An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

Abstract

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2 . CSF is a protein rich fluid that is essential for normal brain development and function [3][4][5][6] .In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8 , while claudin 5a is necessary for tight junction formation 9 . Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab 10 , to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2). This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease. Video LinkThe video component of this article can be found at http://www.jove.com/video/4242/ Protocol 1. Preparing for Microinjection 1. Prepare microinjection needles by pulling capillary tubes using Sutter instruments needle puller. 2. Load microinjection needle with fluorescent dye (FITC-Dextran). 3. Mount needle on micromanipulator and microinjection apparatus. 4. Carefully break microinjection needle using forceps to roughly 2 μm in width, however, this will vary depending on your microinjector setup.For our microinjection needles, this corresponds to the first region of the needle from the tip that does not bend. 5. Measure drop size in oil, adjusting injection time and pressure, so that each injection delivers 1 nl. Example ...

Progressive Differentiation and Instructive Capacities of Amniotic Fluid and Cerebrospinal Fluid Proteomes following Neural Tube Closure

“…7E). These data are consistent with the model that the CSF draws fluid and proteins from the AF (see above; Alonso et al, 1998; Chang & Sive, 2011). Conversely, many proteins were enriched in the E10.5 CSF vs .…”

“…These data suggest that factors available in the AF—possibly including LIF—can reach the CSF by classic diffusion through the neuroepithelium at this age. The zebrafish neuroepithelium is permeable to fluorescent dyes up to 70kDa (Chang and Sive, 2011). …”

The inner CSF–brain barrier: developmentally controlled access to the brain via intercellular junctions

“…Claudin-5 has also been demonstrated in the ventricular zone of zebrafish embryos (Zhang et al, 2010 ). These authors and others (e.g., Chang and Sive, 2012a ) have interpreted this as indicating that the ventricular zone/neuroepithelial cells are joined by tight junctions. The transmission electron micrographs in Zhang et al ( 2010 ) are too low resolution for identification of the nature of the intercellular junctions, but loss of claudin-5 resulted in disruption of these intercellular junctions and allowed passage of lanthanum.…”

“…However, in the study of Fossan et al ( 1985 ) only one sized marker (HRP, 40 kDa) was used and only two developmental stages in fetal sheep were compared. The only other studies of ventricular zone in embryos appear to be those of Chang and Sive ( 2012a , b ) in zebrafish. As in the present report, these authors used a series of different molecular sized dextrans (10–400 kDa FITC-conjugated) injected into the cerebral ventricles of zebrafish embryos as early as 22 h post fertilization.…”

Directional cerebrospinal fluid movement between brain ventricles in larval zebrafish

“…Larvae were anesthetized as described above and the rhombencephalic ventricle was microinjected with 2 nL of Kaede, a photoactivatable protein with fluorescence that changes from green to red upon illumination with UV light [ 36 , 37 ]. After injection, larvae recovered from injection and anesthesia for at least 30 min prior to imaging, mitigating any immediate effects of dye injection.…”