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Summary

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Abstract

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Introduction

A functioning Enteric Nervous System (ENS), which controls motility, nutrient absorption, and local blood flow, is essential to life¹. The ENS is formed by neural crest cells (NCC) that proliferate, migrate and colonize the gut, where they differentiate into ganglia containing neurons and glial cells. Hirschsprung’s Disease (HSCR, Online Mendelian Inheritance in Man), a multigeneic congenital disorder with an incidence of 1 in 4,000 live births, can be considered the prototypic disease for studying disrupted ENS formation. In HSCR, NCC fail to migrate to and colonize variable lengths of the distal hindgut². Additionally, other common gastrointestinal (GI) developmental defects in the pediatric population, such as anorectal malformations, intestinal atresias, and motility disorders are associated with disturbances in basic ENS functions, and are likely associated with subtle, underappreciated, anatomic changes and functional changes in the ENS^3-6. Therefore, techniques that allow us to understand the developmental determinants of ENS formation may shed light on the pathogenesis and potential treatment of pediatric GI tract disorders.

Following migration and colonization, NCC differentiates into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons comprise approximately 60% of enteric neurons⁷, and can be detected by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for the excitatory neurotransmitter acetylcholine. Historically, attempts to visualize cholinergic neurons were confounded by differing antigen specificity of antibodies directed against central nervous system (CNS) ChAT versus peripheral nervous system (PNS) ChAT^8-10. However, antibodies directed against placental ChAT recognize both central and peripheral ChAT^11-13, and we have recently described techniques that allow for visualization of ENS cholinergic neurons with high sensitivity earlier in development than has been achieved with ChAT reporter lines¹⁴.

Here, we present a technique for dissecting, fixing and immunostaining of the murine embryonic GI tract to visualize ENS neurotransmitter expression in neurons. For these studies, we have utilized ChAT-Cre mice mated with R26R:floxSTOP:tdTomato animals to produce ChAT-Cre;R26R:floxSTOP:tdTomato mice (defined as ChAT-Cre tdTomato throughout the manuscript). These animals were then mated with homozygous ChAT-GFP reporter mice, to obtain mice expressing both fluorescent reporters that detect ChAT expression¹⁴. These two reporter animals are on a C57BL/6J background and are commercially available (Jackson Laboratories, Bar Harbor, ME).

Protocol

The University of Wisconsin Animal Care and Use Committee approved all procedures. 1. Preparation of Solutions Use 1x phosphate buffered saline (PBS) as dissection buffer and rinsing solution. Prepare 30% sucrose by weighing 30 g of sucrose and place into a bottle. Add 99 ml of 1x PBS and add 1 ml 10% sodium azide. Mix thoroughly until all of the sucrose is dissolved. Store at 4 °C until required. Prepare Blocking solution by mixing 1x PBS, 3% bovine serum albumin (BSA) and 0.1% Triton-X-100. Mix thoroughly and store in the fridge until needed. Prepare 8% paraformaldehyde (PFA) solution in 1x PBS by weighing the appropriate amount of PFA into 1x PBS and then incubate at 65 °C until it is completely dissolved. Store in 25 ml aliquots in the -20 °C freezer until needed. Dilute to 4% PFA in 1x PBS on the day of use. 2. Embryo and Gut Dissection In accordance with Institutional Animal Care and Use Committee approved protocols, euthanize timed pregnant mouse and transfer the uterus into a 60 mm Petri dish containing ice cold 1x PBS. Under a dissection microscope using sharp scissors, cut the uterine wall open to expose the embryos. Remove the embryos from the uterus and place into a clean 60 mm Petri dish containing ice cold 1x PBS. Euthanize each embryo by decapitation in ice-cold 1x PBS. If you are using transgenic mice containing fluorescent proteins, under fluorescence illumination, identify the positive transgenic embryos. Dissect the GI tract from each embryo using a dissection microscope. Using fine forceps, orient the embryo such that the left side is facing upwards and the right side is against the bottom of the Petri dish. Remove the upper body wall from the embryo to expose the internal organs. Insert fine forceps between the dorsal body wall and the internal organs. Cross the forceps against each other in a scissor-like cutting action to remove the internal organs from the embryo. Further sub-dissect the GI tract away from the surrounding organs and then place each GI tract into a 1.5 ml microcentrifuge tube containing ice-cold 1x PBS. 3. Fixation of GI Tracts Rinse each GI tract 3 times with ice-cold 1x PBS and then replace with 4% PFA. Fix the GI tracts in the 1.5 ml microcentrifuge tubes on a rocking platform at RT for 1.5 hr. Rinse the GI tracts 3 times for 5 min at RT and then for 1 hr on the rocking platform. At this stage, store the GI tracts at 4 °C in 30% sucrose in 1x PBS containing 0.1% sodium azide until needed. NOTE: Alternatively, store the embryos in 30% sucrose for up to one year without any effect on the integrity of the tissues. Storage of the samples in 30% sucrose allows later processing of the samples either for immunostaining or into OCT for cryo-sectioning. Alternatively, proceed with the immunostaining protocol detailed below. 4. Immunostaining Protocol If samples have been stored in 30% sucrose, rinse them 3 times for 20 min in 1x PBS on a rocking platform. Place the GI tracts into blocking solution on a rocking platform for 1h at RT. Remove the blocking solution and incubate the GI tracts with the appropriate amount of primary antibodies diluted in blocking solution for either 4 hr at RT or O/N at 4 °C on a rocking platform. Use 1:1,000 dilution of human anti-Hu antibody (serum obtained from patient), 1:1,000 dilution of chicken anti-green fluorescent protein (GFP) antibody and 1:100 dilution of goat anti-ChAT antibody. NOTE: We utilize a human anti-Hu antibody that was obtained locally from a patient, however, anti-Hu antibodies are commercially available, for example, mouse anti-Hu, (use at 1:500 dilution). Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform. Replace the 1x PBS with secondary antibodies diluted 1:500 in blocking solution on a rocking platform for either 4 hr at RT or O/N at 4 °C. Use donkey anti-human amine-reactive dye such as Dylight, donkey anti-chicken Cy2 and donkey anti-goat Cy5. If using the mouse anti-Hu antibody then use a 1:500 dilution of donkey anti-mouse amine-reactive dye 405. NOTE: The intensity of the endogenous GFP and tdTomato expression and the clarity of immunostaining increase with developmental age. We typically immunostain embryonic guts individually in 0.2 ml tubes with 150 μl of staining solution in order to reduce the volume of antibody used and also to ensure efficient staining of the tissue. Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform. Place a few drops of fluorescence mounting medium with DAPI onto a glass slide. Immerse the GI tract into the fluorescence mounting medium and add a cover glass directly on top of the tissue. The fluorescence mounting medium -G is a water-soluble compound that provides a semi-permanent seal. NOTE: Use fluorescence mounting medium such as Vectashield which is a glycerol based mounting medium that prevents fading and photobleaching of antibodies. Both of these products enable tissues to be stored for long periods of time at 4 °C. Capture images of each of fluorophore using a confocal microscope. Use excitation wavelengths for fluorophores and filters employed presented in Table 3. Perform computer-aided image analysis using appropriate software depending on the type of confocal used to capture images.

Representative Results

We have previously described the generation of mice expressing both GFP and tdTomato fluorescent reporters that detect ChAT expression14. Briefly, ChAT-Cre mice were mated with R26R:floxSTOP:tdTomato animals to produce ChAT-Cre;R26R:floxSTOP:tdTomato mice (called ChAT-Cre tdTomato). These animals were then mated with homozygous ChAT-GFP reporter mice. Embryos were isolated and tissues were dissected prior to being fixed and immunostained as above, with the antibodie…

Discussion

Our laboratory and others have shown that intestinal defects in HSCR are not restricted to the aganglionic colon but extended proximally, even into the ganglionated small intestine^5,15,16. These alterations include changes in ENS neuronal density and neurotransmitter phenotype and may account for dysmotility that has been observed in patients with HSCR. We have utilized the above techniques in our efforts to understand the determinants of ENS formation. Specifically, these techniques have been employed to visu…

Materials

Phosphate Buffered Saline	Oxoid	BR0014G
Sucrose	Fisher	S2
Sodium Azide	Fisher	BP9221
Bovine Serum Albumin	Fisher	BP1605
Triton X-100	Sigma	X100
Paraformaldehyde	Sigma	158127
60 mm Petri dishes	Fisher	FB0875713A
Fluorescence scope	Nikon	SMZ-18 stereoscope
Dissection microscope	Nikon	SMZ-18 stereoscope
Fine forceps	Fine science tools	11252-20
1.5 mL Eppendorf tubes	VWR	20170-038
Fluoromount-G	SouthernBiotech, Birmingham, AL	0100-01
Glass slides	Fisher	12-550-15
Cover glass	VWR	16004-330
Confocal microscope	Nikon	Nikon A1
Nikon Elements	Nikon