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

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The authors have nothing to disclose.

Palatal shelf elevation constitutes a vertical to horizontal remodeling event1,3,4,9,11. It is postulated that this remodeling process requires palatal shelf mesenchymal cells to behave coordinately. The analyses with wildtype MEPM cells show that this cell behavior is intrinsic and can be quantitated21. Thus, these assays can be used to uncover primary palatal shelf elevation defects in new and existing mouse models of cleft palate. The methods outlined in sections 1 and 2 should allow investigators to isolate, culture, and freeze aliquots of primary MEPM cells. These cells can then be used for a wide variety of applications, including the 2D culture and wound-repair assays described here. The 2D culture, when combined with time-lapse imaging, presents a simple method to determine basic cell attributes over a range of cell densities. Presented here is a method to assess cell alignment and cell stream formation, which are attributes of collective cell movement (Figure 4). Wound-closure assays are commonly used to assess cell migration41,42,43. The cell-free region of the &#34;wound&#34; provides a cue for directional movement of cells at the periphery. Time-lapse imaging of this process allowed the determination and assessment of cell trajectories during migration. These cell trajectories, in turn, determine cell migration speed and directionality (Figure 5).
It is important to first optimize conditions for culture and assays using only wildtype MEPM cells. After the palatal shelves have been successfully trypsinized and the resulting single-cell suspension plated overnight, the vast majority (~90%) of cells will readily attach to the growth-surface of the dish. If the cells do not readily adhere, then there may be something wrong with the culture conditions. Initially, the adhered cells will look fairly homogeneous, with a triangular or slightly elongated shape, but as they grow to a high density, they become more spindle-shaped and elongated (Figure 2). If the cells become dramatically large in size or multinucleated, they should not be used for experiments. This optimization will also establish basic wildtype parameters for successful growth (time to confluence) and wound-repair (time to closure). The cells should proliferate by doubling in number almost daily and in time-lapse images, show motility of ~5-10 &#181;m/h. Similarly, if in any subsequent experiment involving a wildtype-mutant comparison, these basic parameters are not met by wildtype cells, the experiment may need to be excluded from analysis. For example, wildtype MEPM cells take 36-40 h to completely close the wound using the 2-well silicone inserts. If in an experiment, wildtype cells take significantly longer than 40 h to close the wound, the entire experiment would be suspect. Poor performance from MEPM cells may be due to 1) poor frozen aliquot quality, 2) poor revival, or 3) excessive differentiation. Differentiated or senescent cells can be detected visually as they grow very large and do not divide. A culture with a large number of such cells should be avoided. In general, there should be no abnormal cells in the field of view of a 10x objective (Figure 3); a cell or two outside the field of view should not materially affect the analysis. Restricting analysis to only two cell passages (above) greatly helps maintain the quality of MEPM cells.
For both the 2D culture and wound-repair assays using primary MEPM cells, the key requirement for success was a high initial cell density. This requirement was first determined during optimization of wound-repair assay in which cells were seeded at &#62;1400 cells/mm2. The density of cells migrating into the wound was then used as the seeding density in 2D culture assays. A seeding density of ~300 cells/mm2 formed cell streams, while still allowing for automated analysis of cell trajectories. Densities higher than 300 cells/mm2 tend to form more vivid streams that can be visualized by eye, but make individual cell tracking difficult. When comparing primary MEPM cells from wildtype and mutant embryos, although wound-closure delay can be assessed without computational analysis, stream formation and directionality differences may not be discernable by eye. If the cell density is too high or image quality is poor/blurred, for example due to loss of focus, automated cell tracking may be difficult. In such a case, it is possible to use manual cell tracking to determine cell trajectories in wound-repair assays using ImageJ. A very low concentration of Hoechst nuclear stain (3 &#181;L/mL of 20 mM solution in MEPM culture medium) can also be used to facilitate cell tracking; however, fluorescent laser toxicity can be an issue with extended use.
For manual tracking, it was easier to track cells in reverse from the end of wound-closure backwards as far as possible until high cell density obscured further tracking. Even partial cell trajectories, when combined for a cell population, were informative. In contrast to wound-repair assays, automated cell tracking is required for 2D cell culture analysis, which is one reason why intermediate cell densities were chosen. Lastly, sensitive primary MEPM-based analyses can be used to identify compounds and pathways that may affect palate elevation. Previous studies indicated that activation of the PI3K-AKT pathway improved both cell speed and directionality of Specc1l mutant MEPM cells21. Other MEPM studies have also used various growth factor or drug treatments to stimulate downstream signaling cascades or to assess proliferation22,29,30,44,45. Thus, MEPM-based analyses offer a quick method to identify many more positive or negative regulatory factors, which can then be validated in vivo.

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

<table><tbody><tr><td>Beaker, 250 mL (x2)</td><td>Fisher Scientific</td><td>FB-100-250</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Matheson Gas</td><td>UN1013</td><td></td></tr><tr><td>Conical tubes, 15 mL (x1)</td><td>Midwest Scientific</td><td>C15B</td><td></td></tr><tr><td>Debian operating system</td><td></td><td></td><td>computational analysis of time-lapse images</td></tr><tr><td>Dulbecco&#039;s Modified Eagles Medium/High Glucose with 4 mM L-Glutamine and Sodium Pyruvate</td><td>Cytiva Life Sciences</td><td>SH30243.01</td><td></td></tr><tr><td>EtOH, 100%</td><td>Decon Laboratories</td><td>2701</td><td></td></tr><tr><td>EVOS FL Auto</td><td>ThermoFisher Scientific</td><td>AMAFD1000</td><td></td></tr><tr><td>EVOS Onstage Incubator</td><td>ThermoFisher Scientific</td><td>AMC1000</td><td></td></tr><tr><td>EVOS Onstage Vessel Holder, Multi-Well Plates</td><td>ThermoFisher Scientific</td><td>AMEPVH028</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>Fine point #5 Stainless Steel Forceps (x2)</td><td>Fine Science Tools</td><td>11295-10</td><td>Dissection</td></tr><tr><td>Instrument sterilizer bead bath</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microcetrifuge tubes, 1.5mL</td><td>Avant</td><td>2925</td><td></td></tr><tr><td>Micro-Dissecting Stainless Steel Scissors, Straight</td><td>Roboz</td><td>RS-5910</td><td>Dissection</td></tr><tr><td>NucBlue (Hoechst) Live Ready Probes</td><td>ThermoFisher Scientific</td><td>R37605</td><td></td></tr><tr><td>Penicillin Streptomycin Solution, 100x</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Silicone Insert, 2-well</td><td>Ibidi</td><td>80209</td><td></td></tr><tr><td>Small Perforated Stainless Steel Spoon</td><td>Fine Science Tools</td><td>MC17C</td><td>Dissection</td></tr><tr><td>Spring Scissors, 4 mm</td><td>Fine Science Tools</td><td>15018-10</td><td></td></tr><tr><td>Sterile 10 cm dishe(s)</td><td>Corning</td><td>430293</td><td></td></tr><tr><td>Sterile 12-well plate(s)</td><td>PR1MA</td><td>667512</td><td></td></tr><tr><td>Sterile 6-well plate(s)</td><td>Thermo Fisher Scientific</td><td>140675</td><td></td></tr><tr><td>Sterile PBS</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Sterile plastic bulb transfer pipette</td><td>ThermoFisher Scientific</td><td>202-1S</td><td></td></tr><tr><td>Trypsin, 0.25%</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr></tbody></table>

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

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

The dissection of palatal shelves is illustrated in Figure 1. The sequence of incisions is designed to minimize slippage of the tissue. Following the removal of the head (Figure 1A,B), the lower jaw is removed (Figure 1B,C). The incision of the upper part of the head (Figure 1C,D) is done to stabilize the tissue when placed upside down (Figure 1E) to visualize (Figure 1E, dotted lines), pinch (Figure 1F), and excise (Figure 1G) the palatal shelves.
The excised palatal shelf pair from a single embryo is&#160;trypsinized and cultured in a 35 mm dish or in a well of a 6-well dish. Larger dishes are not preferred as the cell density is too low for optimal growth. Upon confluence, the cells from each well are trypsinized and passaged into three 35-mm-equivalent wells (passage #1). Confluent cells from passage #1 can then be frozen down into aliquots of 1 &#215; 106 cells/mL. Frozen aliquots are subsequently brought up in a 35-mm-equivalent dish and grown to confluence. The cells are then trypsinized (passage #2) and seeded according to the experiment. Creating and using frozen aliquots helped to normalize conditions, especially with respect to cell density. The use of fresh MEPM cells resulted in more variability in final cell density in experiments, which is believed to be due to a variable proportion of viable or sub-viable cells in fresh cultures. In addition, the number of MEPM cell passages were strictly limited to two (listed above) for these experiments.
Considering that a) cell density was critical, b) MEPM cells from a single embryo were limited, and c) imaging optics were better in a large dish, 2-well silicone inserts were used (Figure 2 and Figure 3). For wound-repair assays, MEPM cells were grown in the 2-well silicone inserts until high confluence, then the inserts were removed, and the wound imaged until closure (Figure 3). However, for 2D culture, the MEPM cells needed imaging as they grew, so the 2-well silicone inserts were simply trimmed to 1/3rd of their height to allow for clear imaging (Figure 2C). Small 3D printed rings40 in 35 mm dishes were also used for 2D motility analysis (Figure 2D).
MEPM trajectories (Figure 4E) are persistent: the direction of cell motility is maintained for several hours. The mean displacement vs. time analysis (Figure 4F) indicates the persistence in the form of displacement being proportional with elapsed time. The typical speed of MEPM cells on tissue culture plastic surface, 10 &#181;m/h, can be also used for quality control of the cell culture. Flow analysis of the motility data reveals that the co-moving clusters of MEPM cells are ~300 &#181;m in size (Figure 5I,J). The representative results also indicate a profound motility difference between wild-type and mutant MEPM cells. In addition to wildtype and mutant comparison, both 2D and wound-repair assays can be combined with various biochemical treatments. For example, PI3K-AKT pathway activators have been used with MEPM cells, as described previously 21.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62151/62151fig01.jpg" /> 
Figure 1: Dissection of embryonic palatal shelves to isolate mesenchymal cells. (A) An E13.5 mouse embryo head is removed by cutting along the neck (red line). (B) Next, the lower jaw and tongue are removed with incisions along the oral cavity, between the upper and lower jaws (yellow line). (C) To stabilize the tissue for palatal shelf removal, the top of the head is excised (green line). (D) The resulting dissected upper jaw region is placed (E) upside down to visualize the palatal shelves (black dotted lines). (F) The excision of individual palatal shelves is depicted schematically, where individual protruding shelves (yellow) are pinched off from the maxilla (blue). (G) Excised pair of palatal shelves from&#160;a single embryo, which can then be trypsinized and cultured in a 35 mm dish or in a 6-well plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62151/62151fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62151/62151fig02.jpg" /> 
Figure 2: Experimental setup of 2D MEPM cell culture. (A) Thaw a frozen aliquot of MEPM cells, and (B) culture the cells in a 35 mm dish or a 6-well plate. When confluent, trypsinize the cells and seed them as described in the protocol in a 35 mm dish either with (C) 2-well silicone inserts that have been trimmed or (D) with 3D-printed rings. A small culture space minimizes the need for large numbers of MEPM cells. The low profile of the trimmed silicone inserts or the 3D-printed rings allows for direct imaging without a halo effect. Time-lapse imaging can continue until the desired cell density is achieved, which can be up to 72 h. Representative images are shown at (E) 0 h, (F) 24 h, and (G) 45 h timepoints. The images were taken using a 4x objective. The scalebars for E, F, G = 300 &#181;m. Abbreviations: 2D = two-dimensional; MEPM = mouse embryonic palatal mesenchyme; 3D = three-dimensional. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62151/62151fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62151/62151fig03.jpg" /> 
Figure 3: Experimental setup of wound-closure assay using MEPM cells. (A) Thaw a frozen aliquot of MEPM cells, and (B) culture cells in a 35 mm dish or a 6-well plate. When confluent, trypsinize the cells and (C) seed them in the 2-well silicone inserts in a 35 mm dish. Culture the cells in the insert until the desired confluence is achieved (~48 h), then remove the silicone insert and image. Representative images are shown (D) immediately after removal of the insert, (E) after 24 h, and (F) after 40 h. Wound closure takes around 36 h. The images were taken using a 10x objective. The scalebar represents 300 &#181;m. Abbreviations: MEPM = mouse embryonic palatal mesenchyme. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62151/62151fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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

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

isolation-time-lapse-imaging-primary-mouse-embryonic-palatal

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

2.2K Views.  University of Kansas Medical Center. Dit protocol stelt onderzoekers in het craniofaciale veld in staat om collectief bewegingsattributen in controle- en mutante cellen kwantitatief te vergelijken als een middel om mesenchymale remodellering te begrijpen tijdens palatale plankverhoging. Het gebruik van primaire muisembryonale palatale mesenchymale cellen en time-lapse beeldvorming bieden een toegankelijke proxymethode voor het beoordelen van palatale schapverhogingsdynamiek. Om palatale schelpen van een muizenembryo te oogsten, ...

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

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

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.

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 ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Neuroscience

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 ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Biology

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo 

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Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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.

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

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