Elsevier

Gene Expression Patterns

Volume 34, December 2019, 119068
Gene Expression Patterns

Expression of transcription factors and signaling molecules in the cerebellar granule cell development

https://doi.org/10.1016/j.gep.2019.119068Get rights and content

Abstract

Cerebellar granule cell precursors (GCPs) and granule cells (GCs) constitute a good model system to investigate proliferation of neural precursors and differentiation of neurons. During development, GCPs proliferate in the outer external granule cell layer (outer EGL) and then exit the cell cycle in the inner EGL to become GCs, which inwardly migrate to the inner granule cell layer (IGL). Misregulation of GCP proliferation or GC differentiation leads to maldevelopment of the cerebellum and the formation of a cerebellar tumor, medulloblastoma. Despite many efforts in this field, the mechanisms underlying GC development remain elusive. In this study, we performed detailed immunostaining in the developing cerebellum, with particular focus on GCPs and GCs, looking at several transcription factors, signaling molecules, cell cycle regulators, some of which are known to regulate neural development. Interestingly, we found distinct distribution patterns of certain proteins within the outer and inner EGL, suggesting the existence of subpopulations of GCPs and GCs in those layers. This study provides a basis for future studies on the cerebellar GC development and medulloblastoma.

Introduction

Tightly regulated developmental events are achieved by complex interactions of various molecules. Although numerous studies have attempted to elucidate these complex developmental processes, they remain unclear. In the developing cerebellum, cerebellar granule cell precursors (GCPs) and granule cells (GCs), form strictly segregated cell layers. During the first two weeks of the postnatal period, GCPs actively proliferate in the outer external granule cell layer (outer EGL) just beneath the pial surface of the cerebellum (Fujita et al., 1966). These proliferating cells subsequently exit the cell cycle and differentiate into GCs in the inner external granule cell layer (inner EGL). GCs then migrate in the molecular layer (ML) along the fibers of Bergmann glia to the internal granule cell layer (IGL) (Komuro and Yacubova, 2003).

With regard to the EGL, some key molecules and/or signaling cascades regulating developmental stages of GCPs have been studied (Chedotal, 2010). For example, it has been reported that Sonic Hedgehog signals promote proliferation of GCPs in the outer EGL (Wallace, 1999) while BMP signals inhibit proliferation of GCPs via degradation of Atoh1, a master regulator of GCP development (Zhao et al., 2008). However, these studies treat the cell population in each cell layer as being composed of a homogeneous cell type, for example, undifferentiated and differentiated or mitotic and postmitotic.

Recent dramatic improvement in next-generation sequencing (NGS) technology allows the analysis of gene expression profiles at the level of single cells (Ramskold et al., 2012). This technology is currently being applied to many tissues and has revealed the existence of small previously uncharacterized subpopulations of cells within seemingly uniform and homogenous populations. Recently, single cell analysis using cerebellar tissue suggested that GCPs are also composed of heterogeneous cell populations (Carter et al., 2018). Thus, the GC development seems more complicated than we thought, however, the underlying molecular mechanisms remain elusive.

In this study, we performed detailed immunostaining in the developing cerebellum, with particular focus on GCPs and GCs, looking at transcription factors, signaling molecules, cell cycle regulators. In particular, we tested molecules that have been reported to play important roles in brain development but whose functions in the cerebellum have been unknown. We also analyzed molecules that were known to play certain roles in the cerebellar development but their precise protein localizations were unknown. This work provides a solid basis for future studies on GC development as well as a cerebellar tumor, medulloblastoma.

Section snippets

Markers to discriminate GCPs and GCs in developing cerebellum

A transcription factor, Pax6, is reported to be expressed in all cells in the granule cell lineage, GCPs and GCs (Engelkamp et al., 1999). In the coimmunostaining with Pax6 and Ki67, double-positive cells correspond to mitotic GCPs that constitute the outer EGL (Fig. 1A). Next we performed coimmunostaining with Ki67 and Dcx, a marker for immature neurons (Fig. 1B) (Francis et al., 1999). Ki67-and Dcx-signals were exclusively observed (Fig.1B,f-g). Therefore, in this study, we used the presence

Discussion

In this study, we evaluated the expression patterns of several proteins with the particular focus on the EGL where GCPs proliferate in the outer region (outer EGL) and differentiate in the inner region (inner EGL). It has been believed that cells in the outer EGL and inner EGL are uniform populations, respectively. However, our immunostaining showed that some proteins were differentially expressed in cells even within the outer EGL and the inner EGL. This suggests subpopulations of GCPs in the

Animals

All animal experiments in this study have been approved by the Animal Care and Use Committee of the National Institute of Neuroscience, NCNP, Japan. ICR mouse at P8 were fixed with 4% PFA and their cerebella were embedded with O.C.T compound (Sakura Finetek). Frozen brains were sagittally sectioned into 16 μm slices with cryostat (Leica Biosystems).

Immunohistochemistry

P8 cerebella were perfusion-fixed with 4% paraformaldehyde (PFA) in PBS and cryoprotected with 30% sucrose in PBS. Next, tissues were embedded in

Conflicts of interest

We declare that we have no conflict of interest.

Declarations of interest

None.

Acknowledgement

We would like to thank Dr. Ruth Yu for comments. This work is supported by Grants-in-Aid for Scientific Research (Grant 18H02538 to M.H.) and Innovative Areas (Grant 16H06528 to M.H.) from MEXT, Strategic Research Program for Brain Sciences from AMED (JP16dm0107085h0001), Takeda Foundation and Intramural Research Grants (Grants 30-9 and 1-4 to M.H.).

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