preparing donor cells and host zebrafish for cell transplantation to study developmental mechanisms

<meta charset="utf8"></head><body>再生・開発研究のための堅牢なゼブラフィッシュ細胞移植

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>著者スポットライト:発生および再生中のゼブラフィッシュ神経系における細胞接続性のマッピング

ゼブラフィッシュの胚および幼生における高分解能細胞移植

Our lab studies how cellular connectivity is patterned in the nervous system during development and regeneration. We examine how environmental signals influence neural behavior by using cell transplantation to address how their behavior changes when they're placed in different genetic spatial or temporal environments. While transplantation is common in zebrafish embryos, it's generally performed during the blastula and gastrula stages when cells are largely undifferentiated.This is valuable for generating genetic mosaics, but it limits the ability of the technique to interrogate how spatial temporal signaling dynamics influence development beyond these stages. Here, we present a protocol that allows for transplantation of cells, including differentiated neurons in embryos and larva up to at least seven days post fertilization. We can transplant as little as one cell with high spatial and temporal resolution and use genetic fluorescent labeling for long-term tracking of transplanted cells in the host animal.This protocol allows us to alter the genetic spatial and temporal environment of cells at many different developmental stages, opening up new opportunities to examine principles of style signaling during development and regeneration.

細胞移植のためのドナー細胞と宿主ゼブラフィッシュの調製と発生メカニズムの研究

preparing-donor-cells-host-zebrafish-for-cell-transplantation-to

Research

JoVE Journal

Developmental Biology

Preparing Donor Cells and Host Zebrafish for Cell Transplantation to Study Developmental Mechanisms

59 ビュー. まず、透明なマニキュアの長方形の輪郭をスライドガラスに塗ります。そのような移植スライドをいくつか準備します。麻酔した動物をマウントするには、パスツールピペットとポンプを使用して、RPTから低融点アガロースまたはLMAに移します。次に、LMAの小さな滴で動物をマニキュアの輪郭内のスライドに移します。LMAが固まる前に、胚...

ビデオ: 細胞移植のためのドナー細胞と宿主ゼブラフィッシュの調製と発生メカニズムの研究

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

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.

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

発生生物学

To begin, apply a rectangular outline of clear nail polish to the glass slide. Prepare several such transplant slides. For mounting the anesthetized animal, use a Pasteur pipette and pump to transfer it from RPT to low-melting agarose or LMA.Then, transfer the animal in a small drop of LMA onto the slide within the nail polish outline. Before the LMA solidifies, use an embryo pusher to orient the animal vertically, with the intended needle insertion site to the right. And let the agarose fully set for approximately five minutes.using a scalpel, cut a straight vertical slice through the agarose drop just to the right of the mounted animal. Wipe off loose agarose from the slide. Employing a scalpel, cut wedges out of the agarose to expose the needle insertion site.Apply RPT to the slide until the agarose drops are fully submerged. And mount the prepared slide on the transplantation microscope. Lower the 40X objective until it breaks the surface of the media and raise it to bring the donor animal into focus.Then, bring the fluorescently labeled donor cells of interest into focus. Move the stage to the left in preparation to locate the transplant needle.

High-Resolution Cell Transplantation in Zebrafish for Nerve Development Studies

To begin, insert a micropipette into the micropipette puller and pull a microinjection needle. Align the needle tip with the divisions of the stage micrometer under a dissecting scope. With the micrometer as a measurement guide, use a sharpened pair of forceps to break the needle to a bore size slightly larger than the cell's diameter.Using a 50-milliliter syringe equipped with a pipette filler, completely fill the needle with the mineral oil. On the transplant apparatus, turn the three-way stopcock so that its long arm faces the microsyringe pump. Depress the reservoir syringe plunger to flush all air bubbles from the hydraulic line.Maintaining light pressure on the plunger, turn the three-way stopcock so that the long arm now faces the reservoir syringe. Then insert the needle into the holder without introducing air bubbles. Adjust the needle so that it faces directly to the left at an angle of 10 to 15 degrees from the horizontal.Using the coarse and fine micromanipulators, maneuver the needle tip under the microscope objective to bring it into focus. If the liquid is flowing into or out of the needle tip, adjust the pressure using the syringe pump until a stable meniscus between the oil and RPT is observed. If an oil bubble remains, move the needle to the right to withdraw its tip from the RPT, then back to the left for repositioning.Move the stage to the right until the donor animal is brought back into view. Recenter and focus on the cells to be transplanted, aligning the needle with the cells just outside the animal. Next, insert the needle into the donor animal, and if necessary, restabilize the oil meniscus in the needle tip with the microsyringe pump.Position the needle tip against the cells of interest and apply gentle suction with the microsyringe pump. After taking adequate cells, remove the needle from the donor and restabilize the material in the needle tip. Reposition the stage to bring the first host animal into view/Center and focus on the area in which the cells are to be placed and align the needle with this region just outside the animal.Then insert the needle into the host animal and restabilize the oil meniscus in the needle tip. Position the needle tip in the deposition site and apply gentle pressure with the microsyringe pump until the correct number of donor cells are released from the needle. Finally, remove the needle from the host.At 12 hours post-transplantation, a donor neuron was successfully observed in the anterior region of the host's vagus motor nucleus, showing signs of healthy neurite protrusions. By two days post-transplantation, the transplanted neuron not only remained correctly positioned but had also begun extending a new axon toward the fourth pharyngeal arch, indicating successful integration within the host.