Distinct cerebellar progenitor populations are established early in the embryo. (A) Expression of ptf1a, DsRed (mRNA) and DsRed (protein) in developing and juvenile Ptf1a:DsRed transgenic fish. Ptf1a, DsRed and DsRed show similar expression patterns; (B) In vivo expression of DsRed and Egfp in the embryonic and juvenile Ptf1a:DsRed and Olig2:egfp transgenic fish. Overlapping Egfp and DsRed expression is seen in the VZ of the cerebellar primordium; (C) Nestin:egfp+ (green) and Ptf1a:DsRed+ (red) progenitors in the cerebellar primordium. The Nestin:egfp labels cells in the URL, while Ptf1a:DsRed line labels cells in the VZ. Two days post-fertilization Nestin:egfp+ and Ptf1a:DsRed+ cells form distinct populations in the URL and VZ of the cerebellar primordium; (D) A dorsal overview of Nestin:egfp+ (green) and Ptf1a:DsRed+ (red) progenitors in the hindbrain of a 5-day-old larval zebrafish. Two distinct progenitor domains are visible in the cerebellum (the junction is indicated with an arrow). Ptf1a:DsRed+ cells localize along the ventricular zone of the IV^th ventricle (the VZ domain is labeled with a hatched orange line), while Nestin:egfp+ cells localize in the URL (hatched green line). MHB: Mid-hindbrain boundary; LRL: Lower rhombic lip.

Proliferative activity of cerebellar progenitors diminishes during juvenile stages. (A) Confocal maximum projection of cerebellar cross sections at a similar level of juvenile, young and adult cerebellum. Proliferating cells are labeled with proliferating cell nuclear antigen (PCNA) in blue. Nestin:egfp+ cells are green, Ptf1a:DsRed+ cells are red and the general tissue morphology is depicted with DAPI staining in grey. Yellow arrows depict the distinct niche in the URL where proliferating Nestin:egfp+/PCNA+ progenitors are located. White arrows depict Ptf1a:DsRed+ cells that localize to the ventral part of the IV^th ventricle and to the cerebellar parenchyma. The majority of the parenchymal Ptf1a:DsRed+ cells are PCNA- while most of the Ptf1a:DsRed+ cells at the VZ close to the ventricle are PCNA+. (B-C) High magnification single confocal planes of the boxed areas in A. (B) A proliferating Ptf1a:DsRed+ /PCNA+ progenitor at the IV^th ventricle (white arrow) and many proliferating Nestin:egfp+/PCNA+ cells in the URL (yellow arrow); (C) A proliferating Ptf1a:DsRed+ cell in the cerebellar parenchyma (white arrow); (D) Confocal maximum projections of juvenile and adult cerebellar cross sections showing a decline in VZ progenitor activity. (E) High magnification of boxed area in D. Proliferating Ptf1a:DsRed+ (red) and Olig2:egfp+ (green) cells are found in VZ and the cerebellar parenchyma in a two-week-old juvenile fish. Note the absence of Ptf1a:DsRed+ and Olig2:egfp+ cells among the PCNA+ cells in the URL. The white arrow depicts the Ptf1a:DsRed+ and Olig2:egfp+cells laminating from the ventricular surface into the parenchyma; (F) Ptf1a:DsRed+ cells adjacent to proliferating PCNA+ cells in a one-month-old fish; (G) Proliferating Ptf1a:DsRed+ and Olig2:egfp+ in the VZ of the fourth ventricle (arrow) in a one-month-old fish. The white arrow shows the Ptf1a:DsRed+ and Olig2:egfp+ cells laminating from the ventricular surface into the parenchyma.

Cerebellar progenitors gives rise to distinct cell lineages. (A) Co-localization (arrow) of Ptf1a:DsRed (blue) and GABA, PV or ZII (green) in inhibitory neurons in the cerebellum of a 5-day-old larvae; (B,C) Ptf1a:DsRed+ cells showing morphologies of differentiating stellate and Golgi neurons; (D) A PV- (red) Ptf1a:DsRed+ (green) and HU C/D+ (blue) stellate cell in the ML; (E) Co-localization (arrows) of Ptf1a:DsRed (blue) and GABA (red) cells in the cerebellum of a 1-month-old juvenile zebrafish; (F) Cross section of the juvenile cerebellum showing abundant co-localization (white arrow) of Ptf1a:DsRed (red) and Pax2 (green); (G) Overview of a cross section of the adult cerebellum showing Pax2 (green) labeled Golgi neurons in the GL and PL. Purkinje neurons in the PL are labeled with parvalbumin (red); (H) Co-localization (arrow) of Olig2:egfp (green) and HU C/D (blue) cell in the PL of an adult zebrafish.

Morphology of Ptf1a:DsRed cells in the VZ. (A) High magnification of a ventricularly located Ptf1a:DsRed+ cell with radial morphology in a juvenile zebrafish; (B) A Ptf1a:DsRed+ cell (red) with a radial morphology located at the ventricle, ventral to the Nestin:egfp+ cells (green) in the URL. Ventricle and apical junctions are outlined with ZO-1 (blue); (C) Ptf1a:DsRed+ and gfap:egfp+ cells (green) at the ventricle of the VZ. Proliferating cells in the progenitor niche are labeled with PCNA (blue) and glia are labeled by gfap:egfp (green). The white arrow depicts a proliferating Ptf1a:DsRed+and PCNA+ cell located lateral to the PCNA+ cells in the URL. The yellow arrow depict a horizontally oriented Ptf1a:DsRed+ and gfap:egfp+ cell at the ventricle; (D) Flat horizontally oriented s100β+ cells (red) are lining the VZ of the progenitor niche in adult zebrafish. The ventricle is outlined with the junctional marker ZO-1 (blue); (E) A Ptf1a:DsRed+ Bergmann glia in the adult cerebellum; (F) A Ptf1a:DsRed+ and Nestin:egfp+ Bergmann glia lateral to the progenitor niche (arrow); (G) Co-localization (arrows) of Nestin:egfp (blue) and NeuroD1 (green) in the cerebellum of a 5 day old larvae; (H) No overlap between Nestin:egfp (green, white arrow) cells and PV+ cells (blue, yellow arrow) is seen in the cerebellum of a 5-day-old larvae; (I) Overlapping expression of Atoh1c and Nestin:egfp in the URL. C: Cerebellum, GL: Granule cell layer, URL: Upper rhombic lip, VZ: Ventricular zone.

Generation of cerebellar cell types over time. (A) Quantification of parvalbumin immunopositive cells in the cerebellum of 7- and 14-day-old juvenile zebrafish shows a significant increase of PV+ cells in the cerebellum between 7- and 14-day-old zebrafish (P = 0.0003, n = 4). The pictures show a top view of the cerebellum and PV + cells; (B) An overview of the BrdU pulse chase experiments. To maximize the labeling of dividing cells the zebrafish were given five consecutive 14 hour pulses of BrdU. The fish were euthanized four weeks after the last BrdU pulse; (C) Confocal maximum projections of juvenile and adult cerebellar cross sections showing a notable production of cells in the juvenile and adult zebrafish cerebellum. PV+ Purkinje cells (red) and their processes are seen in in the PL. Scattered s100β+ glia (blue) are seen in the PL and ML. In the juvenile fish high numbers of BrdU+ cells (green) are found both in the GL (orange arrows) and PL (white and yellow arrows). In the adult cerebellum BrdU+ cells are confined to the GL (orange arrows); (D) High magnification of a single confocal plane of the boxed area in C showing a s100β+ glia co-localizing with BrdU (white arrow) and a PV+ Purkinje neuron co-localizing with BrdU (yellow arrow) in a juvenile zebrafish; (E) High magnification maximum projection of the boxed area in C showing s100β+ Bergmann glia lateral to the URL progenitor niche (yellow hatched line) co-localizing with BrdU (white arrows); (F) High magnification of BrdU+ and Pax6+ granule cells (white arrows) in the GL.

Generation of different cerebellar cell types. (A) Confocal maximum projection of a cerebellar cross section in a juvenile zebrafish showing BrdU labeled GABA+ inhibitory neurons (white arrows) and BrdU+/GABA+/PV+ neurons (yellow arrows); (B) High magnification of a single confocal plane showing a neuron co-localizing with BrdU and GABA (white arrow); (C) High magnification of a single confocal plane showing a neuron co-localizing with BrdU, GABA and PV (white arrow); (D) Confocal maximum projection of a cerebellar cross section showing BrdU labeled ZII+ Purkinje neurons (white arrows) and a Pax2+ Golgi neuron (yellow arrow); (E) High magnification of a single confocal plane showing a Golgi neuron co-localizing with BrdU and Pax2 (white arrow); (F) Magnified single confocal plane showing Purkinje neurons co-localizing with BrdU and ZII (white arrows); (G) A rare proliferating oligodendrocyte progenitor (PCNA/Olig2:egfp+) detected in the cerebellar parenchyma (arrow); (H) A putative oligodendrocyte labeled with BrdU+and Olig2:egfp+ detected in the brain parenchyma six weeks after the BrdU pulse; (I) Quantifications of the cell types produced in the cerebellum of juvenile and adult zebrafish after four weeks BrdU pulse chasing. A significant decline of cerebellar inhibitory neuron and glia production between juvenile and adult stages is detected while granule cell production declines during juvenile stages but is still maintained at a high level in the adult zebrafish (n = 5, P <0.001).