Zebrafish भ्रूण मस्तिष्क ventricles के मैनुअल ड्रेनेज
Summary
हम विधि मस्तिष्कमेरु द्रव (सीएसएफ) को इकट्ठा करने और एक प्रणाली है जो भ्रूण zebrafish मस्तिष्क वेंट्रिकुलर सिस्टम के भीतर सी.एस.एफ. का अभाव बना उपस्थित. यह सी.एस.एफ. और भ्रूण के मस्तिष्क के विकास के दौरान की आवश्यकता संरचना के आगे की परीक्षा के लिए अनुमति देता है.
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 molecules1,2. 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 ventricles3-5. 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 development8,9. 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 neuroepithelium10-20. 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 proliferation21-24. 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
Representative Results
Discussion
इस तकनीक का उपयोग करने के लिए मैन्युअल zebrafish मस्तिष्क ventricles से eCSF नाली विकास दौरान eCSF लिए आवश्यकता का निर्धारण करने के लिए उपयोगी होगा. इसके अलावा, इस तकनीक में भ्रूण के विकास के पाठ्यक्रम पर eCSF प्रोटीन प्रोफ…
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. डॉ. जेन Gutzman, डॉ. Amanda डिकिंसन और अन्य कई उपयोगी चर्चा और रचनात्मक आलोचना के लिए निर्णायक प्रयोगशाला के सदस्यों, और ओलिवर Paugois विशेषज्ञ मछली पालन के लिए विशेष धन्यवाद.
Materials
| Name of Reagent | Company | Catalogue number |
| Eppendorf CellTram Oil | Eppendorf | 516 000.025 |
| Mineral Oil | Sigma | M8410 |
| Tricaine powder | Sigma | A5040 |
| Capillary Tubes | FHC Inc. | 30-30-1 |
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