प्रौढ़ की तैयारी ड्रोसोफिला आंखें पतली सेक्शनिंग और सूक्ष्म विश्लेषण के लिए
Summary
वयस्क तैयार करने के लिए एक मानक दृष्टिकोण<em> ड्रोसोफिला</em> आँखों अर्द्ध पतली सेक्शनिंग और प्रकाश सूक्ष्म विश्लेषण के लिए यहाँ प्रस्तुत किया है. प्रोटोकॉल नेत्र दोष के सकल morphological विश्लेषण के लिए इस्तेमाल किया जा सकता है, या के साथ संकेत समायोजन के आंख की विशिष्ट प्रकार की कोशिकाओं (जैसे कि photoreceptors के प्रतिरूप विश्लेषण) में या इलेक्ट्रॉन सूक्ष्म विश्लेषण के लिए जीन की आनुवंशिक आवश्यकताओं को निर्धारित करने के लिए इस्तेमाल किया जा सकता है.
Abstract
Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface (‘rough eye’ phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells (‘hexagon’ around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six “outer” (R1-6) and two “inner” photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.
Protocol
Discussion
मॉडल जीव, आनुवंशिक जीन सहित मानव उच्च eukaryotes में अत्यधिक संरक्षित संकेत दे रास्ते के अधिकांश के लिए आवश्यक परिवारों के संस्थापक सदस्यों में से कई की पहचान करने के लिए नेतृत्व स्क्रीन के रूप में ड्रोसोफिला का उपयोग करना. के बाद से, प्रयोगशाला परिस्थितियों में, अस्तित्व के लिए एक कार्यात्मक आँख नगण्य है, नेत्र उपन्यास जीन कार्यों की खोज और आनुवंशिक नेटवर्क के आकलन के लिए एक विशेष रूप से अच्छी तरह से अनुकूल ऊतक है. इस प्रकार वर्णित विधि का उपयोग कर उड़ आँख के Ultrastructural विश्लेषण मौलिक विकास और रोग के लिए प्रासंगिक खोजों के लिए नेतृत्व किया. प्रारंभ में, एकल कक्ष clonal विश्लेषण एक्स – रे प्रेरित ज्ञात बारीकी से जुड़े सेल स्वायत्त पीछे हटने का मार्कर के साथ संयुक्त क्लोन का उपयोग कर प्रदर्शन किया था. हाल ही में, FLP / FRT प्रणाली की उपलब्धता को क्लोन उत्पन्न बहुत आँख 6,9 वर्गों में घातक परिवर्तन के प्ररूपी विश्लेषण में मदद की है.
आँख वर्गों के विश्लेषण स्पर्शरेखा यहाँ वर्णित वर्गों तक सीमित नहीं है. अगर वांछित, सिर molds में किसी भी अभिविन्यास में गठबंधन किया जा सकता है और अनुप्रस्थ वर्गों लामिना और मज्जा के रूप में इस तरह के सिर में गहरी परतों का अध्ययन करने के लिए प्राप्त किया जा सकता है. यहाँ वर्णित प्रोटोकॉल इस तरह आंख और सिर संरचनाओं ड्रोसोफिला वयस्कों के विश्लेषण के लिए एक बहुमुखी विधि है.
Disclosures
The authors have nothing to disclose.
Acknowledgements
मैं छवि में चित्रों के लिए डॉ. जेनिफर Curtiss धन्यवाद देना चाहूंगा. 1 और जेरेमी Fagan और समीक्षकों पांडुलिपि पढ़ने के लिए डॉ. फ्लोरेंस मार्लो. हमारा काम NIH अनुदान 1R01GM088202 द्वारा समर्थित है.
Materials
General equipment:
- For general fly husbandry see 10
- Standard dissection microscope (e.g. Nikon SMZ-645 Stereo Zoom Microscope), preferably with a ring light source to prevent casting a shadow of your hands.
- CO2 Fly pad, covered with 3MM Whatman paper to provide protection against dirt during dissection.
- Gelatin coated slides: dissolve 10g gelatin and 1g CrKSO4x 12 H2O in 1L water on a heating plate (bring almost to a boil, takes approximately 2 hrs). Dip well washed (soap) and rinsed (dH2O) slides in holders into gelatin solution and air-dry covered with foil overnight.
- Heating oven (70°C; old vacuum ovens that were used for nitrocellulose filter drying are well suited for this purpose).
- Microtome capable of ultrathin sectioning (0.5-1 μm thick sections), e.g. those used by electron microscopy facilities. We use a Sorvall MT5000 Ultramicrotome with a Histo quality diamond knife. Glass knives also work.
Fixation solutions:
- Prepare a stock solution of 0.2 M Sodium Phosphate buffer, pH 7.2.
- Prepare 2% glutaraldehyde in 0.1M Sodium Phosphate buffer, pH 7.2 (can be stored at 4°C for 4 weeks). Make at least enough to have 200μl per genotype you want to embed. Keep on ice.
- Osmium solution: 2% OsO4 in 0.1M Sodium Phosphate buffer, pH 7.2. Make only enough to have 200μl per genotype you want to embed, since the solution cannot be stored. Keep on ice.
Glutaraldehyde and OsO4 are highly toxic and should be handled with extreme care in a well-ventilated hood. Use filter tips when handling OsO4 to prevent blackening of your pipette.
Dehydration: Make 30%, 50%, 70%, (80%), 90%, and 100% ethanol solutions. Preferably cool 30%, 50%, 70% ethanol on ice before use.
Staining solution:
1% Toluidine-blue in 1% Borax.
| Soft | Hard | |
|---|---|---|
| Resin A | 54g | 50g |
| Hardener B | 44.5g | 50g |
| Accelerator C | 2.5g | 1.75g |
| Plasticizer D | 10g | 0.75g |
Table 1. Resin preparation
To prepare the resin, carefully, but thoroughly mix all ingredients in a plastic beaker using a magnetic stirrer with a large stir bar. Avoid bubbles. After mixing, aliquot in standard scintillation vials or similar containers and freeze at -20°C. Use soft resin for all applications unless your EM facility asks for the harder formulation. Use gloves to handle resin (carcinogenic in its unpolymerized form). To polymerize resin on equipment and waste, bake at 70°C overnight. Waste may then be safely discarded and equipment cleaned and reused. Spilled unpolymerized resin can be cleaned with isopropanol.
| Reagent | Supplier | Catalog number |
|---|---|---|
| Fly pad | e.g. Genesee | 59-119 |
| Glutaraldehyde | Sigma | G7526-10 X 10 ML |
| OsO4 | Polysciences,Inc. | 0972A-20 |
| Propyleneoxide | Fisher | 04332-1 |
| Scalpel handles, for #3 | Fisher | 22080046 |
| Scalpel blades #11 | Fisher | 08-916-5B |
| Transfer pipettes | Fisher | 13-711-7M |
| Durcupan (R) ACM resin | Sigma (Fluka) | 44610-1EA |
| Steel dissecting needle | Fisher | S17346 |
| BEEM flat embedding mold | Electron Microscopy Sci. | 70904-12 |
| Teflon coated razor blades | Electron Microscopy Sci. | 71970 |
| Q-tip (sterile swabs) | Fisher | 14-959-81 |
| Glass slides | Fisher | 12-550-143 |
| Cover slips No 1, 22X60 mm | Fisher | 12-531K |
| Gelatin | Fisher | ICN96010280 |
| Cr(III) K SO4 dodecahydrate | Sigma | 243361 |
| Diamond Histo knife, 6mm | Diatome US | 60-His |
| Toluidine Blue O | Fisher | BP107-10 |
| Borax (Na-Tetraborate) | Fisher | AC20629-1000 |
| DPX mounting medium | Sigma | 44581-100ML |
Table 2. Materials
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