Immunfarvning at Visualiser Murine enteriske nervesystem Udvikling
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
En velfungerende enteriske nervesystem (ENS), som styrer motilitet, absorption af næringsstoffer, og lokal blodgennemstrømning, er afgørende for livet 1. ENS er dannet af neurale kamceller (NCC), der prolifererer, migrerer og koloniserer tarmen, hvor de differentierer til ganglier indeholdende neuroner og gliaceller. Hirschsprungs sygdom (HSCR, Online Mendelsk Arv i Man), en multigeneic medfødt lidelse med en incidens på 1 i 4000 levendefødte, kan betragtes som den prototypiske sygdom for at studere forstyrret ENS formation. I HSCR, NCC undlader at migrere til og kolonisere variable længder af den distale colon, 2. Derudover er andre fælles gastrointestinal (GI) udviklingsmæssige defekter i den pædiatriske population, såsom anorektale misdannelser, tarm atresias og motilitetsforstyrrelser forbundet med forstyrrelser i basale ENS funktioner, og er sandsynligvis forbundet med subtile, underappreciated, anatomiske forandringer og funktionelle ændringer iENS 3-6. Derfor kan teknikker, der tillader os at forstå de udviklingsmæssige determinanter for ENS dannelse belyse patogenesen og potentiel behandling af pædiatriske GI tract lidelser.
Efter migration og kolonisering, NCC differentierer ind neuroner med markører specifikke for deres neurotransmitter fænotype. Cholinerge neuroner udgør ca. 60% af enteriske neuroner 7, og kan påvises ved farvning for cholinacetyltransferase (ChAT), det syntetiserende enzym til excitatoriske neurotransmitter acetylcholin. Historisk har forsøg på at visualisere cholinerge neuroner blev forstyrret af forskellige antigenspecificitet af antistoffer rettet mod det centrale nervesystem (CNS) Chat versus perifere nervesystem (PNS) Chat 8-10. Men antistoffer rettet mod placenta ChAT genkende både centrale og perifere ChAT 11-13, og vi har for nylig beskrevne teknikker, der tillader udstyr og forretnilization af ENS cholinerge neuroner med høj følsomhed tidligere i udvikling, end der er opnået med ChAT reporter linier 14.
Her præsenterer vi en teknik til at dissekere, fastsættelse og immunfarvning af det murine embryonale mavetarmkanalen at visualisere ENS neurotransmitter udtryk i neuroner. Til disse undersøgelser, har vi udnyttet chat-Cre mus parret med R26R: floxSTOP: tdTomato dyr at producere chat-Cre; R26R: floxSTOP: tdTomato mus (defineret som chat-Cre tdTomato hele manuskriptet). Disse dyr blev derefter parret med homozygote chat-GFP reporter mus, til opnåelse af mus, der udtrykker både fluorescerende reportere, der registrerer ChAT ekspression 14. Disse to reporter dyr er på en C57BL / 6J baggrund, og er kommercielt tilgængelige (Jackson Laboratories, Bar Harbor, ME).
Protocol
Representative Results
Discussion
Vores laboratorium og andre har vist, at tarmens defekter i HSCR ikke er begrænset til den aganglionic kolon men udvidede proksimalt, selv ind i den ganglionated tyndtarmen 5,15,16. Disse ændringer omfatter ændringer i ENS neuronal tæthed og neurotransmitter fænotype og kan tegne sig for dysmotilitet der er blevet observeret hos patienter med HSCR. Vi har udnyttet de ovennævnte teknikker i vores bestræbelser på at forstå de afgørende faktorer for ENS-dannelse. Specifikt er disse teknikker blevet anv…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet bythe amerikanske Pediatric Kirurgisk Association Foundation Award (AG) og National Institutes of Health K08DK098271 (AG).
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 |
References
- Gershon, M. D. Developmental determinants of the independence and complexity of the enteric nervous system. Trends Neurosci. 33 (10), 446-456 (2010).
- Amiel, J., Sproat-Emison, E., et al. Hirschsprung disease, associated syndromes and genetics: a review. J Med Genet. 45 (1), 1-14 (2008).
- Erickson, C. S., Barlow, A. J., et al. Colonic enteric nervous system analysis during parenteral nutrition. J Surg Res. 184 (1), 132-137 (2013).
- 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).
- Zaitoun, I., Erickson, C. S., et al. Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung’s disease. Neurogastroenterol Motil. , (2013).
- Margolis, K. G., Stevanovic, K., et al. Enteric neuronal density contributes to the severity of intestinal inflammation. Gastroenterology. 141 (2), 588-598 (2011).
- Qu, Z. -. D., Thacker, M., Castelucci, P., Bagyánszki, M., Epstein, M. L., Furness, J. B. Immunohistochemical analysis of neuron types in the mouse small intestine. Cell Tissue Res. 334 (2), 147-161 (2008).
- Bian, X. -. C., Bornstein, J. C., Bertrand, P. P. Nicotinic transmission at functionally distinct synapses in descending reflex pathways of the rat colon. Neurogastroenterol Motil. 15 (2), 161-171 (2003).
- Johnson, C. D., Epstein, M. L. Monoclonal antibodies and polyvalent antiserum to chicken choline acetyltransferase. J Neurochem. 46 (3), 968-976 (1986).
- Tooyama, I., Kimura, H. A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers. J Chem Neuroanat. 17 (4), 217-226 (2000).
- Koga, T., Bellier, J. -. P., Kimura, H., Tooyama, I. Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis. Acta histochemica et cytochemica. 46 (2), 59-64 (2013).
- Sang, Q., Young, H. M. The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse. Anat Rec. 251 (2), 185-199 (1998).
- Lei, J., Howard, M. J. Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis. Development (Cambridge, England). 138 (21), 4789-4800 (2011).
- Erickson, C. S., Lee, S. J., Barlow-Anacker, A. J., Druckenbrod, N. R., Epstein, M. L., Gosain, A. Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines. Neurogastroenterol Motil. 26 (6), 874-884 (2014).
- Teitelbaum, D. H., Caniano, D. A., Qualman, S. J. The pathophysiology of Hirschsprung’s-associated enterocolitis: importance of histologic correlates. J Pediatr Surg. 24 (12), 1271-1277 (1989).
- Aslam, A., Spicer, R. D., Corfield, A. P. Children with Hirschsprung’s disease have an abnormal colonic mucus defensive barrier independent of the bowel innervation status. J Pediatr Surg. 32 (8), 1206-1210 (1997).
- Puig, I., Champeval, D., De Santa Barbara, P., Jaubert, F., Lyonnet, S., Larue, L. Deletion of Pten in the mouse enteric nervous system induces ganglioneuromatosis and mimics intestinal pseudoobstruction. J Clin Invest. 119 (12), 3586-3596 (2009).
