Spatiotemporal pattern of p19^INK4d and neuron-specific type III β-tubulin expression in the embryonic telencephalon. A–I, Fluorescent photomicrographs of representative parasagittal sections were obtained by confocal microscopy from the dorsal part of the telencephalon at E14 (A, C), E15 (F), E16 (D), and E18 (G, I) and stained with anti-p19^INK4d (recognized by a fluorescein-conjugated secondary antibody) and the neuron-specific antibody TuJ1 (recognized by a rhodamine-conjugated secondary antibody). Neighboring sections were stained with cresyl violet (B, E, H) to demonstrate the corresponding laminae of the developing cerebral cortex. A–C, At E14, on the basis of the pattern of p19^INK4dimmunoreactivity, the VZ can be divided into a p19^INK4d(+) sublamina in the lower (apical) portion of the VZ [VZ lower (VZl)] and a p19^INK4d(−) sublamina in the upper (basal) portion of the VZ [VZ upper (VZu)]. The VZl is a mixture of cells at different stages of the cell cycle, including cells undergoing cytokinesis along the ventricular surface (e.g., B,arrow), progenitor cells that are in the G₁phase of the cell cycle, and newly generated postmitotic neurons. Immature neurons that have recently exited the cell cycle are most likely the p19^INK4d-expressing cells of the VZl. In the VZu, which contains progenitor cells that are in the S phase of the cell cycle and migrating immature postmitotic neurons, there is a low level of p19^INK4d immunoreactivity. This indicates that the cells in the VZu, undergoing DNA synthesis, are primarily devoid of p19^INK4d immunoreactivity. In contrast to the differential pattern of p19^INK4d staining in the sublaminae of the VZ, the staining with TuJ1 is minimal in both the VZu and VZl. The few p19^INK4d(+)/TuJ1(+) cells in the VZ most likely represent newly generated postmitotic neurons destined for the cortical plate (C, arrow). In addition, postmitotic cells of the preplate, which contains the future Cajal Retzius and subplate cells, are also labeled by anti-p19^INK4d. D–I, As development proceeds from E14 to E18, the VZ becomes thinner, and the overlying SVZ becomes thicker. Similar to the telencephalon at E14 (A–C), at E15 and E16 (D–F), and at E18 (G–I) the VZu is predominantly a p19^INK4d(−) sublamina, and the VZl is a p19^INK4d(+) sublamina. However, at progressively older ages, the VZu/VZl ratio becomes smaller (i.e., a greater proportion of VZ is comprised of the VZl). Note the uniform and prominent distribution of p19^INK4d expression by cells of the SVZ. In addition, at E15 (F) and E18 (I), many p19^INK4d (+) neurons of the cortical plate, subplate, intermediate zone, and marginal zone are also immunoreactive for TuJ1 (i.e.,arrows). Note that the p19^INK4dexpression by the postmitotic immature neurons in the VZu appears to be downregulated. After reaching the intermediate zone, the p19^INK4d expression by TuJ1(+) immature neurons appears to be activated again. Also, note that the perinuclear p19^INK4d staining pattern is more extensive in the neurons of the cortical plate than in those of the subplate and intermediate zone where the p19^INK4d has a punctuate perinuclear distribution (also see Fig. 4). CP, Cortical plate; CV, cresyl violet; IZ, intermediate zone; MZ, marginal zone; PP, preplate; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone. Scale bars: A–C, 20 μm; D–I, 50 μm.