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Method Article
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.
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.
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.
All procedures were carried out according to the provisions of the Australian National Health and Medical Research Council code of practice for the care and use of animals and were approved by the institutional ethics committees.
1. Preparation of Zebrafish
2. Preparation for Microinjection
3. Microinjection of Morpholinos and/or mRNA into Single-cell Stage Embryos
NOTE: Microinjections are used to mark all donor cells and to prepare the different host environments (control and gene knockdown) and involve injections of mRNA or morpholino antisense oligonucleotides 20,21.
4. Preparation for Transplantations
NOTE: Transplantations are used to generate chimeric embryos allowing for the effects of different genetic host environments to be assessed within equivalent WT donor progenitors 9,22,23.
5. Chimera Generation via Blastula Transplantation
6. Live Imaging Setup
NOTE: The small size and optical transparency of zebrafish combined with rapid development have allowed it to become a key vertebrate model for in vivo imaging of different cells and organs. Imaging can be performed on a variety of microscopes, which will differ in setup and parameters. The following describes a suitable confocal imaging setup for imaging of retinal development 5,8.
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.
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 ...
The authors have nothing to disclose.
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.
Name | Company | Catalog Number | Comments |
Agarose | Bioline | BIO-41025 | Agarose coated dishes can be prepared a few days prior, sealed with parafilm to prevent drying and stored at 4 °C |
Agarose low melt | Sigma | A9414-100G | Can be prepared in larger volume, microwaved to liquify and then kept molten in a 40 °C water-bath. 1 mL aliquots can be prepared (one for each group of embryos mounted). |
borosillicate glass capillary tube w/o filament | SDR Scientfic | 30-0035 | alternatives with similar diameter can be used |
borosillicate glass capillary tube with filament | SDR Scientfic | 30-0038 | alternatives with similar diameter can be used |
glass petri dishes (60 mm diameter) | Science Supply | 1070506 | any glass alternative of any size |
Injection tube (2 m) | Eppendorf | 524616004 | This connects the needle holder to the syringe during transplantation |
Microinjection mold (plastic mold with wedge-shaped protrusions) | Adaptive Science Tools | PT-1 | alternatives with similar shape can be use |
microneedle holder | Narishige | M-152 | any alternative that fits the outside diameter of the injection needles can be used |
microinjector | Narishige or Eppendorf Femtojet | Pneumatic injector using gas pressure | |
micromanipulator | Coherent Scientific | M330IR | similar alternatives can be used |
microloader tip | Eppendorf | 5242956003 | |
Mineral oil | Sigma | M5904-500ML | |
needle puller | Sutter Instruments | Model P-2000 | 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 |
Parafilm | Interpath | PM996 | any paraffin film |
Pasteur pipette plastic | Samco Scientific | 202 | |
Pasteur pipette glass | Hirschmann Laborgeraete | 9260101 | |
Pronase | Sigma | P5942-25MG | Protease Type XIV |
N-phenyl thiourea | Sigma | P7629-25G | PTU is toxic and can be prepared as a stock solution to avoid frequent exposure to the powder, consult MSDS before purchase/use. |
Qiaquick gel extraction | Qiagen | 28704 | any alternatives to purify DNA can be used |
Rneasy Mini kit | Qiagen | 74104 | any alternatives to purify RNA can be used |
SP6 mMessage kit | Qiagen | AM1340 | any alternatives to transcribe DNA using SP6 |
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