I express great acknowledgment to Fumitoshi Irie and Yu Yamaguchi for their encouragement and kind suggestions in establishing this method. This work was supported by grants-in-aid for scientific research programs from the Japan Society for the Promotion of Science (#19KK0232 to T.I., #20H03896 to T.I.).&#160;The original method for the coating of HA onto glass substrates and in situ HA degradation assays on the substrates was described in Yamamoto et al. (2017)8, while the method for the preparation of HA/Col1 mixed substrates was described in Irie et al. (2021)7.

All procedures were approved by the Animal Ethics Committee of the Osaka University Graduate School of Dentistry.
1. Culture of mouse cranial neural crest cells
NOTE: The neural crest cell line used in this study comprises O9-1 cells, originally derived from Wnt1-Cre; R26R-GFP-expressing cells isolated from E8.5 mouse embryos12 (see discussion). The method described here for culturing O9-1 cells follows a previously established protocol13.
<ol>
	<li>Prepare the basement membrane matrix-coated plate.
	<ol>
		<li>Thaw the basement membrane matrix (see Table of Materials) on ice. Dilute the matrix 1:50 in chilled 1x PBS, and keep on ice.</li>
		<li>Coat a 10 cm culture plate with 10 mL of the diluted basement-membrane matrix solution. Incubate the plate at room temperature for 1 h, and aspirate the matrix before use.</li>
		<li>Wash the plate 3x with 2 mL of PBS.</li>
	</ol>
	</li>
	<li>Culture O9-1 cells on the basement membrane matrix-coated plate.
	<ol>
		<li>Warm the complete embryonic stem (ES) cell medium (see Table of Materials) in a water bath at 37 &#176;C.</li>
		<li>Gently mix the O9-1 cell suspension (see Table of Materials) and count the number of cells using an automated cell counter (see Table of Materials). Adjust the cell concentration to 1.1 &#215; 106/mL with complete ES cell medium.</li>
		<li>Add 8 mL of pre-warmed complete ES cell medium to the matrix-coated 10 cm culture plate, and seed the O9-1 cells at 1.1 x 106 cells/plate. Incubate at 37 &#176;C in a 5% CO2 humidified incubator.</li>
		<li>The next day, replace the medium with fresh complete ES cell medium (pre-warmed to 37 &#176;C). Replace with fresh medium every 2-3 days thereafter. 
		NOTE: When the cells are approximately 80% confluent (3-4 days after plating), they can be dissociated with 0.25% trypsin-EDTA and passaged further or frozen for later use. Representative images of O9-1 cells are shown in Figure 1.</li>
		<li>Wash the culture plate with 2 mL of 1x PBS prior to trypsinization. Add 2 mL of pre-warmed 0.25% trypsin-EDTA and incubate for 5 min at 37 &#176;C. Check for complete cell detachment; gently tap the side of the plate a couple of times if necessary.</li>
		<li>Add 2 mL of pre-warmed complete ES cell medium to the culture plate and transfer the dissociated cells to a 15 mL conical tube. Centrifuge the tube at 300 x g for 5 min.</li>
		<li>Discard the supernatant without disturbing the cell pellet. Then, add 2 mL of pre-warmed complete ES cell medium to the tube, and thoroughly resuspend the cells by pipetting. Seed the cells on a new culture plate at the desired cell density.</li>
		<li>Alternatively, prepare a frozen cell stock by adding 1 mL of cell freezing medium containing 10% dimethyl sulfoxide (DMSO) to the tube in place of complete ES cell medium and thoroughly resuspending the cells. Transfer the cell suspension to cryo tubes, and store at &#8722;80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of the HA/Col1-coated dish
NOTE: The original method of coating HA/Col1 onto glass-bottom dishes was proposed by Irie et al.7.
<ol>
	<li>Carefully add 50 &#181;L of undiluted triethoxysilane to a 3.5 cm glass-bottom dish (see Table of Materials). Incubate for 5 min at room temperature (RT) and protect from light. 
	NOTE: The incubation with triethoxysilane should not exceed 5 min. This may affect the coating efficiency and produce undesirable products.</li>
	<li>Wash the dish quickly 3x with 2 mL of distilled water. Add 50 &#181;L of 0.25% glutaraldehyde, diluted 100x in PBS, per dish, and incubate at RT for 30 min.</li>
	<li>Wash quickly 4x with 2 mL of PBS. Then, coat the dishes with 300 &#181;L of collagen type I in 0.2 N acetic acid at RT for 1 h.</li>
	<li>Wash quickly 3x with 2 mL of PBS. Add 300 &#181;L of 200 &#181;g/mL fluoresceinamine-labeled sodium hyaluronate-H2 (FAHA-H2) (diluted in PBS) to each dish and incubate overnight at RT. Wash again 3x with 2 mL of PBS. After aspirating the PBS, air-dry the plate for 5 min on a clean bench. 
	&#8203;NOTE: FAHA-H2 is a fluoresceinamine-labeled sodium hyaluronate with an average molecular weight ranging from 1,200-1,600 KDa. HA of an appropriate molecular weight can be used according to the research design.</li>
</ol>
3. Migration assay on the HA/Col1-coated dish
NOTE: A wound closure-based assay using defined 500 &#181;m cell-free gaps in Col1/HA substrates was performed using 2-well culture inserts (see Table of Materials). The O9-1 cells express Tmem2, which is required for the adhesion and degradation of HA in the extracellular space9 (Figure 2 and Figure 3).
<ol>
	<li>After drying the coated glass-bottom dish, attach the 2-well culture inserts to the dishes, and fill the inserts externally with 1 mL of PBS.</li>
	<li>Seed the O9-1 cells into the wells at 1 x 104 cells in 100 &#181;L of Dulbecco&#39;s modified Eagle medium (DMEM) containing 2% fetal bovine serum (FBS) per insert. Culture the cells for 2 days at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Remove the inserts carefully from the coated glass-bottom dishes with tweezers. Wash the coated glass-bottom dishes gently with 2 mL of 1x PBS to remove the cells and cell debris. Add 2 mL of fresh DMEM containing 2% FBS into the culture dishes.</li>
	<li>Capture phase-contrast images now as the starting time point using an all-in-one fluorescence microscope in monochrome mode with high-resolution settings (gain at 6 dB, no binning). Objective lenses with magnifications of 4x to 20x were used for imaging in this study (see Table of Materials). 
	NOTE: Capture the phase-contrast images with the corresponding scale bars and save them in TIFF format.</li>
	<li>Culture the cells for an additional 48 h at 37 &#176;C and 5% CO2. Capture phase-contrast images at 24 h and 48 h in culture using the all-in-one fluorescence microscope.</li>
	<li>Fix the cells at 48 h with 1 mL of 4% paraformaldehyde (PFA) for 15 min at room temperature or overnight at 4 &#176;C. Then, wash the dishes 3x for 5 min each with 1 mL of fresh PBS.</li>
	<li>Place a coverslip with mounting medium (see Table of Materials) for further morphological observation. 
	NOTE: The dishes can be imaged immediately or stored for up to 2 months at 4 &#176;C.</li>
	<li>Optional: Immunolabel the cells for proteins of interest. 
	NOTE: A protocol to detect the focal adhesion (FA) complex using a mouse-derived monoclonal anti-vinculin antibody (see Table of Materials) is described here as an example.
	<ol>
		<li>Incubate the dishes (from step 3.6) with 0.5 mL of blocking buffer (5% normal goat serum in PBS) for 60 min. 
		NOTE: Choose an appropriate blocking buffer for the primary antibody. A typical blocking buffer would be 5% normal serum from the same species as the secondary antibody used.</li>
		<li>Prepare the diluted primary antibody in antibody dilution buffer (1% normal goat serum in PBS). Incubate the dishes with 0.5 mL of diluted primary antibody overnight at 4 &#176;C. 
		NOTE: Choose the appropriate antibody dilution buffer. Typically, the antibody is used at a 1:50-1:200 dilution.</li>
		<li>Rinse 3x for 5 min each with 2 mL of PBS. Prepare the diluted goat-derived anti-mouse secondary antibody in the antibody dilution buffer (1% normal goat serum in PBS). Incubate the dishes with 0.5 mL of diluted secondary antibody for 1-3 h at room temperature. 
		NOTE: Typically, the secondary antibody is used at a 1:500-1:1,000 dilution. DAPI can be used for staining the nuclei.</li>
		<li>Rinse 3x with 2 mL of PBS for 5 min each time. Place the coverslip with 50 &#181;L of mounting medium. 
		NOTE: The dishes can be imaged using the all-in-one fluorescence microscope with a GFP filter (excitation: 470/40, emission: 525/50) and a TexasRed filter (excitation: 560/40, emission: 630/75) in monochrome mode with high-resolution settings (gain at 6 dB, no binning). Objective lenses with magnifications of 4x to 20x were used for the imaging in this study. 
		&#8203;NOTE: The slide can be stored for up to 2 months at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Open the ImageJ 1.51s software. In the ImageJ window, select File &#62; Open from the menu bar to open the saved image file.</li>
	<li>For setting the measurement scale, draw a line of the same distance as the scale bar. Go to Analyze &#62; Set scale and type the known distance and units of the line in the appropriate boxes of the Set scale window.</li>
	<li>Draw a straight line between the cell gap, and press Analyze &#62; Measure to transfer the values to a data window. Measure at least five different positions in each sample, and average the distances to obtain the representative sample data. Perform the statistical analyses using appropriate software (see Table of Materials).</li>
</ol>

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The author declares that no competing interests exist.

Various ECM components regulate NCC emigration/migration. For instance, HA positively regulates NCC migration2,15. Interestingly, a study based on genetic mouse models of Tmem2, a cell surface hyaluronidase, elucidated the requirement of HA degradation in NCC migration9. Collagens are also abundant in the ECM surrounding the neural tube16. Decorin, a small leucine-rich proteoglycan, has been shown to regulate NCC mi...

Neural crest cells (NCCs) are a multipotent cell population that is present in developing embryos, and they originate from the neural plate border during neurulation. They contribute to the formation of a variety of tissues, including the peripheral nervous system, cardiovascular system, craniofacial tissues, and the skeleton1. After induction and NCC specification at the neural plate border, NCCs emigrate from the neuroepithelium and migrate toward NCC-derived tissue sites1.
Hyaluronan (HA) is a non-sulfated glycosaminoglycan that is distributed in a variety of tissues as a component of the extracellular matrix (ECM). The importance of HA in embryo development has been demonstrated in model systems through the ablation of genes responsible for hyaluronan metabolism. For instance, mutations in hyaluronan synthase genes (Has1 and Has2) in Xenopus were found to lead to NCC migration defects and craniofacial malformation2. In addition, the HA-binding proteoglycans, aggrecan and versican, have been reported to exert inhibitory effects on NCC migration3. In mice, Has2 ablation leads to severe defects in endocardial cushion formation, resulting in mid-gestation (E9.5-10) lethality4,5,6.
Transmembrane protein 2 (Tmem2), a cell surface hyaluronidase, has been recently demonstrated to play a critical role in promoting integrin-mediated cancer cell adhesion and migration by removing matrix-associated HA at the adhesion sites7,8. More recently, Inubushi et al.9 demonstrated that a deficiency in&#160;Tmem2 leads to severe craniofacial defects due to abnormalities in NCC emigration/migration and survival. In the previous study9, Tmem2 expression was analyzed during NCC formation and migration. Tmem2 expression was observed at the site of NCC delamination and in emigrating Sox9-positive NCCs (Figure 1). Additionally, using Tmem2-depleted mouse O9-1 neural crest cells, the study demonstrated that the in vitro expression of Tmem2 was essential for the O9-1 cells to form focal adhesions and for their migration into HA-containing substrates (Figure 2 and Figure 3)9.
These results strongly indicate that Tmem2 is also important for NCC adhesion and migration through the HA-rich ECM. However, the molecular mechanism of NCC adhesion and migration within the HA-rich ECM is still unclear. It is, therefore, necessary to establish an in vitro culture experimental system to fully explore NCC adhesion and migration within the HA-rich ECM.
Of the numerous approaches employed in testing cell migration, the cell wound closure-based assay is a simple method frequently used in the fields of physiology and oncology10. This approach is useful due to its relevance to the in vivo phenotype and is effective in determining the roles of drugs and chemoattractants during cell migration11. It is possible to evaluate the migration ability of both whole cell masses and individual cells by measuring the cell gap distances over time11. In this manuscript, a modified in vitro wound closure-based assay is introduced to model NCC migration into HA-rich tissues surrounding the neural tube. This procedure is also applicable for studying different ECM components (i.e., collagens, fibronectin, and laminin) to analyze the role of the ECM scaffold in NCC migration.

<table><tbody><tr><td>10cm cell culture dish</td><td>CORNING</td><td>Cat. 353003</td><td></td></tr><tr><td>1X PBS</td><td>Millipore</td><td>Cat. No. BSS-1005-B</td><td></td></tr><tr><td>2-well culture inserts</td><td>ibidi</td><td>Cat. No. 80209</td><td></td></tr><tr><td>Alexa 555-labelled goat anti-mouse IgG</td><td>Invitrogen</td><td>Cat. A21422</td><td>Goat derived anti-mouse secondary antibody</td></tr><tr><td>automated cell counter</td><td>Bio-Rad</td><td>Cat. No. TC20</td><td></td></tr><tr><td>CELLBANKER</td><td>ZENOGEN PHARMA</td><td>Cat. 11910</td><td>Cell freezing medium</td></tr><tr><td>collagen type I</td><td>Sigma</td><td>Cat. No. 08-115</td><td></td></tr><tr><td>Complete ES Cell Medium</td><td>Millipore</td><td>Cat. No. ES-101-B</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>Cat. 10184322</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium&amp;nbsp;</td><td>Gibco</td><td>Cat. 11971025</td><td></td></tr><tr><td>Fetal Bovine serum</td><td>Gibco</td><td>Cat. 10270106</td><td></td></tr><tr><td>fluorescence microscope</td><td>Keyence</td><td>Cat. No. BZ-X700</td><td></td></tr><tr><td>Fluoresent labelled HA</td><td>PG Research</td><td>FAHA-H2</td><td></td></tr><tr><td>Glas bottom dish</td><td>Iwaki</td><td>Cat. 11-0602</td><td></td></tr><tr><td>glutaldehyde</td><td>Sigma</td><td>Cat. No. G5882</td><td></td></tr><tr><td>Matrigel</td><td>Fisher</td><td>Cat. No. CB-40234</td><td>The basement-membrane matrix</td></tr><tr><td>monoclonal anti-vinculin antibody</td><td>Sigma</td><td>Cat. No. V9264</td><td></td></tr><tr><td>mounting media</td><td>Dako</td><td>S3023</td><td></td></tr><tr><td>Normal goat serum</td><td>Fisher</td><td>Cat. 50062Z</td><td></td></tr><tr><td>O9-1 cells</td><td>Millipore</td><td>Cat. No. SCC049</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>Cat. 158127</td><td></td></tr><tr><td>triethoxysilane</td><td>Sigma</td><td>Cat. No. 390143</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Millipore</td><td>Cat. No. SM-2003-C</td><td></td></tr></tbody></table>

in vitro investigation of the effects of the hyaluronan-rich extracellular matrix on neural crest cell migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

A migration assay was performed on mixed substrates composed of Col1 and high-molecular weight HA (average molecular weight: 1,200-1,400 kDa) using the protocol described here. O9-1 cells at the boundary of the gap were found to readily migrate into the HA-rich gap (Figure 4). Immunostaining for a FA marker, vinculin14, confirmed that the O9-1 cells formed focal adhesions (FAs) at the sites of HA degradation (Figure 5).
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In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube ...

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Research

JoVE Journal

Developmental Biology

1.0K Views.  Osaka University Graduate School of Dentistry. Hyaluronzuur is een van de meest voorkomende extracellulaire matrixen in diermodellen. Het belang van hyaluronzuur in de embryonale ontwikkeling is aangetoond. Dit in vitro model is nuttig om te onderzoeken hoe neurale topcellen zich hechten en migreren binnen de hyaluronzuurrijke extracellulaire matrix.Met onze techniek kunnen we de migratie en het HA-degradatievermogen van zowel de groothandelspopulatie als vervolgens individuele cellen in de loop van de tijd evalueren. Bovendien kan...

Video: In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

Neuroscience

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Bioengineering

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Biology

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

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