Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Summary

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Abstract

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkA^taulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkA^taulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkA^taulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

Introduction

Establishment of precise neuronal networks is a complex developmental process essential for the functionality of the nervous system. Disturbance in this process leads to neuronal dysfunction which has been implicated in human neurological diseases^1-3. To study the underlying molecular mechanisms of axon growth and target innervation in mammals, we have developed a protocol to visualize the axonal trajectories of TrkA-expressing sensory neurons using a combination of two genetically modified mouse lines.

TrkA is a receptor for nerve growth factor NGF and is a functional marker of nociceptive sensory neurons⁴. TrkA is hi....

Protocol

NOTE: All procedures comply with the NIH Guide for the Use and Care of Laboratory Animals. The animal protocol was approved by the IACUC at Weill Cornell Medical College.

1. Tissue Preparation

1. Euthanize timed-pregnancy females by cervical dislocation¹⁵. Dissect embryonic E16 - E18 embryos from timed-pregnancy females and place embryos individually in the wells of a 6-well dish, filled with cold phosphate buffered saline (PBS).
2. Rinse embryos in cold PBS.
3. Remove a small portion of the amniotic membrane of the embryo and place in a pre-prepared proteinase K solution for subsequent genotyping. Alternat....

Results

The genotypes of TrkA^WT/taulacZ : Bax^-/- and TrkA^{taulacZ/taulacZ} : Bax^-/- embryos can be unambiguously determined by standard PCR genotyping (Figure 1). X-gal staining displays detailed peripheral axonal arbors subcutaneously in conventionally stained embryos (Figures 2, 3a), and throughout the embryo after tissue clearing (Figures 3b, 4).

We have bred the TrkA^WT/taulacZ : Bax^-/-

Discussion

The above-described X-gal staining procedure of embryonic TrkA^taulacZ mice allows for the rapid and detailed visualization of long distance axon projections in the intact fixed embryo. Because of the Bax null background these mice allow for the probing of signaling mechanisms that may contribute to both axon growth and neuronal survival. Mating with transgenic or knockout mice of interest allows for comprehensive assessment of axonal phenotypes and can serve as useful guide for futur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PFA	Sigma-Aldrich	P6418
PBS	Life Tech	10010-023
Tissue Rinse Solution A	Millipore	BG-6-B
Tissue Rinse Solution B	Millipore	BG-7-B
Tissue Stain Base Solution	Millipore	BG-8-C
X-gal	Sigma-Aldrich	B4252
Glass scintiallation vial	Kimble Chase	74500-20
Incubator	Labline	Model 120
Insect pins	FST	26000-30
DMSO	Sigma-Aldrich	D8418
6-well dish	USA Scientific	CC7672-7506
Primers	IDT	custom DNA primers
Takara dNTP mixture	Takara	4030
Takara LA buffer	Takara	RR002A
Takara LA Taq	Takara	RR002A
PCR machine	Bio-Rad	DNA Engine Dyad
Benzyl alcohol	Sigma-Aldrich	B-1042
Benzyl benzoate	Sigma-Aldrich	B-6630
Dissecting microscope	Leica	M205A
Camera	Leica	DFC310FX
Ring light	Leica	MEB110
Photoshop	Adobe	Photoshop 4.0