Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Podsumowanie

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

Streszczenie

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules^1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles^3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)⁶. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development⁷.

eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development^8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium^10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation^21-24. However, the complex functions of the eCSF are largely unknown.

We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

Protokół

1. Preparing Microinjection Needles and Cell Tram

1. Fill Eppendorf CellTram oil microinjector apparatus with mineral oil according to manufacturer's instructions.
2. Prepare microinjection needles by pulling capillary tubes using Sutter instruments needle puller.
3. Mount needle on micromanipulator connected to Eppendorf CellTram.
4. Carefully break the tip of the needle. For uniform tip size, measure with a micrometer, or compare to a reference needle.
5. Fill the length of the needle with oil, using the CellTram to push oil down the length, being careful to avoid any bubbles.

2. Preparing the Embryos

1. Poke holes in a 1% agarose coated dish with a 1-200 μl pipette tip, and remove agarose plugs. Fill dish with embryo media (made according to Westerfield²⁵).
2. Using forceps, dechorionate embryos under stereomicroscope.
3. Transfer dechorionated embryos into agarose coated dish.
4. To anesthetize embryos, add Tricaine (0.1 mg/ml) to agarose dish until embryos stop moving (made according to Westerfield²⁵).

3. Draining the eCSF

1. Orient the embryos with their tails in the holes of the agarose and their posterior sides closest to the micromanipulator, allowing for visualization of the dorsal side of the brain.
2. Position the needle at the rhombomere 0/1 hinge-point or the widest point of hindbrain ventricle (Figure 1A).
3. Carefully pierce roof plate of hindbrain ventricle being sure not to go through the depth of the brain into the yolk.
4. Drain the eCSF using the CellTram and collect fluid in microinjection needle. Be careful to avoid any cells.

4. Collecting the eCSF for Composition Analysis

1. Once the eCSF is collected in the needle, carefully stop suction from the CellTram.
2. Move the dish out from under the needle and carefully position the needle into a tube containing the desired buffer.
3. Using the CellTram, drain the eCSF out of the needle into the buffer trying to avoid contaminating the solution with oil.
4. In order to remove cells that you have aspirated along with the eCSF, spin eCSF at 10,000 x g. Collect uncontaminated eCSF (supernatant) and store at -80 °C until ready for further analysis.

5. Reintroduction of Selected Factors

1. Drain the eCSF every 2 hr during desired time interval, since the fluid is replenished within that time period. Between drainage time-points, remove the needle and store the embryos at 28.5 °C or desired temperature.
2. Load second micropipette needle with test factor.
3. Place needle into micromanipulator attached to microinjector.
4. Adjust injection volume to 1 nl by measuring the drop size in oil and adjusting injection time and pressure.
5. Inject 1-2 nl into brain ventricles as described previously²⁶.

Wyniki

An example of a drained brain ventricle is shown in Figure 1B-C. Brain ventricles are collapsed as they lack eCSF (Figure 1B vs. C). As seen in dorsal images (Figure 1B-C, and Figure 2A-D) the hindbrain neuroepithelium does retain its characteristic morphology and seems to be open despite lack of eCSF likely due to robust hinge-points. However, lateral views (Figure 2A'-D') demonstrate that the hindbrain ventricle has been drained, consi...

Dyskusje

Use of this technique to manually drain eCSF from zebrafish brain ventricles will be useful for determining the requirement for eCSF during development. In addition, this technique will allow description of the eCSF protein profile over the course of embryonic development. Identification of different proteins during this time will enable further investigation into the function of the CSF and its potential role during brain development. In amniotes, some factors identified in eCSF (IGF2, FGF2, retinoic acid, and apolipop...

Materiały

Name	Company	Catalog Number	Comments
Eppendorf CellTram Oil	Eppendorf	516 000.025
Mineral Oil	Sigma	M8410
Tricaine powder	Sigma	A5040
Capillary Tubes	FHC Inc.	30-30-1