फल मक्खी, ड्रोसोफिला मेलानोगास्टर, के प्रारंभिक विकास सेल आकार में परिवर्तन है कि अच्छी तरह से इमेजिंग दृष्टिकोण के लिए अनुकूल हैं की एक संख्या के द्वारा विशेषता है. यह आलेख ड्रोसोफिला भ्रूण का जीना confocal इमेजिंग के लिए आवश्यक बुनियादी उपकरणों और तरीकों का वर्णन होगा, और एक सेल आकार cellularization बुलाया परिवर्तन पर ध्यान दिया जाएगा.
The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using “embryo glue” to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.
प्रोटोकॉल वर्णित इस के साथ साथ रहते हैं, मक्खी भ्रूण के विकास में सेल आकार में परिवर्तन का एक नंबर के confocal इमेजिंग की अनुमति होगी. इमेजिंग के लिए GFP शेयरों एक व्यक्ति प्रयोगशाला (तालिका 2) द्वारा तैयार किया…
The authors have nothing to disclose.
हम कृतज्ञता एरिक Wieschaus, जो नींव है जिस पर यह प्रोटोकॉल विकसित किया गया था प्रदान स्वीकार करते हैं. हमारा काम जैव रसायन वेरना और Marrs मैकलीन विभाग और आण्विक जीवविज्ञान शुरू हुआ पुरस्कार, चिकित्सा के Baylor कॉलेज के द्वारा समर्थित है.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Slides | Fisherbrand/Fisher Scientific | 12-550-343 | ||
Cover slips 25×25 | Fisher Scientific (Corning #2865-25) | 1 2-524C | ||
Squirt bottles (H2O) | Fisher Scientific | 02-897-11 | ||
50 ml Falcon tubes | Fisher Scientific (BD# 352070) | 14-432-22 | ||
Bulbs for small pipets, 1 mL | Fisherbrand/Fisher Scientific | 03-448-21 | ||
Scintillation vials with caps | VWR (Wheaton #986546) | 66021-533 | ||
Tri-Corn Beakers, 100 mL | Electron Microscopy Sciences | 60970 | ||
BD Falcon Petri dish 60x15mm | Fisher Scientific (BD# 351007) | 08757 100B | ||
BD Falcon Cell strainer | Fisher Scientific (BD#352350) | 08-771-2 | ||
Yellow pipet tips | Ranin | L200 | ||
Stainless steel mesh, 304, 12×24 | Small Parts | CX-0150-F-01 | ||
Glass 5¾ inch Pasteur Pipets | Fisherbrand/Fisher Scientific | 13-678-20B | ||
P4 Filter paper | Fisherbrand/Fisher Scientific | 09-803-6F | ||
Rubber bands | Office Max | A620645 | ||
Scotch double-sided tape, ½ inch | Office Max | A8137DM-2 | ||
Robert Simmons Expression paint brushes E85 round #2 | Jerry’s Artarama | 56460 | ||
Dumont #5 Forceps High Precision Inox | Electron Microscopy Services | 72701-DZ | ||
Razor blades | VWR (BD #214010) | 55411-050 | ||
Halocarbon Oil 27 | Sigma-Aldrich | H 8773 | ||
Heptane | Fisher Chemical/Fisher Scientific | H360-1 | ||
BD Bacto Agar | VWR (BD# 214010) | 90000-760 | ||
Sucrose | Sigma-Aldrich | S7903 | ||
p-Hydroxybenzoic acid | Sigma-Aldrich | H5501 | ||
Red Star Active Dry Yeast | LeSaffre | 15700 | ||
Paper towels, C-fold | Kleenex | |||
Heavy duty aluminum foil | Reynolds Wrap | |||
Bleach | Austin’s A-1 Commercial | |||
100% Apple juice | Ocean Spray or Tree Top |