Name: blastomere explants to test for cell fate commitment during embryonic development
Uploaded: 2013-01-26T13:00:00
Description: Watch this Scientific Journal Video about Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development at JoVE.com

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 M...

<ol>
	<li>Dale, L., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+map+of+the+32-cell+stage+of+Xenopus+laevis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+fates+of+blastomeres+of+the+eight-cell+stage+Xenopus+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+16-cell+stage+Xenopus+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell+stage+Xenopus+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+fate+during+cleavage+of+frog+(Xenopus+laevis)+blastomeres.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+and+roles+of+the+presumptive+organizer+region+in+the+32-cell+embryos+in+normal+development+of+Xenopus+laevis.">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., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+20+Early+events+in+frog+blastomere+fate+determination.">Chapter 20 Early events in frog blastomere fate determination.</a> Cell Fate and Lineage Determination. , 297-321 (1999).</li><li>Moore, K. B., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal-vegetal+asymmetries+influence+the+earliest+steps+in+retinal+fate+commitment+in+Xenopus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+competence+of+Xenopus+blastomeres+to+produce+neural+and+retinal+progeny+is+repressed+by+two+endo-mesoderm+promoting+pathways.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+differentiation+of+dorsal+axial+structures+from+an+animal+cap+cleavage+stage+blastomere+in+Xenopus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+maternal+mRNAs+from+a+dorsal+animal+blastomere+induce+a+secondary+axis+in+host+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+mesoderm+formation+in+the+Xenopus+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+activin-like+signal+activates+a+dorsal-specifying+RNA+between+the+8-+and+16-cell+stages+of+Xenopus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+maternal+influences+on+dorsal-ventral+fate+of+Xenopus+animal+blastomeres.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Development+of+Xenopus+laevis.">Early Development of Xenopus laevis.</a> A Laboratory Manual. , (2000).</li><li>Sullivan, S. A., Akers, L., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=foxD5a,+a+Xenopus+winged+helix+gene,+maintains+an+immature+neural+ectoderm+via+transcriptional+repression+that+is+dependent+upon+the+C-terminal+domain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxD5+plays+a+critical+upstream+role+in+regulating+neural+fate+and+onset+of+differentiation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcortical+rotation+in+Xenopus+eggs:+an+early+step+in+embryonic+axis+specification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendix+A:+Solutions+and+Protocols.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+cleavage+furrow+demarcates+the+dorsal-ventral+axis+in+Xenopus+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close+correlation+between+the+first+cleavage+plane+and+the+body+axis+in+early+Xenopus+embryos.">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>

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 border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Alumina abrasive film</td>
 <td>Thomas Scientific</td>
 <td>#6775E-38 </td>
 <td>Course (12 &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 &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 &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...

Watch this Scientific Journal Video about Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development at JoVE.com

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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