Name: preparing developing peripheral olfactory tissue for molecular and immunohistochemical analysis in drosophila
Uploaded: 2018-06-13T14:30:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila at JoVE.com

This study was supported by a grant from the National Science Foundation to Pelin C. Volkan (DEB-1457690).

The following protocol is consistent with the ethics guidelines established by the Duke University Research Ethics Committee.
1. Tissue Preparation and Developmental Staging of Drosophila melanogaster Pupa
<ol>
	<li>First, identify how many flies will be necessary for the dissection. For RT-PCR and RNAseq, collect 100 - 200 antennal discs and antennae from individuals which are necessary at prepupal, 8 h APF, 40 h APF, and adult stages. For immunohistochemistry, dissect 10 - 20 individual.</li>
	<li>Clean all materials, including dissection pads and forceps, using wipes with 70% EtOH. If the dissected material is to be used for RNA extractions, clean everything with RNase neutralizers and make all solutions using either nuclease-free or diethyl pyrocarbonate (DEPC) treated water. 
	NOTE: This protocol uses silicone dissection pads instead of glass for the dissections. Silicone pads are friendly to ultra-fine forceps and promote fast and high-quality dissections.</li>
	<li>Collect pupa at the 0 h APF/prepupal stage.
	<ol>
		<li>To ensure the most precise staging, monitor the larvae at 30 min to 1 h intervals. 
		NOTE: Wandering third instar larvae in a moderately crowded bottle will likely pupate within a few hours.</li>
		<li>Once the larvae become immobile and are surrounded by a very light (almost white) colored pupal case, transfer them into a small 2-in Petri dish with a moist filter paper.</li>
		<li>Continue for 1 - 2 h, occasionally monitoring, identifying, and transferring prepupae. 
		NOTE: Alternatively, clear a bottle of all pupae and allow the larvae to develop for 2 h. All pupae collected after 2 h will be within a 0 - 2 h APF range.</li>
	</ol>
	</li>
	<li>Immediately move to step 3.1 to dissect the prepupal antennal disc for the 0 h APF stage. If the prepupa needs to be aged, collect them into a dish, cover the dish and place a paraffin film around the edges to inhibit dryness, and place the Petri dish at 25 &#176;C. 
	NOTE: The pupae can now be aged to eclosion.</li>
	<li>For pupae collected in a 2 h range, age the prepupae for 2 h less than the desired age in order to ensure that the pupae do not overage.</li>
	<li>Use ultra-fine forceps (Dumont 55) as the tissue is very small and fragile.</li>
</ol>
2. Tissue Preparation and Developmental Staging of Pupa from Other Drosophila Species
<ol>
	<li>To determine the length of the pupal development in other Drosophila species, place the samples at the optimum temperature, record the dates when prepupae are observed, sort them into a fresh vial, and record the number of days from prepupa to adulthood.</li>
	<li>Record the ratio of time for pupal development for non-melanogaster species and for melanogaster. Multiply the ratio of the developmental time by the number of hours used for staging melanogaster to obtain the number of hours to age the non-melanogaster species.</li>
</ol>
3. Dissection of D. melanogaster Pupal Antennal Disc
Note: All the aging times and dissection protocols are described for Drosophila melanogaster. For all other species, a modification of the aging protocol based on species-specific developmental time points will be required, as described above.
<ol>
	<li>For pupal antennal disc dissections, collect 50 - 100 0 - 2 h or 6 - 8 h old pupae using a spatula and place them on a dissection pad.</li>
	<li>Pipette 150 &#181;L of RNA isolation solution into an RNase free 1.5-mL microfuge tube using a 200-&#181;L pipette and place the tube on ice.</li>
	<li>Dissect the pupae in 150 - 200 &#181;L of 1x PBS (phosphate buffered saline, nuclease-free). Place multiple droplets of PBS (150 - 200 &#181;L per droplet) on the dissection pad.</li>
	<li>On the dissection pad, place the 0 - 2 h or 6 - 8 h aged pupae (one pupa per drop) to be dissected in one of the 1x PBS droplets. Use forceps in one hand to stabilize the body.</li>
	<li>For the 0 - 2 h aged pupae, using forceps in the other hand, hold on to the most anterior part (the mouth hooks) of the prepupa and pull it out to expose the brains and the imaginal discs attached to the anterior pupal case.</li>
	<li>For the 8 h APF pupae, first gently peel off the pupal case from the most anterior quarter of the pupa, exposing the head within the membrane sack that surrounds the body. 
	NOTE: The body, at this stage, looks like a degenerating larva.</li>
	<li>Remove the head at the neck joint. 
	NOTE: This ensures that the antennal discs will not be lost, which at this stage are connected primarily to the optic lobes of the brain.</li>
	<li>Transfer the tissue with the brains and the imaginal discs to another 1x PBS droplet using forceps or a 20-&#181;L pipette.</li>
	<li>Identify the eye-antennal disc connected to the brain at the optic lobes. Using forceps, disconnect the eye-antennal disc from the brain and the pupal case. Transfer it to a new 1x PBS droplet using forceps or a 20-&#181;L pipette. 
	NOTE: The prepupal eye-antennal disc is a flat tissue. However, by the 8 h pupae, the eye discs are rotated cupping the antennal discs, which sits anterior to the central brain.</li>
	<li>Using forceps, cut the tissue at the site of connection between the eye and the antennal disc. Use a 20-&#181;L pipette to gently transfer the dissected tissue into the RNA isolation solution reagent. Minimize the amount of liquid transferred by pipetting an amount as small as possible.</li>
	<li>Repeat until all pupae have been dissected.</li>
</ol>
4. Dissection of D. melanogaster Mid-pupal and Adult Antennae
Note: By 40 h APF, the majority of the metamorphosis is complete, and the adult structures are becoming apparent, but are still white and nondescript. At this developmental time point, the adult antenna has taken its final shape yet still is transparent and the majority of the olfactory receptors, except for a few, have not started expression.
<ol>
	<li>For pupal antennal dissections, collect 50 - 100 prepupae as described in step 1 and age them for 40 h at 25 &#176;C.</li>
	<li>Pipet 150 &#181;L of RNA isolation solution into an RNase free 1.5-mL microcentrifuge tube using a 200-&#181;L pipet and place the tube on ice.</li>
	<li>On the dissection pad, place the pupa to be dissected into one of the 1x PBS droplets. Use forceps in one hand to stabilize the body. Using forceps in the other hand, gently peel off the pupal case from the most anterior quarter of the pupa, exposing the head within the membrane sack that surrounds the body.</li>
	<li>Using forceps, carefully cut off the head from the rest of the pupal body by holding the body with one set of forceps and ripping the head off at the neck joint with another set of forceps. Gently transfer the head into another 1x PBS droplet using forceps. Pipette up and down to clean out the contents of the head (the brain, etc.) to obtain a clear membrane that used to surround the developing Drosophila head. 
	NOTE: At 40 - 50 h APF, the antennae are connected to the anterior-most section of the membrane. They will become apparent for dissection when the contents of the head are cleared with pipetting.</li>
	<li>Using forceps, disconnect the pupal antennae from the membrane. Disconnect the 2nd segment of the antenna from the 3rd using forceps to slice at the segmental joint, as only the 3rd segment will house the olfactory receptor neurons.</li>
	<li>Use a 20-&#181;L pipette to gently transfer the dissected tissue into the RNA isolation solution reagent. Minimize the amount of liquid transferred by pipetting an amount as small as possible. Repeat until all pupae have been dissected.</li>
	<li>For adult antennal dissections, collect approximately 50 adults and age them for a week post-eclosion. 
	NOTE: The expression of olfactory receptor genes and other genes peak a week after eclosion. The adults are aged for a week to ensure biologically relevant genes with low abundance transcripts to rise above the detection threshold.</li>
	<li>For RNA extractions, dissect flies while they are still alive on a CO2 pad. Treat the CO2 pad with RNase inhibitors. For confocal imaging, dissect the flies on a dissection pad in 1x PBS or PBS + 0.2% Triton.</li>
	<li>First, put the adults to sleep on the CO2 station pad under the dissection scope. On the CO2 station pad, using forceps in one hand to stabilize the body, use forceps in the other hand to gently pull/pinch out the third antennal segment from the second.</li>
	<li>Transfer the antennae into an RNA isolation solution, using forceps to place the antennae directly into the liquid. Repeat until all adults have been dissected. 
	NOTE: Ensure that the antennae are submerged in RNA isolation solution. The antennae may become stuck on the side of the tube. This will cause an increased degradation of the RNA, reducing RNA quality and quantity.</li>
	<li>Directly proceed to the RNA extraction or place the tube at -80 &#176;C for long-term storage.</li>
</ol>
5. RNA Isolation from Dissected Tissues
<ol>
	<li>Remove all samples from the -80 &#176;C storage and thaw them on ice.</li>
	<li>Briefly spin (5 - 6 s, ~6,000 x g) each sample to collect the material at the bottom of the tube.</li>
	<li>Grind each sample using an autoclaved plastic pestle and an electric motor for 10 s on ice.</li>
	<li>Repeat steps 5.2 and 5.3 4.</li>
	<li>Perform the RNA extraction using commercially available kits and following the manufacturer&#39;s protocols for an optimal yield.</li>
	<li>Perform qRT-PCR using commercially available reagents and specific primers against genes of interest and following the manufacturer&#39;s protocols.
	<ol>
		<li>Carry out all PCR reactions using the following conditions: 95 &#176;C denaturation for 10 min followed by 40 cycles of 95 &#176;C for 15 s, 55 &#176;C for 30 s, and 60 &#176;C for 30 s. 
		NOTE: Primer pairs for DIP and dpr genes are listed in Table 1, and primers for olfactory receptor genes are listed in Hueston CE et al.8.</li>
	</ol>
	</li>
</ol>
6. Fixation of the Pupal Antennal Discs and Antennae for Immunohistochemistry
NOTE: Fix the tissues in batches from 10 - 20 individuals to ensure the tissues are fixed for the same amount of time. Minimize the amount of time the samples stay in PBT (PBS with detergent) prior to transferring them to a fixative to maximize the integrity of the tissues.
<ol>
	<li>Collect the dissected antennal discs or pupal antennae in a 200-&#181;L PCR tube containing 200 &#181;L of 0.2% PBT on ice to maintain tissue integrity, and to avoid the sample from sticking to the wall of 200-&#181;L PCR tubes which will lead to an incomplete fixation. 
	Note: Pupal antennae and antennal discs can be difficult to see as they are quite translucent. It is advisable to use a dissection scope for all washing steps to prevent any loss of tissue. Alternatively, 96-well Terasaki plates may be used for the fixation and staining to best track the tissue.</li>
	<li>Fix the dissected antennal disc and antennal samples from pupae in approximately 200 &#181;L of 4% PFA (Paraformaldehyde) for 30 min at room temperature. For this and subsequent steps, keep the tubes with the samples on a nutator to properly mix the samples in the solution. Progress to step 6.7.</li>
	<li>For the adult antenna, dissect the entire head first and fix it in approximately 200 &#181;L of 4% PFA (paraformaldehyde) for 1 h at room temperature.</li>
	<li>Wash 3x with 200 &#181;L of 0.2% PBT for 10 min each. Keep the samples on the nutator during the incubation.</li>
	<li>Go back to the dissection microscope for a finer dissection of the second antennal segment from the fixed heads. Using forceps to stabilize the head, gently pull/pinch out the third antennal segment from the second.</li>
	<li>Transfer the dissected third antennal segments into approximately 200 &#181;L of 4% PFA (paraformaldehyde) and fix them for 30 min at room temperature.</li>
	<li>Wash 3x with 200 &#181;L of 0.2% PBT for 10 min each. Keep the samples on the nutator during the incubation.</li>
	<li>Store the samples at 4 &#176;C or continue directly with whole mount immunohistochemistry. 
	Note: The efficiency of staining might change when the antibody is being used. This method should be appropriate for less abundant protein tags or weaker antibodies. However, at times, it might require an optimization of the protocol (either the identity or amount of the fixative or the time of fixation).</li>
</ol>

<ol>
	<li>Barish, S., Volkan, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+olfactory+receptor+neuron+specification+in+Drosophila.">Mechanisms of olfactory receptor neuron specification in Drosophila.</a> Wiley Interdisciplinary Reviews. Developmental Biology. 4 (6), 609-621 (2015).</li><li>Pan, J. W., Volkan, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+development+and+evolution+of+the+insect+olfactory+system.">Mechanisms of development and evolution of the insect olfactory system.</a> Cell and Developmental Biology. 2, 130 (2013).</li><li>Ramdya, P., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+olfactory+systems+on+the+fly.">Evolving olfactory systems on the fly.</a> Trends in Genetics. 26 (7), 307-316 (2010).</li><li>Bellen, H. J., Tong, C., Tsuda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=100+years+of+Drosophila+research+and+its+impact+on+vertebrate+neuroscience:+a+history+lesson+for+the+future.">100 years of Drosophila research and its impact on vertebrate neuroscience: a history lesson for the future.</a> Nature Reviews Neuroscience. 11 (7), 514-522 (2010).</li><li>Knecht, Z. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+combinations+of+variant+ionotropic+glutamate+receptors+mediate+thermosensation+and+hygrosensation+in+Drosophila.">Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila.</a> eLife. 5, 44-60 (2016).</li><li>Enjin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humidity+sensing+in+Drosophila.">Humidity sensing in Drosophila.</a> Current Biology. 26 (10), 1352-1358 (2016).</li><li>Li, Q., Barish, S., Okuwa, S., Volkan, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+endogenous+rotund+expression+and+function+in+developing+Drosophila.+olfactory+system+using+CRISPR-Cas9-mediated+protein+tagging.">Examination of endogenous rotund expression and function in developing Drosophila. olfactory system using CRISPR-Cas9-mediated protein tagging.</a> G3: Genes|Genomes|Genetics. 5 (12), 2809-2816 (2015).</li><li>Hueston, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modulatory+proteins+and+olfactory+receptor+signaling+in+the+refinement+and+maintenance+of+fruitless+expression+in+olfactory+receptor+neurons.">Chromatin modulatory proteins and olfactory receptor signaling in the refinement and maintenance of fruitless expression in olfactory receptor neurons.</a> PLoS Biology. 14 (4), 1002443 (2016).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+rules+of+precursor+specification+underlying+olfactory+neuron+diversity.">Combinatorial rules of precursor specification underlying olfactory neuron diversity.</a> Current Biology. 23 (24), 2481-2490 (2013).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functionally+conserved+gene+regulatory+network+module+governing+olfactory+neuron+diversity.">A functionally conserved gene regulatory network module governing olfactory neuron diversity.</a> PLoS Genetics. 12 (1), 1005780 (2016).</li><li>Pan, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+transcriptional+parallelism+and+variation+in+the+developing+olfactory+system+of+Drosophila+species.">Patterns of transcriptional parallelism and variation in the developing olfactory system of Drosophila species.</a> Scientific Reports. 7 (1), 8804 (2017).</li><li>Pan, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+behavioral+and+transcriptional+variation+underlying+CO2+sensory+neuron+function+and+development+in+Drosophila.">Comparative analysis of behavioral and transcriptional variation underlying CO2 sensory neuron function and development in Drosophila.</a> Fly. 11 (4), 239-252 (2017).</li><li>Stern, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+transgenic+reagents+for+Drosophila+simulans,+D.+mauritiana,+D.+yakuba,+D.+santomea,+and+D.+virilis.">Genetic and transgenic reagents for Drosophila simulans, D. mauritiana, D. yakuba, D. santomea, and D. virilis.</a> G3: Genes|Genomes|Genetics. 7 (4), 1339-1347 (2017).</li></ol>

The authors have nothing to disclose.

This protocol is a useful resource for labs interested in identifying the genetic and transcriptional programs operating in developing Drosophila olfactory tissue, as well as a comparative analysis of these programs across Drosophila species. It is especially useful if systems-level transcriptional profiles from different developmental stages are needed as a primer for future studies. The protocol described here demonstrates how to stage and isolate developing olfactory tissue from Drosophila t...

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior studies, and behavioral evolution1,2,3,4. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons1,5,6. The overall goal of this manuscript is to demonstrate how to stage and dissect developing pupal and adult olfactory tissue in Drosophila species. The rationale for this technique is to generate samples from different pupal stages for molecular and developmental analyses of the peripheral olfactory system, such as RNAseq and quantitative RT-PCR, in addition to in vivo tissue labeling techniques. This technique can be especially useful to identify dynamics of transcriptional profiles in the developing adult olfactory system from non-melanogaster Drosophila species, which do not have all the transgenic reagents for cell type specific labeling and analyses. In this manuscript, we demonstrate how to dissect antennal discs and antennae from pupal and adult Drosophila species. First, we show how to identify Drosophila 0 h of puparium formation (APF, white pupae or prepupae) for developmental staging and species-specific aging. Next, we show how to dissect antennal discs and the third antennal segment; specifically, how to dissociate them from the eye disc and second antennal segment for the tissue purity required for RNAseq or RT-PCRs. The stages in D. melanogaster described in this protocol roughly correspond to the pre-patterned antennal disc (prepupae), the selection of precursors from the antennal disc (8 h APF), the start of the terminal differentiation for ORNs (40 h APF), and adult antenna. Finally, we give examples for a quantitative RT-PCR from adult antennae isolated using the method, and for in vivo immunohistochemistry. This method can be used to generate samples for an RNA/DNA analysis and for immunohistochemistry on developing peripheral olfactory tissues from different Drosophila species.

<table><tbody><tr><td>10X Phosphate-buffered Saline</td><td>Gibco</td><td>70011-044</td><td>Dilute to 1X in RNase free water</td></tr><tr><td>CO&lt;sub&gt;2 &lt;/sub&gt;tank</td><td>Air Gas</td><td>CD R200</td><td></td></tr><tr><td>Two sharp forceps (Dumont #55)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-026</td><td>RNA isolation solutions</td></tr><tr><td>Dissecting Microscope</td><td></td><td></td><td></td></tr><tr><td>Small bucket with ice</td><td></td><td></td><td></td></tr><tr><td>P20 and P200 micropipettes and tips</td><td></td><td></td><td></td></tr><tr><td>1.7 mL Microfuge tubes</td><td></td><td></td><td>Purchase nuclease free for best results</td></tr><tr><td>0.2 mL PCR tubes</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde powder</td><td>Polysciences</td><td>380</td><td></td></tr><tr><td>10% Triton X-100</td><td>Teknova</td><td>T1105</td><td>Dilute to 0.2% v/v in 1X PBS</td></tr><tr><td>2&quot; petri dishes</td><td></td><td></td><td></td></tr><tr><td>55mm filter paper</td><td>Whatman</td><td>1001-055</td><td>Wet with water and place in a petri dish to keep pupae moist</td></tr><tr><td>25 C incubator</td><td></td><td></td><td></td></tr><tr><td>deionized H2O</td><td></td><td></td><td>Purchase nuclease free or treat with DEPC</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td></td><td>to be used for making dissection pads from Terizaki plate covers.</td></tr><tr><td>MicroWell Mini Trays with lids</td><td>Nunc</td><td>438733</td><td>Lids can be used to make silicone dissection pads</td></tr><tr><td>Rabbit anti-GFP antibody</td><td>MBL international corpor</td><td>PM005</td><td>primary antibody</td></tr><tr><td>Mouse anti RAT CD2</td><td>AbD seroTec</td><td>MCA154RHDL</td><td>primary antibody</td></tr><tr><td>ADL195 (anti lamin, mouse)</td><td>Dev studies hydoma ban</td><td>ADL195-s</td><td>primary antibody</td></tr><tr><td>Alexa Fluor&amp;reg; 488 goat anti-rabbit IgG (H+L)</td><td>invitrogen</td><td>A11008</td><td>Secondary antibody</td></tr><tr><td>Cy3 Goat Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>115-166-003</td><td>Secondary antibody</td></tr><tr><td>Triton X-100, Protein Grade Detergent, 10% Solution, Sterile-Filtered</td><td>CALBIOCHEM</td><td>648463</td><td>detergent</td></tr><tr><td>Lab Rotator</td><td>Thermo scientific</td><td></td><td>Rotator</td></tr><tr><td>Nuclease Free Water</td><td>Growcells.com</td><td>NUPW-0125</td><td>Water</td></tr><tr><td>Light-Duty Tissue Wipers</td><td>VWR</td><td>82003-822</td><td>For cleaning dissection supplies</td></tr><tr><td>Superscript II</td><td>invitrogen</td><td>18064014</td><td>Reverse Transcriptase&amp;nbsp;</td></tr><tr><td>QIAshredder (50)</td><td>RNA extraction</td><td>QIAGEN</td><td>79654</td></tr><tr><td>Oligo(dT)12-18 Primer</td><td>RT</td><td>life technologies</td><td>18418012</td></tr><tr><td>RNeasy MinElute Cleanup Kit (50)</td><td>RNA extraction</td><td>QIAGEN</td><td>74204</td></tr><tr><td>FASTSTART UNIV SG MASTER (5 ML)(500 RXN)</td><td>qPCR</td><td>Roche</td><td>04913850001</td></tr><tr><td>FASTSTART ESSENTIAL GREEN DNA MASTER&amp;nbsp;</td><td>qPCR</td><td>Roche</td><td>06402712001</td></tr><tr><td>LIGHTCYCLER 480 - MULTIWELL PLATE 96</td><td>qPCR</td><td>Roche</td><td>04729692001</td></tr><tr><td>NEBNext High-Fidelity 2X PCR Master Mix</td><td>qPCR</td><td>NEB</td><td>M0541S</td></tr></tbody></table>

preparing developing peripheral olfactory tissue for molecular and immunohistochemical analysis in drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

The dissected developing olfactory tissue can be used for RNA extraction followed by RT-PCR to assess gene expression or can be taken through immunohistochemistry protocols to determine its expression pattern, and the sub-cellular localization of genes of interest. In this section, we present representative results from either protocol. Figure 1 is the representative quantitative RT-PCR showing the transcription of cell surface receptor and olfactory receptor...

Watch this Scientific Journal Video about Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila at JoVE.com

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

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5.7K Views.  Duke University. Hier presenteren we een protocol om te stagen en ontleden olfactorische weefsel van Drosophila diersoorten te ontwikkelen. Het ontleed weefsel kan later worden gebruikt voor moleculaire analyses, zoals kwantitatieve RT-PCR (Reverse transcriptie-Polymerase Chain Reaction) of RNA sequencing (RNAseq), evenals in vivo analyses uitgevoerd, zoals immunohistochemistry of in situ hybridisatie.

Video: Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Neuroscience

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

Behavior

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

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.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

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 ...

Biology

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Olfactory Experience-Induced Synaptic Remodeling in Drosophila's Juvenile Brain

Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent, temporally restricted, and transiently reversible only in a short, well-defined critical period. The directionality of brain circuit synaptic connectivity remodeling is determined by the specific odorant acting on the respondent receptor class of olfactory sensory neurons. In general, each neuron class expresses only a single odorant receptor and innervates a single olfactory synaptic glomerulus. In the Drosophila genetic model, the full array of olfactory glomeruli has been precisely mapped by odorant responsiveness and behavioral output. Ethyl butyrate (EB) odorant activates Or42a receptor neurons innervating the VM7 glomerulus. During the early-life critical period, EB experience drives dose-dependent synapse elimination in the Or42a olfactory sensory neurons. Timed periods of dosed EB odorant exposure allow investigation of experience-dependent circuit connectivity pruning in juvenile brain. Confocal microscopy imaging of antennal lobe synaptic glomeruli is done with Or42a receptor-driven transgenic markers that provide quantification of synapse number and innervation volume. The sophisticated Drosophila genetic toolkit enables the systematic dissection of the cellular and molecular mechanisms mediating brain circuit remodeling.

Author Spotlight: Exploring Glial Influence in Experience-Dependent Synaptic Pruning During Critical Periods

We describe here methods for inducing and analyzing olfactory experience-dependent remodeling of antennal lobe synaptic glomeruli in the Drosophila juvenile brain.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

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