We thank Rayhan Arif and Hassaan Shahawy for camera work, Tripti Gupta and Frederike Alwes for assistance refining the cell ablation technique, and Extavour lab members for feedback on the data, video and manuscript. This work was partially supported by the Harvard Stem Cell Institute (Seed Grant number SG-0057-10-00) the Ellison Medical Foundation (New Scholar Award number AG-NS-07010-10) to CGE, and Harvard College Research Program awards to ARN.

Comments that may be helpful in executing certain steps are indicated in italics.
1. Day 1: Preparation of Materials
<ol>
	<li>Prepare the following materials (see Table of Materials and Equipment):
	<ul>
		<li>15 cm and 22 cm Pasteur pipettes</li>
		<li>Diamond scribe</li>
		<li>filtered artificial seawater (salinity between 0.0018-0.0020) containing 1 mg/ml Amphotericin B (1:100 of a 100 mg/ml stock solution), 100 units/ml penicillin and 100 mg/ml streptomycin (1:50 of a stock solution containing 5,00...

<ol>
	<li>Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3.">Chapter 3.</a> Emerging Model Organisms: A Laboratory Manual Volume. , 373-404 .</li><li>Sander, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+insect+embryogenesis:+the+evolution+of+concepts+and+mechanisms.">Pattern formation in insect embryogenesis: the evolution of concepts and mechanisms.</a> Int. J.Insect Morphol. Embryol. 25, 349-367 (1997).</li><li>Regier, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthropod+relationships+revealed+by+phylogenomic+analysis+of+nuclear+protein-coding+sequences.">Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences.</a> Nature. 463, 1079-1083 (2010).</li><li>Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+isolated+blastomeres+with+respect+to+germ+cell+formation+in+the+amphipod+crustacean+Parhyale+hawaiensis.">The fate of isolated blastomeres with respect to germ cell formation in the amphipod crustacean Parhyale hawaiensis.</a> Dev. Biol. 277, 387-402 (2005).</li><li>Browne, W. E., Price, A. L., Gerberding, M., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+in+the+amphipod+crustacean,+Parhyale+hawaiensis.">Stages of embryonic development in the amphipod crustacean, Parhyale hawaiensis.</a> Genesis. 42, 124-149 (2005).</li><li>Gerberding, M., Browne, W. E., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+of+the+amphipod+crustacean+Parhyale+hawaiensis+reveals+an+early+restriction+of+cell+fates.">Cell lineage analysis of the amphipod crustacean Parhyale hawaiensis reveals an early restriction of cell fates.</a> Development. 129, 5789-5801 (2002).</li><li>Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+and+dissection+of+Parhyale+hawaiensis+embryos.">Fixation and dissection of Parhyale hawaiensis embryos.</a> Cold Spring Harbor Protoc. 2009, (2009).</li><li>Price, A. L., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+divergent+mechanisms+of+mesoderm+development+in+arthropods:+the+expression+of+Ph-twist+and+Ph-mef2+in+Parhyale+hawaiensis.">Investigating divergent mechanisms of mesoderm development in arthropods: the expression of Ph-twist and Ph-mef2 in Parhyale hawaiensis.</a> J. Exp. B. Dev. Evol. 310, 24-40 (2008).</li><li>Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+of+labeled+RNA+probes+to+fixed+Parhyale+hawaiensis+embryos.">In situ hybridization of labeled RNA probes to fixed Parhyale hawaiensis embryos.</a> Cold Spring Harbor Protoc. 2009, (2009).</li><li>Browne, W. W., Schmid, B. G. M., Wimmer, E. A., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+otd+orthologs+in+the+amphipod+crustacean,+Parhyale+hawaiensis.">Expression of otd orthologs in the amphipod crustacean, Parhyale hawaiensis.</a> Dev. Genes Evol. 216, 581-595 (2006).</li><li>Prpic, N. M., Telford, 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=Expression+of+homothorax+and+extradenticle+mRNA+in+the+legs+of+the+crustacean+Parhyale+hawaiensis:+evidence+for+a+reversal+of+gene+expression+regulation+in+the+pancrustacean+lineage.">Expression of homothorax and extradenticle mRNA in the legs of the crustacean Parhyale hawaiensis: evidence for a reversal of gene expression regulation in the pancrustacean lineage.</a> Dev. Genes Evol. 218, 333-339 (2008).</li><li>Kizil, G., Havemann, J., Gerberding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cells+in+the+crustacean+Parhyale+hawaiensis+depend+on+Vasa+protein+for+their+maintenance+but+not+for+their+formation.">Germ cells in the crustacean Parhyale hawaiensis depend on Vasa protein for their maintenance but not for their formation.</a> Dev. Biol. 327, 320-239 (2009).</li><li>Liubicich, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+Parhyale+Ultrabithorax+recapitulates+evolutionary+changes+in+crustacean+appendage+morphology.">Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in crustacean appendage morphology.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 13892-13896 (2009).</li><li>Pavlopoulos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+evolution+of+appendage+specialization+by+Hox+gene+misexpression+in+an+emerging+model+crustacean.">Probing the evolution of appendage specialization by Hox gene misexpression in an emerging model crustacean.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 13897-13902 (2009).</li><li>Vargas-Vila, M. A., Hannibal, R. L., Parchem, R. J., Liu, P. Z., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prominent+requirement+for+single-minded+and+the+ventral+midline+in+patterning+the+dorsoventral+axis+of+the+crustacean+Parhyale+hawaiensis.">A prominent requirement for single-minded and the ventral midline in patterning the dorsoventral axis of the crustacean Parhyale hawaiensis.</a> Development. 137, 3469-3476 (2010).</li><li>Simanton, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+arthropod+midline+netrin+accumulation+revealed+with+a+cross-reactive+antibody+provides+evidence+for+midline+cell+homology.">Conservation of arthropod midline netrin accumulation revealed with a cross-reactive antibody provides evidence for midline cell homology.</a> Evol. Dev. 11, 260-268 (2009).</li><li>Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+staining+of+Parhyale+hawaiensis+embryos.">Antibody staining of Parhyale hawaiensis embryos.</a> Cold Spring Harbor Protoc. 2009, (2009).</li><li>Pavlopoulos, A., Averof, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+genetic+transformation+for+comparative+developmental+studies+in+the+crustacean+Parhyale+hawaiensis.">Establishing genetic transformation for comparative developmental studies in the crustacean Parhyale hawaiensis.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 7888-7893 (2005).</li><li>Kontarakis, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+strategy+for+gene+trapping+and+trap+conversion+in+emerging+model+organisms.">A versatile strategy for gene trapping and trap conversion in emerging model organisms.</a> Development. 138, 2625-2630 (2011).</li><li>Zeng, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+assembly+and+characterization+of+a+maternal+and+developmental+transcriptome+for+the+emerging+model+crustacean+Parhyale+hawaiensis.">De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis.</a> BMC Genom. 12, 581 (2011).</li><li>Zeng, V., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASGARD:+an+open-access+database+of+annotated+transcriptomes+for+emerging+model+arthropod+species.">ASGARD: an open-access database of annotated transcriptomes for emerging model arthropod species.</a> Database. , (2012).</li><li>Nestorov, P., Battke, F., Levesque, M. P., Gerberding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Maternal+Transcriptome+of+the+Crustacean+Parhyale+hawaiensis+Is+Inherited+Asymmetrically+to+Invariant+Cell+Lineages+of+the+Ectoderm+and+Mesoderm.">The Maternal Transcriptome of the Crustacean Parhyale hawaiensis Is Inherited Asymmetrically to Invariant Cell Lineages of the Ectoderm and Mesoderm.</a> PLoS ONE. 8, (2013).</li><li>Alwes, F., Hinchen, B., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+cell+lineage,+movement,+and+migration+from+germ+layer+specification+to+gastrulation+in+the+amphipod+crustacean+Parhyale+hawaiensis.">Patterns of cell lineage, movement, and migration from germ layer specification to gastrulation in the amphipod crustacean Parhyale hawaiensis.</a> Dev. Biol. 139, 110-123 (2011).</li><li>Ewen-Campen, B., Schwager, E. E., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+machinery+of+germ+line+specification.">The molecular machinery of germ line specification.</a> Mol. Reprod. Dev. 77, 3-18 (2010).</li><li>Price, A. L., Modrell, M. S., Hannibal, R. L., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoderm+and+ectoderm+lineages+in+the+crustacean+Parhyale+hawaiensis+display+intra-germ+layer+compensation.">Mesoderm and ectoderm lineages in the crustacean Parhyale hawaiensis display intra-germ layer compensation.</a> Dev. Biol. 341, 256-266 (2010).</li><li>Fraser, S. E., 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=The+molecular+metamorphosis+of+experimental+embryology.">The molecular metamorphosis of experimental embryology.</a> Cell. 100, 41-55 (2000).</li><li>Olsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clash+of+traditions:+the+history+of+comparative+and+experimental+embryology+in+Sweden+as+exemplified+by+the+research+of+G&#246;sta+J&#228;gersten+and+Sven+H&#246;rstadius.">A clash of traditions: the history of comparative and experimental embryology in Sweden as exemplified by the research of G&#246;sta J&#228;gersten and Sven H&#246;rstadius.</a> Theory Biosci. 126, 117-129 (2007).</li><li>Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+Parhyale+hawaiensis+blastomeres+with+fluorescently+labeled+tracers.">Injection of Parhyale hawaiensis blastomeres with fluorescently labeled tracers.</a> Cold Spring Harbor Protoc. 2009, (2009).</li><li>Chaw, R., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+migration+of+cell+populations+in+the+early+gastrulation+of+the+amphipod+crustacean+Parhyale+hawaiensis.">Independent migration of cell populations in the early gastrulation of the amphipod crustacean Parhyale hawaiensis.</a> Dev. Biol. 371, 94-109 (2012).</li><li>Schmitz, E. H., Harrison, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crustacea+Microscopic+Anatomy+of+Invertebrates.">Crustacea Microscopic Anatomy of Invertebrates.</a> Microscopic Anatomy of Invertebrates. 9, 443-528 (1992).</li></ol>

We describe a protocol for manual ablation and complete physical removal of single blastomeres from early cleavage stages of the amphipod P. hawaiensis. We demonstrate use of this protocol by removing the single germ line precursor cell g from an eight-cell stage embryo, and show that ablation has been successful by confirming absence of g&#8217;s daughter cells in later embryogenesis. This protocol can be used to remove any of the micromeres from the embryo at early cleavage st...

The amphipod crustacean Parhyale hawaiensis has emerged over the last decade as a promising model organism with great potential for use in evolutionary developmental biology research1. Among the arthropods, most model systems are insects, and the most extensively studied of these is the fruit fly Drosophila melanogaster. D. melanogaster is a member of the insect order Diptera, and as such displays many embryological features that are derived with respect to those of basally branching insects2. Moreover, insects are nested within the subphylum Pancrustacea3, meaning that insects have their closest relatives within the long-standing &#8220;natural&#8221; group called pancrustaceans, and that this group is paraphyletic. This suggests that in addition to basally branching insect models, studies of other crustaceans are required to gain a broader view of the evolutionary history of the developmental traits and molecular mechanisms that have been so well studied in D. melanogaster. However, very few crustaceans have been well established for experimental laboratory analysis of development. The amphipod P. hawaiensis is a highly tractable laboratory model system, amenable to a range of experimental techniques. Amphipods display many unique features within their parent superorder Peracarida (beach hoppers, scuds, and well shrimps), and are therefore thought to be relatively derived within this group of crustaceans. Nevertheless, the relative ease of embryological and functional genetic manipulation offered by Parhyale make this amphipod a valuable addition to the current inventory of model organisms.
As a laboratory animal, P. hawaiensis offers many advantages. Animals are tolerant to a wide range of temperatures and salinities, and survive well in large cultures of artificial seawater1. It is easy to distinguish between males and females based on clear morphological differences, most notably, the large, hooked, anterior trunk appendages that males use to grasp the females during mating. For embryological and developmental work, P. hawaiensis has several very appealing features. Embryogenesis lasts approximately 10 days and the time to sexual maturity is approximately six weeks at 28 &#186;C (but note that Parhyale survives well at temperatures ranging from approximately 20-30 &#186;C, and that detailed developmental staging information is available for embryos raised at 18 &#186;C4, 25 &#186;C4, and 26 &#186;C5,6). Adults mate all year round in the laboratory, so embryos are available at any time of year. Females lay 2-20 (depending on the age of the female) fertilized eggs into a ventral brood pouch located between the first several pairs of legs (Figures 1A and&#160;1B), and it is possible to gather these embryos very early in development without killing the female or damaging the embryos (Figure 1C). The embryos survive in filtered artificial sea water through to hatching, can be fixed for subsequent gene expression or histological analysis7, and a detailed staging table allows accurate identification of the progress through development5. Robust protocols have been used to perform gene expression analysis by in situ hybridization8-15 or immunostaining4,16,17, functional knockdown by RNA interference13,15 or morpholinos12, and stable germ line transgenesis18. Using the transgenesis system, inducible expression14 and enhancer trap19 methods can also be used to investigate gene function in P. hawaiensis. While a publicly available genome sequence is not currently available, a transcriptome containing transcripts produced during oogenesis and embryogenesis has been de novo assembled and annotated20, and deposited in a searchable database21, facilitating gene discovery. In sum, P. hawaiensis is a highly tractable model organism suitable for multiple experimental and genetic approaches to understanding development.
Unlike the early syncytial cleavages of D. melanogaster, P. hawaiensis embryos cleave holoblastically following fertilization (Figure 2A). Lineage tracing analysis has shown that by third cleavage, each of the third cleavage blastomeres is specifically fated to give rise to one of the three germ layers or the germ line6 (Figure 2B). These data, together with microarray data22, cell lineage analyses6,23, and blastomere isolation experiments4 have suggested that developmental potentials are segregated to at least some third cleavage blastomeres by asymmetric inheritance of cell fate determinants. Accordingly, in blastomere ablation experiments in which the germ line precursor (termed &#8220;g&#8221; in the Parhyale cell lineage nomenclature6) was removed at the eight cell stage, embryos lacked germ cells at later developmental stages4, as indicated by the absence of cells expressing the protein Vasa, which is a germ line marker in most metazoans24. In contrast, somatic blastomere ablation experiments showed that P. hawaiensis embryos also possess significant regulatory capabilities, such that the fates of mesoderm or ectoderm precursor blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres25. How regulative cell fate replacement can occur, and the extent of autonomous cell fate adoption by somatic blastomeres, remains unknown. Experimental embryological techniques such as blastomere ablation can be useful in understanding the relative autonomy and nonautonomy of cell fate decisions26,27&#160;and are therefore of interest in the study of P. hawaiensis embryogenesis.
In the experiments that demonstrated regulative replacement of ectodermal and mesodermal lineages, blastomere ablation was performed by injection28 and subsequent excitation of phototoxic dyes25. While this technique is effective at killing the injected blastomere(s), it does not completely remove the dead cell body from the embryo. In addition, differences have been observed between cell lineage data gathered through to gastrulation stages of embryogenesis by injecting blastomeres with fluorescent lineage tracers28,29, and data gathered by following unperturbed blastomeres through development of the same embryonic stages23. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications.
We previously published the results of cell lineage analyses of embryos in which single cells were manually ablated23. However, the delicate operations required to remove single blastomeres from early cleavage stage embryos have not yet been fully described. Here we present a protocol for collection of P. hawaiensis embryos and manual ablation of a single blastomere from an eight-cell stage embryo. The goal of this method is to achieve complete removal of the cell body from the embryo, allowing observation of the cellular behaviors and cell fate competencies of the remaining cells during embryogenesis and post-embryonic development. Our protocol shows removal of the germ line precursor g (Figure 2C), but can be applied to any cell at the eight-cell stage, or to blastomeres of earlier cleavage stages. In principle, this protocol could be applied to remove single cells from early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

<table border="0" cellpadding="0" cellspacing="0" width="1060">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">15 cm Pasteur pipette</td>
			<td style="width:164px;">Wheaton</td>
			<td style="width:123px;">53499-630</td>
			<td style="width:495px;">For transferring intact and blastomere-ablated embryos from dish to dish. 
			VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">22 cm Pasteur pipette</td>
			<td style="width:164px;">Wheaton</td>
			<td style="width:123px;">53499-632</td>
			<td style="width:495px;">For making mouth pipette tips. 
			VWR</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">3 cm petri dishes</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:123px;">25382-334</td>
			<td style="width:495px;">Use some unaltered to collect and store embryos; coat some with a layer of Sylgard (see below) to create a working surface for blastomere ablations. 
			VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">48-well plates</td>
			<td style="width:164px;">Greiner Bio-One</td>
			<td style="width:123px;">82051-004</td>
			<td style="width:495px;">For culturing embryos following ablation. 
			VWR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Bottle top filters 0.2 micron</td>
			<td style="width:164px;">Nalge Nunc International</td>
			<td style="width:123px;">28199-296</td>
			<td style="width:495px;">For creating FASW (See below) 
			VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Bunsen burner</td>
			<td style="width:164px;">VWR</td>
			<td style="width:123px;">89038-530</td>
			<td style="width:495px;">For creating tungsten wire tool and fine tip of mouth pipette. 
			VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Diamond scribe</td>
			<td style="width:164px;">Musco Sports Lighting</td>
			<td style="width:123px;">52865-005</td>
			<td style="width:495px;">For creating wide-mouthed Pasteur pipettes for collecting couples. 
			VWR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:164px;">Forceps</td>
			<td style="width:164px;">Fine Science Tools</td>
			<td style="width:123px;">11050-10</td>
			<td style="width:495px;">For holding anaesthetized females during removal of embryos from brood pouch. These do not have to be fine-tipped Dumont forceps - the tips can be at least as blunt as that in the forceps for which the catalogue number is listed here. 
			Fine Science Tools</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Fungizone-Amphotericin B</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:123px;">A2942</td>
			<td style="width:495px;">Add to FASW to a final concentration of 1mg/ml, and use this solution to culture blastomere-ablated embryos. 
			Sigma Aldrich</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Glass capillaries 4 inches long, 1/0.58 OD/ID (mm)</td>
			<td style="width:164px;">World Precision Instruments</td>
			<td style="width:123px;">1B100-4</td>
			<td style="width:495px;">need to include glass capillary and needle puller machine? 
			World Precision Instruments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Glass watchglass 300 mm diameter</td>
			<td style="width:164px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">70543-30</td>
			<td style="width:495px;">For use in removing embryos from the brood pouch of anaesthetized females. 
			Electron Microscopy Sciences</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Instant Ocean Artificial Sea Water (ASW)</td>
			<td style="width:164px;">Instant Ocean</td>
			<td style="width:123px;">IS160</td>
			<td style="width:495px;">Make ASW by dissolving salt in deionized water to a salinity of X. 
			Aquatic EcoSystems</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Instant Ocean Filtered Artificial Sea Water (FASW)</td>
			<td style="width:164px;">Instant Ocean</td>
			<td style="width:123px;">IS160</td>
			<td style="width:495px;">Use 0.2 micron filters to sterilize ASW. Make up in small amounts (&#60; 250 ml) as needed. 
			Aquatic EcoSystems</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Kimwipes</td>
			<td style="width:164px;">Kimberly-Clark</td>
			<td style="width:123px;">21905-026</td>
			<td style="width:495px;">For safely breaking off the fine end of Pasteur pipettes modified for collecting couples. 
			VWR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Mouth pipette adaptor</td>
			<td style="width:164px;">Drummond Labware</td>
			<td style="width:123px;">A5177-5EA</td>
			<td style="width:495px;">Insert the fine pulled pasteur pipette tip into this end, to be used for removing extruded cell contents during the ablation procedure. 
			Sigma Aldrich</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Mouth pipette tubing</td>
			<td style="width:164px;">Aldrich</td>
			<td style="width:123px;">Z280356</td>
			<td style="width:495px;">Cut this to be long enough to comfortably hang around your neck during the ablation procedure. 
			Sigma Aldrich</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Needle Puller</td>
			<td style="width:164px;">Sutter Instrument Company</td>
			<td style="width:123px;">P97</td>
			<td style="width:495px;">For making needles from glass capillaries for puncturing the blastomere to be ablated. 
			Sutter Instrument Company</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Penicillin-Streptomycin Solution</td>
			<td style="width:164px;">Mediatech</td>
			<td style="width:123px;">45000-650</td>
			<td style="width:495px;">Add to FASW to a final concentration of 100 units/ml penicillin and 100 mg/ml streptomycin, and use this solution to culture blastomere-ablated embryos. 
			VWR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Rubber bulb</td>
			<td style="width:164px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">100488-418</td>
			<td style="width:495px;">For using Pasteur pipettes to transfer embryos from dish to dish. 
			VWR</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Sylgard 184</td>
			<td style="width:164px;">K. R. Anderson, Inc.</td>
			<td style="width:123px;">NC9659604</td>
			<td style="width:495px;">Make up according to manufacturer&#39;s instructions. Pour a layer 2-5 mm thick into a 3cm petri dish to create a working surface for blastomere ablations. 
			Fisher Scientific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Tungsten wire 0.004 inches diameter</td>
			<td style="width:164px;">A-M Systems</td>
			<td style="width:123px;">719000</td>
			<td style="width:495px;">Use this to make a tool for removing embryos from the brood pouch of anaesthetized females. 
			A-M Systems</td>
		</tr>
	</tbody>
</table>

ablation of a single cell from eight-cell embryos of the amphipod crustacean parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Following successful ablation of single third cleavage P. hawaiensis micromeres as described in this protocol, the remaining micromeres gradually shift their positions slightly so as to partially occupy the space formerly occupied by the ablated blastomere. For example, when g is removed, the neighboring blastomeres mr and ml shift slightly and come to share the lateral cell borders that were formerly in direct contact with g (compare&#160;<stro...

Watch this Scientific Journal Video about Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis at JoVE.com

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

ablation-single-cell-from-eight-cell-embryos-amphipod-crustacean

Research

JoVE Journal

Biology

12.4K ビュー.  Harvard University. 端脚Parhyaleのhawaiensisは甲殻類発生学および比較節足動物発展と進化の研究のための有望なモデル生物である。このプロトコルは、Parhyaleの初期卵割期胚からの単一割球を手動で除去する方法を説明します。 

ビデオ: Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...