January 1, 2016;
The evolution of basal progenitors in the developing non-mammalian brain.
The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain
structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon
(basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2
(+) intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium
show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN
(Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2
(+) cells are distinct cell lineages in the developing avian telencephalon
. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain
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Fig. 1. Distinct characteristics of basal mitotic cells and Tbr2+
cells in the developing chicken pallium. (A,B) Distribution of phosphorylated histone H3 (PH3)+ cells in the developing mouse (A, E15.5) and chick (B, E8) telencephalon. Arrows indicate mitotic cells on the basal side of the pallia. (C,D) The proportion of the PH3+ cells that were also Pax6+, Sox2+ or EdU+ on the basal side of the chicken pallium. Arrows, indicate PH3+/Pax6+ cells (C, magnified by insets) and Sox2+/EdU+ cells (D). Mean±s.d. (E,F) Distribution of Tbr2+ cells in the developing mouse (E, E12.5) and chick (F, E8) telencephalon. (G-J) Labeling of Tbr2+ cells by electroporation (EP) with a GFP expression vector. Electroporation was performed at E4 and the chick embryos were collected at E7. Arrowheads (I,J), GFP+/Tbr2+ cells. (K-P) Characterization of Tbr2+ cells in the developing chick pallium. The sections were stained with anti-Pax6 (K), anti-BrdU (L,M), anti-PH3 (N) or anti-NeuN (O,P) antibodies and an anti-Tbr2 antibody. Arrowhead (N), a PH3+ cell that does not overlap with Tbr2. DP, dorsal pallium; DVR, dorsal ventricular ridge; MP, medial pallium; NCx, neocortex; GE, ganglionic eminence; SVZ, subventricular zone; VZ, ventricular zone. Scale bars: 200 µm in B (for A,B), E (for E,F), G; 50 µm in C (for C,D), K (for K-P); 10 µm in I (for I,J).
Fig. 2. Radial glial morphology of chicken basal mitotic cells. (A-G) Visualization of basal mitotic cells in the developing chicken pallium with the pCALNL-GFP vector (A-C) or a membrane-bound GFP vector (D-G). Electroporation was performed at E4 and samples were collected at E6. Note that PH3+ basal mitotic cells (purple arrow) extended radial fibers toward both the basal and apical sides of the brain (red arrowheads in A). (D-G) Oblique cleavage plane of basal mitotic cells. (H) Classification of basal mitotic cells based on the presence of apical and/or basal radial fibers. (I) Quantification of the cleavage plane angle relative to the ventricular surface in APs and BPs. Bars indicate mean. *P<0.05, **P<0.01; Student's t-test. NL, neuronal layer; VZ, ventricular zone. Scale bars: 10 µm in A (for A-C), D (for D-G).
Fig. 3. Experimental amplification of basal mitotic cells and Tbr2+ cells in the developing chick pallium. (A) Diagram of the LGN and LGN-C proteins. C-terminal TPR repeats were replaced with mCherry in LGN-C. (B,C) LGN-C increased the number of PH3+ cells on the basal side of the chicken pallium at the expense of APs (arrowheads in B). (D-G) Overexpression of Cdk4 and cyclin D1 (collectively 4D) in the developing chick pallium. (D) The structures of the 4D vectors. Co-expression of GFP and RFP in the same cells was induced by electroporation of 4D vectors. (E-G) Overexpression of 4D vectors increased the number of Tbr2+ cells. (H-J) Overexpression of Tbr2 in the developing chicken pallium. (H) Structure of the expression vectors containing GFP and Tbr2. GFP and Tbr2 were co-expressed in the same cells upon electroporation. (I,J) Overexpression of Tbr2 suppressed Sox2 expression in APs but did not change the number of APs and BPs. All bar charts show mean±s.d. **P<0.01 (Student's t-test); n.s., not significant. Scale bars: 50 µm in B,E (for E,F); 25 µm in D; 15 µm in H; 10 µm in I.
Fig. 4. Comparative analysis of basal mitotic cells in various tetrapod species. (A,B) Distribution of PH3+ cells in the developing opossum cortex (M. domestica, P1). PH3+ basal mitotic cells (arrowheads) expressed Pax6 (A) or Sox2 (B). (C,C′) A PH3+ basal mitotic cell (arrowhead) was Sox2+ in the developing turtle cortex. (D,E) Distribution of PH3+ cells in developing Xenopus (D,D′) and axolotl (E). Arrowheads indicate in green (D′) indicate PH3+ cells at the ventricular surface; arrowheads in yellow (D,D′) and white (E) indicate PH3+ cells at the basal side of the ventricular zone. (F) The proportion of basal mitotic cells among all pallial mitotic cells in various tetrapods. Mean±range (opposum, turtle, axolotl) or mean±s.d. (Xenopus). Scale bars: 100 µm in A,C,D; 50 µm in B,E.
Fig. 5. A possible scenario of changes in pallial progenitor subtypes during amniote evolution. In mammals, apical progenitors (APs) proliferate and generate basal progenitors (BPs), which include Tbr2+ intermediate progenitors (IPs) and other types of BPs [such as basal radial glial cells (bRGs)]. In birds, both BPs and Tbr2+ cells exist, but these cells are distinct populations. We propose that a few subapical mitotic cells that have already evolved in ancestral amniotes are at the origin of BPs, although de novo BPs appeared in several taxa independently.
Evolution of cortical neurogenesis.