1; Flammable liquids, categories 1,2,3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Absolute ethanol</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">CAUTION: Hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation. Slightly hazardous in case of skin contact (permeator), of ingestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Lactic acid &#62;98%</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td>CAUTION: Corrosive to metals, category 1 Skin corrosion, categories 1A,1B,1C Serious eye damage, category 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium chloride (NaCl)</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">CAUTION: Acute toxicity (oral, dermal, inhalation), category 4 Skin irritation, category 2 Eye irritation, category 2 Skin sensitisation, category 1 Specific Target Organ Toxicity &#8211; Single exposure, category 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">This material can be an irritant, contact with eyes and skin should be avoided. Inhalation of dust may be irritating to the respiratory tract.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">4&#8242;,6-Diamidine-2&#8242;-phenylindole dihydrochloride (DAPI)</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">Cell permeable fluorescent minor groove-binding probe for DNA. Causes skin irritation. May cause an allergic skin reaction. May cause respiratory irritation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ice</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">NA</td>
			<td>The DAPI solution has to be kept on ice.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fluorescent microscope</td>
			<td>AxioScope A1, Axiocam ICm1 from Zeiss or other</td>
			<td style="width:209px;"></td>
			<td>Any fluorescent microscope can be used for determining meristem length.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microscopic slide</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cover glass</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Tweezers</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">Tweezers are needed for unfolding the rolled maize leaf right after harvesting in order to cut a proper sample for cell length and meristem length measurements.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Image-analysis software</td>
			<td>Axiovision (Release 4.8) from Zeiss</td>
			<td style="width:209px;">NA</td>
			<td style="width:813px;">The software can be downloaded at: http://www.zeiss.com/microscopy/en_de/downloads/axiovision.html. Other softwares such as ImageJ (https://imagej.nih.gov/ij/) could be used as well.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microscope equipped with DIC</td>
			<td>AxioScope A1, Axiocam ICm1 from Zeiss or other</td>
			<td style="width:209px;"></td>
			<td>Any&#160; microscope, equipped with differential interference contrast (DIC) can be used to measure cell lengths.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">R statistical analysis software</td>
			<td>R Foundation for Statistical Computing</td>
			<td style="width:209px;">NA</td>
			<td>Open source; Could be downloaded at https://www.r-project.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:315px;">R script</td>
			<td style="width:333px;">NA</td>
			<td style="width:209px;">NA</td>
			<td style="width:813px;">We use the kernel smoothing function locpoly of the Kern Smooth package (Wand MP, Jones MC.&#160; Kernel Smoothing: Chapman &#38; Hall/CRC (1995)). The script is available for Mac and Windows upon inquire with the corresponding author. We have versions for Mac and Windows.</td>
		</tr>
	</tbody>
</table>

kinematic analysis of cell division and expansion: quantifying the cellular basis of growth and sampling developmental zones in zea mays leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Here, we show a comparison between well-watered plants (control, 54% soil water content, (SWC)) and plants subjected to drought stress conditions (drought, 34% SWC) in terms of their leaf growth. All plants were grown in a growth chamber under controlled conditions (16 hr day/8 hr night, 25 &#176;C/18 &#176;C day/night, 300-400 &#181;Em-2sec-1 photosynthetically active radiation (PAR). The drought conditions were established by withholding water until the correct SWC...

Watch this Scientific Journal Video about Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves at JoVE.com

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

The overall goals of this analysis, are to determine the cellular basis of maize leaf growth, and to use these data to investigate molecular growth regulatory mechanisms by performing developmental, stage specific sampling strategies. This method can help to address a key question in plant developmental biology. How do cell division and cell expansion contribute to differences in whole organ growth?This approach allows the investigation and spacial mapping of biochemical molecular and physiological processes, underlying the growth of the whole plant organ. Though this system can provide insight into maize leaf growth, the technique can be used for other systems. Including other grass species, and organs.Visual demonstration of these methods is critical. At the harvesting, sample bi-blation and measurementive steps are difficult to perform without that demonstration. Demonstrating the procedure, will be Katrien Sprangers, and Viktoriya Avramova.Two PHD students from my laboratory. To perform a full chenematic analysis of leaf growth in monocots, grow at least 15 plants for each treatment and genotype, under controlled conditions in a growth room. When a leaf of interest appears, use a ruler to measure the length of the leaf, that is the length from the soil to the tip of the leaf, daily until the leaf is fully expanded.At the developmental stage of interest, cut the above ground part of the plant from at least five representative plants as close to the roots as possible, to keep the meristematic region intact. Then, starting from the outer leaves, gently unroll all of the leaves up to the leaf of interest, one by one. Removing a few extra millimeters from the base to detach the leaves as necessary.Next, remove the apex and the small leaves enclosed by the leaf of interest, and cut a three centimeter segment from the leaf, starting from the base on one side of the mid vein. It is crucial to cut the leaf right where it is connected to the stem. As cutting to high, would lead to an under estimation of the size of the meristem.Store the segment in a 1.5 milliliter test tube, filled with three two one volume by volume absolute ethanol acetic acid solution, at four degrees Celsius for 24 hours up to several months. From the other side of the vein, cut an 11 centimeter segment from the base, and store this segment in a 15 milliliter tube filled with absolute ethanol, at four degrees Celsius for at least six hours to remove the pigments. After a second 24 hour round in absolute ethanol at four degrees Celsius, replace the ethanol with pure lactic acid, and store the segment at four degrees Celsius for another 24 hours, or until further analysis.To measure the meristem, first transfer the three centimeter leaf segment into freshly prepared rinsing buffer for 20 minutes. At the end of the incubation, transfer the segment into a container of Dapi staining solution, for two to five minutes on ice in the dark. Then, quickly mount the tissue onto a glass microscope slide, and cover the segment with a cover glass.Confirm the fluoresces of the epidermal cells under a microscope, using UV fluorescence. Then, mount the sample in a drop of rinsing buffer on a new microscope slide with a new glass cover. Using a 20 times magnification, return the segment to the microscope, and scroll through the sample to locate proliferative mitotic figures.Avoid any formative cell division of the developing stemmata, and then define where the most distal mitotic figure is located. Using and image analysis software program, measure the full length of the image frame. In this example, 645 micrometer.Then count the number of frames that cover the full meristem length from the most distal mitotic figure to the leaf base, and multiply this number by the length of one frame to obtain the full meristem length. To obtain a cell length profile, transfer the 11 centimeter segment from the lactic acid, to the bench, and use a scalpel to cut the tissue into 10 one centimeter segments. Mount the resulting leaf segments onto a single microscope slide, and a small drop of lactic acid.Placing all of the pieces with the same side up. And then, transfer the slide under a microscope equipped with differential interference contrast optics. Beginning with the segment nearest to the leaf base, use an appropriate image analysis software program to measure the length of at least 20 replicate epidermal cells and files directly adjacent to the stemmatal files, to ensure the consistent selection of the same cell type at equally spaced positions along each of the segments.Take are to wright down the corresponding position for each measurement throughout the leaf. Then determine the average cell length at each millimeter along the leaf axis using a local polynomial smoothing procedure implemented in R script that is available as supplementary file one. Be careful not to over smooth.The smoothing should remove the noise in the celling data, but not affect the overall shape of the curve. Here, a comparison between well watered plant, and plants subjected to drought stress conditions in terms of their leaf growth are show. The final leaf length of the control plants, was 40 percent lower than that of the drought stressed plants, due to a lower leaf elongation rate.The leaf elongation rate of the drought stressed plants, is 73 percent lower than that of well watered controls, due to a reduced cell production. Which in tern, is mainly caused by a reduced cell division rate. Kinematic analysis provides a map of the leaf growth zone localization, allowing sampling with a subzonal resolution for molecular and biochemical analysis.For example, MDA quantification along the growth zone of the maize leaf in plant subjected to well watered control and drought conditions, demonstrates a significant increase in this product of lipid breakdown throughout the growth zone in response to water withholding conditions. Due to leaf shortening in response to the drought, developmental zones did not correspond in the control and in the stressed plants in this experiment. Using the cell length profile, however, the length of the developmental zones can be determined, allowing the MDA levels in the individual zones to be compared.Once mastered, a single sample can be processed in four hours, if the technique is performed properly. While attempting this procedure, it is important to remember to keep the meristem intact during it's harvesting. Following this procedure, other techniques can be applied, such as the determination of molecular and biochemical parameters, to study their involvement in growth regulation.After watching this video, you should have a good understanding of how to quantify cell division and cell expansion in growing maize leaf, determine the size of the different growth zones, and interpret molecular and biochemical data accordingly. Don't forget that working with lactic and acetic acids, and EDTA can be hazardous. Therefore, precautions such as wearing gloves, and cleaning the microscope each stage after use, should always be taken when performing this procedure.

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Research

JoVE Journal

Biology

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...