Origins, Development, and Compartmentation of the Granule Cells of the Cerebellum

Abstract

Granule cells (GCs) are the most numerous cell type in the cerebellum and indeed, in the brain: at least 99% of all cerebellar neurons are granule cells. In this review article, we first consider the formation of the upper rhombic lip, from which all granule cell precursors arise, and the way by which the upper rhombic lip generates the external granular layer, a secondary germinal epithelium that serves to amplify the upper rhombic lip precursors. Next, we review the mechanisms by which postmitotic granule cells are generated in the external granular layer and migrate radially to settle in the granular layer. In addition, we review the evidence that far from being a homogeneous population, granule cells come in multiple phenotypes with distinct topographical distributions and consider ways in which the heterogeneity of granule cells might arise during development.

Keywords: Bergmann glial fibers; cerebellum; compartmentation; external granular layer; granule cell; radial migration; upper rhombic lip.

Figure 1

Schematic representation of the cerebellar cytoarchitecture. The cerebellar cortex is organized in three laminae: molecular layer, Purkinje cell (PC) layer, and granular layer (GL). Purkinje, Golgi, stellate, and basket cells are inhibitory neurons; granule and unipolar brush cells are excitatory. Granule cells project their axons into the molecular layer, giving rise to the parallel fibers, which synapse with the dendrites of all inhibitory neurons of the molecular layer and with those of Golgi cells. Afferents are of two main types: mossy and climbing fibers. Both are excitatory. The former originate from brainstem nuclei and the spinal cord, whereas climbing fibers come from the contralateral inferior olive. Most mossy fibers synapse directly on the dendrites of four to five granule cells in specialized synaptic glomeruli (blue-gray circle), which also receive inhibitory feedback from Golgi cell axons. A small mossy fiber subset, first synapses on unipolar brush cells that then relay amplified excitatory signals to granule cells. Each PC receives a connection from a single climbing fiber. Purkinje cell axons carry inhibitory signals to the cerebellar nuclei, whereas mossy and climbing fiber collaterals provide the cerebellar nuclei with excitatory afferents. The large glutamatergic neurons of the CN project their axons to nuclei located in the brainstem and diencephalon.

Figure 2

Granule cell development. Schematic representation of granule cell development between E11 and P30. See text for discussion. Abbreviations: EGL, external granular layer; GCPs, granule cell progenitors; GL, granular layer; iEGL, inner lamina of the EGL; oEGL, outer lamina of the EGL; PCs, Purkinje cells; URL, upper rhombic lip.

Figure 3

Stages and stage-specific genes of granule cell development. Schematic representation of some genes playing roles in granule cell birth, proliferation, differentiation, and migration. (A) Atoh1+ granule cell progenitors of the external upper rhombic lip (eURL) derive from ill-defined stem cells of the interior URL (iURL) and migrate to give rise to the EGL thanks to a combination of attractive and repulsive cues (see also Figure 4C), and expand in number between E12.5 and E16.5. (B) After populating the oEGL, GCPs start dividing mostly symmetrically in a process called clonal expansion, promoted by Purkinje cell-secreted sonic hedgehog. Postmitotic GCs form the interior EGL (iEGL) and undertake tangential and radial migration (see text) after extending two axons in the frontal plane (prospective parallel fibers). (C) As they begin their descent into the molecular layer (ML), GC somata extend a radially oriented axon and dendrite, and once in the granular layer, stack with an inside-out progression such that the early-born GCs occupy deeper locations in the GL and project their axons to deeper locations in the ML. Abbreviations: eURL, exterior upper rhombic lip; iURL, interior upper rhombic lip; EGL, external granular layer; oEGL, outer lamina of the EGL; iEGL, inner lamina of the EGL; LM, leptomeninges; ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer.

Figure 4

Formation of the external granular layer. (A) Schematic representation of the embryonic hindbrain (dorsal view, circa E12.5). The view illustrates the departure of GCPs from the URL and the invasion of the EGL (arrowheads). (B) The same process is seen in a sagittal view to show the formation of the EGL. (C) A sketch of extracellular signals and their receptors controlling GCP migration from the URL into the EGL. The URL is represented as a triangle on the left. The location of the isthmic organizer and mesencephalon is on the right side of each box. CXCL12 is released by the leptomeninges (horizontal black line); SLIT2 is released by the URL (triangle); netrin is secreted by the mesencephalic ventral midline. Abbreviations, URL, upper rhombic lip; EGL, external granular layer; VZ, ventricular zone; ChP, choroid plexus; Cb, cerebellar anlage.

Figure 5

(A–K) Distribution of eleven transcripts in the embryonic cerebellar primordium. Sagittal sections hybridized in situ with antisense riboprobes specific for genes, cited in the text, that play important roles in the early stages of cerebellar development. Positive territories are labeled black. All images show E13.5 cerebellar primordia, except (G), which shows an E15.5 section. Image credit: Allen Institute. © 2008 Allen Institute for Brain Science. Allen Developing Mouse Brain Atlas. Available online at: https://developingmouse.brain-map.org/. Abbreviations: Cb, cerebellar primordium; ChP, choroid plexus; EGL, external granular layer; NTZ, nuclear transitory zone; URL, upper rhombic lip; VZ, ventricular zone. Scale Bar in (K) = 200 μm and applies to all panels.

Figure 6

Distribution of nine transcripts in the P4 cerebellum. Sagittal sections hybridized in situ with antisense riboprobes specific for genes cited in the text that play important roles at early stages of cerebellar development. Image credit: Allen Institute. © 2008 Allen Institute for Brain Science. Allen Developing Mouse Brain Atlas. Available online at: https://developingmouse.brain-map.org/. Abbreviations: Cb, cerebellum; iEGL, inner EGL; oEGL, outer EGL. (C,E,J) are magnifications of areas in panels (B,D,I), respectively. Scale bar in panel (A; 400 μm) applies to (B,D,F,H,J); scale bar in panel (C; 100 μm) applies to (E,G,I,K–O).

Figure 7

Transverse boundaries established by genes involved in cerebellar development. (A) Schematic representation of a midsagittal cerebellar section and its subdivision into PC transverse zones [see text for details: some genes are included that are not expressed in GCs (nor cited in the text) but serve to indicate the locations of transverse boundaries (for a review, see Armstrong and Hawkes, 2013]; PC-specific genes are in red in the text boxes. Ebf2 is expressed both in GC progenitors and in a PC subset. Lines point to boundaries between adjacent zones identified by the borders of Purkinje cell and granule cell gene expression domains. Abbreviations: AZ, anterior zone; CZa, anterior central zone; CZp, posterior central zone; PZ, posterior zone; NZ, nodular zone. (B) An example of the above: a sharp posterior boundary established by GCs derived from Ebf2+ GCPs populating from URL at E11.5.