Immunostaining to Visualize Murine Enteric Nervous System Development

1Department of Surgery, University of Wisconsin School of Medicine and Public Health, 2Department of Neurosciences, University of Wisconsin School of Medicine and Public Health, 3Department of Pediatrics, University of Wisconsin School of Medicine and Public Health
Developmental Biology

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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.

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Barlow-Anacker, A. J., Erickson, C. S., Epstein, M. L., Gosain, A. Immunostaining to Visualize Murine Enteric Nervous System Development. J. Vis. Exp. (98), e52716, doi:10.3791/52716 (2015).

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 ENS3-6. 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) ChAT8-10. However, antibodies directed against placental ChAT recognize both central and peripheral ChAT11-13, 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).

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Protocol

The University of Wisconsin Animal Care and Use Committee approved all procedures.

1. Preparation of Solutions

  1. Use 1x phosphate buffered saline (PBS) as dissection buffer and rinsing solution.
  2. 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.
  3. 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.
  4. 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

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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

  1. 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

  1. If samples have been stored in 30% sucrose, rinse them 3 times for 20 min in 1x PBS on a rocking platform.
  2. Place the GI tracts into blocking solution on a rocking platform for 1h at RT.
  3. 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).
  4. Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.
  5. 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.
  6. Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.
  7. 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.
  8. Capture images of each of fluorophore using a confocal microscope. Use excitation wavelengths for fluorophores and filters employed presented in Table 3.
  9. Perform computer-aided image analysis using appropriate software depending on the type of confocal used to capture images.

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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 antibodies listed in Table 1. The distal small intestine (SI) and proximal colon were analyzed at E11, E13.5 and E16.5

At E11, Hu-positive neurons are present within the distal SI, but NCC have not yet reached the proximal colon (Figure 1). At this time point, the majority of Hu-positive neurons demonstrate ChAT immunoreactivity as well as GFP positivity. Interestingly, at this time point, expression of the ChAT-Cre tdTomato construct is not detectable. By E13.5, the majority of Hu-positive neurons are both ChAT-IR and ChAT-GFP positive, and a small number of ChAT-Cre tdTomato-positive neurons are seen. The number of ChAT-Cre tdTomato-positive neurons increases by E16.5, but continues to be a small fraction of the number of ChAT-IR neurons.

Figure 1
Figure 1. Representative Images of Cholinergic Neurons in the Distal Small Intestine and Colon at Embryonic Day 11. At E11, in the distal SI (labeled “SI”) and proximal colon (labeled “Co”), Hu-positive neurons (blue) are only present within the SI at this stage. Most Hu-positive neurons are co-immunostained with ChAT-IR (white) and ChAT-GFP (green). However, at this time point, none of these neurons are ChAT-Cre tdTomato positive (red). Scale bar = 100 μm. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Representative Images of Cholinergic Neurons in the Distal Small Intestine and Colon at Embryonic Days 13.5 and 16.5. Image shows cholinergic neurons in the distal small intestine at E13.5 (upper panels) and E16.5 (lower panels). At E13.5 the majority of Hu-positive neurons were ChAT-IR and ChAT-GFP positive (upper panels). A small number of these co-expressed ChAT-Cre tdTomato. By E16.5, more ChAT-Cre tdTomato neurons were found in nascent ganglia (lower panels). Scale bar = 100 μm. Please click here to view a larger version of this figure.

Primary Antibodies
Antigen Immunogen Dilution Supplier & Details
Hu (Human neuronal protein) Hu protein 1:1,000 Serum from human patient (Madison, WI)
GFP (Green Fluorescent Protein) GFP protein, IgY fraction 1:1,000 Aves Lab, Inc. (Tigard, OR), Chicken polyclonal, GFP-1020
ChAT (Choline Acetyltransferase) Human placental enzyme 1:100 Millipore (Billerica, MA), Goat polyclonal, AB144P
Secondary Antibodies
Name Fluorophore Dilution Supplier & Details
Donkey anti-Human Dylight 405 1:500 Jackson ImmunoResearch Laboratories (West Grove, PA), 709-475-149
Donkey anti-Chicken Cy2 1:500 Jackson ImmunoResearch Laboratories (West Grove, PA), 703-225-155
Donkey anti-Goat Cy5 1:500 Jackson ImmunoResearch Laboratories (West Grove, PA), 705-175-147

Table 1. Details of primary and secondary antibodies used in the study. The primary and secondary antibodies used in this study, along with their supplier and the dilutions utilized.

Primary Antibodies
Antigen Dilution Supplier Secondary Antibody
Sox10 1:50 Santa Cruz Biotechnology, Dallas, TX Donkey anti-Goat
p75 1:250 Promega, Madison WI Donkey anti-Rabbit
PGP9.5 1:1,000 Abcam, Cambridge, MA Donkey anti-Rabbit
BFABP 1:500 Millipore, Billerica, MA Donkey anti-Rabbit
S-100 1:500 DAKO, Carpinteria, CA  Donkey anti-Rabbit
GFAP 1:500 DAKO, Carpinteria, CA  Donkey anti-Rabbit
nNOS 1:500-1,000 Emson, Cambridge, UK Donkey anti-Sheep
VIP (Vasoactive Intestinal Polypeptide) 1:500 Epstein & Paulsen, Madison, WI Donkey anti-Rabbit
Substance P 1:200-400 DiaSorin, Stillwater, MN Donkey anti-Sheep
Secondary Antibodies
Name Dilution Supplier & Details Fluorophore
Donkey anti-Human 1:500-1,000 Jackson ImmunoResearch Laboratories, West Grove, PA Dylight 488
Donkey anti-Human 1:1,000 Jackson ImmunoResearch Laboratories, West Grove, PA Cy3
Donkey anti-Rabbit 1:500 Jackson ImmunoResearch Laboratories, West Grove, PA Dylight 488
Donkey anti-Rabbit 1:500 Jackson ImmunoResearch Laboratories, West Grove, PA Cy3
Donkey anti-Sheep 1:500 Jackson ImmunoResearch Laboratories, West Grove, PA Cy2
Donkey anti-Sheep 1:1,500 Jackson ImmunoResearch Laboratories, West Grove, PA Cy3
Donkey anti-Goat 1:500 Jackson ImmunoResearch Laboratories, West Grove, PA Cy3

Table 2. Additional primary and secondary antibodies of use in studying ENS development. The primary and secondary antibodies that we have previously employed in our studies of ENS development.

Excitation Emission
Laser Line Fluorophore Type Examples Photomultiplier Tube Filter Options
408 nm Blue Alexa Fluor 405, Cascade Blue, Coumarin 30, DAPI, Hoechst, Pacific Blue, most quantum dots 1 BP 425-475 nm
488 nm Green Alexa Fluor 488, ATTO 488, Calcein, Cy2, eGFP, FITC, Oregon Green, YO-PRO-1 2 BP 500-550 nm
561 nm Red Alexa Fluor 546, 555, 568, and 594, Cy3, DiI, DsRed, mCherry, Phycoerythrin (PE), Propidium Iodine (PI), RFP, TAMRA, tdTomato, TRITC 3 BP 570-620 nm
638 nm Far Red Alexa Fluor 633 and 647, Allophycocyanin (APC), Cy5, TO-PRO-3 4 BP 663-738 nm

Table 3. Confocal imaging excitation wavelengths, fluorophores and filters. The excitation wavelengths, detectable fluorophores, and filters employed are presented. This information can be used in choosing combinations of secondary antibodies for multicolor immunofluoresence.

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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 intestine5,15,16. 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 visualize neuronal nitric oxide synthase (nNOS) neurons, vasoactive intestinal polypeptide neurons (VIP), as well as choline acetyltransferase (ChAT) neurons (Table 2)5. A key advantage of this technique is the ability to store samples in 30% sucrose for later processing, either for immunostaining or for embedding and cryosectioning.

NCC colonization of the gut occurs as an advancing wavefront from E9.5 to E14.5. Immunostaining with Hu, which identifies neurons, as described above, coupled with computer-aided image analysis, allows for quantitative comparisons of neuronal density. Reductions in neuronal density are found portions of the ganglionated bowel of HSCR5, as well as in other models of intestinal dysmotility13. Mice with a conditional deletion of Hand2 demonstrate changes in the proportions of neurons expressing different neurotransmitters as well as reduced neuronal numbers. Additionally, animals with over-expression of Noggin or PTEN demonstrate increased neuronal density along their bowel6,17.

The balance of neurotransmitter phenotypes among neurons of the ENS is tightly regulated, resulting in coordinated contraction of the bowel wall for movement of luminal contents, regulation of blood flow and nutrient absorption. Once NCC acquire a neuronal identity, there is a narrow window of time before committing to a neurotransmitter phenotype14. We have recently shown that immunostaining identifies a ChAT neurotransmitter phenotype at E10.5, an earlier embryonic stage than observed with genetic reporter models (e.g., ChAT-Cre or ChAT-GFP)14. Additionally, ChAT immunoreactive neurons reach adult levels (as a proportion of all ENS neurons) early in development, a result which suggests that ChAT neurons may have a role in regulating further neuronal differentiation in the ENS.

Nitric oxide synthase (NOS) is the primary relaxation neurotransmitter found in the ENS, and is proportionally increased in ENS neurons along the bowel in HSCR5. Additionally, alterations in vasoactive intestinal peptide (VIP), a neurotransmitter that also participates in mediating muscle relaxation and regulating blood flow along the bowel wall, are found in HSCR models5. These data suggest that, in order to understand how the components of the ENS regulate its function, a systematic examination of the different neurotransmitters should be undertaken. The techniques presented here can be readily adapted to allow this type of analysis by use of the appropriate antibodies.

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported bythe American Pediatric Surgical Association Foundation Award (AG) and the National Institutes of Health K08DK098271 (AG).

Materials

Name Company Catalog Number Comments
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 microscope 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

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References

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  4. Erickson, C. S., Zaitoun, I., Haberman, K. M., Gosain, A., Druckenbrod, N. R., Epstein, M. L. Sacral neural crest-derived cells enter the aganglionic colon of Ednrb(-/-) mice along extrinsic nerve fibers. J Comp Neurol. 20, (3), 620-632 (2011).
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