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.
Highlights
The cerebellum is composed of distinct types of neurons that have unique morphological features and synaptic connectivities [1]
Deep nuclei (DN) neurons, Purkinje cells, inhibitory interneurons and granule cells are generated sequentially [5,6,7,8], the final division of granule cells occurs in the external granular layer (EGL) [9,10,11] and that of Golgi cells, stellate cells and basket cells in the white matter (WM) [12,13]
We show that Purkinje cells, Golgi cells, unipolar brush cells (UBCs), large-diameter DN neurons, and DN astrocytes almost directly arise from labeled progenitors at respective specific developmental stages, whereas precursors of granule cells, stellate/basket cells, Bergmann glia, oligodendrocytes, and cortical astrocytes stay in the upper rhombic lip (uRL)/ventricular zone (VZ) and continue proliferation over a protracted period of embryonic development
Summary
The cerebellum is composed of distinct types of neurons that have unique morphological features and synaptic connectivities [1]. Deep nuclei (DN) neurons, Purkinje cells, inhibitory interneurons and granule cells are generated sequentially [5,6,7,8], the final division of granule cells occurs in the external granular layer (EGL) [9,10,11] and that of Golgi cells, stellate cells and basket cells in the white matter (WM) [12,13]. Recent genetic fate mapping (GFM) studies showed that excitatory neurons like large-diameter DN neurons and granule cells and inhibitory neurons like Purkinje, Golgi, stellate, and basket cells respectively originate from spatially distinct regions, the upper rhombic lip (uRL) [14,15,16,17,18] and the adjacent ventricular zone (VZ) [8,19]
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