Development of cerebellar neurons and glias revealed by in utero electroporation: Golgi-like labeling of cerebellar neurons and glias

PLoS One. 2013 Jul 23;8(7):e70091. doi: 10.1371/journal.pone.0070091. Print 2013.

Abstract

Cerebellar cortical functions rely on precisely arranged cytoarchitectures composed of several distinct types of neurons and glias. Studies have indicated that cerebellar excitatory and inhibitory neurons have distinct spatial origins, the upper rhombic lip (uRL) and ventricular zone (VZ), respectively, and that different types of neurons have different birthdates. However, the spatiotemporal relationship between uRL/VZ progenitors and their final phenotype remains poorly understood due to technical limitations. To address this issue, we performed in utero electroporation (IUE) of fluorescent protein plasmids using mouse embryos to label uRL/VZ progenitors at specific developmental stages, and observed labeled cells at maturity. To overcome any potential dilution of the plasmids caused by progenitor division, we also utilized constructs that enable permanent labeling of cells. Cerebellar neurons and glias were labeled in a Golgi-like manner enabling ready identification of labeled cells. Five types of cerebellar neurons, namely Purkinje, Golgi, Lugaro and unipolar brush cells, large-diameter deep nuclei (DN) neurons, and DN astrocytes were labeled by conventional plasmids, whereas plasmids that enable permanent labeling additionally labeled stellate, basket, and granule cells as well as three types of glias. IUE allows us to label uRL/VZ progenitors at different developmental stages. We found that the five types of neurons and DN astrocytes were labeled in an IUE stage-dependent manner, while stellate, basket, granule cells and three types of glias were labeled regardless of the IUE stage. Thus, the results indicate the IUE is an efficient method to track the development of cerebellar cells from uRL/VZ progenitors facing the ventricular lumen. They also indicate that while the generation of the five types of neurons by uRL/VZ progenitors is regulated in a time-dependent manner, the progenitor pool retains multipotency throughout embryonic development.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Astrocytes / cytology
  • Cerebellar Cortex / cytology*
  • Cerebellar Cortex / embryology
  • Electroporation
  • Embryo, Mammalian
  • Female
  • Gene Transfer Techniques
  • Golgi Apparatus / chemistry
  • Mice
  • Mice, Inbred C57BL
  • Mice, Inbred ICR
  • Neural Stem Cells / cytology*
  • Neuroglia / cytology*
  • Neurons / cytology*
  • Pregnancy

Grant support

This work was supported by Grant-in-Aid for Scientific Research on Priority from the Ministry of Education, Science, Sports and Culture of Japan (MEXT KAKENHI22200045002, http://www.mext.go.jp/english/). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.