1Department of Biology, Massachusetts Institute of Technology, 2Whitehead Institute of Biomedical Research
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Chang, J. T., Sive, H. An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium. J. Vis. Exp. (68), e4242, doi:10.3791/4242 (2012).
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 function3-6.
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 lab10, 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.
1. Preparing for Microinjection
2. Preparing the Embryos
3. Injecting the Brain Ventricles
5. Quantification of Dye Movement
6. Representative Results
An example of results obtained in a neuroepithelial permeability assay using wild type embryos is shown in Figure 1B-D. To accurately differentiate permeability, it is useful to test dyes with different molecular weightsto identify a size that is only slightly leaky in wild type or control embryos (Figure 2). This allows for identification of genetic mutants or environmental conditions that either increase or decrease permeability (Figure 1D, green and red lines respectively). For the 24 hpf zebrafish neuroepithelium, 70 kDa FITC Dextran leaks slowly over 2 hr, whereas 2,000 kDa does not and 10 kDa almost immediately leaks out. Therefore 70 kDa is the ideal molecular weight to identify conditions that both increase and decrease neuroepithelial permeability.
If the needle misses the ventricular lumen, fluorescence will appear outside the brain at t=0 (for an example see Gutzman and Sive, 200910). These embryos should be discarded since the injected dye was not initially contained within the brain and no clear conclusion regarding movement of the dye and permeability of the neuropeithelium can be made.
Finally, if embryos have small ventricles or un-inflated brain ventricles, pre-injection of ventricles with a saline solution can be done prior to injection of the fluorescent dye. This inflates the ventricles making subsequent visualization of the ventricles easier when injecting with the fluorescent dye. Proper controls must be performed to determine whether injection of saline disrupts normal neural tube development.
Figure 1. Time course of different molecular weight dyes. (A) Experimental Diagram. First, fluorescent dye is injected into the ventricles. X = position of needle for injection. Next dorsal images are captured over time. Finally, the distance moved by the dye front from the forebrain hinge-point is measured (represented by a red line). (B-C) Merged brightfield and fluorescent dorsal images at 22 hpf (t = 0 min, B) and 24 hpf (t =120 min, C). White line indicates distance of the dye front from forebrain ventricle. (D) Hypothetical sample permeability data. Blue = wild type or controls, red = sample with decreased permeability relative to control, and green = sample with increased permeability relative to control.
Figure 2. Measurement of neuroepithelial permeability to different molecular weight dyes. (A-E) Dorsal merged brightfield and fluorescent images of 22 hpf wild type embryos at t=0 min after injection with FITC-dextran of the following molecular weights: 10 kDa (A), 40 kDa (B), 70 kDa (C), 500 kDa (D) and 2,000 kDa (E). (A'-E') Same embryo as in (A-E) at t=120 min at 24 hpf. Anterior to left. F= forebrain, M= midbrain, H= hindbrain. Asterisk = ear.
We demonstrate the ability to quantify permeability of the living embryonic zebrafish brain as determined for an injected dye of a given molecular weight. Our observation that the embryonic zebrafish neuroepithelium is differentially permeable to dyes of differing molecular weights suggests that the dye is moving via paracellular permeability. However, we cannot rule out the possibility of a transcellular contribution to the observed permeability. This technique could be applied to any other tubular structure as long as both the inside and outside of the tube can be seen and the lumen can be injected. However, identification of the ideal molecular weight for other organs will need to be determined as this may differ between tissues and developmental stages .
This assay will enable further investigation into the role of epithelial permeability during lumen inflation and the regulation of lumen size. In addition, this technique will help characterize alterations in epithelial permeability associated with disorders such as hydrocephalus and polycystic kidney disease.
No conflicts of interest declared.
This work was supported by the National Institute for Mental Health, and National Science Foundation. Special thanks to Sive lab members for many useful discussions and constructive criticism, and to Olivier Paugois for expert fish husbandry.
|Dextran, Fluorescein, Anionic, Lysine Fixable||Invitrogen||D7136, D7137, D1822, D1820, D1845|
|Capillary Tubes||FHC Inc.||30-30-1|