Evaluering af Planar-Cell-polaritet fænotyper i Ciliopathy Mouse Mutant Cochlea
Summary
Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.
Abstract
In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.
Introduction
Primære cilier er lange mikrotubulus-baserede vedhæng, som strækker sig fra overfladen af de fleste pattedyrceller. Primære cilier forveksles ofte med motile cilier, hvoraf der er altid flere per celle, og hvis formål er at bevæge fluid tværs membranoverflader. Primære cilier, derimod vedtage sensoriske roller og er derfor også betegnet som sensorisk cilia. Når længe glemt, dette organel er for nylig blevet "genopdaget" som et resultat af sin tilknytning til et væld af genetiske sygdomme hos mennesker 1. Ideelt placeret som en signalering organel, har den primære cilium blevet vist at regulere en lang række signalveje, hvoraf mange er vigtigt ikke blot i vævshomeostase og sygdom, men også under udvikling 2.
En af de første signalveje vist at være forbundet med cilia dysfunktion var den ikke-kanoniske Wnt signalvejen, også kendt som den plane cellepolaritet (PCP) pathway <sup > 3. Denne signaleringskaskade oprindeligt identificeret i Drosophila, er afgørende for embryogenese; især for konvergenskriterierne og udvidelse processer og for den korrekte orientering af celler i flyet af epithel 4. Den sekventielle signalering af en kerne sæt regulatoriske proteiner oversætter retningsbestemte signaler, som i sidste ende fører til cytoskeletale omrokeringer og resultere i en koordineret polarisering af epitelceller i et plan 5. Processen med konvergens og udvidelse er absolut nødvendig for cochlear kanal til aflang og for korrekt cellulære mønster 6. Da disse er reguleret via aktivering af PCP pathway, en af de mest slående fænotyper af cochlea PCP mutanter er en forkortet cochlear kanal med uorganiseret sensoriske epithel 7. Tilsvarende mus mutanter, som mangler cilier, også udviser en sådan konvergens og udvidelse fænotype 8,9, men præcist, hvordan det er reguleret mangler at blive belyst.
ve_content "> Fordi konvergenskriterierne og udvidelse processer er afgørende for udvækst af cochlear kanalen, og cellulære mønster af den sensoriske epithel i cochlear kanalen, udviklingslandene cochlea er en ideel organ til at gennemgå PCP signalering under hvirveldyr udvikling. orgel Corti udtrykket givet til den specialiserede sensoriske epithel at linjer cochlear kanal, består af ikke-sensoriske understøttende celler og mechanosensory hårceller, som skal ensartet orienteret for cochlea til at fungere 10. de mechanosensory hårceller er såkaldte grund af den stereociliary bundter, der strækker sig fra den kutikulære plade (apikale overflade) af hvert sensorisk hår celle 11. Disse fungerer som primære transducere mechanosensation og trods deres nomenklatur som stereocilia, faktisk består af modificeret actin filament-baserede microvilli. Inden for hver chevron-formede hår bundt, tre rækker af stereocilia er organiseret i en meget ordnet og regelmæssig pattern i en trappe-lignende måde. Rigtig mikrotubuli-baserede cilier, betegnes kinocilia, er nødvendige for udviklingen og orienteringen af de stereociliary bundter 12. Ved hvert hår celle, er en enkelt kinocilium fysisk anbragt på stereocilia bundt, placeret centralt ved siden af højeste række af stereocilia. Den præcise funktion af kinocilium er uklar, og en hypotese er, at kinocilium "trækker" den stereocilia i facon, som de modne fra mikrovilli 12. I hvirveldyr, kinocilia i cochlea er kun til stede kortvarigt og trækkes tilbage fra hårcellerne i mus før begyndelsen af hørelse 11,13,14.Komplet tab af cilier i udviklingslandene cochlea resulterer i alvorligt forkortede cochlear kanaler, mis-formet og mis-orienterede stereociliary bundter samt mis-placerede basale organer 8,9. En funktionel cilium er ikke kun består af den ciliære axoneme. Mange proteiner forbundet med cilierfunktion forekommer i komplekser lokaliseret til cilia-relaterede underdomæner såsom basale krop, overgangszone eller ciliaere axoneme 15. Den basale legeme, der stammer fra moderen centriole af centrosom, er også en mikrotubulus-organiserende center for mikrotubuli strækker sig bort fra cilium i cellen kroppen og kan regulere intracellulær transport samt ciliær trafficking. Den ciliaere overgangszonen er en anden region, hvor ciliaere funktion er reguleret i forhold til at organisere import og eksport af ciliære forbindelser 16.
Flere studier har identificeret en sammenhæng mellem cilier og ikke-kanonisk Wnt (PCP signalering), selvom den præcise mekanisme er uklar 17. Redundans af ciliære og PCP gener og følsomheden af cellepolaritet til generelle cellulære abnormaliteter, gør det vanskeligt at direkte forbinde en mutation til PCP-specifikke underskud. En af de læste outs af PCP signalering er positioneringen af den basale legeme og Primary cilium derfor adskillelse den primære fra de sekundære defekter er udfordrende. Nogle undersøgelser i zebrafisk og mus mutanter har foreslået nogen forbindelse mellem cilier og Wnt signalering 18-20. Uoverensstemmelser i dataene kan afspejle arter, væv eller tidsmæssige-afhængige forskelle i ciliære bidrag til Wnt signalering. Desuden kan normal Wnt lydhørhed bibeholdes, hvis basale organer forbliver funktionelt. En dybere indsigt i den rolle, ciliære proteiner i cellulære signalveje og andre biologiske fænomener er afgørende for vores forståelse af cellulær og udviklingsmæssige biologi, samt til udvikling af målrettede behandlingsstrategier.
Protocol
Representative Results
Discussion
Ved udarbejdelsen cochlear væv til analyse, er der et par vigtige punkter at huske på. For det første kan forskelle i genetisk baggrund ændre cochlear fænotype, hvilket gør det nødvendigt at analysere og sammenligne kun kuld kontroller. For det andet er fuldstændig fjernelse af tectorial kræves for at opnå de bedste billeder med immunhistokemi og er afgørende for SEM. Den tectorial er en uigennemsigtig struktur og kan tilsløre cellerne i sensoriske epithel direkte under den, hvilket gør billeddannelse mere …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatteren vil gerne takke Matthew Kelley, Tiziana Cogliati, Jessica Gumerson, Uwe Wolfrum, Rivka Levron, Viola Kretschmer og Zoe Mann og for deres kritiske evaluering af manuskriptet. Dette arbejde blev finansieret af Sofja Kovalevskaya Award (Humbodlt Foundation) og Johannes-Gutenberg Universitet, Mainz, Tyskland.
Materials
| Tools/Equipment | |||
| Silicone elastomere – Sylgard 184 | Sigma-Aldrich | 761028-5EA | See Note 2 |
| Micro dissecting scissors-straight blade | Various | ||
| Fine forceps (no. 5 and 55) and blunt forceps | Various | ||
| Dissecting microscope. | Various | ||
| Uncoated glass microscope slides | Various | ||
| Microscope cover slips (22 mm × 40 mm × 0.15 mm) | Various | ||
| Transfer pipettes | Various | ||
| Minutien pins | Fine Science Tools | 26002-10 | |
| SEM sample holder | tousimis | 8762 | |
| Scanning electron microscopy studs | TED PELLA | 16111 | |
| PELCO Tabs: Carbon adhesive | TED PELLA | 16084-3 | |
| Fluorescent Microscope | Various | ||
| Critical Point Dryer | Various | ||
| Scanning Electron Microscope | Various | ||
| Glass microscope slides | Various | ||
| Glass coverslips | Various | ||
| Kimwipe Tissue | Various | ||
| Fine Paint Brush | |||
| Reagents | |||
| 1× Phosphate buffered saline (PBS) | Gibco/Life Technologies | 10010023 | |
| Paraformaldehyde (PFA) (EM Grade Required for EM) | Various | Prepare a 4% solution in 1× PBS made fresh each time. EM Grade Required for EM. | |
| 2.5% Glutaraldehyde Grade1 | Sigma-Aldrich | G5882 | |
| Tris-HCl (pH 7.5) | Various | ||
| NaCl | Various | ||
| CaCl 2 | Various | ||
| Triton X-100 | Various | ||
| Normal Goat Serum | Various | ||
| AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 715-007-003 | |
| Fluoromount-G Mounting media | SouthernBiotech | 0100-01 | |
| 10× Hanks’ Balanced Salt Solution (HBSS) | Gibco/Life Technologies | 14065 | |
| Hepes | Gibco/Life Technologies | 15630-080 | |
| Osmium tetroxide (OsO4 ) | Sigma-Aldrich/Fluka Analytical | 75632 | |
| Tannic acid | Sigma-Aldrich | 403040 | |
| Ethanol 200 proof | Various | ||
| Antibodies | |||
| anti Arl13b | Protein Tech | 17711-1-AP | Suggested concentration 1:1000 |
| anti acetylated tubulin (611-B1) | Sigma-Aldrich | T6793 | Suggested concentration 1:800 |
| anti gamma tubulin (GTU-88) | Sigma-Aldrich | T6557 | Suggested concentration 1:200 |
| anti Zo_1 | Invitrogen | 40-2300 | Suggested concentration 1:500 |
| Myosin VI | Proteus Biosciences | 25-6791 | Suggested concentration 1:1000 |
| Myosin VIIa | Proteus Biosciences | 25-6790 | Suggested concentration 1:1000 |
| anti Vangl2 | Merk Millipore | ABN373 | Suggested concentration 1:250 |
| anti Gαi3 | Sigma-Aldrich | G4040 | Suggested concentration 1:250 |
| Alexa Fluor® 488 Phalloidin | Invitrogen/Life Technologies | A12379 | Suggested concentration 1:300-1000 |
| Alexa Fluor® 568 Phalloidin | Invitrogen/Life Technologies | A12380 | Suggested concentration 1:300-1000 |
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