पारगम्यता के लिए Zebrafish भ्रूण Neuroepithelium की एक परख
Summary
हम एक जीवित भ्रूण zebrafish मस्तिष्क की पारगम्यता के लिए पूरे पशु मात्रात्मक माप का वर्णन. तकनीक न्यूरल ट्यूब लुमेन के भीतर मस्तिष्कमेरु और विभिन्न आणविक भार के तरल पदार्थ अणुओं को बनाए रखने की क्षमता का विश्लेषण करती है और उनके आंदोलन ventricles के बाहर quantifies. इस विधि उपकला और विकास और रोग के दौरान पारगम्यता परिपक्वता में मतभेद के बारे में फैसला लेने के लिए उपयोगी है.
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 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.
Protocol
Discussion
हम करने के लिए रहने वाले भ्रूण zebrafish मस्तिष्क के पारगम्यता यों के रूप में एक दिया आणविक वजन के एक इंजेक्शन डाई के लिए निर्धारित करने की क्षमता प्रदर्शित करता है. हमारी अवलोकन है है कि भ्रूण zebrafish neuroepithelium विभि?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
इस काम के मानसिक स्वास्थ्य के लिए राष्ट्रीय संस्थान, और राष्ट्रीय विज्ञान फाउंडेशन द्वारा समर्थित किया गया. कई उपयोगी चर्चा और रचनात्मक आलोचना, विशेषज्ञ मछली पालन के लिए और ओलिवर Paugois के लिए निर्णायक प्रयोगशाला के सदस्यों के लिए विशेष धन्यवाद.
Materials
| Name of Reagent | Company | Catalogue number |
| Dextran, Fluorescein, Anionic, Lysine Fixable | Invitrogen | D7136, D7137, D1822, D1820, D1845 |
| Tricaine powder | Sigma | A5040 |
| Capillary Tubes | FHC Inc. | 30-30-1 |
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