Zebrafish Brain Ventricle Injection

Summary

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Abstract

Proper brain ventricle formation during embryonic brain development is required for normal brain function. Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid. In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation. In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue. However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space. Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy. The brightfield and the fluorescent images are then processed and superimposed in Photoshop. This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image. Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages. We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

Protocol

Protocol Part 1: Preparing for microinjection Prepare for the injection by pulling capillary needles using Sutter Instruments needle puller. Fill the needle with a fluorescent dye (such as Texas-Red Dextran). Mount the needle in a micromanipulator and microinjection apparatus. Cut the needle to the appropriate size at an angle to create a beveled tip. Measure the drop size and check that it is between 1-2nl per injection in oil. <p cl…

Discussion

In this video we demonstrate how to inject fluorescent dye (Texas-Red Dextran) into developing zebrafish brain ventricles. This method is used to visualize the brain ventricle space in contrast to the surrounding neuroepithelium and is extremely useful for determining the shape of the ventricle space as well as the shape of the surrounding brain tissue. It allows us to better understand the process of brain ventricle formation and brain morphogenesis over time in the live embryo. This technique is an excellent tool for…

Materials

Material Name	Type	Company	Catalogue Number	Comment
Forceps	Tool	Fine Science Tools	11252-20
Capillary Tubes (needles)	Tools	Frederick Haer and Co.	30-30-1
Agarose-Coated dishes	Tools	Agarose (Seakem GTG; Lonza) Dishes (Corning)	Agarose (50027) Dishes (430166)	Make agarose approximately 2-3 mm deep in the dish depending on the stage of embryo you are injecting
Dissecting Microscope	Microscope	Zeiss Stereomicroscope Stemi SV 6
Microscope Camera with Fluorescence capabilities and with image capture software	Tools
1-200 μ pipette tips	Tools	Tip One, US Scientific	1111-0806	These are the perfect size to poke the holes in the agarose and have the embryos fit perfectly.
Photoshop	Image Analysis	Adobe
Embryo Loop	Tools			Make these yourself with a glass pipette and hair or fishing line and seal with beeswax.
Microinjector	Tools	Medical Systems Research Products Harvard Apparatus	PL1-100
Needle Puller	Tools	Sutter Instruments		Settings (Heat 785, Pull 70, Velocity 40, Time 70).
Micromanipulator	Tools	Narishige
Fluorescent dye Texas-Red Dextran 70,000MW	Reagent	Molecular Probes	D1830	Other Molecular weights are available if desired.
Tricaine (3-aminobenzoic acid ethyl ester)	Reagent	Sigma	A-5040
Embryo Media	Reagent			According to Westerfield (5)