This work was supported by an Ehrman fellowship to H-Y. H., a Jane Coffin Childs Memorial Fund for Medical Research postdoctoral fellowship to R.J.J., NIH Grant F32EY016309 to D.V., a New York University Dean's Dissertation Fellowship to D.J., NIH GrantR01 EY13010 to C.D. and a DFG fellowship to J.R. (RI 2208/1-1). We thank Nina Vogt and Pamela Boodram for comments on the manuscript.

1. Introduction
In this video, we describe methods for retinal dissections and immunohistochemistry at three defined developmental periods: the third instar larval, the midpupal and the adult stage. Although our protocol also works for other pupal stages (for details on earlier stages, see 16), we chose the midpupal stage, as it is optimal for imaging all PRs in one focal plane and their nuclei are easily identifiable, which facilitates the visualization of transcription factor expres...

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No conflicts of interest declared.

1. Troubleshooting
In our experience, dissections require practice (up to several weeks) and are facilitated by achieving a comfortable hand position 21 by resting the elbows and forearms on the table and with the fingers making contact with the dissection dish. That way, only the thumbs, index and middle fingers perform subtle movements.
Removing the lamina without damaging the photoreceptors is probably the most challenging step. Practice with red-eyed w...

<table border="1">
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline (PBS1x, pH 7.4)</td>
 <td>Sigma</td>
 <td></td>
 <td>Prepare 10x stock solution 20. Dilute with distilled water to obtain 1x PBS and store at room temperature. Cool on ice before dissections. </td>
 </tr>
 <tr>
 <td>Triton-X 100</td>
 <td>Sigma</td>
 <td>9002-93-1</td>
 <td>Caution: Irritant! Wear gloves. Prepare 50 ml 1xPBS with 0.3% Triton X-100 (PBST). Store at room temperature.</td>
 </tr>
 <tr>
 <td>37% formaldehyde solution </td>
 <td>Fisher Scientific</td>
 <td>F75P1GAL </td>
 <td>Caution: Toxic, probable human carcinogen! Wear gloves. Before the fixation step, freshly prepare 3.7% solution in a chemical fume hood by diluting with PBS, store on ice. </td>
 </tr>
 <tr>
 <td>5% normal horse serum </td>
 <td>Jackson Immuno Research</td>
 <td>008-000-001</td>
 <td>Prepare 5% v/v dilution in PBST. Store at four degrees.</td>
 </tr>
 <tr>
 <td>Primary and secondary antibodies (e.g. Donkey anti-sheep Alexa Fluor 488, Donkey-anti rabbit Alexa Fluor 555, Donkey anti-mouse Alexa Fluor 647) </td>
 <td>Invitrogen Molecular Probes</td>
 <td>A11015 A31572 A31571</td>
 <td>Dilute secondary antibodies 1:800 in PBST and store at four degrees. </td>
 </tr>
 <tr>
 <td>Alexa Fluor 488 Phalloidin</td>
 <td></td>
 <td>A12379 </td>
 <td>Dilute 1:100 in secondary antibody solution.</td>
 </tr>
 <tr>
 <td>Slowfade Gold Antifade reagent</td>
 <td>Invitrogen Molecular Probes</td>
 <td>S36936</td>
 <td>Mounting medium. Store at -20 degrees.</td>
 </tr>
 <tr>
 <td>Glycerol </td>
 <td>Fisher Scientific</td>
 <td>G31-1 </td>
 <td>For mounting. Prepare 10 ml of 50% dilution with distilled water, store at room temp.</td>
 </tr>
 <tr>
 <td>CO2</td>
 <td></td>
 <td></td>
 <td>For anesthetizing adult flies.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Two sharp forceps (Dumont #55)</td>
 <td>Fine Science Tools</td>
 <td>11255-20 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sylgard 184 Silicone Elastomer Kit</td>
 <td>Dow Corning</td>
 <td></td>
 <td>Prepare Sylgard dissection dish by filling a plastic Petri dish with Sylgard mixture.</td>
 </tr>
 <tr>
 <td>Three-well glass dissection dishes</td>
 <td>Fisher Scientific</td>
 <td>21-379 </td>
 <td></td>
 </tr>
 <tr>
 <td>Two minutien dissecting pins (0.1 mm diameter) and two pinholders (12 cm)</td>
 <td>Fine Science tools </td>
 <td>26002-10</td>
 <td>Insert one minutien pin in each of the two pinholders and bend one of the pins to form a hook.</td>
 </tr>
 <tr>
 <td>Microscope cover slips (22x22-1 and 24x40-1)</td>
 <td>Fisher Scientific</td>
 <td>12-542A</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope slides (25x75x1.0 mm; precleaned)</td>
 <td>Fisher Scientific</td>
 <td>12-550-143</td>
 <td></td>
 </tr>
 <tr>
 <td>1.5 ml microcentrifuge tubes</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Clear nail polish or Scotch tape</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Orbital shaker</td>
 <td>Bellco</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Slide holder box</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>Bemis</td>
 <td>PM996</td>
 <td></td>
 </tr>
 <tr>
 <td>Thin paintbrush </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>Dissecting microscope</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Small bucket with ice</td>
 <td></td>
 <td></td>
 <td>For cooling the glass well plates, dissected retinas and solutions.</td>
 </tr>
 </table>

dissection and immunohistochemistry of larval, pupal and adult drosophila retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Watch this Scientific Journal Video about Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas at JoVE.com

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

dissection-immunohistochemistry-larval-pupal-adult-drosophila

Research

JoVE Journal

Neuroscience

25.0K visualizações.  New York University. O Drosophila é uma estrutura cristalina do tipo composto por um pequeno número de tipos de células que são geradas de um modo estereotipado 1. Sua receptividade à análise genética sofisticada permite o estudo de complexos programas de desenvolvimento. Este protocolo descreve dissecações e imunohistoquímica de retinas em três distintas fases de desenvolvimento, com foco na diferenciação de fotorreceptores.