Name: in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences
Uploaded: 2017-03-22T15:00:00.000+00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

This work was supported by an ARC DECRA to PRJ (DE120101311) and by a Deutsche Forschungsgemeinschaft (DFG) research grant to LP (PO 1440/1-1). The Australian Regenerative Medicine Institute is supported by funds from the State Government of Victoria and the Australian Federal Government. We acknowledge Dr Jeremy Ng Chi Kei, who conducted experiments described here as published in Kei et al., 2016. We are grateful for provisions of transgenic fish from Prof. Higashijima and thank Profs. Turner and Rupp for the provision of the pCS2+ plasmid and Dr. Wilkinson for generating H2B-RFP and H2A-GFP constructs. We thank FishCore facility staff (Monash University) for taking care of our animals.

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The authors have nothing to disclose.

Understanding the extent to which neighboring cells influence timing of the expression of crucial cell fate determining factors is essential when aiming to efficiently instruct embryonic or induced multipotent stem cells to differentiate into a specific post-mitotic cell type or even patterned tissue. Furthermore, examining these molecular events in the developing cells of the living animal additionally provides relevant dynamic (temporal and spatial) information on the particular cellular contexts associated with these events in vivo. Such studies cannot be easily achieved in all vertebrate models.
In the presented protocol, the amenable zebrafish embryo is exploited as a vertebrate model to address these questions in vivo, in the relatively simple, yet highly organized three-dimensional patterned tissue of the vertebrate retina. This is exemplified by testing the hypothesis that depleting a particular retinal cell type influences gene expression timing and developmental fate of retinal progenitor cells. We can additionally utilize the donor cell marker to track specific cells back in time to assess, for instance, how long before the expression of a transgene a postmitotic cell underwent its last division, or assess the general behavior of this cell (e.g., migration) at various stages prior to the transgene expression in different environments. For these studies, a combination of relatively easy to perform techniques are presented, which include morpholino-mediated gene knock down, transplantation and live imaging. For the transplantation method, the protocol introduces a novel, relatively easy and inexpensive transplantation rig and demonstrates the ease of this operating setup.
This protocol is not restricted to the retina. It can also be applied to study a number of different dynamic cellular processes, that require disentangling of cell-autonomous developmental programs from cell non-autonomous environmental influences. However, while little troubleshooting is required, a number of critical steps need to be taken into account prior to starting an experiment. One important prerequisite for the successful application of the technique is that the organ or tissue of choice must be easily targeted in the early embryo using the fate map. If the efficiency of tissue targeting has not been previously tested, this needs to be first optimized and standardized. The number of microinjected and transplanted embryos can be increased and screened prior to live imaging to obtain sufficient embryos with donor cells correctly integrated and positioned within the relevant tissue. Additionally, while this protocol can be easily applied to early embryonic zebrafish stages, application to later stages such as the growing larvae become more difficult for two reasons. Firstly, the efficiency of gene knockdown obtainable with morpholinos decreases over time (depending on each morpholino, it is generally most efficient before 3 dpf). To override this problem, the use of genetic mutants as donor embryos for transplantation is preferable. Secondly, as the zebrafish grows, deeper parts of the larvae become less accessible to imaging with a confocal microscope, especially at higher magnifications (high power objectives) where the working distance of the microscope objective is more limited. Thus, each tissue of interest at relevant age needs to be first assessed for its amenability to imaging.
The time-lapse imaging can be performed either on an inverted or upright confocal microscope.
There are a number of advantages of the upright microscope, if available. At the required temperature, evaporation of the immersion substance used (e.g., water or immersion oil with water refractive index) on an inverted microscope can lead to complete drying out. This requires constant monitoring and eventually refilling, with the risk of placing the sample out of focus. This can be partially avoided by using automation to lower the objective a defined distance, adding the immersion substance and raising the objective again, though in our experience, refocusing is often still required, which should happen before the beginning of the next time point of imaging. Secondly, the largest coverslip bottom dishes we sourced are still much smaller (50 mm in diameter) than the standard 90 mm diameter Petri dishes utilized for upright microscopy. This means that the number of embryos that can be mounted and the embryo medium volume added are more limited when using inverted microscopes. Nevertheless, the time-lapse parameters usually constitute the main limiting factor for the number of embryos that can be imaged and therefore the size of the dish is still suitable. When using very high magnification objectives, inverted microscopy may allow a greater depth in the z-dimension as the eye and objective are only separated by a thin coverslip. When combining very high magnification with upright microscopy, the agarose drop covering the embryo must be kept as thin as possible. Overall, the data obtained using upright or inverted microscopy is of equivalent quality.
For generation of high quality data, there are additional critical steps that need to be considered. Firstly, selection of brightly labeled donors is crucial for obtaining transplanted embryos with an appropriate number of transplanted cells in the eye at the start of the time-lapse movies (a few, but not too many). The protease enzymatic dechorionation is a critical step and incubation in protease should be kept to a minimum followed by quick, but gentle rinses to not affect the subsequent health of the embryos. Age matching of donor and host embryos allows for accurate tissue targeting according to the fate map and for efficient integration of the cells. A well-shaped transplantation needle is important, ensuring that the tip is small and sharp enough to insert into host embryos without causing much damage, but large enough in internal diameter to not cause mechanical damage to the cells being transplanted. During transplantation, if the equipment is not fully sealed (especially at connection points), it is very difficult to manually control the movement of E3 medium and cells into and out of the transplantation needle. This is most obvious when there is flow either into or out of the transplantation needle without manually changing the syringe volume. In this case all joints must be re-checked and resealed. This should be tested in advance of the experiment.
Lastly, during the elapsed time between transplantation and time-lapse setup, it is crucial that the transplanted embryos are allowed to develop at low density whilst meticulously cleaning out any unhealthy embryos. This is particularly important, when the timing of development or gene expression is being investigated. When assessing the timing of any process (e.g., gene expression), controls must be used to rule out non-specific side effects of the morpholino technology. In our work, we use the timing of transgene expression in an unrelated tissue (e.g., the muscle) unaffected by the specific gene targeted by the morpholino.

The ability to visualize important developmental processes in an in vivo vertebrate has contributed to making the zebrafish a key model for studying normal and disease conditions (reviewed in 1,2). In particular, the neural retina is an accessible part of the central nervous system. The retina lends itself to easily perform studies of neurogenesis due to its highly organized, yet relatively simple structure, and its highly conserved neuron types across vertebrate species 3. Dynamics of cellular behaviors such as proliferation, cell cycle exit, asymmetric cell division, fate specification, differentiation, and neural circuitry formation can be followed throughout the entire process of retinogenesis, which is completed in the central retina of the zebrafish by 3 d postfertilization (dpf) 4,5,6,7.
Furthermore, the functional requirements of different genes in each of the above mentioned stages can be concomitantly assessed in the zebrafish retina, providing an advantage over other vertebrate models in which phenotypes resulting from application of gene knockout techniques can only be assessed upon post-mortem examination of fixed tissues. In particular, the use of transgenic lines in which we can visualize and monitor the expression of fluorescent proteins as reporter transgenes in the retina, allows us to obtain temporal resolution of gene expression that underlies the genesis of a particular neuronal cell type. Due to the rapid development of zebrafish, these events can be visualized during the entire developmental period, thereby providing deeper insights into the temporal importance of gene expression in relation to neuronal cell identity acquisition and cell behavior.
Finally, these approaches can be combined efficiently in the zebrafish with the generation of chimera via transplantations, resulting in insights into two key aspects of gene function. Firstly, examining cells transplanted from a donor embryo, in which a particular gene was knocked down while they develop in an unlabeled wild type environment, allows us to obtain relevant information about gene function in a cell-autonomous manner. This leads to important insights about the function of retinal fate determinant factors within the progenitor cells they are normally expressed in. This is exemplified by the examination of the developmental fate outcome of progenitors that can no longer generate functional protein from these genes 4,6,8,9. Utilizing this approach, we have shown that many fate determinant factors (e.g., Vsx1, Atoh7, Ptf1a, Barhl2) act cell-autonomously to drive specific retinal neuronal fates; the lack of gene expression primarily leads to a fate switch, such that the cells with gene knockdown remain viable by adopting an alternate cell fate 4,6,7,10. Secondly, such chimeric experiments can be used to assess how wild type progenitors behave when they develop within different genetic environments. For example, by comparing the development of WT cells that usually express a gene of interest (and reporter transgene) in a WT versus manipulated host environment (e.g., gene knockout / knockdown), the resulting effects on gene expression and cell fate can be assessed. The lack of certain neuron types in the host environment, for instance, has been shown to influence wild type progenitor behavior in a cell non-autonomous manner, to bias them towards differentiating into the underrepresented or missing neuron types 4,7,11,12. Given that retinal neurons are born in a conserved histogenic order by the sequentially timed expression of specific neuronal fate determinant genes (fate gene expression) (reviewed in 13), we used these methods to demonstrate how the timing of fate gene expression in wild type progenitors is affected when such progenitors develop in retinal host environments with induced aberrant cellular compositions. Here, we outline these approaches as evidence for how the combination of relatively standard and widely used techniques enables the examination of the timing of fate gene expression in developing retinal progenitors 8,9.
This protocol describes an experimental approach combining time-lapse imaging with the ease of performing transplantation in the ex vivo developing zebrafish embryo to follow individual mosaically labeled cells throughout the entire period of developmental retinogenesis. By performing functional gene manipulations either in the host embryo, donor embryo, both or neither, one can assess the cell autonomy of gene function. This approach can be adapted widely to similar research questions in any other system for which the individual components outlined here are suitable.

<table><tbody><tr><td>Agarose</td><td>Bioline</td><td>BIO-41025</td><td>Agarose coated dishes can be prepared a few days prior, sealed with parafilm to prevent drying and stored at 4 &amp;#176;C</td></tr><tr><td>Agarose low melt</td><td>Sigma</td><td>A9414-100G</td><td>Can be prepared in larger volume, microwaved to liquify and then kept molten in a 40 &amp;#176;C water-bath. 1 mL aliquots can be prepared (one for each group of embryos mounted).</td></tr><tr><td>borosillicate glass capillary tube w/o filament</td><td>SDR Scientfic</td><td>30-0035</td><td>alternatives with similar diameter can be used&amp;nbsp;</td></tr><tr><td>borosillicate glass capillary tube with filament</td><td>SDR Scientfic</td><td>30-0038</td><td>alternatives with similar diameter can be used&amp;nbsp;</td></tr><tr><td>glass petri dishes (60 mm diameter)</td><td>Science Supply</td><td>1070506</td><td>any glass alternative of any size</td></tr><tr><td>Injection tube (2 m)</td><td>Eppendorf</td><td>524616004</td><td>This connects the needle holder to the syringe during transplantation</td></tr><tr><td>Microinjection mold (plastic mold with wedge-shaped protrusions)</td><td>Adaptive Science Tools</td><td>PT-1</td><td>alternatives with similar shape can be use</td></tr><tr><td>microneedle holder</td><td>Narishige</td><td>M-152</td><td>any alternative that fits the outside diameter of the injection needles can be used</td></tr><tr><td>microinjector</td><td>Narishige or Eppendorf Femtojet</td><td></td><td>Pneumatic injector using gas pressure</td></tr><tr><td>micromanipulator</td><td>Coherent Scientific</td><td>M330IR</td><td>similar alternatives can be used</td></tr><tr><td>microloader tip</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M5904-500ML</td><td></td></tr><tr><td>needle puller</td><td>Sutter Instruments</td><td>Model P-2000</td><td>Settings used for this puller are: H 430, Fil 4, Vel 50, Del 225, Pul 75. Any needle puller can be used if it results in appropriate tip dimension</td></tr><tr><td>Parafilm</td><td>Interpath</td><td>PM996</td><td>any paraffin film</td></tr><tr><td>Pasteur pipette plastic</td><td>Samco Scientific</td><td>202</td><td></td></tr><tr><td>Pasteur pipette glass</td><td>Hirschmann Laborgeraete</td><td>9260101</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>P5942-25MG</td><td>&amp;nbsp;Protease Type XIV</td></tr><tr><td>N-phenyl thiourea</td><td>Sigma</td><td>P7629-25G</td><td>PTU is toxic and can be prepared as a stock solution to avoid frequent exposure to the powder, consult MSDS before purchase/use.</td></tr><tr><td>Qiaquick gel extraction</td><td>Qiagen</td><td>28704</td><td>any alternatives to purify DNA can be used</td></tr><tr><td>Rneasy Mini kit</td><td>Qiagen</td><td>74104</td><td>any alternatives to purify RNA can be used</td></tr><tr><td>SP6 mMessage kit</td><td>Qiagen</td><td>AM1340</td><td>any alternatives to transcribe DNA using SP6</td></tr></tbody></table>

in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

This work presents an experimental protocol to assess changes in gene expression timing when wild type retinal progenitors develop within a morphant host embryo. The experimental host embryos are Ptf1a morphants, which lack the intermediate born horizontal and amacrine interneurons of the retina 7,26. These have been compared to control host embryos, which were injected with a standard control morpholino.
Since neurogenesis of different types of neurons across the central nervous system (including the retina) occurs in a highly conserved histogenic order 27,28,29,30,31 the progression of the subsequent fate may depend on cell non-autonomous feedbacks from accumulating earlier born neuron types. This hypothesis was tested by assessing the timing of the generation of later born neural types in transplanted progenitor cells developing in the absence of intermediate born neuron types. As a control, the timing of early born cells was also quantified, which should be unaffected independently of whether the intermediate cells are generated or not. To this end, the Tg(atoh7:RFP) or Tg(atoh7:gapGFP) lines were used to visualize the onset of the first born ganglion cell neurogenesis while the Tg(vsx1:GFP) line was used to analyze the onset of the late born bipolar population. Other than additionally expressing the reporter transgenes, the donor cells from these embryos are genetically WT.
Microinjection of 1 nL&#160;mRNA (to label donor cells) into the yolk of single-cell stage embryos results in fluorescent reporter protein expression by 3 hpf, at which stage the brightest donors are selected (Figure 1). Prepare transplantation setup when embryos are around 2 hpf (Figure 2). At 24 hpf, embryos with transplanted donor cells in the relevant retinal locations are chosen and mounted in 1% low melt agarose in groups of five (Figure 3). After saving the embryo position, setting the z-stack and the time parameters, time-lapse imaging is performed for 16 - 24 h. Timing of first born ganglion cells is indicated by the onset of the Atoh7:RFP or Atoh7:gapGFP transgene and timing of the last born bipolar generation is indicated by the strong upregulation of the Vsx1:GFP transgene, which is also expressed at distinctly lower brightness levels in the developing progenitors. The timing of first transgene expression, such as Atoh7:gapGFP is identified in individual cells at each timepoint of the imaging (white arrowheads, Figure 4). Atoh7:gapGFP and ganglion cell differentiation occurs at equivalent times regardless of whether the host embryo is WT or a Ptf1a morphant lacking intermediate (i.e. after ganglion cell birth) horizontal and amacrine cells.
The data presented demonstrate that this experimental approach provides a powerful tool to assess the role of particular retinal environments on gene expression timing, with relevant implications, not only for understanding normal developmental processes but also for future applications of cell reprogramming strategies in normal and diseased conditions.
<img alt="Figure 1" src="/files/ftp_upload/55490/55490fig1.jpg" /> 
Figure 1: Microinjection into the Yolk of Single-cell Stage Embryos used to Label Donor Cells and to Generate Different Host Environments. A) Embryos are aligned against the edge of a glass slide with most of the surrounding liquid removed. B) The needle is inserted into the center of the yolk and either H2A-GFP or H2B-RFP mRNA (for donor) or morpholino (standard MO or Ptf1a MO for host) are injected. C) The injection needle is generated using a needle puller that generates two symmetrical needles from each capillary. Each pulled needle subsequently needs to have the tip broken with forceps to open the needle whilst providing a sharp angled tip (not blunt) that can be easily inserted into embryos. D) Donor embryos injected with H2B-RFP mRNA at the single-cell stage start expressing the fluorescent reporter proteins by 2.5 hpf, at which stage the brightest and most evenly labeled embryos can be selected. E) Around 2.5 - 3 hpf, donor and host embryos are enzymatically dechorionated using proteases. Swirling of the Erlenmeyer flask provides the mechanical force necessary to break open weakened chorions and brings dechorionated embryos into the center. As soon as the first one (asterisk) or a few dechorionated embryos are observed, rapid but gentle rinsing ensures that the protease solution is removed. F) For transfer of dechorionated embryos, glass transfer pipettes are utilized. These can be initially shortened (optional) to any length that is comfortably maneuvered by the user, by scratching the glass at the appropriate position with a diamond knife until it snaps off. This results in a sharp cut tip, which needs to be fire-polished to smoothen any rough edges that may damage cells being sucked into the pipette during transfer. Scale bars A, B, D, E = 1 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55490/55490fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55490/55490fig2.jpg" /> 
Figure 2: Transplantation Setup. A) The transplantation consists of a transplantation needle mounted in a micropipette holder attached to a micromanipulator. The end of the micropipette holder is attached to tubing that leads to a 1 mL&#160;syringe, which is manually operated to suck up or deposit cells. B) The transplantation tubing shown indicates how potential leaks at joints are simply prevented using moldable putty and paraffin film. C) A higher magnification view of the connection between the injection tubing and any suitable tubing to connect to the syringe. While any tubing can be used, the tube utilized here has a diameter that tightly fits a 10 &#956;L&#160;pipette tip. Use paraffin film to ensure a tight seal. D) The mold has 150 (6 x 25) wedges to generate the triangular divots in the agarose injection plate. E) Approximate dimensions of each wedge shape are shown. The company measurements are proprietary. F) Prepared agarose injection plates with wedge-shaped indentations are used with donor embryos added to the first column and columns 2 - 6 filled with host embryos. Transplantations are performed across the row, whereby cells collected from one donor in each row can be transplanted into 5 host embryos. Within each mold, cells from up to 25 donors can be transplanted into up to 125 host embryos. Note the mold is placed asymmetrically with the deeper end of the wedge closer to the Petri dish edge. This allows the transplantation needle from the right hand side without hitting the Petri dish edge. G) The transplantation needle is pulled with the same parameters as the injection needle to result in a long tapered tip shown here. The needle must be broken to generate a larger internal diameter that can take up cells without deforming them. The higher power inset shows that the tip of the transplantation needle should be angled and ideally have a &#34;flute&#34; shape as shown in this example. H) Higher power view shows orientation of host embryos with cells at the top. The transplantation needle is inserted into the cell mass of the blastula stage host embryo laterally (white asterisks) and pushed into the future eye as shown here. Donor cells can be seen in the transplantation needle (arrow) and thus the rough number of cells transplanted can be controlled. I) Two examples (left and right) showing that immediately after transplantation, embryos with successfully transplanted cells in the correct location can immediately be sorted out using the donor label (e.g., H2B-RFP). A fluorescent channel was added to the brightfield using &#34;linear dodge (add)&#34; option under the layer menu in the raster graphics editor. Scale bar (for H, I) = 500 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55490/55490fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55490/55490fig3.jpg" /> 
Figure 3: Embryo Mounting for Time-lapse Imaging. A) Petri dish (90 mm) showing multiple low melt agarose drops containing five embryos each connected by agarose to form a firm network to prevent individual drops from detaching. B) Higher power view of 24 hpf embryos aligned laterally and embedded within solidified agarose. For the ideal angle, the two eyes should be positioned on top of each other (in the z-dimension). Depending on the gene of interest, mounting should be delayed until just prior to relevant timing (e.g., 26 hpf for ganglion cell differentiation or 35 hpf for the start of the bipolar cell differentiation). C) Confocal image of a single optical z-section showing overlay of brightfield and red channels (using &#34;linear dodge (add)&#34; option under the layer menu in the raster graphics editor) of the developing eye with a few labeled donor cells (red) within an otherwise unlabeled host environment. Scale bar B = 1 mm, scale bar C = 50 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55490/55490fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55490/55490fig4.jpg" /> 
Figure 4: Timing of Neurogenesis can be Visualized in Transgenic Donor Cells Transplanted into Different Host Environments. Micrographs from time-lapse image of the developing WT donor cells from Tg(atoh7:gapGFP) transgenic embryos transplanted into unlabeled WT host. Transgene expression onset is indicated for a few cells by white arrowheads. Scale bar = 10 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55490/55490fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.6K Views.  The University of Melbourne.  Live volgen van individuele WT retinale voorlopers in verschillende genetische achtergronden maakt voor de beoordeling van de bijdrage van de cel niet-zelfstandige signalering tijdens de neurogenese. Hier, een combinatie van gen knockdown, chimeer generatie via embryo transplantatie en in vivo time-lapse confocale beeldvorming werd gebruikt voor dit doel. 

Video: In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuroscience

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Biology

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Robust Zebrafish Cell Transplantation for Regeneration and Development Research

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.

Author Spotlight: Mapping Cellular Connectivity in the Zebrafish Nervous System During Development and Regeneration

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 ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

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