Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Summary

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Abstract

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Introduction

Spinal MNs are the part of the central nervous system but innervate peripheral muscles to control movement. In the developing spinal cord, MN progenitors (pMNs) are established according to the signals emanating from the notochord and adjacent somites. All differentiated post-mitotic MNs are then generated from pMNs, eventually giving rise to a series of MN subtypes along the rostrocaudal axis of the spinal cord^1,2. Spinal MNs are topographically and anatomically well organized. Their morphological arrangement correlates with the position of their respective target in the periphery³. Up....

Protocol

All of the live animals were kept in a specific pathogen free (SPF) animal facility, approved and overseen by IACUC of Academia Sinica.

1. Fixation

1. Collect embryos of embryonic day 12.5 (E12.5) then decapitate and eviscerate them.
2. Fix the embryos individually in 24-well plates with 1 mL/well of freshly prepared 4% paraformaldehyde in 1x phosphate buffer saline (PBS) overnight. Incubate at 4 °C on a shaker.
3. Wash the fixed embryos at least 3x, each for 5-10 min, with 1 mL of 1x PBS and incubate overnight at 4 °C on a shaker to remove the residual paraformaldehyde.

2. W....

Results

LSFM provides detailed 3D visualization of MN trajectory and axon arborization in mouse embryos. Under bright field, tissues appear completely transparent after being immersed in the commercial clearing reagent. No shrinkage or swelling of the sample was noticed after up to one week of storage in clearing reagent before imaging. Under fluorescence channel, motor neurons are labeled with transgenically expressed GFP (Movie 1). In some instance, details of the axon structur.......

Discussion

Several steps in the protocol may be subjected to changes under certain circumstances. For example, the duration of fixation depends on the age of the embryos, varying from 2 h to 1 or more days using freshly-made paraformaldehyde. Since fixation is carried out prior to whole mount immunostaining, for antibodies that are sensitive to protein crosslinking, methanol can be used as an alternative fixative agent. For a high signal-to-noise ratio upon staining, it is necessary to optimize the detergent concentration (between .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hb9::GFP	The Jackson labortory	005029	Collect embryos of embryonic day 12.5 (E12.5)
4% Paraformaldehyde (PFA)			For 200ml: Add 20ml 10X PBS, 8g PFA in ddH2O. Adjust pH to 7.4 with NaOH (10N). Filter sterilize and store at -20 °C.
Phosphate buffer saline 10X (PBS 10X)			For 1L: Add 80 g NaCl, 2 g KCl,14.4g Na2HPO4, 2.4 g KH2PO4 and top up with ddH2O. Autoclave and store at RT.
Triton X-100	Sigma	X100-500ML
Fetal Bovine Serum	ThermoFisher	26140079
Sheep polyclonal anti-GFP	AbD Serotec	4745-1051	1:1000
Alexa Fluor 488 donkey anti-sheep	Invitrogen	A-11015	1:1000
RapiClear 1.49 clearing reagent	SunJin Lab	RC149001
1.5ml micro tube	Sarstedt	72.690.001
24 wells plate	ThermoFisher	142475
5 SA Tweezer	ideal-tek	3480641
Iris Scissors striaght sharp/sharp	Aesculap	BC110R
Microsurgery Scalpels, single use	Aesculap	BA365
Dissecting microscope	Nikon	SMZ800
Shaker	TKS	RS-01
Lightsheet Z.1 microscope	Carl Zeiss Microscopy
Imaris 8.4.0 image analysis software	Bitplane, Zurich, Switzerland
B6.Cg-Tg(Hlxb9-GFP)1Tmj/J (Hb9::GFP mice)	The Jackson Laboratory	005029