الوقت الفاصل بين التصوير حية من خلايا ذات صلة Clonally السلف العصبية في الدماغ المقدم اسماك الزرد النامية

Summary

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Abstract

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function^1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

Protocol

1. Preparation of Zebrafish Embryos Two days before electroporation, set up mating cages of wild type fish using dividers to separate males from females. One day before electroporation, pull the dividers in mating cages from the previous day. Collect embryos and incubate 50 fertilized embryos in 30 ml embryonic medium containing 0.003% phenylthiourea (PTU) at 28.5°C. On the day of electroporation, dechorionate the embryos manually with fine forceps (Inox 5, Dumont Electronic…

Discussion

In this video, we demonstrate a method for time-lapse live imaging of neural progenitor cells at a clonal level in the developing zebrafish forebrain. We tested and modified the existing electroporation protocols^4-6 to genetically and fluorescently label individual neural progenitor cells. A lineage tree composed of clonally related progeny cells can be established since only a few cells were sparsely labeled with a relative low voltage of electroporation. In addition to EGFP, the neural progenitor cells can b…