Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

Summary

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Abstract

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Introduction

During development of the nervous system, axons of presynaptic neurons navigate through diverse regions of the brain to reach their target areas. When axons invade their target tissues, they establish synaptic connections with postsynaptic target neurons. In many types of neurons, axons increase the number and spatial extent of synaptic connections they can make by elaborating networks of terminal branches or arbors¹. The retino-tectal projection of tadpoles of the aquatic frog Xenopus laevis is a powerful vertebrate model for examining mechanisms underlying terminal axon arborization and synaptic connectivity²<....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Touro University California (Protocol # TUCA003TE01X).

1. Obtaining X. laevis Embryos

1. Obtain X. laevis embryos by natural mating of pairs of male and female adult frogs primed with human chorionic gonadotropin (HCG), by in vitro fertilization of eggs shed from female adult frogs primed with HCG, or by ordering directly (Table of Materials).<.......

Discussion

In this article, we demonstrate how to express exogenous DNA constructs in single or small numbers of optic neurons and how to image individual GFP expressing optic axonal arbors with normal and altered molecular signaling in intact, living tadpoles of the frog X. laevis. We also explain how to reconstruct and quantify the morphology of GFP expressing optic axonal arbors from images captured in vivo. To express exogenous DNA plasmids in small number of optic neurons, we microinject a DNA/lipofection reagent mixt.......

Materials

Name	Company	Catalog Number	Comments
3.5" Micropipettes	Drummond Scientific	3-000-203 - G/X
μ-manager software (Version )			www.micro-manager.org
CCD camera	Scion Corporation	CFW-1312 M
Chorulon (Human Chorionic Gonadotropin)	AtoZ Vet Supply	N/A
Cysteine	Sigma-Aldrich	168149-100G
DOTAP	Sigma-Aldrich	11202375001
Dumont Forceps #5	Fine Science Tools	11250-10
Eclipse E800 epifluoresence microscope	Nikon		Objectives: Nikon Plan Apo 20X/0.75, Nikon Plan Fluor 40/0.75
GNU Image Manipulation Program (Version 2.10.10)	GIMP
Illustrator (2017 Creative Cloud)	Adobe
Image J (Version 1.46r)	NIH
Microfil	World Precision Instruments	MF 34G-5
Micromanipulator with universal adaptor and support base	Drummond Scientific	3-000-024-R
		3-000-025-SB
		3-000-024-A
Micropipette Puller	Sutter Instrument	P-30
Miniprep Kit	Qiagen	27104
Motorized z-stage	Applied Scientific Instrumentation	MFC-2000
Nanoject II injector	Drummond Scientific	3-000-204
Powerpoint (Version 15.31)	Microsoft
Xenopus laevis embryos	Nasco	LM00490