Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Summary

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.

Abstract

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.

Protocol

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 Emb....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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