Mitotic granule cell precursors undergo highly dynamic morphological transitions throughout the external germinal layer of the chick cerebellum

Abstract

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL). The EGL is comprised solely of granule cell precursors, whose progeny migrate inwardly to form the internal granule cell layer. While a range of cell morphologies in the EGL has long been known, how they reflect the cells’ differentiation status has previously only been inferred. Observations have suggested a deterministic maturation from outer to inner EGL that we wished to test experimentally. To do this, we electroporated granule cell precursors in chick with plasmids encoding fluorescent proteins and probed labelled cells with markers of both proliferation (phosphohistone H3) and differentiation (Axonin1/TAG1 and NeuroD1). We show that granule cell precursors can display a range of complex forms throughout the EGL while mitotically active. Overexpression of full length NeuroD1 within granule cell precursors does not abolish proliferation, but biases granule cells towards precocious differentiation, alters their migration path and results in a smaller and less foliated cerebellum. Our results show that granule cells show a greater flexibility in differentiation than previously assumed. We speculate that this allows the EGL to regulate its proliferative activity in response to overall patterns of cerebellar growth.

Heterochronic Developmental Shifts Underlying Squamate Cerebellar Diversity Unveil the Key Features of Amniote Cerebellogenesis

“…In particular, at 25 dpo mitotic cells are found at different levels along the rostrocaudal EGL extent, including its rostral edge ( Figure 3D). As cerebellum develops, cell divisions become more prominent and, like recently observed in chicken (Hanzel et al, 2019), also occur in more internal EGL regions ( Figure 3E). Moreover, actively dividing cells are still detected even far from the URL region by 60 dpo (Figure 3F).…”

BMP signalling facilitates transit amplification in the developing chick and human cerebellum

“…In its absence, cells fail to cling to the basal membrane as they migrate and fail to form a transit-amplifying layer. Similar results are found when NeuroD1, which marks the differentiation of granule cells, is overexpressed at the rhombic lip (Butts et al, 2014b;Hanzel et al, 2019) suggesting that the ability of granule cell precursors to suspend differentiation is intimately linked to the subpial location of granule cell precursors within the EGL. This germinal niche facilitates the mesenchymal-like cell behaviour that is seen in transit amplifying progenitors, including an absence of polarisation (Singh & Solecki, 2015) that is regulated by both transcriptional (Singh et al 2016) and post-transcriptional (Famulski et al 2010;Trivedi et al 2017;Kullman et al 2020) mechanisms.…”

“…We first re-examined the normal time course of granule cell differentiation from our control embryos electroporated with tomato at E4 and harvested at E7. Granule cell precursors exhibit a variety of morphologies consistent with the reported steps of proliferation and early migration within the EGL (Hanzel et al, 2019), with migrating granule cell precursors being predominantly bipolar or unipolar (Figure 4a). However, no cells at this age had yet entered the internal granule cell layer and adopted the T-shape axonal morphology that characterises definitive granule cells after their exit of the EGL.…”

“…In Cajal's scheme, after terminal migration cells exit the EGL, migrating inwards with a leading dendritic process, leaving their T-shape axons behind to extend through the molecular layer. While this sequence of events may be less stereotyped than Cajal presumed (Hanzel et al, 2019), development of granule cells takes place in the absence of these steps in ray-finned fish which do not exhibit a transient transit amplifying layer within the cerebellum (Kaslin et al, 2009;Chaplin et al, 2010;Butts et al, 2014c).…”

The Eya1 Phosphatase Mediates Shh-Driven Symmetric Cell Division of Cerebellar Granule Cell Precursors

“…To do so, we injected a GFP-expressing retrovirus into the EGL of P1 and P4 mice; 48 h later, at P3 and P6, we generated and imaged organotypic cerebellar slices. The proportion of GFP labeled cells in the IGL with the characteristic bipolar morphology of migrating postmitotic granule neurons [54] in P6 slices (54/110; 49%) was increased compared to P3 (25/101; 25%), suggesting a progressive increase in neurogenic divisions by GCPs that were infected at the later postnatal developmental stage (online suppl. Fig.…”