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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • النتائج
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

تم تنفيذ مزيج من التقنيات البصرية المتقدمة في ليزر المسح المجهري مع الطول الموجي الفوتوني متعدد الأسفار الإثارة منذ وقت طويل لالتقاط التصوير عالية الدقة، ثلاثي الأبعاد، في الوقت الحقيقي للهجرة crest العصبي في تيراغرام (sox10:EGFP) والأجنة الزرد تيراغرام (foxd3:GFP).

Abstract

العين الخلقية والشذوذ الجمجمة تعكس اضطرابات في قمة العصبية، عدد سكانها عابر للخلايا الجذعية المهاجرة التي تؤدي إلى العديد من أنواع الخلايا في جميع أنحاء الجسم. فهم بيولوجيا crest العصبي كان محدودا، مما يعكس عدم وجود نماذج أصعب وراثيا التي يمكن دراستها في فيفو وفي الوقت الحقيقي. الزرد نموذج إنمائي أهمية خاصة لدراسة السكان خلية المهاجرة، مثل قمة العصبية. لدراسة الهجرة crest العصبي في العين النامية، تم تنفيذ مزيج من التقنيات البصرية المتقدمة في ليزر المسح المجهري مع الإثارة fluorescence فوتون متعدد الطول الموجي الطويل التقاط الفيديو عالية الدقة، ثلاثي الأبعاد، في الوقت الحقيقي للعين النامي في الأجنة المعدلة وراثيا الزرد، هما تيراغرام (sox10:EGFP) وتيراغرام (foxd3:GFP)، كما قد ثبت في العديد من النماذج الحيوانية لتنظيم المبكر crest العصبي التمايز والمرجح أن تمثل علامات للخلايا العصبية كريست sox10 و foxd3 . تصوير الوقت الفاصل بين فوتون متعددة استخدمت لتمييز السلوك وأنماط الهجرة اثنان السكان خلية crest العصبي تسهم في الإسراع بتطوير العين. هذا البروتوكول يوفر المعلومات لتوليد الوقت الفاصل بين أشرطة الفيديو أثناء الهجرة crest العصبي الزرد، على سبيل مثال، ويمكن تطبيقها أيضا على تصور التطور المبكر للعديد من الهياكل في الزرد وغيرها من الكائنات نموذج.

Introduction

أمراض العيون الخلقية يمكن أن تسبب عمي الطفولة وغالباً ما تكون بسبب شذوذ قمة العصبية الجمجمة. هي الخلايا العصبية كريست عابرة من الخلايا الجذعية التي تنشأ من الأنبوب العصبي وتشكل أنسجة عديدة في جميع أنحاء الجسم. 1 , 2 , 3 , 4 , 5 الخلايا العصبية كريست، المستمدة من بروسينسيفالون وميسينسيفالون، تؤدي إلى العظام والغضاريف ميدفيس في المناطق الأمامية، وايريس، القرنية، ميشوورك ترابيكولار، والصلبة في الجزء الأمامي من العين. 4 , 6 , 7 , 8 خلايا crest العصبي من النموذج رهومبينسيفالون بلعومية الأقواس والفك، والمسالك تدفق القلب. 1 , 3 , 4 , 9 , 10 قد أبرزت الدراسات بمساهمات قمة العصبية للعين والتنمية بيريوكولار، مؤكدا على أهمية هذه الخلايا في التنمية عين الفقاريات. وفي الواقع، انقطاع الهجرة الخلية كريست العصبية والتمايز تؤدي إلى الشذوذ الجمجمة والعين كما هو ملاحظ في متلازمة أكسينفيلد-ريجر ومتلازمة بيترز بالإضافة إلى. 11 , 12 , 13 , 14 , 15 , 16 , 17 تقديم فهم شامل للهجرة، وانتشار والتفريق بين هذه الخلايا العصبية كريست هكذا، في نظرة ثاقبة التعقيدات الكامنة وراء أمراض العيون الخلقية.

الزرد كائن نموذج قوية لدراسة التنمية العين، هياكل العين الزرد مماثلة لنظرائهم في الثدييات، وتقحم حفظت العديد من الجينات بين الزرد والثدييات. 18 , 19 , 20 ، وباﻹضافة إلى ذلك، الأجنة الزرد شفافة والاباضه، تسهيل تصور التنمية العين في الوقت الحقيقي.

التوسع في الأعمال المنشورة سابقا، وصفت6،،من720 نمط الهجرة من الخلايا العصبية كريست استخدام فوتون متعدد الأسفار الوقت الفاصل بين التصوير على خطوط الزرد المعدلة وراثيا المسمى مع البروتين الفلورية الخضراء (بروتينات فلورية خضراء) تحت سيطرة جمهورية يوغوسﻻفيا (تحديد الجنس منطقة Y) النسخي-مربع 10 (sox10) أو "مربع فورخيد" D3 المناطق التنظيمية الجينات (foxd3). 21 , 22 , 23 , 24-فوتون متعدد الأسفار الوقت الفاصل بين التصوير هو تقنية قوية أن يجمع بين التقنيات البصرية المتقدمة لليزر المسح المجهري مع طويلة الطول الموجي الفوتوني متعدد الأسفار الإثارة لالتقاط الصور ذات الدقة العالية، وثلاثي الأبعاد للعينات الموسومة fluorophores. 25 , 26 , 27 استخدام الليزر متعددة فوتون مزايا متميزة أكثر مجهرية [كنفوكل] القياسية، بما في ذلك اختراق الأنسجة المتزايدة وتبيض فلوروفوري انخفض.

باستخدام هذا الأسلوب، كان السكان مميزة اثنين من الخلايا العصبية كريست متفاوتة في توقيت الهجرة ومسارات الهجرة crest العصبي الذين يتعرضون للتمييز، وهي: foxd3-إيجابية الخلايا في ميسينتشيمي بيريوكولار والعين النامية وكريست العصبية الإيجابية sox10 خلايا mesenchyme الجمجمة. مع هذا الأسلوب، وهو عرض نهج لتصور الهجرة الهجرة crest العصبي العين والجمجمة في الزرد، مما يجعل من السهل لمراقبة الهجرة الخاضعة للتنظيم العصبي كريست في الوقت الحقيقي أثناء التطوير.

هذا البروتوكول يوفر المعلومات لتوليد الوقت الفاصل بين أشرطة الفيديو أثناء تطوير العين مبكرا في تيراغرام (sox10:EGFP) والزرد المعدلة وراثيا تيراغرام (foxd3:GFP)، على سبيل مثال. يمكن تطبيق هذا البروتوكول كذلك للمرئيات عالية الاستبانة، ثلاثي الأبعاد، في الوقت الحقيقي للإسراع بتطوير أي بنية العين والجمجمة المستمدة من الخلايا العصبية كريست في الزرد. وعلاوة على ذلك، يمكن كذلك تطبيق هذا الأسلوب لتصور تطوير الأنسجة والأعضاء في الزرد وغيرها نماذج حيوانية أخرى.

Protocol

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.

النتائج

تصوير الوقت الفاصل بين فوتون متعدد الأسفار ولدت سلسلة من أشرطة الفيديو التي كشفت عن أنماط الهجرة الجمجمة العصبي كريست الخلايا التي تؤدي إلى الهياكل الجمجمة والجزء الأمامي من العين في تيراغرام (sox10:EGFP) وتيراغرام (foxd3:GFP) خطوط الزرد. على سبيل مثال، ترحيل sox10خلا...

Discussion

تصوير الوقت الفاصل بين فوتون متعددة يمكن التتبع في فيفو السكان خلية العابرة والمهاجرة. يمكن استخدام هذه التقنية القوية لدراسة العمليات الجنينية في الوقت الحقيقي، وفي هذه الدراسة، نتائج هذا الأسلوب تعزيز المعارف الراهنة للهجرة الخلية crest العصبي والتنمية. عادة استخدام الدراسات الوقت...

Disclosures

وكان هذا العمل الدعم المالي من خلال المنح المقدمة من المعهد الوطني للعين للمعاهد الوطنية للصحة (K08EY022912-01) ورؤية البحوث الأساسية (P30 EY007003).

Acknowledgements

يشكر المؤلفون توماس شيلينغ للاهداء يرجى الأسماك تيراغرام (sox10:eGFP)، وماري هالوران للاهداء يرجى الأسماك تيراغرام(foxd3:GFP) .

Materials

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

References

  1. Barembaum, M., Bronner-Fraser, M. Early steps in neural crest specification. Sem Cell Dev Biol. 16, 642-646 (2005).
  2. Gage, P. J., Rhoades, W., Prucka, S. K., Hjalt, T. Fate maps of neural crest and mesoderm in the mammalian eye. Invest Ophthalmol Vis Sci. 46 (11), 4200-4208 (2005).
  3. Minoux, M., Rijli, F. M. Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development. Development. 137, 2605-2621 (2010).
  4. Trainor, P. A. Specification of neural crest cell formation and migration in mouse embryos. Sem Cell Dev Biol. 16, 683-693 (2005).
  5. Johnston, M. C., Noden, D. M., Hazelton, R. D., Coulombre, J. L., Coulombre, A. Origins of avian ocular and periocular tissues. Exp Eye Res. 29, 27-43 (1979).
  6. Bohnsack, B. L., Kahana, A. Thyroid hormone and retinoic acid interact to regulate zebrafish craniofacial neural crest development. Dev Biol. 373, 300-309 (2013).
  7. Chawla, B., Schley, E., Williams, A. L., Bohnsack, B. L. Retinoic acid and pitx2 regulate early neural crest survival and migration in craniofacial and ocular development. Birth Defects Res B Dev Reprod Toxicol. , (2016).
  8. Trainor, P. A., Tam, P. P. L. Cranial paraxial mesoderm and neural crest cells of the mouse embryo-codistribution in the craniofacial mesenchyme but distinct segregation in branchial arches. Development. 121 (8), 2569-2582 (1995).
  9. Hong, C. S., Saint-Jeannet, J. P. Sox proteins and neural crest development. Sem Cell Dev Biol. 16, 694-703 (2005).
  10. Steventon, B., Carona-Fontaine, C., Mayor, R. Genetic network during neural crest induction: from cell specification to cell survival. Sem Cell Dev Biol. 16, 647-654 (2005).
  11. Dressler, S., et al. Dental and craniofacial anomalies associated wth Axenfeld-Rieger syndrome with PITX2 mutation. Case Report Med. , 621984 (2010).
  12. Ozeki, H., Shirai, S., Ikeda, K., Ogura, Y. Anomalies associated with Axenfeld-Rieger syndrome. Graefes Arch Clin Exp Ophthalmol. 237 (9), 730-734 (1999).
  13. Strungaru, M. H., Dinu, I., Walter, M. A. Genotype-phenotype correlations in Axenfeld-Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations. Invest Ophthalmol Vis Sci. 48, 228-237 (2007).
  14. Tumer, Z., Bach-Holm, D. Axenfeld-Rieger syndrome and spectrum of Pitx2 and Foxc1 mutations. Eur J Hum Genet. 17, 1527-1539 (2009).
  15. Schoner, K., et al. Hydrocephalus, agenesis of the corpus callosum, and cleft lip/palate represent frequent associations in fetuses with Peters plus syndrome and B3GALTL mutations. Fetal PPS phenotypes, expanded by Dandy Walker cyst and encephalocele. Prenat Diagn. 33 (1), 75-80 (2013).
  16. Aliferis, K., et al. A novel nonsense B3GALTL mutation confirms Peters plus syndrome in a patient with multiple malformations and Peters anomaly. Ophthalmic Genet. 31 (4), 205-208 (2010).
  17. Lesnik Oberstein, ., S, A., et al. Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase. Am J Hum Genet. 79 (3), 562-566 (2006).
  18. Brittijn, S. A., et al. Zebrafish development and regeneration: new tools for biomedical research. Int J Dev Biol. 53, 835-850 (2009).
  19. Bohnsack, B. L., Kasprick, D., Kish, P. E., Goldman, D., Kahana, A. A zebrafish model of Axenfeld-Rieger Syndrome reveals that pitx2 regulation by retinoic acid is essential for ocular and craniofacial development. Invest Ophthalmol Vis Sci. 53 (1), 7-22 (2012).
  20. Williams, A. L., Eason, J., Chawla, B., Bohnsack, B. L. Cyp1b1 regulates ocular fissure closure through a retinoic acid-independent pathway. Invest Ophthalmol Vis Sci. 58 (2), 1084-1097 (2017).
  21. Curran, K., Raible, D. W., Lister, J. A. Foxd3 controls melanophore specification in the zebrafish neual crest by regulation of Mitf. Dev Biol. 332 (2), 408-417 (2009).
  22. Dutton, K., Dutton, J. R., Pauliny, A., Kelsh, R. N. A morpholino phenocopy of the colourless mutant. Genesis. 30 (3), 188-189 (2001).
  23. Dutton, K. A., et al. Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates. Development. 128 (21), 4113-4125 (2001).
  24. Kucenas, S., Takada, N., Park, H. C., Woodruff, E., Broadie, K., Appel, B. CNS-derived glia ensheath peripheral nerves and mediate motor root development. Nat Neurosci. 11, 143-151 (2008).
  25. Denk, W., Strickler, J., Webb, W. Two-photon laser scanning fluorescence microscopy. Science. 248 (4951), 73-76 (1990).
  26. Helmchen, F., Denk, W. Deep tissue two-photon microscopy. Nat Methods. 2 (12), 932-940 (2005).
  27. Denk, W., Delaney, K. Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy. J Neurosci Methods. 54 (2), 151-162 (1994).
  28. Bohnsack, B. L., Gallina, D., Kahana, A. Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle developmnt to changes in retinoic acid and insulin-like growth factor signaling. PLoS ONE. 6, e22991 (2011).
  29. Gfrerer, L., Dougherty, M., Laio, E. C. Visualization of craniofacial development in the sox10:kaede transgenic zebrafish line using time-lapse confocal microscopy. J Vis Exp. (79), e50525 (2013).
  30. Lopez, A. L. R., Garcai, M. D., Dickinson, M. E., Larina, I. V. Live confocal microscopy of the developing mouse embryonic yolk sac vasculature. Methods Mol Biol. 1214, 163-172 (2015).
  31. McGurk, P. D., Lovely, C. B., Eberhart, J. K. Analyzing craniofacial morphogenesis in zebrafish using 4D confocal microscopy. J Vis Exp. (83), e51190 (2014).
  32. Nowotschin, S., Ferrer-Vaquer, A., Hadjantonakis, A. K. Imaging mouse development with confocal time-lapse microscopy. Methods Enzymol. 476, 351-377 (2010).

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