Comparative developmental timescale of the progression of cerebellar foliation in the chick, mouse and human and planes of sections used throughout this study .

(A) In the chick (blue), granule cell precursors are born from the rhombic lip around embryonic day 6 (E6), forming the EGL by E7, transit amplification ensues from E8 with the onset of foliation around E10 and its completion by 10 days post-hatching (P10). In the mouse (green), granule cells are derived from embryonic day 12 (E12), are proliferative by E15 with foliation beginning around E19 and continuing until postnatal day 19 (P19). In humans (pink), granule cells start to be generated around 9 post-conception weeks (9PCW), with the cerebella foliation starting between 13-17PCW and continuing until around 8 months (8 mth) postnatally. Dotted lines show the timeline of peak EGL proliferation in each species. Schematic not to scale. (B) A schematic showing the anatomy of the developing chick hindbrain (rl; rhombic lip, rp; roof plate, fb; forebrain, mb; midbrain and hb; hindbrain), how samples were orientated for whole mount (i) and the planes of section used throughout this study for samples between E6-E9 (ii) and greater than E10 (iii), which were either sagittal (blue) or transverse (red). These symbols are used throughout the figures to orientate the reader.

Characterisation of BMP activity and expression throughout the development of the chick cerebellum.

An antibody against the conserved phosphorylated 3’ serine residues of Smad1/5/9 (A; red-circled residues) show the spatial requirements of BMP activity at the E5 rhombic lip (B, n=3), during EGL establishment at E8 (C, n=3), at the onset of proliferation in the E10 EGL as folia develop (D, n=2) and at highly proliferative and foliated stages of cerebellum development (E, F; E14, n=6. (G) Quantification of pSmad expression in the different layers (EGL; external granule layer, PCL; Purkinje cell layer and IGL; inner granule layer) of the crests (blue bars) and fissures (grey bars) shows that BMP activity is significantly higher in the EGL of the folia crests compared to the fissures (G; p= 0.0015; mean ± SEM = 3.521e-005 ± 8.116e-006), however no significant differences in activity were observed between the PCL and IGL (n=6 sections from 4 cerebella). (H) A summary of BMP activity during EGL formation and folia development in the chick (E5-E14) (I) mRNA expression of BMP ligands (Bmp2, Bmp4) and receptors (BmpR1a, BmpR1b) at E10 (n=3) and E14 (n=4). Scale bars B, E; 25µm, C-D; 50µm.

Characterisation of BMP activity, Purkinje cell development and Shh signalling during human cerebellar development.

BMP signalling, here determined by pSmad expression, is observed throughout the 13 pcw human cerebellum (A, n=2) folia crests (B) and fissures (C). BMP activity in the 19 pcw human cerebellum (D, n=2) is observed in the EGL of the folia crests (E) and to a lesser extent in the EGL of the fissures (F). Calbindin expression in the 13 pcw cerebellum (D) folia crests (E) and fissures (F) shows the early migration of Purkinje cell precursors. Calbindin expression in the 19 pcw cerebellum (J) shows a less organised Purkinje cell layer in the folia crests (K) when compared to the fissures (L). (M) Quantification of the PCL between tips and troughs of pcw 19 human and E13 chick cerebella. The number of cell soma per dorsal-ventral column were counted. For E13 chick tips, n=85 columns from n=4 sections and troughs n=75 columns from n=4 sections, p=<0.0001; mean ± SEM1.637 ± 0.1486. For human pcw 19, tips n= 79 columns from n=1 section and troughs n=42 from n=1 section, p=0.0002; mean ± SEM 0.9575 ± 0.2483. Expression of the proliferation marker Ki67, Sonic Hedgehog (SHH) and SHH receptor PATCHED1 (PTCH1) during EGL establishment at 10 pcw (N; n=2) and during early foliation at 12 pcw (O; n=1). Scale bars N, O; 500µm.

BMP signalling is required for establishing the external granule layer.

(A) E3 hindbrains were electroporated with pCAß-GFP alone (n=3), or in combination with pCAß-Smad1EVE-IRES-GFP (Smad1; BMP ; n=3) or pCAß-Smad6 (Smad6; BMP ; n=3), incubated to E5, then embedded in gelatine and sectioned sagittally at 100µm. Sections were immunolabelled using an antibody against phosphorylated Smad1/5/9 (pSmad) to confirm either no change in BMP activity (WT), or up- or down-regulation of BMP signalling at the rhombic lip in Smad1EVE and Smad6 electroporated embryos, respectively. (B) Percentage of cells co-expressing tomato and pSmad following tomato, Smad1EVE or Smad6 electroporation (n= no. cells counted per condition). E4 embryos were electroporated with pCAß-tdTomato alone (C, n=3), or in combination with Smad1 (BMP ; D, n=7) or Smad6 (BMP ; E, n=7), further incubated to E7 and then sagittally sectioned. Dotted white lines A; rhombic lip, C-E; pial surface. Scale bars A; 50µm, C-E; 100µm, C’-E”; 25µm.

BMP signalling is required for the initial tangential migration of rhombic lip derivatives.

(A) Electroporation of tomato, Smad1EVE (Smad1) or Smad6 at the E3 rhombic lip affects migration of early rhombic lip derivatives at E5 (n=3 per condition). (B) The ‘gray’ value plotted against distance from the rhombic lip (coloured lines in A), shows cell density and is indicative of migration from the rhombic lip (n=1 per condition). (C) Sagittal sections from E7 embryos electroporated at E4 with tdTomato control DNA (tomato) show migrating or proliferative granule precursor cells (cell traces C, C’) towards the pial surface (dotted white line). (D) To visualise granule cell morphologies at a foliated stage of development, embryos were electroporated at E2 with tol2::GFP and tol2-transposase and sectioned transversely at E14. Cell traces from the E14 cerebellum show classic T-shaped axon and parallel fibre morphology (D’). (E) Resulting morphologies show varied maturation at E7 following upregulation of BMP signalling at E4, including spherical progenitors (cells 1-6), bilateral progenitors (cells 7-9) and T-shaped axon and fibres (cells 10-15). Mature morphologies are observed at E7 following knock-down of BMP signalling at E4 (F). (G) A summary of morphologies observed in each plane of section for each condition (n= no. cells analysed per condition from n=3 cerebellums per condition, all cells with traceable morphologies were selected from compressed Z-stacks). Scale bars A; 200µm, C, D; 25µm. C’-F cell traces not to scale.

Upregulation of BMP signalling reveals a temporal switch in GCP responsiveness at E8, coinciding with the onset of SHH-induced proliferation.

(A) Proliferation, determined by immunolabelling of PH3, at the pial surface greatly increases between E7 and E8, which coincides with the onset of Shh signalling at E8, as determined by expression of Ptch1 mRNA (B). The nuclei density within the EGL (C) significantly increases between E7 and E8, as determined by the number of DAPI-positive nuclei per 20µm bin of the EGL (n=6 bins per sample from n=7 for E7 and n=7 for E8; p<0.0001, mean ± SEM = 30.37 ± 3.556). Quantification of PH3 immunolabelling (D) of the whole-mounted cerebella (A) shows that there is a significant increase in proliferative activity between E7 and E8 (p=0.0465, mean ± SEM = 0.0005322 ± 0.0002344; n=6 for both E7 and E8). In a control cerebellum electroporated with tdTomato at E4 and sagittally sectioned at E8 (E; n=3), granule cell precursors still exhibit migrating or proliferative morphologies (F). In embryos electroporated at E4 with Smad1 and sectioned sagittally at E8 (G; n=4), an anterior-posterior gradient of EGL loss is observed, with a molecular layer forming in place of the EGL (G’), within which fibres can be seen extending (Smad1, cyan) and sparse DAPI labelling. (H) Granule cells in the E4-Smad1-electroporated cerebellum exhibit mature granule cell morphologies at E8. (I) To show the extent of the depletion of the EGL in control (wt), Smad1 (Bmp ↑) and Smad6 (Bmp ↓) electroporated cerebella, the fluorescent intensity of DAPI labelling at the pial surface was quantified (n=3 samples quantified for each condition at each developmental stage). Significant losses of DAPI density were observed at both E7 and E8 for Smad1 (E7; p=0.0176, mean ± SEM = 92.33 ± 12.41, E8; p=0.0073; mean ± SEM = 173.7 ± 14.97, n=3 per condition) and Smad6 (E7; p=0.0046, mean ± SEM = -124.4 ± 8.446, E8; p=0.0248; mean ± SEM =165.8 ± 26.58, n=3 per condition). Scale bars A; 200µm, E, G’; 25µm. Cell traces in F and H not to scale.

Following BMP-manipulated cell behaviour in a tol2::gfp wildtype background.

E2 embryos were electroporated with tol2::gfp and tol2-transposase in order to permanently label the progenitors and their progeny within the developing cerebellum with tol-2::gfp. The same embryos were then electroporated for a second time at E4 with either tomato alone (A-C) or in combination with Smad6 (D) or Smad1EVE (not shown) and incubated until E7, whereby both electroporations can be visualised on the intact cerebellum (A). In sections from cerebella electroporated with tomato only at E4, rhombic lip derivatives expression both tol2-gfp and tomato are observed migrating across the pial surface (B”). In cerebella electroporated with Smad6 at E4, cells expressing gfp only are observed migrating along the pial surface (D’, coloured arrows), whereas cells expressing both tomato and smad6 migrate ventrally following their specification from the rhombic lip, and prematurely differentiate (D’, cells 1-4).

Tightly regulated spatiotemporal activity of the BMP signalling pathway is required for proper development of the external granule layer.

In control cerebella, granule cell precursors (grey circles) migrate tangentially from the rhombic lip (rl) across the pial surface to form the external granule layer (blue), where they undergo massive transit amplification before they exit the cell cycle, differentiate, and migrate radially towards the inner granule layer, eventually leaving behind a molecular layer (pink) in which their parallel fibres extend. In cerebella with increased levels of BMP signalling (Smad1, BMP ↑), granule cells are initially recruited to the pial surface, however coinciding with the onset of Shh at E8 (in chick), the excess of BMP signalling forces granule cells to exit the cell cycle prematurely and migrate, leaving behind a molecular layer and a depleted cerebellum. Following inhibition of BMP signalling at the rhombic lip (Smad6, BMP ↓) cells fail to be recruited to the pial surface, and instead immediately migrate ventrally and radially, before extending parallel fibres in a disorganised arrangement into the molecular layer that forms at the pial surface.

DNA plasmids

Antibodies used in this study.

mRNA riboprobes used in this study.