Fate Mapping with Lipophilic Membrane Dyes

The Peripheral Neuropathy Solution

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The lipophilic membrane dye DiI is the first-choice dye for following the fate of relatively small groups of neighboring cells in ovo. It is expensive at first glance ($190 for 100 mg), but 100 mg does go a long way.

DiI, like the other carbocyanine membrane dyes, such as DiO and DiA (Molecular Probes D-275 and D-291), is lipophilic, and thus following application to a tissue, it readily and preferentially diffuses into cell membranes rather than remaining in and diffusing through the aqueous extracellular spaces.

There are a number of ways of applying DiI to tissues, most of which initially involve dissolving the dye in a suitable solvent. Using 3 mg DiI in 1 mL of DMF in the first instance is suggested. DMSO or alcohol can be used as an alternative solvent if you suspect DMF is toxic for your cells.

3.1.1. Preparation of Embryos

Incubate eggs on their sides. Punch a small pinhole through the blunt end of the shell, and window eggs. The window needs to be large enough to enable easy access of a micropipet to the embryo. Inject ink beneath the embryo. Make a small hole in the vitelline membrane directly over the part of the embryo to be labeled, and carefully add a small drop of saline onto the embryo to prevent it from drying out. Use alcohol-cleaned instruments and sterile solutions.

3.1.2. Pressure Injection

This is the most traditional method of applying DiI to tissues in the chick embryo. It is a good way to label many cells in a small region of the embryo, but is not as suitable for analyzing the fate of small numbers of neighboring cells. Although you can use a mouthpiece to control the application of the dye, it is best to use a pressure injection device, such as the Picospritzer II, made by General Valve Corporation (Fairfield, NJ). You will also need a micromanipulator, which is firmly attached either to a base plate or directly to the microscope being used to visualize the embryo. A good stereomicroscope is essential to target accurately the embryonic tissue. To check the accuracy of application, it is a good idea to use an epifluorescent microscope fitted with a long working distance objective. If the dye deposits are not checked each time at the time of application, then it is essential that some embryos be sacrificed immediately after injection so that the initial spread of the dye can be assessed in fixed and cleared preparations. Dye is delivered via micropipets manufactured on a micropipet puller. The author uses 1-mm diameter, thin-walled borosilicate capillary glass with internal filament and pulls these capillaries to make pipets with a tip opening of approx 1.5 pm. Small aliquots of DiI can be delivered from these pipets using a pressure of 50 psi and a pulse duration of about 5 ms (see Notes 1-4).

3.1.3. Iontophoresis

This is the easiest method for labeling very small numbers of adjacent cells. It can be used to label between 1 and 10 cells. It is not recommended as a way of labeling many cells. DiI is a charged molecule and can thus be driven out of a micropipet by applying a potential difference across the pipet and using it as a microelectrode. In addition to a micropipet puller, good micromanipulator, and stereomicroscope, all you need is a 9-V battery. The negative pole of the battery should be connected to the albumin in the egg. This is easily achieved by a flexible cable attached to a thin silver wire, which is inserted into the hole in the shell created at the time of windowing the egg. The positive pole is attached to the back of the microelectrode via an electrode holder. Contact with the Dil in the electrode tip is acheived via a 1 M lithium chloride solution, which is backfilled into the electrode. The author routinely uses an electrode with a 1.5-^m tip diameter to label a few cells in the neural plate although electrodes with submicron tips also work well. The author uses 1.2-mm diameter, thin-walled borosilicate glass with internal filament. First insert the negative silver wire electrode into the albumin, and then manipulate the dye-laden microelectrode tip onto the cells to be labeled before completing the electrical circuit (see Note 4). Between 1 and 5 s of current are sufficient to label a few cells brightly (see Notes 5 and 6). The amount of dye expelled is very small and may not be visible without the aid of an epifluorescent microscope, but this technique is very effective and so can be confidently used without epifluorescence. Of course, if you want to check or measure exactly where the dye is deposited, an epifluorescent microscope with long working distance objective becomes essential.

3.1.4. Application as a Solid

To label superficial cells exclusively (e.g., ectoderm), rather than deep cells is difficult using micropipets and microelectrodes, because their sharp tips can readily slip through superficial cell layers. A better approach is to manipulate small crystals of DiI directly onto the surface cells. This can be acheived by first recrystalizing the dissolved Dil onto the blunt tip of a tiny glass rod and then touching this glass rod onto the surface to be labeled. Tiny glass rods can be made from micropipets by carefully melting and sealing their tips in a small flame or using a microforge. To recrystalize the dye onto the tip, place a small drop of Dil dissolved in either DMF of alcohol onto a clean plastic surface and wait for most of the solvent to evaporate off. As the solution becomes increasingly more concentrated and sticky, it will simply adhere to the tip of a glass rod when one is dipped into it. Gently manipulate the Dil-loaded glass rod onto the cells, and the dye will redissolve into the cell membranes.

3.1.5. Fixation, Mounting, and Viewing

The maximum survival time for the embryo and the dye will depend on the rate of dye dilution by cell division. The fate of rapidly dividing cells can readily be studied for up to 3 d, and less rapidly dividing systems for at least a week. Embryos should then be fixed in 3.5% paraformaldehyde and stored in this solution in the refrigerator until viewing. The material can be viewed as a whole-mount if the fluorescent cells are close to the surface, or it can be sectioned either

Fig. 1. (A) Cells labeled with DiI and DiA in the chick embryo neural plate and viewed immediately after iontophoretic applications. About four cells are labeled with DiI (red) and one cell with DiA (green). Bar is 10 |im. (B) After 48 h of development, the descendants of cells labeled as in (A) are still visible and their two colors distinct. Bar is 50 |im. (C) Motor neurons retrogradely labeled from their peripheral nerves in fixed tissue. One nerve was labeled with DiI, and the adjacent nerve with DiA. Bar is 40 |im. (See color plate 2 appearing after p. 368.)

Fig. 1. (A) Cells labeled with DiI and DiA in the chick embryo neural plate and viewed immediately after iontophoretic applications. About four cells are labeled with DiI (red) and one cell with DiA (green). Bar is 10 |im. (B) After 48 h of development, the descendants of cells labeled as in (A) are still visible and their two colors distinct. Bar is 50 |im. (C) Motor neurons retrogradely labeled from their peripheral nerves in fixed tissue. One nerve was labeled with DiI, and the adjacent nerve with DiA. Bar is 40 |im. (See color plate 2 appearing after p. 368.)

on a cryostat or vibratome. To increase the transparency of the tissue, the material can be cleared in 90% glycerol in PBS containing 2.5% DABCO. Observe and photograph the material as soon as possible for the best results.

3.1.6. Two Color Fate Mapping

If you want to examine the relative movements of two cell populations directly in the same embryo, then each population should be labeled with a differently colored dye. A good combination is to label one set with DiI and the other with DiA (Molecular Probes, cat. no. D-291). DiA fluoresces over a broad range of wavelengths when it is incorporated into cell membranes, but is most intense as a green emission. Using appropriate filter sets, it is thus readily distinguished from the red/orange fluorescence of DiI (Fig. 1). DiA performs better than the other commonly used green-fluorescing dye DiO (Molecular Probes, cat. no. D-275), because it is more soluble, it iontophoreses more efficiently, and it fluoresces more intensely.

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