Widespread proliferation impairment and hypocellularity in the cerebellum of fetuses with down syndrome

Abstract

Evidence in mouse models for Down syndrome (DS) and human fetuses with DS clearly shows severe neurogenesis impairment in various telencephalic regions, suggesting that this defect may underlie the cognitive abnormalities of DS. As cerebellar hypotrophy and motor disturbances are part of the clinical features of DS, the goal of our study was to establish whether these defects may be related to neurogenesis impairment during cerebellar development. We found that in fetuses with DS (17-21 weeks of gestation) the cerebellum had an immature pattern, a reduced volume and notably fewer cells (-25%/-50%) in all cerebellar layers. Immunohistochemistry for Ki-67, a marker of cycling cells, showed impaired proliferation (-17%/-50%) of precursors from both cerebellar neurogenic regions (external granular layer and ventricular zone). No differences in apoptotic cell death were found in DS vs. control fetuses. The current study provides novel evidence that in the cerebellum of DS fetuses there is a generalized hypocellularity and that this defect is due to proliferation impairment, rather than to an increased cell death. The reduced proliferation potency found in the DS fetal cerebellum, in conjunction with previous evidence, strengthens the idea that the trisomic brain is characterized by widespread neurogenesis disruption.

Figure 1

Comparison of the anatomy of the cerebellum in Down syndrome (DS) and control fetuses. A–D. Nissl‐stained parasagittal sections across the cerebellum of a control (A, C) and a DS (B, D) fetus at gestational week (GW) 20. Sections in (A) and (B) and sections in (C) and (D) were taken close to the midline and approximately 2 mm from the midline, respectively. The white arrows in (A) and (B) indicate the major cerebellar fissures and the black arrow indicates a deep germinative region, called here the ventricular pocket (VP; see Results) near the roof of the fourth ventricle. The white dots in (A–D) indicate the cerebellar lobuli. E–G. Depth of the cerebellar fissures (E), number of lubuli (F) and total cerebellar volume (G) in control and DS fetuses. Values are mean ± SD. *P < 0.05 (two tailed t‐test). Calibration bar in D = 2000 µm refers to A–D. Abbreviations: c = caudal; Ch Pl = choroid plexus; d = dorsal; IV = fourth ventricle; l, = lateral; L1‐L5 = cerebellar lobes; m = medial; MB = midbrain; pc = preculminate fissure; pl = posterolateral fissure; pr = primary fissure; r = rostral; sec = secondary fissure; v = ventral.

Figure 2

Comparison of the thickness of the cerebellar layers in Down syndrome (DS) and control fetuses. A–E. Nissl‐stained sagittal section close to the cerebellar midline of a control fetus [gestation week (GW) 18], showing the cytoarchitectonics of the cerebellar cortical layers in different lobes. Images in (A) and (C) correspond to the regions enclosed by a black square in (B). Images in (D) and (E) correspond to the regions enclosed by a white rectangle in (A) and (C), respectively. F–I. Thickness of the external granular layer (F), molecular layer (G), Purkinje cell layer (H) and internal granular layer (I) in control and DS fetuses. Values are mean ± SD. *P < 0.05 (two‐tailed t‐test). Calibration bars: B = 2000 µm; A,C = 500 µm; D,E = 50 µm. Abbreviations: c = caudal; d = dorsal; EGL = external granular layer; IGL = internal granular layer; l = lateral; LD = lamina dissecans; L1‐L5 = cerebellar lobes; m = medial; MOL = molecular layer; PURK = Purkinje cell layer; r = rostral; v = ventral, WM = white matter.

Figure 5

Cell proliferation in the VP of Down syndrome (DS) and control fetuses. A, B. Nissl‐stained sagittal sections across the cerebellum of a control (A) and a DS (B) fetus [gestation week (GW) 20]. Images at the bottom are higher magnifications of the region enclosed by squares. Note a mass of darker cells (enclosed by the stippled white line) in the region of the fifth lobe overlying the roof of the fourth ventricle. This region is called the VP here. C, D. Examples of sections across the VP of a control (A) and a DS (B) fetus (GW 20) immunostained for Ki‐67 and counterstained with Mayer's hematoxylin. Cells immunostained for Ki‐67 appear labeled in brown. E–G. Mean surface area (E), cell density, expressed as number of cells/mm² (F) and number of Ki‐67‐positive cells, expressed as number of cells over total cell number (labeling index: LI) (G) in the VP of control and DS fetuses. Values are mean ± SD. **P < 0.01; ***P < 0.001 (two‐tailed t‐test). Calibration bars in (A,B): low magnification images = 1000 µm; higher magnification images = 500 µm. Calibration bars in (C,D) = 50 µm; Abbreviations: IV = fourth ventricle; L4 and L5 = cerebellar lobes; VP = ventricular pocket.

Figure 3

Comparison of cell density in the cerebellum of Down syndrome (DS) and control fetuses. A–H. Examples of Nissl‐stained sagittal sections from the vermis region of the cerebellum of a control (A, C, E, G) and a DS (B, D, F, H) fetus [gestation week (GW) 20]. Images in (E,F) show that the PURK is populated by migrating granule cell precursors (empty arrow) and large cells (white arrow). Images in (G,H) show that the IGL is populated by cerebellar granule cells (empty arrow) and larger ovoid cells (white arrow). I–M: Cell density, expressed as number of cells/mm² in the EGL (I), MOL (J), PURK (K), IGL (L) and perspective white matter (M). Values are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (two‐tailed t‐test). Calibration bars = 20 µm. Calibration in (D) refers to (A–D) and calibration in (H) refers to (E–H). Abbreviations: CGCs = cerebellar granule cells; EGL = external granular layer; GCPs = granule cell precursors; IGL = internal granular layer; LD = lamina dissecans; MOL = molecular layer; OCs = other cells; PURK = Purkinje cell layer; TOT = total cell number; WM = white matter.

Figure 4

Cell proliferation in the cerebellar cortex of Down syndrome (DS) and control fetuses. A, B. Sagittal sections across the cerebellum of a control (A) and a DS (B) fetus [gestation week (GW) 19] immunostained for Ki‐67 and counterstained with Mayer's hematoxylin. Images on the left in (A) and on the right in (B) are higher magnifications of the regions enclosed by boxes. Cells immunostained for Ki‐67 appear labeled in brown. Proliferating cells were present in the EGL (see, for instance, the cells indicated by the white arrow in the EGL) and in the IGL (see, for instance, the cells indicated by the red arrow in the IGL). C, D. Density of Ki‐67‐positive cells, expressed as number of cells over total cell number (labeling index: LI) in the EGL (C) and IGL (D) of control and DS fetuses. Values are mean ± SD. ***P < 0.001 (two‐tailed t‐test). Calibration bars: low magnification images = 50 µm; higher magnification images = 10 µm. Abbreviations: EGL = external granular layer; IGL = internal granular layer; MOL = molecular layer; PURK = Purkinje cell layer.

Figure 6

Vimentin immunoreactivity in the cerebellum of fetuses with Down syndrome (DS). A, B. Examples of sections processed for immunofluorescence with an anti‐vimentin antibody from a control (A) and a DS (B) fetus [gestation week (GW) 20]. Radial glia processes radiate across all cerebellar layers. C, D. Examples of sections processed for immunofluorescence with an anti‐vimentin antibody (red) from a control (C) and a DS (D) fetus. Cell nuclei were stained by Hoechst dye (blue). The white arrows in (A–D) indicate processes of radial glia cells. Calibration bars = 25 µm. Abbreviations: EGL = external granular layer; IGL = internal granular layer; MOL = molecular layer; PURK = Purkinje cell layer.