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

Summary

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.

Abstract

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.

Introduction

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 spe....

Protocol

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

1. Adult fish pairing
   1. Set up adult fish as pairs (or two pairs) in appropriately sized breeding tanks the evening prior to use.
   2. To keep the female separated from the male, use a divider to enable the fish to see, but not touch each other.
   3. In the morning, after switching on the light, remove the dividers and allow the fish to mate undisturbed.

Results

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.

Discussion

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 .......

Materials

List of materials used in this article

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