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

Michalina Hanzel,Richard J T Wingate,Victoria Rook

doi:10.1038/s41598-019-51532-y

Michalina Hanzel, Richard J T Wingate + Show 1 more

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https://doi.org/10.1038/s41598-019-51532-y

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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.

Highlights

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL)
This three phase sequence is a rational, parsimonious interpretation of the array of morphologies revealed by Golgi staining and is a graphical narrative that has had a profound influence on how morphology and differentiation status of granule cell precursors (GCPs) has been assessed in many species and systems[7,8,9,10,11,12,13,14,15,16,17], including the chick cerebellum[10]
Www.nature.com/scientificreports tipped with growth cones and decorated with spines. These spines are retracted as bipolar cells’ cell bodies transition seamlessly into an inward radial migration towards the inner granular layer (IGL), leaving behind the parallel fibres, the cells’ axons (Fig. 1A, stage c). Does this deterministic and linear interpretation of morphology capture the diversity of GCP behaviour? Given that the neuroblasts of the rostral migratory stream (RMS), for example, retain their ability to divide as they migrate towards the olfactory bulb[19,20,21,22] and express markers characteristic of postmitotic neurons[23,24,25], we explored the possible presence of similar developmental features in GCPs in the developing cerebellum

Summary

Introduction

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL). An accepted view of EGL assembly, established by Cajal[18], is that GCPs, which accumulate in this transient secondary epithelium after their birth at, and migration from, the rhombic lip, undergo determined sequential phases of proliferation, morphological elaboration, followed by tangential and radial migration into the inner granular layer (IGL) (Fig. 1A) This three phase sequence is a rational, parsimonious interpretation of the array of morphologies revealed by Golgi staining and is a graphical narrative that has had a profound influence on how morphology and differentiation status of GCPs has been assessed in many species and systems[7,8,9,10,11,12,13,14,15,16,17], including the chick cerebellum[10]. The consequence is a smaller and unfoliated cerebellum with mislocalised cells of abnormal morphologies

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