Studere Wnt Signaling Under mønster af ledende Airways
Summary
Anvendelsen af reporter mus koblet til hele mount og sektion farvning, mikroskopi og in vivo assays letter analysen af mekanismer bag den normale mønsterdannelse i luftvejene. Her beskriver vi, hvordan disse teknikker bidraget til analyse af Wnt-signalering under tracheal udvikling.
Abstract
Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.
Introduction
Udvikling luftvejene er initieret af embryonale dag 9 (E9) med fremkomsten af Nkx2.1 positive celler i den ventrale endodermal fortarm 1,2. Esophageal-trachealtube separation vil løse ved E11.5 når rørene kan skelnes som særskilte enheder, hver omgivet af mesenkymale væv 3. Wnt signalering spiller en central rolle i specifikationen af luftvejene som sletning af Wnt2 og Wnt2b ved splanknisk mesenkym og sletning af β-catenin fra endodermal respiratorisk epitel udtrykt vil resultere i lunge agenesi 4,5. Vores tidligere undersøgelser fastslået, at sletning af WLS, en last receptor medierer sekretion af alle Wnt-ligander, fra endodermale resultater luftveje i lungehypoplasi, defekter i pulmonal vaskulær udvikling og mis-mønster af tracheal mesenchym 6,7. Disse data understøtter betydningen af epitel-mesenchymale cross taler i celledifferentiering og specifikation, som det også er blevet vist i andre undersøgelser 8,9.
Undersøgelsen af de tidligste stadier af lunge udvikling er afhængig af genetiske, in vitro og ex vivo teknikker, der har givet os mulighed for bedre at forstå mekanismerne kørsel respiratorisk identitet 10-16. Hele lunge eksplantatkulturer på Air Liquide interfase er ofte blevet anvendt til at undersøge virkningerne af vækstfaktorer i tidlige stadier af pulmonal forgrening morfogenese 10,17,18. Mens denne metode anvendes som udlæsning af morfologiske ændringer, såsom forgrening morfogenese og genekspression modulation, det er begrænset til studiet af tidlige stadier af udviklingsprocessen, da kulturen ikke selv støtte udviklingen af karrene 17. Udvikling af tracheal brusk kræver længere inkubationstider, der kan være ikke kompatibel med denne kultur teknik.
at analyze rolle Wnt signalering under dannelsen luftveje, har vi tilpasset standardteknikker til at opfylde de behov, vores embryonale undersøgelser. Vi har ændret volumener, farvning gange, forarbejdning cykling for paraffin indlejring og timing for rydning af tracheal-lungevæv. Det vigtigste mål for at optimere de beskrevne teknikker i nærværende undersøgelse var at analysere de tidligste stadier af trachea udvikling hos mus, der finder sted fra E11 til E14.5. Brug af reporter mus linje Axin2LacZ vi præcist bestemte steder i Wnt / β-catenin aktivitet i udviklingslandene tracheal mesenkym. Vi har også tilpasset lectin farvningsprocedure for hele mount luftrør væv. Således var vi i stand til at visualisere mesenkymale fortætninger og forudsige steder, hvor chondrogenese vil finde sted. Farvning af hele mount og sektioner af fostervæv opnået fra WlsShhCre mus, kombineret med avancerede mikroskopi-teknikker, tilladt os at afsløre rolle Wnt ligander produceret af traCheal epitel i tracheal mønster.
Protocol
Representative Results
Discussion
Arrangementer ligger til grund for morfogenese i luftvejene forstås ikke til fulde, navnlig de processer, der er nødvendige for mønsterdannelse af de ledende luftveje. Tidligere undersøgelser har anvendt ex vivo-teknikker, hvor udviklingslande eksplantater dyrkes ved luft-væske interfase eller indlejret i matrigel 21,22. Disse undersøgelser har vist, hvordan vækstfaktorer påvirker mønsterdannelse af den udviklende luftrøret og dannelsen af tracheal brusk. En begrænsning til disse und…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi anerkender bistand fra Mike Muntifering og Matt Kofron med konfokal billedbehandling og Gail Macke med histologiske procedurer. Dette arbejde blev delvist støttet af National Institutes of Health-NHLBI (K01HL115447 til DS).
Materials
| Anti Sox9 ab. | Millipore | AB5535 | 1:400 , rabbit |
| Anti Sox9 ab. | Santa Cruz | Sc-20095 | 1:50, rabbit |
| Anti Smooth Muscle Actin ab. | Sigma | A5228 | 1:2k, mouse |
| Anti NKX2.1 ab. | Seven Hills | n/a | 1:100, guinea pig |
| Anti NKX2.1 ab. | Seven Hills | n/a | 1:400, mouse |
| Anti Brdu ab. | Abcam | AB1893 | 1:200, sheep |
| Anti Brdu ab. | Santa Cruz | Sc-32323 | 1:4k, mouse |
| PNA Lectin | Sigma | L 7381 | |
| Secondary antibodies | Life technologies | Alexa fluor Molecular probes | |
| K3Fe(CN)6 | Sigma | P8131 | |
| K4Fe(CN)6 | Sigma-Aldrich | P3289 | |
| MgCl2 | Sigma-Aldrich | M9272 | |
| NaDOC | Life Technologies | 89905 | |
| NP4O | Life Technologies | 85124 | |
| Alcian Blue 8GX | Sigma | A-3157 | |
| Fisher brand super-frost plus | Fisher | 12-550-15 | |
| PFA (16%) | EMS | 15710 | |
| PBS | Gibco | 70011-044 | |
| Fetal Calf Serum | Sigma | 11K413 | |
| Blocking reagent | Invitrogen | Component of TSA kit #2 ( T20932) | |
| BrDu | Sigma | B5002-5g | |
| Vectashield mounting medium | Vector labs | H-1000 | |
| Permount | Fisher | SP15-500 | |
| Tissue-loc cassettes Histoscreen | Fisher | C-0250-GR | |
| Biopsy cassettes | Premiere | BC0109 | Available in different colors |
| Nuclear fast red Kernechtrot 0.1% | Sigma | N3020 | |
| Citric acid | Sigma | C1909-500G | |
| Sodium citrate tribasic dihydrate | Sigma | S4641-1Kg | |
| Trizma hydrochloride | Sigma | T5941-500G | |
| Xylene | Pharmco-AAPER | 399000000 | |
| Ethanol | Pharmco-AAPER | 111000200 | |
| Micro knives | FST | 10318-14 | |
| Dumont #5 ceramic coated | FST | 11252-50 | |
| Dumont #5CO | FST | 11295-20 | |
| Dumont # 5 | FST | 91150-20 | |
| Thermo/Shandon Excelsior ES | Thermo Fisher | ||
| Microtome | Leica | RM2135 | |
| Nikon i90 | Nikon | Wide field microscope | |
| NikonA1Rsi | Nikon | Confocal microscopy. Settings:NikonA1 plus camera, scanner: Galvano, detector:DU4. Optics Plan Apo lambda 10x. Modality: Widefield fluorescence laser confocal. | |
| Leica MS 16 FA | Leica | Fluorescence Dissecting microscope | |
| Zeiss | Zeiss | Automated fluorescence microscope | |
| Leica Application suite | Leica | Leica imaging software | |
| NIS | Nikon | Nikon imaging software | |
| IMARIS | Bitplane | Imaging processing software |
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