The expression of aristaless-related homeobox in neural progenitors of gyrencephalic carnivore ferrets

Abstract

Aristaless-related homeobox (ARX) has important functions in the development of various organs including the brain. Mutations of the human ARX gene have been associated with malformations of the cerebral cortex such as microcephaly and lissencephaly. Although the expression patterns of ARX in the lissencephalic cerebral cortex of mice have been intensively investigated, those in expanded gyrencephalic brains remained unclear. Here, we show the expression patterns of ARX in the developing cerebral cortex of gyrencephalic carnivore ferrets. We found that ARX was expressed not only in intermediate progenitor (IP) cells but also in outer radial glial (oRG) cells, which are neural progenitors preferentially observed in the gyrencephalic cerebral cortex. We found that the majority of ARX-positive oRG cells expressed the proliferating cell marker Ki-67. These results may indicate that ARX in oRG cells mediates the expansion of the gyrencephalic cerebral cortex during development and evolution.

Keywords: ARX; Ferret; Intermediate progenitor cells; Neural progenitors; Outer radial glial cells.

Fig. 1

The distribution of ARX expression in the germinal zones of the ferret cerebral cortex. (A, B) Coronal sections of the ferret cerebrum at E40 were stained using anti-ARX antibody (red) and Hoechst 33342 (blue). Lower magnification images (A) and higher magnification images of the germinal zones of the cerebral cortex (B) are shown. (C) Coronal sections of the ferret cerebrum at E40 were stained using anti-ARX antibody (green), anti-reelin antibody (red) and Hoechst 33342 (blue). High magnification images of the marginal zone are shown. (D) Coronal sections of the ferret cerebral cortex at P16 were stained using anti-ARX antibody (red) and Hoechst 33342 (blue). Higher magnification images of the germinal zones of the cerebral cortex are shown. MZ, marginal zone; CP, cortical plate; GZ, germinal zone; GE, ganglionic eminence; VZ, ventricular zone; ISVZ, inner subventricular zone; OSVZ, outer subventricular zone. Scale bars = 1 mm (A), 200 μm (B, D) and 30 μm (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

ARX expression in neural progenitors in the ferret OSVZ. (A) Coronal sections of the ferret cerebral cortex at E40 were stained using Hoechst 33342, anti-ARX (green), anti-Pax6 (red) and anti-Tbr2 (cyan) antibodies. Images corresponding to the germinal zones are shown. (B) High magnification images of the OSVZ. Arrowheads and arrows indicate ARX-positive oRG cells and ARX-positive IP cells, respectively. (C) Coronal sections of the ferret cerebral cortex at E40 were stained using Hoechst 33342, anti-ARX antibody (green) and anti-Sox2 antibody (red). High magnification images of the OSVZ are shown. Arrows indicate ARX- and Sox2-positive cells. (D) The percentages of oRG cells and IP cells co-expressing ARX. Bars represent mean ± SD. Student's t-test; **p < 0.01; n = 4 for each condition. Scale bars = 50 μm (A), 10 μm (B) and 25 μm (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The expression of Ki-67 in ARX-positive neural progenitors in the ferret OSVZ. (A) Coronal sections of the ferret cerebral cortex at E40 were stained using anti-ARX (green), anti-Pax6 (red), anti-Tbr2 (cyan) and anti-Ki-67 (blue) antibodies. Images corresponding to the germinal zones are shown. (B) High magnification images of the OSVZ. Arrowheads and arrows indicate ARX-positive oRG cells and ARX-positive IP cells co-expressing Ki-67. (C) The percentages of ARX-positive oRG cells and ARX-positive IP cells co-expressing Ki-67. (D) Coronal sections of the ferret cerebral cortex at E40 were stained using Hoechst 33342, anti-ARX antibody (green) and anti-pHH3 antibody (red). Images corresponding to the germinal zones are shown. (E) High magnification images of the OSVZ. Bars represent mean ± SD. Scale bars = 50 μm (A), 10 μm (B), 200 μm (D) and 25 μm (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)