נתיחה והתקנה<em> דרוזופילה</em> גלמי דיסקי העין
Summary
The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.
Abstract
The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.
Introduction
תחומי ביולוגיה התפתחותית ותא כבר הושפע רב מאורגניזם המודל: דרוזופילה melanogaster. בתוך מודל זה, מחקרים של דיסק העין תרמו רב איתות ידע הנוגע ל, ביולוגיה של תא ובאזורים אחרים. דיסק הזחל השלישי מאוחר עין instar נחקר בהרחבה והוא מודל רב עוצמה לנצל, כפי שהוא נותן תמונת מצב של סדרה של תקופות התפתחותיות, כל אחד עם המולקולות שלו האיתות ייחודית ותהליכים, כמו תלם המוךפו"גנטי מתקדם פני דיסק העין 8. עם זאת, יש צורך להרחיב עוד יותר את ההבנה של תהליכים התפתחותיים שלנו לשלב גלמים של פיתוח. אמנם היו מחקרים על דיסק עין גלמי 3-7, הידע שלנו לא מתקרב לרוחבה של עבודה שבוצעה על דיסק עין instar השלישי. זאת בשל, בחלקו, לקושי הגדול יותר בלנתח את דיסק עין גלמים. לכן, הצגתשיטה נכונה של נתיחה יכולה להרחיב מאוד את המחקר בתחום זה.
אמנם יש שלבים בהתפתחות דיסק עין גלמים שבקלות גזורים, במיוחד סביב תקופת אמצע גלמים-, תקופות זמן אחרות הן הרבה יותר מאתגר לנתח. פרוטוקול זה מייצג שיטה אחת ללנתח דיסקי עין גלמים שניתן להשתמש בי אוניברסלי לכל מסגרות הזמן התפתחותי גלמים. פרוטוקול זה יכול לשמש כתחליף לעוד פרוטוקול 9 שמראה שיטה קלה ומהירה יותר לנתח דיסקי עין מנקודות זמן midpupal. פרוטוקול זה צולם במקור ופיתח להכשרת סטודנטים לתואר ראשון מתקדמים באוניברסיטת UCLA לתואר ראשון קונסורציום המחקר בגנומיקה פונקציונלית (URCFG) 10,11 בטכניקה של נתיחת עין גלמים. סטודנטים לתואר ראשון רבים היו מסוגלים לנצל את וידאו ובשיטה זו כדי ללמוד את הטכניקה מאתגרת זו.
Protocol
Representative Results
Discussion
While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou…
Declarações
The authors have nothing to disclose.
Acknowledgements
We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.
Materials
| Phosphate-buffered saline (PBS, pH 7.4) | 80g NaCl, 2g KCl, 14.4g Na2HPO4, 2.4g KH2PO4, Bring volume to 1 l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1X PBS with autoclaved ddH2O before using. | ||
| Triton X-100 | Promega | H5142 | Caution: Irritant! Wear gloves. |
| 0.3% PBT | 1.5 ml of Triton X-100, 500 ml 1X PBS. | ||
| 37% formaldehyde solution | Fisher Scientific | F75P1GAL | Caution: Toxic, probable human carcinogen! Wear gloves. |
| Fix Solution | (≈4% Formaldehyde in PBS) 50 μl of 37% Formaldehyde, 450 μl 1XPBS, make fresh before use | ||
| Normal goat serum | Rockland antibodies & assays | B304 | Aliquot in 1 ml volumes and store at -80C |
| Block Solution | 10% NGS in PBT. This can be made and stored at 4 °C for a few days prior to use. | ||
| DAPI stock solution | Life Technologies | D3571 | For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH2O. |
| VectaShield Mounting Medium | Vector Labs | H-1000 | Mounting medium |
| Glycerol | Sigma | G5516 | For mounting. Prepare 70% dilution with ddH2O. |
| Equipment | |||
| Nutating mixer | VWR | 82007-202 | Used to rock tissue in 3 well glass dish |
| SylGard 182 Silicone Elastomer Kit | Krayden | NC9897184 | Used to make silicone dissection dish |
| Silicone dissecting dish | Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 °C incubator. | ||
| 3 well glass dish | Corning | 7220-85 | The 3 well variety of these are no longer available, this is the 9 well product. |
| 72 well microwell minitray | Nunc | 438733 | |
| Sharp forceps (Dumont #55) | Fine Science Tools | 11255-20 | |
| Vannas-type Micro Scissors, Straight, 5mm blade | Ted Pella | 1346 | |
| 100 mm Borosilicate glass capillaries | World Precision Instruments | 1B100-4 | Pull with needle puller to make fine point tip that allows a small stream of PBS to flow. |
| Disposable Transfer Pipets, Fine Tip | Samco Scientific | 231 | |
| Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches | |||
| PVC tubing (1/8 x 3/16 x 1/32) | Nalgene | 8000-0010 | Use these with pulled needle to assemble the blower tube as shown in Figure 2. |
| Tygon Silicone tubing (3/32 x 5/32 x 1/32) | Saint Gobain Performance Plastics | ABW00004 | |
| Tygon Silicone tubing (1/32 x 3/32 x 1/32) | Saint Gobain Performance Plastics | ABW00001 | |
Referências
- Ready, D. F., Hanson, T. E., Benzer, S. Development of the Drosophila retina, a neurocrystalline lattice. Dev Biol. 53 (2), 217-240 (1976).
- Wolff, T., Ready, D. F. The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave. Development. 113 (3), 841-850 (1991).
- Carthew, R. W. Pattern formation in the Drosophila eye. Curr Opin Genet Dev. 17 (4), 309-313 (2007).
- Bao, S., Cagan, R. Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye. Dev cell. 8 (6), 925-935 (2005).
- Grzeschik, N. A., Knust, E. IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin. Development. 132 (9), 2035-2045 (2005).
- Sprecher, S. G., Desplan, C. Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons. Nature. 454 (7203), 533-537 (2008).
- Zuker, C. S., Cowman, A. F., Rubin, G. M. Isolation and structure of a rhodopsin gene from D. melanogaster. Cell. 40 (4), 851-858 (1985).
- Voas, M. G., Rebay, I. Signal integration during development: insights from the Drosophila eye. Dev Dyn. 229 (1), 162-175 (2004).
- Hsiao, H. Y., et al. Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas. J Vis Exp. , 4347 (2012).
- Call, G. B., et al. Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes. Genética. 177 (2), 689-697 (2007).
- Chen, J., et al. Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers. PLoS Biol. 3 (2), 59 (2005).
- Wolff, T. Dissection techniques for pupal and larval Drosophila eyes. CSH Protoc. 2007, (2007).
- Williamson, W. R., Hiesinger, P. R. Preparation of developing and adult Drosophila brains and retinae for live imaging. J Vis Exp. 37, 1936 (2010).
- Xu, T., Rubin, G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development. 117 (4), 1223-1237 (1993).
- Duffy, J. B. GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis. 34 (1-2), 1-15 (2002).
