Cerebellar granule cell precursors can extend processes, undergo short migratory movements and express postmitotic markers before mitosis in the chick EGL

Cerebellar granule cell precursors (GCPs) form a secondary germinative epithelium, the external germinal layer (EGL) where they proliferate extensively to produce the most numerous cell type in the brain. The morphological sequence of events that characterizes the differentiation of GCPs in the EGL is well established. However, morphologies of individual GCP and their differentiation status have never been correlated. Here, we examine the morphological features and transitions of GCPs in the chicken cerebellum by labelling a subset of GCPs with a stable genomic expression of a GFP transgene and following their development within the EGL in fixed tissue and using time-lapse imaging. We use immunohistochemistry to observe cellular morphologies of mitotic and differentiating GCPs to better understand their differentiation dynamics. Results reveal that mitotic activities of GCPs are more complex and dynamic than currently appreciated. While most GCPs divide in the outer and middle EGL, some are capable of division in the inner EGL. Some GCPs remain mitotically active during process extension and tangential migration and retract their processes prior to each cell division. The mitotically active precursors can also express differentiation markers such as TAG1 and NeuroD1. Further, we explore the result of misexpression of NeuroD1 on granule cell development. When misexpressed in GCPs, NeuroD1 leads to premature differentiation, defects in migration and reduced cerebellar size and foliation. Overall, we provide the first characterisation of individual morphologies of mitotically active cerebellar GCPs in ovo and reaffirm the role of NeuroD1 as a differentiation factor in the development of cerebellar granule cells.


Introduction
Transit amplification of basal progenitors is an important feature of the vertebrate brain development that allows for a rapid expansion of specific cell populations and has facilitated the extraordinary evolution of foliated structures such as the cortex and the cerebellum 1-3 .
Secondary proliferation also allows dedicated progenitors to respond to local environmental conditions to populate neural structures as required during development and repair. The process is found, for example, in the progenitors in the subventricular zone (SVZ) that generate the migrating neuroblasts of the rostral migratory stream (RMS) 4 , the basal neocortical progenitors 1,5,6 , and the progenitors in the external germinal layer (EGL) of the cerebellum. In addition to potentiating exponential growth, the adaptive behaviour of secondary proliferative populations may also underlie developmental plasticity.
The most well studied secondary proliferative population in the brain is the EGL. The traditional view of EGL assembly, established by Cajal 7 , is that granule cell precursors (GCPs) born at the rhombic lip populate the dorsal cerebellar anlagen where they undergo determined sequential phases of proliferation, morphological elaboration, followed by tangential and radial migration into the inner granular layer (IGL) (Fig. 1A). This view has had a profound influence on how morphology and differentiation status of GCPs is interpreted and has been observed in many species and systems [8][9][10][11][12][13][14][15][16][17][18] , including chick cerebella 11 (Fig. 1B). However, does this deterministic and linear interpretation of morphology capture the Cerebellar granule cell precursors (GCPs) form a secondary germinative epithelium, the external germinal layer (EGL) where they proliferate extensively to produce the most numerous cell type in the brain. The morphological sequence of events that characterizes the differentiation of GCPs in the EGL is well established. However, morphologies of individual GCP and their differentiation status have never been correlated. Here, we examine the morphological features and transitions of GCPs in the chicken cerebellum by labelling a subset of GCPs with a stable genomic expression of a GFP transgene and following their development within the EGL in fixed tissue and using time-lapse imaging. We use immunohistochemistry to observe cellular morphologies of mitotic and differentiating GCPs to better understand their differentiation dynamics. Results reveal that mitotic activities of GCPs are more complex and dynamic than currently appreciated. While most GCPs divide in the outer and middle EGL, some are capable of division in the inner EGL. Some GCPs remain mitotically active during process extension and tangential migration and retract their processes prior to each cell division. The mitotically active precursors can also express differentiation markers such as TAG1 and NeuroD1. Further, we explore the result of misexpression of NeuroD1 on granule cell development. When misexpressed in GCPs, NeuroD1 leads to premature differentiation, defects in migration and reduced cerebellar size and foliation. Overall, we provide the first characterisation of individual morphologies of mitotically active cerebellar GCPs in ovo and reaffirm the role of NeuroD1 as a differentiation factor in the development of cerebellar granule cells.
diversity of GCP behaviour? Given that the neuroblasts of the RMS, for example, retain their ability to divide as they migrate towards the olfactory bulb [19][20][21][22] and express postmitotic markers [23][24][25] , we decided to explore the possible presence of similar developmental features in GCPs in the cerebellum.
Given the number of studies of EGL development, there is a surprising dearth of time-lapse movies of GCP behaviour in an intact EGL, and the few performed do not explore progenitor morphology 26,27 . One reason for this could be the high density of cell packing in the EGL that makes visualisation of detailed morphology difficult. To overcome this, we electroporated a GFP transgene into chick rhombic lip cells in early development and allowed the cerebellum to grow in ovo for ten days. This resulted in sparse labelling of EGL cells and allowed detailed morphological examination of GCPs. We find, in fixed tissue and ex ovo time-lapse imaging of cerebellar organotypic slices, that GCPs retain their ability to divide in all layers of the EGL, are highly motile between cell divisions, elaborate long and complex cellular processes that are retracted prior to cytokinesis, and can express postmitotic markers before undergoing mitosis.

Results
To observe the morphologies of individual GCPs in the chick EGL, we electroporated embryonic day 5 (E5) embryos with a plasmid encoding a GFP transgene flanked by Tol2 sites, and a plasmid encoding a Tol2 transposase, resulting in a stable genomic expression of GFP in a subset of granule cells born at the rhombic lip.
At E14, the peak period of GCPs proliferation in the chick EGL, we sacrificed the embryos and examined fixed tissue with immunohistochemistry, as well as performed time-lapse imaging of the living cells in the EGL in organotypic cerebellar slices. We find that there are sparsely labelled rhombic-lip derived cells in the EGL at this stage, representing granule cells at various stages of development ( Fig. 2A-B). Proliferating GCPs are traditionally only expected to localise to the upper half of the EGL and 'have a round soma without any long processes' 16 . We therefore characterised the morphologies of electroporated cells staining for PH3 located in the different EGL layers. We found that the cells located in the outer EGL were indeed mostly round and lacking long cellular processes (e.g.   GCs in some studies 35 . We therefore tested whether misexpression of NeuroD1 in GCPs will force them out of proliferation. To this end, we electroporated a plasmid encoding a full-length NeuroD1 conjugated to GFP at E6, the time of GCP birth at the rhombic lip, and observed the effect on proliferation two days later, at E8, in the forming EGL. We observed that many GCPs misexpressing NeuroD1 nevertheless express PH3 at E8 and therefore are able to undergo mitosis ( Fig. 7A-B). However, misexpression of NeuroD1 leads to premature differentiation of GCPs and a depleted EGL (Fig. 7C-D) and results in a smaller and unfoliated cerebellum at E11   of a dividing granule cell precursor illustrates that they can migrate short distances before division after which their daughter cells migrate in the opposite directions A selection of time-frames from a time-lapse movie of cerebellar slices. Small red arrows point to processes that extend from the cell body. Thick red arrows point to the cell of interest and its two daughter cells after division. The panel below shows a surface of each cell for visualisation. Dotted line in A represents the pial surface A) At the start of observation, the cell is extending one leading process. Whether it has a trailing process cannot be determined due to obstruction from another cell. B) Within 2 hours, the cell has migrated a small distance in a medio-lateral direction and extends two small, thin processes. The leading process seems to bifurcate and a growth cone is visible at the tip of one of the processes. C) The cell continues to extend its leading process, which has become longer and thinner. D) The cell has migrated further, and extends a thicker and shorter process. E) Within 3 hours the cell once again shows two long, thin processes from both sides prior to F) the cell retracting all processes and rounding up for cell division. G) within an hour of division, the cell divides perpendicularly to the pial surface. H) Both daughter cells extend their own processes in the opposite directions. One daughter cell extends a process with a very broad growth cone, whereas the other daughter cell has a longer and much thicker process. I) The daughter cells separate from each other as each migrates in a different direction. J-L ) One of the daughter cells seems to extend two processes that bifurcate into a T-shaped process, resembling formation of a parallel fibre. The other daughter cell extends a long process towards the pial surface with a large, very motile growth cone. At the end of the time-lapse, the cell seems to turn its process back towards the inner EGL. Scale bar = 20μm Basal attachment has been proposed to be essential for   7 | NeuroD1-misexpressing GCPs are able to undergo mitosis but have a limited proliferative potential and differentiate early A) A plasmid encoding full length NeuroD1 and GFP was electroporated into E6 embryos which were fixed two days later, at E8, and stained for PH3. A sagittal cut through the forming EGL reveals co-expressing cells (arrows). B) Higher magnification of EGL cells co-expressing NeuroD1-GFP and PH3 (arrows). C and D) Embryos were electroporated with a control GFP plasmid (C) or NeuroD1-GFP (D) and fixed five days later, at E11. In the control electroporation there is a thick EGL (box, insert) whereas after NeuroD1 misexpression, the EGL is nearly devoid of cells (box, insert), which are located in the IGL close to the rhombic lip (RL). E-G) Three examples of whole cerebella electroporated with NeuroD1-GFP on one side of the rhombic lip only (red arrow). The electroporated side of the cerebellum shoes reduced size and lack of foliation pattern seen on the unelectroporated side. Scale bar= A,C,D= 100μm B=10μm were chosen from the whole z-stack, depending on the best combination to observe specific cell morphologies.
Histology and Immunohistochemistry. E14 chicken cerebellum was fixed in 4% PFA/PBS overnight. The tissue was then washed in PBS 3 times for 15 min and transferred into 10% sucrose (Sigma) in PBS for 30min until the tissue was perfused. The tissue was then transferred to 20% sucrose solution until perfused and finally was transferred into 30% sucrose solution and allowed to perfuse overnight. The tissue was then transferred into OCT compound (VWR) in moulds and placed on dry ice or liquid nitrogen to freeze. The tissue was stored at -80˚C overnight. For sectioning, the blocks were placed at -20˚C an hour before cutting to raise their temperature. The tissue was mounted on cryostat chucks using OCT compound and cut using a Zeiss Microm HM 560 cryostat at 50µm thickness and transferred onto Superfrost Plus slides (VWR). The cut sections were allowed to air dry for two hours and were stored at -80˚C long term and -20˚C short term. Cryostat sections were defrosted for at least 30min at room temperature. The slides were then washed three times for 5 min in PBS.
Slides were then covered in 500-800µl blocking solution (1% normal goat serum, 0.2% Triton in PBS) and incubated for 30min at RT. Primary antibody was diluted

Figure 8 | Proposed new model of GCP differentiation in the EGL
A) The current model of GCP morphological transitions postulates that mitotically active, polyhedral or round cells, with a possible basal attachment to the pial surface proliferate in the outer layer of the EGL. Only after the cell becomes postmitotic, it extends small horizontal processes and begins tangential migration. The processes continuously extend until the cell makes a switch to radial migration, at which point the horizontal processes are considered to be nascent parallel fibres. We suggest that this model is incomplete. The proposed model retains and confirms the morphological features of GCs in the EGL, but suggest that cells previously considered postmitotic, can in fact be proliferative. Divisions of GCPs occur in all layers of the EGL and cells with long horizontal processes are able to undergo mitosis by retracting all their processes and rounding up before division. In the proposed model, white shading represents a highly proliferative precursor, and black shading denotes a postmitotic cell. As cells migrate closer towards the inner EGL, the proliferative potential of the cells decreases, which means that the number of times the cell divides differs in the different layers, with many mitoses in the oEGL and few mitoses in the iEGL. (Purkinje cells are shown in light grey.) B) The suggested transcriptome of developing granule cells resembles the one found in SVZderived neuroblasts in the RMS where their gene expression gradually shifts hence proliferation and differentiation genes can overlap. Genes responsible for granule cell precursors proliferation are most highly expressed in the outer EGL and are continuously downregulated as the cells migrate through the EGL layers and towards the IGL. Genes involved in tangential migration are upregulated in the middle and inner EGL. Differentiation-related genes start to be expressed in the middle and inner EGL and are strongly upregulated during radial migration and final stages of differentiation in the IGL.
at an appropriate concentration in the blocking solution.
After the blocking solution was removed from the slides, 150-200µl of the antibody solution was added onto the slide and covered with parafilm to prevent drying.
Incubation was performed overnight at 4 o C. The next day, primary antibody was washed off with PBS three times for 5 mins. Secondary antibody was diluted in block solution and put onto the slides for 2hrs at RT. The slides were then washed with PBS three time for 5mins and covered with a coverslip using Fluoroshield mounting medium with DAPI (Abcam). Fluorescent confocal images were taken with Zeiss LSM 800 microscope. Zstack projections were compiled using ImageJ. Z-stacks were taken at 1-202µm intervals.