Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism

Abstract

Many cerebral cortical neurons and glia are produced by apical progenitors dividing at the ventricular surface of the embryonic dorsal telencephalon. Other neurons are produced by basal progenitor cells, which are derived from apical progenitors, dividing away from the ventricular surface. The transcription factor Pax6 is expressed in apical progenitors and is downregulated in basal progenitors, which upregulate the transcription factor Tbr2. Here we show that Pax6(-/-) cells are under-represented in the cortex of Pax6(+/+)<-->Pax6(-/-) chimeras early in corticogenesis, indicating that Pax6 is required for the production of normal numbers of cortical cells. We provide evidence that this underproduction is attributable to an early depletion of the progenitor pool caused by greater than normal proportions of newly divided cells exiting the cell cycle. We show that most progenitor cells dividing away from the ventricular surface in Pax6(-/-) embryos fail to express the transcription factor Tbr2 and that Pax6 is required cell autonomously for Tbr2 expression in the developing cortex of Pax6(+/+)<-->Pax6(-/-) chimeras. Transcription factors normally expressed ventrally in the telencephalic ganglionic eminences (Mash1, Dlx2 and Gsh2) are upregulated cell autonomously in mutant cells in the developing cortex of Pax6(+/+)<-->Pax6(-/-) chimeras; Nkx2.1, which is expressed only in the medial ganglionic eminence, is not. These data indicate that early functions of Pax6 in developing cortical cells are to repress expression of transcription factors normally found in the lateral ganglionic eminence, to prevent precocious differentiation and depletion of the progenitor pool, and to induce normal development of cortical basal progenitor cells.

Fig. 1

Pax6^−/− cells are under-represented in the telencephalon of E12.5 Pax6^−/−↔Pax6^+/+ chimeras. Plastic embedded sections of (A) Pax6^+/+ and (B) Pax6^−/− E12.5 cortex. Boxed area in panel A is an enlargement of preplate of wild-type neocortex showing a cell undergoing mitosis (closed arrowhead) next to a cell undergoing apoptosis (open arrowhead). Mitotic figures were seen at the ventricular surface and at subventricular locations in both Pax6^+/+ and Pax6^−/− cortex (e.g., arrow in panel B). (C) Pax6 protein localization in the E12.5 Pax6^+/+ telencephalon. Pax6 is expressed in a ^highlateral to ^lowmedial gradient in the ventricular zone of the developing neocortex (ctx), hippocampus (h) and dorsal lateral ganglionic eminence (dLGE). (D) High power image of medial telencephalon showing boundary of Pax6 expression between hippocampus and cortical hem (ch). (E) Pax6^−/−↔Pax6^+/+ chimera (with a global contribution of 53% Pax6^−/−, Tg+ cells determined by GPI1 analysis) showing under-representation of Pax6^−/−, Tg+ cells (brown spots in nucleus) in the Pax6-expressing hippocampus but not in the cortical hem. (F) Composition of telencephalic regions of E12.5 Pax6^+/+↔Pax6^+/+ and Pax6^−/−↔Pax6^+/+ chimeras. Mean ratios ± SEM of corrected percentages of Tg+ cells in each tissue to global GPI1B percentage in the embryo (Oc/E) are shown for both Pax6^+/+↔Pax6^+/+ (light bars) and Pax6^−/−↔Pax6^+/+ (dark bars) chimeras. At E12.5, there is a significant reduction in the number of Pax6^−/− cells contributing to hippocampus (Student's t-test, p < 0.001), neocortex (p < 0.03) and dLGE (p < 0.02) in Pax6^−/−↔Pax6^+/+ (dark bars) chimeras. Significant differences are marked with an asterisk (*). Scale bars: (A, B) = 20 μm, (C) = 200 μm, (D) = 100 μm, (E) = 50 μm.

Fig. 2

Analysis of cell cycle characteristics using IdU/BrdU co-labeling. (A) Injection at T0 h with IdU and T1.5 h with BrdU followed by sacrifice at T2.0 h allows identification of three cell types: those within S-phase at T0–1.5 but within G2/M at T1.5–2.0 (L_cells: IdU-labeled only—green in panels B–E); cells entering S-phase at T1.5–2.0 (BrdU-labeled only—red in panels B–E); cells within S-phase at both T0 and T1.5 (IdU/BrdU co-labeled—yellow in panels B–E). By counting the numbers of cells in each of these three populations, cell cycle characteristics can be determined as described in Methods. Cell cycle characteristics were examined at E10.5 (B, C) and E12.5 (D, E) in wild-type (B, D) and Pax6^−/− (C, E) telencephalon. Scale bar = 50 μm.

Fig. 3

Assessment of proportions of cells exiting the cell cycle using cumulative BrdU labeling. (A) Section through the neocortex of an E12.5 Pax6^+/+ embryo showing BrdU-labeled cells after a 12.5-h pulse of BrdU-labeled cells are either heavily (closed arrowhead) or lightly labeled (open arrowhead). (B) Diagrammatic representation of the cell cycle indicating how proportions of heavily and lightly labeled cells would vary for progressively longer pulses of BrdU. After a pulse shorter than the length of time between S-phase and M-phase (example 1), a single population of heavily labeled cells would be identified (black circles). After a longer pulse (example 2), a second population of labeled cells with a lower mean intensity of label would emerge as cells progress through M-phase (grey symbols). As the pulse lengthens further (example 3) cells in the second, lightly labeled population would either re-enter S-phase and replenish their BrdU load and become heavily labeled or would exit the cell cycle, enter G0 and remain lightly labeled. (C–E) Graphs illustrate frequency distributions for intensity of BrdU label in cells in histological sections after pulses of BrdU lasting (C) 0.5 h and (D, E) 12.5 h in wild-type (C, D) and mutant (C, E) neocortex. Each set of differently shaded bars corresponds to an individual animal. (C) At 0.5 h in both mutant and wild type all cells are heavily labeled with BrdU. (D, E) After a 12.5-h pulse with BrdU, heavily and lightly labeled cell populations can be identified but more lightly labeled cells are present in the mutant (E) than in wild type (D).

Fig. 4

Precocious neurogenesis in the E12.5 Pax6^−/− telencephalon. β-III-tubulin expression in (A) wild-type and (B, C) Pax6^−/− neocortex. (A) In wild-type telencephalon at E12.5, β-III-tubulin-expressing cells are present in the preplate. (B) In Pax6^−/− telencephalon, β-III-tubulin-expressing neurons can be observed both in the preplate and abnormally located within the ventricular zone (white arrowheads in panel C). (C) Enlargement of area outlined in panel B. (D–F) Flow cytometric analysis of β-III-tubulin expression in dissociated (E) Pax6^+/+ and (F) Pax6^−/− E12.5 neocortical cells. (D) No primary (NP) control reaction: gating was established (boxed region in panels D–F) by PI staining for cells containing a diploid DNA content (cells in G1/G0). Plots shown are from a single representative experiment for each genotype. The proportion of cells expressing β-III-tubulin are within the boxed region. (F) Pax6^−/− neocortex shows an increase in the number of β-III-tubulin-expressing neurons. Scale bars = (A, B) 80 μm; (C) = 40 μm.

Fig. 5

The transcription factor Tbr2 is downregulated in the Pax6^−/− telencephalon. (A) Western blot for Tbr2 protein from E12.5 and E14.5 cortex of Pax6^+/+ and Pax6^−/− embryos. A significant reduction in Tbr2 protein is observed in the homozygous mutant lateral cortex at both E12.5 and E14.5. (B–E) Tbr2 localization in Pax6^+/+ (B, D) and Pax6^−/− (C, E) telencephalon at E12.5. In Pax6^+/+ telencephalon, Tbr2-expressing cells are located in the preplate (pp) and within the ventricular and subventricular zone (vz). In the Pax6^−/− telencephalon, almost all Tbr2-expressing cells are present in the preplate. (F) Quantification of telencephalic cell density and (G) Tbr2-expressing cell density in hippocampus (h), dorsal cortex (dcxt), central neocortex (cctx) and lateral neocortex (lctx) (boxed areas in panel B) at E12.5 in Pax6^+/+ (open bars) and Pax6^−/− mutant (filled bars) telencephalon. (F) No significant difference is observed in cell density between mutant and wild-type animals in any telencephalic region examined. (G) A significant increase is observed in the density of Tbr2-expressing cells present in the Pax6^−/− hippocampus and a significant decrease in the densities observed in the Pax6^−/− central neocortex and lateral neocortex compared to wild types. Significant differences (Student's t-test, p < 0.05) are marked with an asterisk (*).

Fig. 6

Tbr2-expressing postmitotic neurons are present in the Pax6^−/− telencephalon. (A) In the proliferative layer of E12.5 wild-type neocortex, Tbr2 labels cells that are β-III-tubulin negative (red arrowheads). Some of these cells appear to be dividing indicating they are BPCs (red arrowhead containing white m). In the preplate, Tbr2/β-III-tubulin co-expressing postmitotic neurons are observed (yellow arrowhead) as well as β-III-tubulin-expressing neurons which are down regulating or have down regulated Tbr2 expression (white arrowhead). (B) In the proliferative layer of E12.5 Pax6^−/− neocortex, many Tbr2-positive cells are β-III-tubulin-positive (yellow arrowheads) while some are not (red arrowhead). In the preplate, co-expressing (yellow arrowhead) and β-III-tubulin-positive/Tbr2 negative (white arrowhead) cells are seen. (C, D) A 2-h BrdU pulse was used to label cells that are actively cycling in the E12.5 Pax6^+/+ and Pax6^−/− telencephalon. (C) In Pax6^+/+ telencephalon, Tbr2/BrdU co-labeled BPCs can be observed in the basal ventricular zone and preplate (white arrowheads). (D) In the Pax6^−/− telencephalon, very few of the BrdU-labeled cells express Tbr2 (white arrows). Scale bars: (A, B) = 20 μm; (C, D) = 40 μm.

Fig. 7

Few abventricular mitoses express Tbr2 in Pax6^−/− mutant telencephalon. Immunohistochemistry for Tbr2 and phosphohistone-3 (pH3) in panels A, C–E Pax6^+/+ and (B, F–H) Pax6^−/− E12.5 telencephalon. pH3 labels mitotic cells in late G2/M-phase. (A, C–E) In wild-type telencephalon, cells strongly reactive for pH3 can be observed both at the apical ventricular surface (avs) and within the basal ventricular zone (bvz); all pH3-expressing cells observed in the bvz are also Tbr2-expressing (yellow arrowheads in panels C–E). (B, F–H) In the Pax6^−/− neocortex, there are very few BPCs expressing both Tbr2 and pH3 (yellow arrowhead). In contrast, many pH3-expressing, Tbr2-non-expressing cells (light blue arrowheads) are present within the proliferating zone. Scale bars (A, B) = 100 μm, (C–H) = 80 μm.

Fig. 8

Mash1 is ectopically expressed in some Pax6^−/− neocortical abventricular mitotic cells. (A) At E12.5 in Pax6^+/+ telencephalon, Mash1 is restricted to the ganglionic eminences (lge, lateral ganglionic eminence), abutting Tbr2 expression in the developing neocortex (lctx, lateral cortex) at the pallial–subpallial boundary. (B) In E12.5 Pax6^−/− telencephalon, many cells throughout the cerebral cortex (ctx) express Mash1. Tbr2-expressing cells of the mutant preplate do not express Mash1. (C) In E14.5 Pax6^+/+ telencephalon, Mash1 expression is restricted to the ganglionic eminences. (D) In E14.5 Pax6^−/− telencephalon, dorsal and ventral telencephalic cells express Mash1. (E,G) Abventricular mitoses in the lateral ganglionic eminence of Pax6^+/+ telencephalon express Mash1 (yellow arrows; panel G is the same field as panel E in green channel alone). (F, H) Most pH3-expressing cells undergoing mitosis in abventricular locations in the Pax6^−/− neocortex do not express Mash1 (red arrows); a minority of these cells do express low levels of Mash1 (yellow arrows; panel H is the same field as panel F in the green channel alone). Scale bars (A, B) = 80 μm; (C, D) = 160 μm; (E–H) = 40 μm.

Fig. 9

Pax6 is required cell autonomously for Tbr2 expression and repression of lateral ganglionic eminence markers in cortical cells. (A, B) Sections of cortex of Pax6^−/−↔Pax6^+/+ chimera double-labeled for Tbr2 protein (green) and Pax6^−/− cells (red dots resulting from DNA–DNA in situ hybridization to a reiterated β-globin transgene, β-glb). Most Pax6^−/− cells do not express Tbr2 (Panel A, rostral section; Panel B, central section). (C, D) Sections of cortex (C) and lateral ganglionic eminence (D) of Pax6^−/−↔Pax6^+/+ chimera double-labeled for Mash1 protein (grey) and Pax6^−/− cells (brown dots). In cortex, Mash1 expression is in Pax6^−/− cells but not in wild-type cells. In the lateral ganglionic eminence, Mash1 is expressed in both wild-type and Pax6^−/− cells. (E, F) Sections of rostral (E) and caudal (F) cortex of Pax6^−/−↔Pax6^+/+ chimera double-labeled for Gsh2 protein (grey) and Pax6^−/− cells (brown dots). Gsh2 expression coincides with the Pax6^−/− cells. (G) Dlx2-positive cells in the cortex of a Pax6^−/−↔Pax6^+/+ chimera. (H) Dlx2-positive cells in the lateral ganglionic eminence (lge), medial ganglionic eminence (mge) and cortex (ctx) of a Pax6^−/−↔Pax6^+/+ chimera; (I) Dlx2-positive cells in the cortex are Pax6^−/− (i.e., Tg-positive). (J) Nkx2.1-positive cells in the medial ganglionic eminence (mge) and around the internal capsule (ic) of a Pax6^−/−↔Pax6^+/+ chimera; (K) Pax6^−/− (Tg+) cortical cells are Nkx2.1-negative. Scale bars: (A, B, G, K) = 40 μm; (C–F, I) = 20 μm; (H, J) = 60 μm.

Fig. 10

Pax6^−/− cells in the medial regions of the cortex downregulate Tbr2 and upregulate ventral telencephalic markers. (A) Hippocampus of Pax6^−/−↔Pax6^+/+ chimera double-labeled for Tbr2 protein (green) and Pax6^−/− cells (red dots, as in Fig. 9). (B) Hippocampus of Pax6^−/−↔Pax6^+/+ chimera double-labeled for Mash1 protein (grey) and Pax6^−/− cells (brown dots). Scale bar = 20 μm.