Fig 1.
Neurospheres display circadian rhythms in mPer1 gene expression during early differentiation.
DG neurospheres grown in stem cell medium were transferred to serum medium (SM) immediately after a synchronizing forskolin treatment ending at time zero. Shown are 5-point running averages of intensity measurements from images collected hourly. Note that rhythms can be observed as early as the second day in SM. ADUs: analog-to-digital units of the camera.
Table 1.
Cell types identified by markers for stem cells and differentiated cells after 1 or 4 days in serum medium.
Fig 2.
NSPCs are the dominant cell types during the first four days of DG neurosphere differentiation.
SOX2+ (green) cells after A: 1 day in SM, B: 4 days in SM. Msi1+ (green) cells after C: 1 day in SM and D: 4 days in SM. E: Nestin+/GFAP+(yellow) cells at day 4 in SM indicating that radial glial-like cells persist after 4 days of differentiation in SM. Also shown are Nestin+/GFAP- (green) cells and very few Nestin-/GFAP+ (red) cells. Neurospheres lack immature and mature neuronal cells after differentiating 4 days in SM as shown by F: BetaIII+ (green) and G: NeuN+ (green). Nuclei were stained with propidium iodide (red). H: The source of the bioluminescence signal indicated by mPER1+ (green) cells in the neurosphere core after 3 days in SM. All nuclei were stained with Hoechst3342 (blue) unless specified. Scale bar = 50 μm.
Fig 3.
Bmal1-/- neurospheres show altered growth patterns and increased cell death.
A: brightfield image of Bmal1-/- DG neurosphere (arrows indicate large and small lacunae). Live/dead stain using propidium iodide (red) and Hoechst3342 (blue) shows higher cell death near the lacunae in B: Bmal1-/- DG neurospheres when compared to C: WT controls. After 2 days of differentiation in SM, caspase–3+ cells were detected in D: WT neurospheres and Bmal1-/- neurospheres at E: 20x and at F: 40x magnification. Nuclei were stained with propidium iodide in (D-F). G: Percentage of caspase3+ and propidium iodide-positive cell staining in DG neurospheres from Bmal1-/- and WT littermates. Scale bar = 50 μm.
Fig 4.
Cry1-/-, 2-/- neurospheres have reduced proliferation and growth in culture.
The average size of DG and SVZ neurospheres from Cry1-/-, 2-/-, WT, and Cry1-/- mice were compared at days 14 and 35 in vitro while in SCM. Shown is the total area of all spheres of each dish (as μm2). Asterisk indicates significant difference from the respective control (p<0.05).
Fig 5.
Neuronal commitment is diminished in Bmal1-/- DG neurospheres.
In WT DG neurospheres the sequence of cell types during differentiation in B27 medium parallels events during in situ neurogenesis. A: Immature neurons expressing BetaIII-tubulin (green) at day 7 and lacking GFAP co-localization (red). B: Mature neurons expressing NeuN (green) at day 14 and lacking GFAP (red). In contrast, Bmal1-/- neurospheres displayed reduced neuronal differentiation and increased astrocyte proliferation. C: Lack of BetaIII-tubulin expression (green) shown with GFAP (red) in a Bmal1-/- neurosphere at day 7. D: Lack of NeuN (green) shown with GFAP (red) at day 14. All nuclei were stained with Hoechst (blue). Scale bar = 50 μm. E: Percentage of positive cells for DCX (neuroblasts), BetaIII-tubulin, and NeuN at days 4, 7, and 14 after differentiation in B27 medium, respectively.
Table 2.
Percent positive mature neuronal and glial cells after 14 days of differentiation in B27 medium.