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In questo articolo

  • Riepilogo
  • Abstract
  • Introduzione
  • Protocollo
  • Risultati
  • Discussione
  • Divulgazioni
  • Riconoscimenti
  • Materiali
  • Riferimenti
  • Ristampe e Autorizzazioni

Riepilogo

Una combinazione delle tecniche ottiche avanzate di scansione microscopia con lungo l'eccitazione di fluorescenza multi-fotone lunghezza d'onda del laser è stata implementata per catturare immagini ad alta risoluzione, tridimensionale, in tempo reale della migrazione dalla cresta neurale in embrioni di zebrafish di Tg (foxd3:GFP) e Tg (sox10:EGFP).

Abstract

Occhio congenita e anomalie craniofacciali riflettono imprevisti nella cresta neurale, una popolazione transitoria di migratori cellule staminali che danno origine a numerosi tipi di cellule in tutto il corpo. Comprensione della biologia della cresta neurale è stata limitata, che riflette una mancanza di modelli geneticamente trattabili che possono essere studiati in vivo e in tempo reale. Zebrafish è un modello di sviluppo particolarmente importante per lo studio di popolazioni di cellule migratorie, come la cresta neurale. Per esaminare la migrazione dalla cresta neurale nell'occhio in via di sviluppo, una combinazione delle tecniche ottiche avanzate di scansione microscopia con eccitazione di fluorescenza di multi-fotone lunghezza d'onda del laser è stata implementata per catturare video ad alta definizione, tridimensionali, in tempo reale dell'occhio in via di sviluppo negli embrioni di zebrafish transgenici, vale a dire Tg (sox10:EGFP) e Tg (foxd3:GFP), come sox10 e foxd3 è stati dimostrati in numerosi modelli animali di regolare differenziazione iniziale dalla cresta neurale e probabilmente rappresentano gli indicatori per le cellule della cresta neurale. Time-lapse di immagini multi-fotone è stato utilizzato per discernere il comportamento e schemi migratori delle due popolazioni di cellule neurali della cresta che contribuiscono al precoce sviluppo dell'occhio. Questo protocollo fornisce informazioni per la generazione di video time-lapse durante la migrazione dalla cresta neurale di zebrafish, come esempio e possa essere applicato per visualizzare lo sviluppo precoce di molte strutture in zebrafish e altri organismi di modello.

Introduzione

Malattie congenite dell'occhio possono causare la cecità di infanzia e spesso sono dovuti ad anomalie della cresta neurale cranica. Cellule della cresta neurale sono cellule staminali transitorie che derivano dal tubo neurale e formano numerosi tessuti in tutto il corpo. 1 , 2 , 3 , 4 , 5 cellule della cresta neurale, derivate dal prosencefalo e mesencefalo, danno luogo all'osso e la cartilagine del midface e regioni frontali e l'iride, cornea, trabecolato e sclera nel segmento anteriore dell'occhio. 4 , 6 , 7 , 8 cellule neurali della cresta da romboencefalo forma che il pharyngeal arches, mascella e tratto di efflusso cardiaco. 1 , 3 , 4 , 9 , 10 studi hanno evidenziato i contributi della cresta neurale all'oculare e perioculare sviluppo, sottolineando l'importanza di queste cellule in sviluppo dell'occhio dei vertebrati. Infatti, la rottura di differenziazione e migrazione delle cellule neurali della cresta condurre ad anomalie cranio-facciali ed oculari come osservato nella sindrome di Axenfeld-Rieger e sindrome di Peters Plus. 11 , 12 , 13 , 14 , 15 , 16 , 17 in tal senso, una comprensione globale della migrazione, proliferazione e differenziazione di queste cellule della cresta neurale fornirà approfondimenti le complessità sottostanti malattie congenite dell'occhio.

Zebrafish è un organismo potente modello per studiare lo sviluppo oculare, come le strutture dell'occhio zebrafish sono simili alle loro controparti dei mammiferi, e molti geni sono evolutivamente conservate tra zebrafish e mammiferi. 18 , 19 , 20 inoltre, embrioni di zebrafish sono trasparenti e ovipari, facilitando la visualizzazione di sviluppo dell'occhio in tempo reale.

Espansione sul lavoro precedentemente pubblicato,6,7,20 il modello migratore delle cellule della cresta neurale è stata descritta usando la fluorescenza multi-fotone time-lapse di imaging su linee di zebrafish transgenici etichettati con la proteina fluorescente verde (GFP) sotto il controllo trascrizionale di SRY (regione dideterminazione Y)-scatola 10 (sox10) o Forkhead Box D3 nelle regioni regolatrici del gene (foxd3). 21 , 22 , 23 , 24. fluorescenza multi-fotone time-lapse imaging è una tecnica potente che combina le avanzate tecniche ottiche di microscopia con lungo l'eccitazione di fluorescenza di multi-fotone lunghezza d'onda di catturare immagini ad alta risoluzione, tridimensionale di esemplari con fluorofori etichettati di scansione laser. 25 , 26 , 27 l'uso del laser multi-fotone presenta chiari vantaggi rispetto la microscopia confocale standard, tra cui aumentata del tessuto vaginale e candeggio fluoroforo in diminuzione.

Utilizzando questo metodo, due popolazioni distinte delle cellule della cresta neurale varia in tempi di migrazione e percorsi migratori erano cellule della cresta neurale discriminata, vale a dire foxd3-positivo nel mesenchima periocular e occhio in via di sviluppo e le cellule di cresta neurale sox10-positivi nel mesenchima craniofacial. Con questo metodo, viene introdotto un approccio per visualizzare la migrazione della migrazione oculare e craniofacial cresta neurale in zebrafish, che lo rende facile osservare regolamentato dalla cresta neurale migrazione in tempo reale durante lo sviluppo.

Questo protocollo fornisce informazioni per la generazione di video time-lapse durante lo sviluppo iniziale di occhio in Tg (sox10:EGFP) e zebrafish transgenici Tg (foxd3:GFP), ad esempio. Questo protocollo possa essere applicato per la visualizzazione ad alta risoluzione, tridimensionale, in tempo reale dello sviluppo iniziale di qualsiasi struttura oculare e craniofacial derivato dalle cellule della cresta neurale in zebrafish. Inoltre, questo metodo possa essere applicato per la visualizzazione dello sviluppo di altri tessuti ed organi in zebrafish e altri modelli animali.

Protocollo

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).

1. Embryo Collection for Time-lapse Imaging

  1. Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating.
    NOTE: The Tg(sox10:EGFP) and Tg(foxd3:GFP) fish, kind gifts from Thomas Schilling and Mary Halloran, respectively, were crossed into the Casper (roy -/-, nacre -/-) background to decrease auto-fluorescence and interference with pigmentation.
    1. Assemble the breeding tank (slotted inner tank + solid outer tank) and fill it with reverse osmosis (RO) system water.
    2. Transfer up to 3 females and 3 males into opposing sides of the tank separated by a divider; up to 6 fish can be bred pairwise in a single breeding tank.
    3. The next morning, remove the divider shortly after the lights turn on in the vivarium.
      NOTE: In the present study, the lights are on a 14-hour light (on at 9 am) and 10-hour dark (off at 11 pm) cycle.
    4. Allow undisturbed mating for 20 min or until sufficient numbers of embryos are produced; the embryos will be at the bottom of the breeding tank. When eggs are observed, lift the slotted inner tank out along with the fish and quickly place them (fish and inner tank) into a clean solid outer tank filled with RO water.
  2. Prepare standard 1X embryo medium by adding the following per 1 L of RO water: 0.287 g (4.9 mM) NaCl, 0.127 g (0.17 mM) KCl, 0.048 g (0.33 mM) CaCl2.2H2O, 0.04 g (0.33 mM) MgSO4 (anhydrous). Stir solution until the salts are completely dissolved.
    1. Add 100 µL of 0.1% methylene blue as a fungicide. Add 0.25 g of sodium bicarbonate to adjust the pH to 7.75. Store medium at room temperature.
  3. Collect eggs from the outer tank using an egg-collecting screen, and transfer the eggs to Petri dishes (~50 embryos per dish) containing 30 mL of 1X embryo medium. Incubate the collected eggs at 28.5 °C.
  4. Embryo preparation
    1. Tg(sox10:EGFP) embryos.
      1. For Tg(sox10:EGFP) embryos, at 12 h post-fertilization (hpf), remove the dead eggs and assess the developmental stage of the embryos. Screen for GFP-positive embryos using a dissecting fluorescent stereomicroscope with a standard 460-490 nm band-pass excitation filter. Using forceps, remove the chorions from 3-4 embryos that express GFP, and transfer these embryos to a separate Petri dish containing 30 mL of fresh 1X embryo medium.
    2. Tg(foxd3:GFP) embryos.
      1. For Tg(foxd3:GFP) embryos, at 22-24 hpf, remove the dead eggs and assess the developmental stage of the embryos. Screen for positive embryos using a dissecting fluorescent stereomicroscope with a standard 460-490 nm band-pass excitation filter. Using forceps, remove the chorions from 3-4 embryos that express GFP.
      2. Prepare 1000X Phenylthiourea (PTU) stock solution by dissolving 0.75 g (3%) of PTU in 25 mL of Dimethylsulfoxide (DMSO). Aliquot and store the stock solution at 4 °C. Add 30 µL of 1000X PTU (3%) stock solution to 30 mL of 1X embryo medium to generate 0.003% PTU solution.
      3. Place the screened and dechorionated GFP-positive embryos in 0.003% PTU solution to inhibit pigmentation. Do not initiate treatment of the embryos with PTU prior to 20 hpf, as early treatment (at <20 hpf) can have adverse effects on neural crest and neuroepithelial development.28
    3. Prior to conducting the time-lapse experiment, monitor the embryos through live imaging using a standard stereomicroscope at 12-24 h intervals in age-matched comparisons as a control for temperature drift.

2. Mounting of Embryo for Time-lapse Imaging

  1. Preparation of time-lapse embryo media
    1. Prepare standard 0.4% tricaine stock solution by adding 0.004 g of tricaine to 100 mL of RO water. Aliquot (~1 mL) the stock solution into fresh 1.5-mL centrifuge tubes and store at -20 °C.
    2. Prepare time-lapse embryo medium (50 mL of 1X embryo medium, 0.016% tricaine) by adding 2 mL of 0.4% tricaine solution to 48 mL of 1X embryo medium. If using embryos >22 hpf, then also add 50 µL of 3% PTU stock solution (final concentration.003% PTU) to the embryo medium.
  2. Preparation of 2% low-melt agarose
    1. Add 0.4 g of low-melt agarose powder to 20 mL of 1X embryo medium. Heat for 1-2 min or until the solution is clear and all particles are dissolved.
      NOTE: Increasing the percentage of the agarose gel decreases the porosity of the matrix, which may physically impede the growth and development of the embryo. Therefore, the agarose solution can be lowered to 1.5% to minimize these effects. However, when the excitation beam is held stationary during laser-scanning microscopy, greater heating can occur, increasing rapidly in a logarithmic relationship with time. In this protocol, heating due to fluorophore absorption is highly localized to the focal region. Thus, in the region of interest, the temperature could increase to a value high enough (≥ 30 °C) to melt agarose solutions made at percentages lower than 1.5, thereby directly exposing the embryo to the laser or enabling conditions in which the embryo floats out of the focal plane. For this reason, the use of agarose solutions lower than 1.5% is not recommended.
    2. Aliquot (~1 mL) the agarose solution into fresh 1.5-mL centrifuge tubes for storage. Store excess agarose solution in liquid form on a heat block (60-70 °C) for 2-3 weeks.
  3. Mounting the Embryo
    1. To set up the open bath chamber, place a small amount of high vacuum grease on the base of the open bath chamber. Place a circular glass coverslip onto the base and screw the top of the open bath chamber onto the base until tight ( Figure 1A, B).
    2. Pipet (~500-700 µL) of 2% agarose solution (60-70 °C) into the open bath chamber until the base is ~3/4 filled ( Figure 1C). Note that once the agarose solution is in the open bath chamber on the lab bench, the temperature of the solution decreases approximately 1 °C/s.
    3. Wait 30 s to allow the agarose to cool slightly (~30-40 °C), without completely polymerizing, and subsequently transfer a single embryo to the center of the base ( Figure 1D, E).
    4. Under a fluorescent stereomicroscope, position the embryo using a 1-10 µL micropipette tip, making sure that the embryo is placed near the bottom of the agarose, as the embryo may float away when covered with embryo media if it is placed too near the surface of the agarose.
      NOTE: In the presented videos (Videos 1-3), the embryos are oriented laterally, but depending on the area of interest, the embryos can be oriented ventrally or dorsally.
    5. Monitor and reposition the embryo until the agarose has set. Once the agarose has set, use a transfer pipet to fill the assembled open chamber entirely with time-lapse embryo media ( Figure 1F).
    6. Place the open bath chamber in the quick exchange platform, which fits onto the stage adapter ( Figure 1G). Place the entire setup (embryo, open bath chamber, quick exchange platform and stage adaptor) onto the stage of the microscope immediately above the condenser ( Figure 1H).

3. Microscope Set-up for Time-lapse Imaging

  1. Determining laser settings
    1. Determining laser wavelength to use. Use a wavelength that is twice the excitation wavelength of the fluorophore of interest (e.g. wavelength between 880 and 940 nm for GFP).
      NOTE: In the present study, the wavelength setting for GFP was between 880 and 940 nm. The higher the wavelength, the lower the output power of the laser.
    2. Determine laser transmission. A high percent of laser transmission will kill the embryo, use the lowest level of transmission (recommended). For 24 to 48 h time-lapse studies, as presented herein, keep transmission below 5%.
    3. Determine microscope detection systems and ensure that the correct filters for the fluorophore are in place.
      NOTE: For multi-photon microscopes, there are multiple detection systems with various sensitivities for the emitted fluorescence. In general, an internal detection system has less sensitivity than an external detection system. For transgenic lines with high levels of GFP expression, the internal detection system is adequate. For transgenic lines with low levels of GFP expression or with other fluorophores (e.g., red fluorescent protein), an external detection system may be required to maintain the percent of laser transmission at a reasonable level. Regardless the detection system used, the correct filters for the fluorophore must be in place.
  2. Adjusting software settings on the multi-photon microscope
    1. Using the 5X objective, locate the embryo. Manually raise the stage to the highest position, and use the fine focus to position the embryo in the middle of the microscope range.
    2. Manually lower the stage, and change the 5X objective to the 25X water immersion objective (numerical aperture NA, 0.95). Carefully raise the stage to bring the embryo back into focus.
    3. In the software, click on the "xyzt" mode for obtaining multiple images at time intervals (t) in the x-y plane over a depth of "z". Use the epifluorescence or brightfield view to find the depth of focus in the area of interest, which will demarcate the Z-stack.
      1. In the software, click on "begin" button; for these experiments, the lateral edge of the eye was the beginning of the Z-stack. Click on "end" button; the midline of the embryo was the end of the Z-stack.
        NOTE: The step size was 0.3-0.6 µm and there was a total of ~200 steps for a z-stack size of 60 to 120 µm).
    4. Click on the menu for adjusting acquisition time and imaging frequency. For the present system, ~200 steps requires approximately 5 min for each z-stack acquisition. For adequate recovery of the fluorophore and survival of the embryo, allow for a ratio of at least 1:3 between z-stack acquisition (laser power on) and recovery time (laser power off).
      NOTE: For example, z-stacks are acquired every 20 min with 5 min of z-stack acquisition and 15 min of recovery. For this protocol, larger z-stacks can be obtained, but would appropriately increase the time between z-stack acquisitions, resulting in fewer images over the time-lapse course.
      1. With appropriate time for embryo recovery, set the time between z-stacks in the designated window. Set the total length of time for the experiment in the appropriate window.
    5. Final software, laser, and embryo adjustments.
      1. Turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission (see 3.1.2), gain and offset slider bars within the software to optimize the fluorescent image. Also, adjust the orientation of the embryo, as needed, depending on the length of the experiment, anticipated growth of the embryo, etc. Make sure that the area of interest remains within the frame through the duration of the experiment.
      2. Turn off the epifluorescent light source as it is no longer needed during time-lapse acquisition. Cover the stage with the laser safety box ( Figure 1I). When using the internal detection system, the laser safety box is adequate for protection against background light. Press "start".
        NOTE: However, with more sensitive external detection systems, the laser safety box does not block enough background light, and additional covers are required to prevent the disruption of image acquisition.

4. During Time-lapse Acquisition

  1. Refill the open bath chamber with time-lapse embryo media every 8-12 h (at least 2 times per day) during time-lapse acquisition through the sliding doors on the laser safety box ( Figure 1I).
  2. Before opening the doors of the laser safety box, ensure that the microscope is not actively acquiring an image.
    NOTE: The use of heaters and circulating media systems is not necessary for time-lapse imaging experiments lasting 24 to 48 h. Indeed, the temperatures of both stage and in-line heaters are difficult to control, and during image acquisition the embryo exhibits an adequate development rate at a temperature range from 25-28 °C. Moreover, circulating media systems tend to overflow and potentially damage the equipment. Thus, all embryos are routinely staged post-acquisition.

5. Post-acquisition processing

  1. In the software, click on the "file" menu and choose "save".
  2. Open the file in image processing software (see the Table of Materials). Highlight the correct image series. In the software, choose the "Process" menu. Click on 3D Deconvolution and "Apply" to deconvolve each z-stack.
    NOTE: The file is large; therefore, this step may take many hours.
  3. In the software, under the "Process" menu, click on "maximum projection." Click on "Apply" to initiate maximum projection to generate 1 image per z-stack. Export each maximum projected file (1 image per z-stack) as a tiff.
  4. Import individual tiff files into video processing software. Select all tiff files and drag them into the video editor. Adjust length of each image within the video to 0.1s. Export video as mov or mp4 file.

Risultati

Time-lapse imaging multi-fotone di fluorescenza generata una serie di video che ha rivelato i modelli di migrazione delle cellule neurali craniche della cresta che danno origine alle strutture craniofacial e segmento anteriore dell'occhio nel Tg (sox10:EGFP) e Tg (foxd3:GFP) linee di zebrafish. Ad esempio, sox10 -positivo dalla cresta neurale cellule tra 12 e 30 hpf migrano dal bordo del tubo neurale nella regione craniofacial (Video 1,

Discussione

Time-lapse di immagini multi-fotone consente il monitoraggio in vivo di popolazioni cellulari transitori e migratori. Questa potente tecnica può essere utilizzata per studiare i processi embrionali in tempo reale, e nello studio presente, i risultati di questo metodo ha migliorato la conoscenza attuale di sviluppo e migrazione delle cellule neurali della cresta. Precedenti studi di imaging time-lapse in genere utilizzano la microscopia a scansione laser confocale. 29 ,

Divulgazioni

Questo lavoro è stato sostenuto finanziariamente attraverso borse di studio dal National Eye Institute del National Institutes of Health (K08EY022912-01) e Vision Research Core (P30 EY007003).

Riconoscimenti

Gli autori ringraziano Thomas Schilling per gifting gentilmente il pesce di Tg (sox10:eGFP) e Mary Halloran per gifting gentilmente il pesce(foxd3:GFP) Tg.

Materiali

NameCompanyCatalog NumberComments
Breeding Tanks with DividersAquaneeringZHCT100Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert
M205 FA Combi-ScopeLeica Microsystems CMS GmbHStereofluorescence Microscope - FusionOptics and TripleBeam
Sodium ChlorideMillipore (EMD)7760-5KGDouble PE sack. CAS No. 7647-14-5, EC Number 231-598-3
Potassium ChlorideMillipore (EMD)1049380500Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.
Calcium Chloride DihydrateFisher ScientificC79-500Poly bottle; 500 g. CAS No. 10035-04-8
Magnesium Sulfate (Anhydrous)Millipore (EMD)MX0075-1Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2
Methylene BlueMillipore (EMD)284-12Glass bottle; 25 g. Powder, Certified Biological Stain
Sodium BicarbonateMillipore (EMD)SX0320-1Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8
N-PhenylthioureaSigmaP7629-25G>98%. CAS Number 103-85-5, EC Number 203-151-2
DimethylsulfoxideSigmaD8418-500MLMolecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3
Tricaine MethanesulfonateWestern Chemical Inc.MS222Tricaine-S
Low-Melt AgaroseISC BioexpressE-3112-25GeneMate Sieve GQA Low Melt Agarose, 25 g
Open Bath ChamberWarner InstrumentsRC-40HPHigh Profile
Glass CoverslipsFisher Scientific12-545-102Circle cover glass. 25 mm diameter
High Vacuum GreaseFisher Scientific14-635-5C2.0-lb. tube. DOW CORNING CORPORATION
1658832
Quick Exchange PlatformWarner InstrumentsQE-135 mm
Stage AdapterWarner InstrumentsSA-20LZ-AL16.5 x 10 cm
TC SP5 MP multi-photon microscopeLeica Microsystems CMS GmbH
Mai Tai DeepSee Ti-Sapphire LaserSpectraPhysics
Laser Safety BoxLeica Microsystems CMS GmbH
Leica Application Suite X (LAS X)  SoftwareLeica Microsystems CMS GmbH
Photoshop CS 6 Version 13.0 x64 SoftwareAdobe
iMovie Version 10.1.4 SoftwareApple

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