The authors would like to thank Dr. Louis Reichardt for the TrkAtaulacZ mice and Dr. Annette Markus for insightful discussion and suggestions. This work was supported by startup funds from the Burke Foundation as well as Whitehall Foundation research grant 2010-08-61, a research grant from Wings for Life Foundation (WFL-US-028/14), grant ZB1-1102-1 from the Christopher &#38; Dana Reeve Foundation, and grants 1R01EY022409 and 3R01EY022409-01S1 from the National Eye Institute, to JZ. KJO is a Goldsmith fellow.

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
<ol>
	<li>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).</li>
	<li>Rinse embryos in cold PBS.</li>
	<...

<ol>
	<li>Verze, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+innervation+in+hereditary+sensory+and+autonomic+neuropathy+type+IV.">Cutaneous innervation in hereditary sensory and autonomic neuropathy type IV.</a> Neurology. 55, 126-128 (2000).</li><li>Sethna, N. F., Meier, P. M., Zurakowski, D., Berde, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+sensory+abnormalities+in+children+and+adolescents+with+complex+regional+pain+syndromes.">Cutaneous sensory abnormalities in children and adolescents with complex regional pain syndromes.</a> Pain. 131, 153-161 (2007).</li><li>Uceyler, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+fibers+in+Fabry+disease:+baseline+and+follow-up+data+under+enzyme+replacement+therapy.">Small fibers in Fabry disease: baseline and follow-up data under enzyme replacement therapy.</a> J Peripher Nerv Syst. 16, 304-314 (2011).</li><li>Reichardt, L. F., Mobley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+the+distance,+or+not,+with+neurotrophin+signals.">Going the distance, or not, with neurotrophin signals.</a> Cell. 118, 141-143 (2004).</li><li>White, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+onset+of+NGF+and+TrkA+survival+dependence+in+developing+dorsal+root+ganglia.">Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia.</a> J Neurosci. 16, 4662-4672 (1996).</li><li>Farinas, I., Wilkinson, G. A., Backus, C., Reichardt, L. F., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neurotrophin+and+Trk+receptor+functions+in+developing+sensory+ganglia:+direct+NT-3+activation+of+TrkB+neurons+in+vivo.">Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo.</a> Neuron. 21, 325-334 (1998).</li><li>Markus, A., Zhong, J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf+and+akt+mediate+distinct+aspects+of+sensory+axon+growth.">Raf and akt mediate distinct aspects of sensory axon growth.</a> Neuron. 35, 65-76 (2002).</li><li>Kuruvilla, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neurotrophin+signaling+cascade+coordinates+sympathetic+neuron+development+through+differential+control+of+TrkA+trafficking+and+retrograde+signaling.">A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling.</a> Cell. 118, 243-255 (2004).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf+kinase+signaling+functions+in+sensory+neuron+differentiation+and+axon+growth+in+vivo.">Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo.</a> Nature. 10, 598-607 (2007).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+olfactory+sensory+map+develops+in+the+absence+of+normal+projection+neurons+or+GABAergic+interneurons.">An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons.</a> Neuron. 21, 1273-1282 (1998).</li><li>Moqrich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expressing+TrkC+from+the+TrkA+locus+causes+a+subset+of+dorsal+root+ganglia+neurons+to+switch+fate.">Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate.</a> Nature. 7, 812-818 (2004).</li><li>Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax-deficient+mice+with+lymphoid+hyperplasia+and+male+germ+cell+death.">Bax-deficient mice with lymphoid hyperplasia and male germ cell death.</a> Science. 270 (5233), 96-99 (1995).</li><li>Lentz, S. I., Knudson, C. M., Korsmeyer, S. J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+support+the+development+of+diverse+sensory+axon+morphologies.">Neurotrophins support the development of diverse sensory axon morphologies.</a> J. Neurosci. 19, 1038-1048 (1999).</li><li>Patel, T. D., Jackman, A., Rice, F. L., Kucera, J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sensory+neurons+in+the+absence+of+NGF/TrkA+signaling+in+vivo.">Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.</a> Neuron. 25, 345-357 (2000).</li><li>Donovan, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-RAF+kinase+drives+developmental+axon+growth+and+promotes+axon+regeneration+in+the+injured+mature+CNS.">B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS.</a> The Journal of experimental medicine. 211, 801-814 (2014).</li><li>Mercer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+endogenous+oncogenic+V600EB-raf+induces+proliferation+and+developmental+defects+in+mice+and+transformation+of+primary+fibroblasts.">Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts.</a> Cancer research. 65, 11493-11500 (2005).</li><li>Tronche, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+glucocorticoid+receptor+gene+in+the+nervous+system+results+in+reduced+anxiety.">Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.</a> Nature genetics. 23, 99-103 (1999).</li><li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nat Neurosci. 15, 793-802 (2012).</li><li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Schmidt, H., Rathjen, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiI-labeling+of+DRG+neurons+to+study+axonal+branching+in+a+whole+mount+preparation+of+mouse+embryonic+spinal+cord.">DiI-labeling of DRG neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord.</a> J Vis Exp. , (2011).</li></ol>

The authors declare that there are no competing interests.

The above-described X-gal staining procedure of embryonic TrkAtaulacZ 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...

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 diseases1-3. 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 highly expressed in nociceptive neurons during early development and mediates NGF-dependent neuron survival, axon growth, arborization and target innervation5-9. In TrkAtaulacZ mice, the wild type TrkA gene is replaced by a taulacZ expression cassette10, such that the axonal morphology of putative TrkA-positive neurons can be visualized by &#946;-gal (X-gal) staining11. Using a heterozygous TrkAtaulacZ/WT line, we can examine factors that may regulate or interfere with the development of sensory afferent projections in vivo.
Moreover, TrkA expression is absent in homozygous TrkAtaulacZ/taulacZ mice, which can therefore be used to assess the axon growth promoting mechanisms in the absence of NGF/TrkA signaling. Since nociceptive neurons depend on NGF/TrkA signaling not only for axon growth, but also for survival, we employ another mouse line, lacking the pro-apoptotic Bax gene, to inhibit apoptosis in embryonic DRG neurons, rescuing them from cell death that is otherwise observed in the absence of TrkA signaling. The Bax-/- background12 thus allows for the molecular dissection of signaling pathways that specifically affect axon growth7-9,13-15. In TrkA-/- : Bax-/- mice, DRG neurons survive, but sensory afferent innervation in the skin is completely abolished14,15. We can selectively activate signaling pathways to determine their respective contributions to the development of axon projections. The utility of this method is that it allows the assessment of changes in axonal growth phenotypes when different genetic modifications are bred onto the TrkAtaulacZ/taulacZ : Bax-/- or TrkAtaulacZ/WT : Bax-/- backgrounds.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

whole-mount imaging of mouse embryo sensory axon projections

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 TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ 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 TrkAtaulacZ/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.

The genotypes of TrkAWT/taulacZ : Bax-/- and TrkAtaulacZ/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 TrkAWT/taulacZ : Bax-/-</sup...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Research

JoVE Journal

Neuroscience

10.0K Views.  Weill Medical College of Cornell University. 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.