<meta charset="utf8"></head><body>Robusto trapianto di cellule di pesce zebra per la ricerca sulla rigenerazione e lo sviluppo

<ol>
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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL &#34;reservoir syringe&#34;</td>
			<td>Fisher Scientific</td>
			<td>14-955-459</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mL disposable vacuum filter, .2 &#181;m, PES</td>
			<td>Corning</td>
			<td>431153</td>
			<td></td>
		</tr>
		<tr>
			<td>20 x 12 mm heating block</td>
			<td>Corning</td>
			<td>480122</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Braun Medical Inc.</td>
			<td>455991</td>
			<td></td>
		</tr>
		<tr>
			<td>3 x 1 Frosted glass slide</td>
			<td>VWR</td>
			<td>48312-004</td>
			<td></td>
		</tr>
		<tr>
			<td>40x water dipping objective</td>
			<td>Nikon</td>
			<td>MRD07420</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Coarse Manipulator</td>
			<td>Narishige</td>
			<td>MN-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom microsyringe pump</td>
			<td>University of Oregon</td>
			<td>N/A</td>
			<td>Manufactured by University of Oregon machine shop (tsa.uoregon@gmail.com). A commercially available alternative is listed below.</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>1129500</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse FN1 &#34;Transplant Microscope&#34;</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>electrode handle</td>
			<td>World Precision Instruments</td>
			<td>5444</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Sterile Surgical Blade, #11</td>
			<td>VWR</td>
			<td>21899-530</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine micromanipulator, Three-axis Oil hydraulic&#160;</td>
			<td>Narishige</td>
			<td>MMO-203</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES pH 7.2</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>High Precision #3 Style Scalpel Handle</td>
			<td>Fisher Scientific</td>
			<td>12-000-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Disposable Borosilicate Pasteur Pipette, Wide Tip, 5.75 in</td>
			<td>DWK Life Sciences</td>
			<td>63A53WT</td>
			<td></td>
		</tr>
		<tr>
			<td>KIMBLE Chromatography Adapter&#160;</td>
			<td>DWK Life Sciences</td>
			<td>420408-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>M3516-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>LSE digital dry bath heater, 1 block, 120 V</td>
			<td>Corning</td>
			<td>6875SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual microsyringe pump</td>
			<td>World Precision Instruments</td>
			<td>MMP</td>
			<td>Commercial alternative to custom microsyringe pump</td>
		</tr>
		<tr>
			<td>Microelectrode Holder</td>
			<td>World Precision Instruments</td>
			<td>MPH310</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroFil Pipette Filler</td>
			<td>World Precision Instruments</td>
			<td>MF28G67-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish</td>
			<td>Electron MIcroscopy Sciences</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>VWR</td>
			<td>82007-334</td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Flaming/Brown Type Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>p4458-100ML</td>
			<td>5,000 units penicillin and 5 mg streptomycin/mL</td>
		</tr>
		<tr>
			<td>pipette pump 10 mL</td>
			<td>Bel-Art</td>
			<td>37898-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Super Glue</td>
			<td>Loctite</td>
			<td>LOC1365882</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polystyrene Test Tubes</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage micrometer</td>
			<td>Meiji Techno America</td>
			<td>MA285</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes without Needle, 50 mL</td>
			<td>BD Medical</td>
			<td>309635</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine Methanosulfonate</td>
			<td>Syndel USA</td>
			<td>SYNCMGAUS03</td>
			<td></td>
		</tr>
		<tr>
			<td>Trilene XL smooth casting Fishing line</td>
			<td>Berkley</td>
			<td>XLFS6-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing, polyethylene No. 205</td>
			<td>BD Medical</td>
			<td>427445</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>Invitrogen</td>
			<td>16520050</td>
			<td></td>
		</tr>
		<tr>
			<td>Wiretrol II calibrated micropipettes</td>
			<td>Drummond</td>
			<td>50002010</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution cell transplantation in embryonic and larval zebrafish

Cell transplantation has a long and storied history as a foundational technique in developmental biology. Around the turn of the 20th century, approaches using physical manipulations to perturb the developmental process, including transplantation, transformed embryology from an observational science into an experimental one1,2. In one landmark experiment, Hans Spemann and Hilde Mangold ectopically transplanted the dorsal blastopore lip of a salamander embryo onto the opposite side of a host embryo, inducing the nearby tissue to form a secondary body axis3. This experiment showed that cells could induce other cells to adopt certain fates, and subsequently, transplantation developed as a powerful method for interrogating critical questions in developmental biology regarding competence and cell fate determination, cell lineage, inductive ability, plasticity, and stem cell potency1,4,5.
More recent scientific advances have expanded the power of the transplantation approach. In 1969, Nicole Le Douarin&#39;s discovery that nucleolar staining could distinguish species of origin in quail-chick chimeras allowed for the tracking of transplanted cells and their progeny6. This concept was later supercharged by the advent of transgenic fluorescent markers and advanced imaging techniques5, and has been leveraged to track cell fate6,7, identify stem cells and their potency8,9, and track cell movements during brain development10. Additionally, the rise of molecular genetics facilitated transplantation between hosts and donors of distinct genotypes, supporting precise dissection of autonomous and non-autonomous functions of developmental factors11.
Transplantation has also made important contributions to the study of regeneration, particularly in organisms with strong regenerative abilities such as planarians and axolotl, by elucidating the cellular identities and interactions that regulate the growth and patterning of regenerating tissues. Transplant studies have revealed principles of potency12, spatial patterning13,14, contributions of specific tissues15,16, and roles for cellular memory12,17 in regeneration.
Zebrafish are a leading vertebrate model for the study of development and regeneration, including in the nervous system, due to their conserved genetic programs, high genetic tractability, external fertilization, large clutch size, and optical clarity18,19,20. Zebrafish are also highly amenable to transplantation at early developmental stages. The most prominent approach is the transplantation of cells from a labeled donor embryo to a host embryo at the blastula or gastrula stage to generate mosaic animals. Cells transplanted during the blastula stage will scatter and disperse as epiboly begins, producing a mosaic of labeled cells and tissues across the embryo21. Gastrula transplants allow for some targeting of transplanted cells according to a rough fate map as the shield forms and the A-P and D-V axes can be determined21. The resulting mosaics have been valuable in determining whether genes act cell autonomously, testing cell commitment, and mapping tissue movement and cell migration throughout development5,11. Mosaic zebrafish can be generated in several ways, including electroporation22, recombination23, and F0 transgenesis24 and mutagenesis25, but transplantation provides the greatest manipulability and precision in space, time, and number and types of cells. The current state of zebrafish transplantation is largely constrained to progenitor cells at early stages, with a few exceptions including transplantation of spinal motor neurons26,27, retinal ganglion cells28,29, and neural crest cells in the first 10-30 h post fertilization (hpf)30, and of hematopoietic and tumor cells in adult zebrafish5,31. Expanding transplantation methods to a broad range of ages, differentiation stages, and cell types would greatly enhance the power of this approach to provide insights into developmental and regenerative processes.
Here, we demonstrate a flexible and robust technique for high resolution cell transplantation effective in zebrafish embryos and larvae up to at least 7 days post fertilization. Transgenic host and donor fish expressing fluorescent proteins in target tissues can be used to extract single cells and transplant them with near-perfect spatial and temporal resolution. The optical clarity of zebrafish embryos and larvae allows for the transplanted cells to be imaged live as the host animal develops or regenerates. This approach has previously been used to examine how spatiotemporal signaling dynamics influence neuronal identity and axon guidance in the embryo32, and to examine the logic by which intrinsic and extrinsic factors promote axon guidance during regeneration in larval fish33. While we focus here on the transplantation of differentiated neurons, our method is widely applicable to both undifferentiated and differentiated cell types across many stages and tissues to address questions in development and regeneration.

<meta charset="utf8"></head><body>Autore in primo piano: Mappatura della connettività cellulare nel sistema nervoso del pesce zebra durante lo sviluppo e la rigenerazione

Trapianto di cellule ad alta risoluzione in zebrafish embrionale e larvale

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

high-resolution-cell-transplantation-in-embryonic-and-larval-zebrafish

Research

JoVE Journal

Developmental Biology

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

562 Views.  University of Minnesota. Il nostro laboratorio studia come la connettività cellulare è modellata nel sistema nervoso durante lo sviluppo e la rigenerazione. Esaminiamo come i segnali ambientali influenzano il comportamento neurale utilizzando il trapianto di cellule per affrontare il modo in cui il loro comportamento cambia quando vengono collocate in diversi ambienti genetici, spaziali o temporali. Sebbene il trapianto sia comune negli embrioni di zebrafish, viene generalmente ese...

Video: Autore in primo piano: Mappatura della connettività cellulare nel sistema nervoso del pesce zebra durante lo sviluppo e la rigenerazione

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.