The authors would like to acknowledge the GWU Harlan Fellowship for support of Paaqua Grant and the GWU Luther Rice Fellowship for support of Mona Herold. This work was supported by the National Science Foundation grant MCB-1121711.

1. Preparation of Instruments, Culture Media and Dishes
<ol>
 <li>Sharpen four forceps using Alumina abrasive film. Do this under a dissecting microscope to monitor the tip size. One pair of forceps serves as a back-up in case a tip is damaged during the procedure. Autoclave all four forceps and store in a sterile container.</li>
 <li>Make 500 ml each of 0.1X and 1.0X culture medium (either Marc's Modified Ringers [MMR] or Modified Barth's Saline [MBS]; recipes in 15) and filter sterilize. We routinely use MBS (1X = 88 mM NaCl; 1 mM KCl, 0.7 mM CaCl2, 1 mM MgSO4, 5 mM HEPES [pH 7.8], 2.5 mM NaHCO3). Store at 14-18 &deg;C for months.</li>
 <li>Make a 2% agarose solution in culture medium (2 g electrophoresis grade agarose in 100 ml 1X culture medium in a screw cap glass bottle). Autoclave to dissolve agarose. This can be stored at 4 &deg;C for months, and microwaved to liquefy the agarose when it is next needed.</li>
 <li>While the agarose is liquid, pour about 0.5 ml onto the bottom of each well of a sterile 24-well culture plate. This will prevent the explants from sticking to the plastic.</li>
 <li>While the agarose is liquid, pour about 2-3 ml onto the bottom of two to three 60 mm Petri dishes, and swirl gently to make sure the bottom is covered. These will serve as dissection dishes and the agarose prevents embryos from sticking to the plastic once their membranes are removed.</li>
 <li>When the agarose has cooled and hardened, flame the tip of a 6" Pasteur pipette until it melts into a ball, and lightly touch it on the surface of the agarose in each well of the culture plate. This will create a shallow depression into which the explant will be placed.</li>
 <li>Fill each well of the culture plate with 1X sterile culture medium.</li>
 <li>When the agarose has cooled and hardened, fill the dissection dish with diluted culture medium (0.1X MMR or 0.1X MBS) to facilitate blastomere separation.</li>
</ol>
2. Selection of Embryos
<ol>
 <li>Obtain fertilized eggs and remove the jelly coats according to standard protocols 15. Transfer them to 0.5X culture medium in a 100 mm Petri dish.</li>
 <li>When embryos reach the 2-cell stage, sort those in which the first cleavage furrow bisects the lightly pigmented area in the animal hemisphere (Figure 1) into a separate dish. These will be the donors for the explants. Keep the remaining 2-cell embryos in a separate dish next to the donor embryos throughout the procedure to serve as sibling controls to stage the explants.</li>
</ol>
3. Preparation of Explants
<ol>
 <li>Place 5-10 embryos in a dissection dish, but only work on one embryo at a time.</li>
 <li>Position the first embryo so the transparent vitelline membrane can be seen; it is separated by a clear space (perivitelline space) above the surface of the animal pole of the embryo. Using a sharpened forceps in one's subdominant hand (left hand if one is right handed), grasp the vitelline membrane above the perivitelline space. Using a sharpened forceps in the other hand, grasp the membrane close to the first forceps tip, and gently pull in opposite directions to peel the membrane away. Make the initial grasp at a distance from the cell that is to be dissected, so that the target cell is not damaged during removal of the membrane. One can tell that the membrane has been removed because the embryo will flatten.</li>
 <li>Grab one neighboring cell with the forceps in the subdominant hand and use this cell as a "handle" so the cell to be dissected is not directly touched. With the forceps in the other hand, gently pull the remaining neighboring cells away from the desired blastomere. If midline cells, which share the same fate, are targeted, they can be removed together as a pair. Finally, dissect away the "handle" cell.</li>
 <li>Pick up the blastomere (or blastomere pair), with a sterile, glass Pasteur pipette, avoiding air bubbles and excessive suction. The pipette can be fire-polished to remove sharp edges that can damage the cell, but we do not routinely do this. Place the tip of the Pasteur pipette under the surface of the culture medium in a well of the explant culture dish, and gently expel the blastomere. It should slide into the shallow depression by gravity. The same blastomere from more than one embryo can be combined into a single explant.</li>
 <li>Repeat this procedure with the remaining embryos in the dissection dish, one-by-one. After about 10 embryos, the dissection dish will be filled with cellular debris. When this occurs, change to a fresh dissection dish.</li>
 <li>After all dissections are done, check whether the explants are in the shallow depressions. If not, they can be gently pushed into the depression with a hair loop that has been sterilized in 70% ethanol and air dried.</li>
 <li>About an hour after the last dissection, remove debris surrounding the healed explants with a sterile, glass Pasteur pipette.</li>
 <li>Culture the plate of explants at 14-20 &deg;C next to the Petri dish containing the sibling, control embryos. Sibling embryos will indicate the stage of development of the blastomere explants.</li>
</ol>
4. Harvesting Explants for Analysis
<ol>
 <li>Harvest the explants when the siblings reach the desired developmental stage. These explants survive quite well even if some of the cells disintegrate. Therefore, if there is a cloudy mass in the culture well, explore it with a hair loop or forceps to determine if a healthy explant is buried within.</li>
 <li>Pick up explants in a small volume of culture solution with a glass Pasteur pipette, and gently expel them into a fixative or lysis buffer appropriate for the assay to be conducted.</li>
</ol>

<ol><li>Dale, L., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fate+map+of+the+32-cell+stage+of+Xenopus+laevis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Fate map of the 32-cell stage of Xenopus laevis.</a> Development. 100, 279-295 (1987).</li>
<li>Masho, R., Kubota, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+fates+of+blastomeres+of+the+eight-cell+stage+Xenopus+embryo%5BTitle%5D)+AND+%22Dev.+Growth%2C+Diff%22%5BJournal%5D)">Developmental fates of blastomeres of the eight-cell stage Xenopus embryo.</a> Dev. Growth, Diff. 30, 347-359 (1988).</li>
<li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fates+of+the+blastomeres+of+the+16-cell+stage+Xenopus+embryo%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Fates of the blastomeres of the 16-cell stage Xenopus embryo.</a> Dev. Biol. 119, 560-578 (1987).</li>
<li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fates+of+the+blastomeres+of+the+32-cell+stage+Xenopus+embryo%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Fates of the blastomeres of the 32-cell stage Xenopus embryo.</a> Dev. Biol. 122, 300-319 (1987).</li>
<li>Moody, S. A., Kline, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Segregation+of+fate+during+cleavage+of+frog+%28Xenopus+laevis%29+blastomeres%5BTitle%5D)+AND+%22Anat.+Embryol%22%5BJournal%5D)">Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres.</a> Anat. Embryol. 182, 347-362 (1990).</li>
<li>Takasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fates+and+roles+of+the+presumptive+organizer+region+in+the+32-cell+embryos+in+normal+development+of+Xenopus+laevis%5BTitle%5D)+AND+%22Dev.+Growth%2C+Diff%22%5BJournal%5D)">Fates and roles of the presumptive organizer region in the 32-cell embryos in normal development of Xenopus laevis.</a> Dev. Growth, Diff. 29, 141-152 (1987).</li>
<li>Sullivan, S. A., Moore, K. B., Moody, S. A. Chapter 20 Early events in frog blastomere fate determination. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSullivan%2C+SA+%22Cell+Fate+and+Lineage+Determination%22">Cell Fate and Lineage Determination</a>. Moody, S. A. , Academic Press. 297-321 (1999).</li>
<li>Moore, K. B., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal-vegetal+asymmetries+influence+the+earliest+steps+in+retinal+fate+commitment+in+Xenopus%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Animal-vegetal asymmetries influence the earliest steps in retinal fate commitment in Xenopus.</a> Dev. Biol. 212, 25-41 (1999).</li>
<li>Yan, B., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+competence+of+Xenopus+blastomeres+to+produce+neural+and+retinal+progeny+is+repressed+by+two+endo-mesoderm+promoting+pathways%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">The competence of Xenopus blastomeres to produce neural and retinal progeny is repressed by two endo-mesoderm promoting pathways.</a> Dev. Biol. 305, 103-119 (2007).</li>
<li>Gallagher, B. C., Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autonomous+differentiation+of+dorsal+axial+structures+from+an+animal+cap+cleavage+stage+blastomere+in+Xenopus%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus.</a> Development. 112, 1103-1114 (1991).</li>
<li>Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenopus+maternal+mRNAs+from+a+dorsal+animal+blastomere+induce+a+secondary+axis+in+host+embryos%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Xenopus maternal mRNAs from a dorsal animal blastomere induce a secondary axis in host embryos.</a> Development. 116, 347-3345 (1992).</li>
<li>Godsave, S. F., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+analysis+of+mesoderm+formation+in+the+Xenopus+embryo%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Single cell analysis of mesoderm formation in the Xenopus embryo.</a> Development. 111, 523-530 (1991).</li>
<li>Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+activin-like+signal+activates+a+dorsal-specifying+RNA+between+the+8-+and+16-cell+stages+of+Xenopus%5BTitle%5D)+AND+%22Dev.+Genetics%22%5BJournal%5D)">An activin-like signal activates a dorsal-specifying RNA between the 8- and 16-cell stages of Xenopus.</a> Dev. Genetics. 19, 210-221 (1996).</li>
<li>Pandur, P. D., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+maternal+influences+on+dorsal-ventral+fate+of+Xenopus+animal+blastomeres%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">Multiple maternal influences on dorsal-ventral fate of Xenopus animal blastomeres.</a> Dev. Dyn. 225, 581-587 (2002).</li>
<li>Sive, H. L., Grainger, R. M., Harland, R. M. Early Development of Xenopus laevis. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSive%2C+HL+%22A+Laboratory+Manual%22">A Laboratory Manual</a>. , Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. (2000).</li>
<li>Sullivan, S. A., Akers, L., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((foxD5a%2C+a+Xenopus+winged+helix+gene%2C+maintains+an+immature+neural+ectoderm+via+transcriptional+repression+that+is+dependent+upon+the+C-terminal+domain%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent upon the C-terminal domain.</a> Dev. Biol. 232, 439-457 (2001).</li>
<li>Yan, B., Neilson, K. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FoxD5+plays+a+critical+upstream+role+in+regulating+neural+fate+and+onset+of+differentiation%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">FoxD5 plays a critical upstream role in regulating neural fate and onset of differentiation.</a> Dev. Biol. 329, 80-95 (2009).</li>
<li>Vincent, J. -P., Gerhart, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcortical+rotation+in+Xenopus+eggs%3A+an+early+step+in+embryonic+axis+specification%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification.</a> Dev. Biol. 123, 526-539 (1987).</li>
<li>Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Appendix+A%3A+Solutions+and+Protocols%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Appendix A: Solutions and Protocols.</a> Methods in Cell Biology. 36, 657-662 (1991).</li>
<li>Klein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+first+cleavage+furrow+demarcates+the+dorsal-ventral+axis+in+Xenopus+embryos%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos.</a> Dev. Biol. 120, 299-304 (1987).</li>
<li>Masho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Close+correlation+between+the+first+cleavage+plane+and+the+body+axis+in+early+Xenopus+embryos%5BTitle%5D)+AND+%22Dev.+Growth+Diff%22%5BJournal%5D)">Close correlation between the first cleavage plane and the body axis in early Xenopus embryos.</a> Dev. Growth Diff. 32, 57-64 (1990).</li>
</ol>

No conflicts of interest declared.

The most critical steps for successful culturing of individual blastomeres are: 1) correctly identifying the blastomere of interest; 2) maintaining a sterile, healthy culture; 3) dissecting cells at the correct part of the cell cycle; and 4) developing the necessary manual dexterity to prevent damage during dissection and transfer to the culture well.
To manipulate specific blastomeres, it is essential to be able to identify the cardinal axes. In Xenopus embryos, the dorsal sid...

Xenopus laevis embryos have been utilized extensively to identify the mechanisms by which embryonic cells acquire their specific fates because their eggs are large enough to permit microsurgical approaches. Additionally, they develop externally without the need for nutritional supplementation of the culture medium because each cell contains a rich intracellular supply of yolk platelets that provide an intrinsic energy store. An important asset for studying the mechanisms by which cell fate is determined is the comprehensive set of fate maps of the cleavage stage blastomeres (from 2- through 32-cell stages) 1, 2, 3, 4, 5, 6. These maps were constructed by microinjecting a detectable molecule into a single, identifiable blastomere and monitoring later in development which tissues are populated by the labeled progeny. Consistent fate maps are possible because the cardinal axes of the embryos can be reliably identified in many embryos. First, in all wild type embryos the animal hemisphere is pigmented, whereas the vegetal hemisphere is not. Second, the entry of the sperm at fertilization causes a contraction of the animal hemisphere pigmentation towards the future ventral side; in many embryos a pigmentation difference therefore can be used to discriminate between dorsal and ventral sides. Third, the first cleavage furrow approximates the mid-sagittal plane in most embryos, and thus can be used to identify the right and left sides of the embryo. The fate maps also rely on the fact that naturally fertilized eggs frequently cleave in regular patterns that make each blastomere identifiable across a large population of embryos. While there is variability within and between clutches of eggs regarding pigmentation and cleavage patterns, using selection procedures described herein allows cells with prescribed fates to be identified with about 90% accuracy.
Cell fates can be determined during embryogenesis by several mechanisms. Intrinsic factors, such as differentially inherited cytoplasmic mRNAs or proteins, contribute to several aspects of early patterning. For example, specific maternal mRNAs determine which cells will contribute to the germ line, become the endoderm, or contribute to the dorsal body axis (reviewed in 7). Extrinsic factors provided locally by neighboring cells or more distantly from an embryonic signaling center, are responsible for inducing specific tissue types and patterning nearly every organ system. Examples of signaling centers include the organizer/node in the gastrula that induces the neural ectoderm and patterns the mesoderm, and the zone of polarizing activity that patterns the anterior-posterior axis of the limb bud. Although fate maps identify the precursors of the different organs and reveal the developmental path taken by their descendants in the normal embryo, they cannot distinguish between intrinsic and extrinsic influences on those cells. They also do not reveal the full developmental potential of a cell, whose descendants differentiate in the complex signaling environment of the embryo. Two experimental approaches can test whether a cell's fate is determined by intrinsic factors or is subsequently influenced by external factors: 1) transplantation of the cell to a novel location in the embryo; or 2) removal of the cell from the embryo followed by culture in the absence of exogenous signals.
Both experimental approaches have been feasible in Xenopus because the cells are large enough to be manually separated. For example, numerous studies have deleted single cells from embryos (to change cell-cell interactions) or transplanted cells to novel locations in host embryos to test for fate changes (reviewed in 7, 8, 9). In addition, the second approach of explanting small numbers of cells from different regions of the embryo into culture to elucidate inductive tissue interactions in the absence of exogenous factors is possible because the cells of the Xenopus embryo are filled with an intracellular nutrient store, the yolk platelets. Therefore, they can be cultured for a few days in a defined salt medium without nutritional or growth factor supplementation of the culture medium. We have used this approach to show that dorsal animal blastomeres have an autonomous ability to produce neural and dorsal mesodermal tissues due to maternally inherited mRNAs 10, 11, and others showed that mesoderm specification of 32-cell blastomeres relies on both intrinsic and extrinsic information 12. An advantage of culturing cells as explants is that the medium can also be supplemented with defined signaling factors to determine which cell-to-cell communication pathway might influence the fate of the explanted cell 12, 13, 14. In addition, one can inject the blastomere prior to culture with mRNA to over-express a gene, or with anti-sense oligonucleotides to prevent the translation of endogenous mRNAs. These, gain- and loss-of-function analyses can identify which molecules are required for an autonomously expressed fate. To analyze the fate of the explant, identification of specific cell types can be performed by standard gene expression (e.g., in situ hybridization, RT-PCR) and immunocytochemical assays. This protocol provides a simple, yet powerful, way to distinguish between intrinsic and extrinsic mechanisms that regulate how an embryonic cell develops into specific tissues.

<table><tbody><tr><td>Alumina abrasive film</td><td>Thomas Scientific</td><td>#6775E-38</td><td>Course (12 &amp;mu;m), for major repairs of forceps tips</td></tr><tr><td>Alumina abrasive film</td><td>Thomas Scientific</td><td>#6775E-46</td><td>Medium (3 &amp;mu;m), for fine sharpening of forceps tips</td></tr><tr><td>Alumina abrasive film</td><td>Thomas Scientific</td><td>#6775E-54</td><td>Fine (0.3 &amp;mu;m), for polishing of forceps tips</td></tr><tr><td>Forceps: Dumont, Dumoxel Biologie #5</td><td>Fine Science Tools</td><td>#11252-30</td><td>These have the fine tips that do not need sharpening when first purchased. Corrosive resistant so they can be autoclaved.</td></tr><tr><td>Gentamicin solution</td><td>Sigma</td><td>G1397</td><td>Add to medium on same day as use</td></tr></tbody></table>

blastomere explants to test for cell fate commitment during embryonic development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The ability of this assay to accurately assess the developmental potential of the cell relies upon dissecting out the correct blastomere based on the fate maps 1, 2, 3, 4, 5, 6. Therefore, it is critical to choose embryos with the correct pigmentation pattern at the 2-cell stage, as illustrated in Figure 1, that subsequently conform to regular cleavage patterns, as illustrated in Figure 2. If one observes the sorted embryos as they reach the required cleavage stage and s...

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Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

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15.2K Views.  The George Washington University. Het lot van een individuele embryonale cel kan worden beïnvloed door erfelijke moleculen en / of signalen van naburige cellen. Gebruik te maken van het lot kaarten van de splitsing podium Xenopus embryo, kan enkel blastomeren worden geïdentificeerd voor cultuur in isolatie om de bijdragen van erfelijke moleculen te beoordelen ten opzichte van cel-cel interacties.

Video: Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Developmental Biology

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Preparation of Single-Somite Explants from Zebrafish Embryos

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

We present a protocol for isolating single somites from zebrafish embryos, the dynamics of which can be followed in culture for several hours by fluorescence time-lapse microscopy, thus providing a methodology to quantify tissue-scale shape changes at single-cell resolution.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular ...

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 ...

Explant Culture for Developmental Studies

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo approach allows researchers to manipulate and observe developing tissues in ways that are not possible in vivo. Once established, explant culture is frequently used to understand the role of genes and signaling molecules in organogenesis.
This video will first introduce the basic principles of explant culture and demonstrate a protocol to isolate and grow explanted mammalian tissues. Common genetic and molecular methods of manipulating explant cultures will then be discussed. Finally, the viewers will learn about how explant techniques are currently being applied to study organ development.

Explant Culture for Developmental Studies and Morphogenesis

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

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