Analysis of Auts2 protein sequence and cellular localization. A: alignment of Auts2 amino acid sequence from Mus Musculus (MM), Homo Sapiens (HS) and Gallus Gallus (GG). Conserved residues are represented in black, similar residues in gray, and differently charged residues are not marked. Nuclear localization sequences (NLS) are highlighted in red. A’: Upper trace shows putative SH2 and SH3 domains. Lower trace shows surface accessibility plot with putative NLS (red). B: The specificity of Auts2 antibody was tested by Western blotting on E14 protein mouse cortical extract, revealing a band at 139.8 kDa. C–D: Auts2 protein was evaluated by transfection of membrane tomato (red) alone (C) or with Auts2 (green, D) in 293T cells. Auts2 was detected in the nuclei of cells transfected with the Auts2 expression plasmid. E–F: Expression of Auts2 (green) in postnatal day (P) 21 cortex showed no obvious colocalization with GFAP (red, E). However, Auts2 was detected in neurons, as shown by colocalization with NeuN (red, P21, F). Scale bars: C–D–E–F: 10 µm.

Auts2 mRNA expression patterns at different embryonic and postnatal ages revealed by in situ hybridization (ISH). NCX: neocortex; V: ventricle; PP: preplate; VZ: ventricular zone; CP: cortical plate; DT: dorsal thalamus; CB: cerebellum; LC: locus ceruleus; OB: olfactory bulbs; FCX: frontal cortex; HIP: hippocampus; PT: pretectum; SC: superior colliculus; IC: inferior colliculus; ATN: anterior thalamic nuclei; VL/VM: ventrolateral/ventromedial nuclei; VN: vestibular nuclei. Scale bars: A: 500 µm; B: 200 µm; C–D–F–G: 1mm; d: 100 µm; E: 200 µm, e: 75 µm, H: 500 µm, h’ (for h’–h”) 100 µm. Ages: A: E11, B: E12, C: E13, D: E14, E: E16, F: E19, G: P21.

Auts2 mRNA expression in the developing cortex, revealed by ISH (green, pseudocolor). The pattern of expression is studied in association with other markers detected by immunohistochemistry (red) in the same section. A, a, E, e, G: Tbr1; B, b: calretinin; C, c: Pax6; D, d: Tbr2; F, f, H: Ctip2; I: Cux1. CP: cortical plate; IZ: intermediate zone. Scale bars: A: 50 µm (applies to A, B); a: 20 µm (applies to a, b); C: 50 µm (applies to C–F); c: 20 µm (applies to c–f); G: 100 µm (applies to G–I). Ages: A–B, a–b: E13, C–F, c–f: E14, G–I, g–i: P0.

Auts2 mRNA and protein expression in the developing hippocampus. A–B: At P0, Auts2 expression, as detected by ISH (gray or green), was apparent throughout the developing hippocampus where it colocalized with Tbr1 immunofluorescence (red). C–H: Auts2 protein, detected by immunofluorescence at P21, was apparent throughout the granule cell layer (GCL) of the dentate gyrus. Coexpression with specific markers of progenitor cells and differentiating neurons was assessed by double immunofluorescent labeling with C) Sox2; D) PCNA; E) NeuroD1; F) calbindin (CalB); G–G”) DCX; H–H”) calretinin (CalR). Scale bars: A 100 µm (applies to A, B); C: 50 µm (applies to C–F) G: 50 µm (applies to G–H”).

Expression of Auts2 mRNA in brain regions outside the cortex at different stages of development. A–a) Double labeling for Auts2 mRNA revealed by in situ hybridization (green) and Tbr1 protein by immunofluorescence (red) in the E14 cerebellum. NTZ: nuclear transitory zone; EGL: external granular layer; CTZ: cortical transitory zone; RL: rhombic lip; VZ: ventricular zone. B) Postnatal day (P) 21 cerebellum labeled with Auts2 (green) in relationship to Tbr1 (red); b) expression of Auts2 protein (green, immunofluorescence) in Purkinje cells identified by calbindin immunofluorescence (red). C) Auts2 mRNA (green) was expressed in the dorsal thalamus (DT), in a complementary fashion compared to the eminentia thalami (ET), stained by Tbr1 (red, immunofluorescence). D) Auts2 mRNA (green) was localized in the P21 olfactory bulb mitral cells (MC) and the periglomerular cells (PGC); overlap with Tbr1 (red) was found in some MC. Also, some overlap of Auts2 mRNA (green) with GAD67 (red, d), a marker of GABAergic neurons, was detected in the GC (arrows). E) Strong coexpression of Auts2 protein (green) and TH (red) was found in the substantia nigra (SN) and the ventral tegmental area (VTA). Scale bars: A: 100 µm; a: 50 µm; B: 100 µm; b: 50 µm; inset b: 20 µm; C: 200 µm; D: 100 µm; d: 30 µm; E: 100 µm; inset E: 20 µm. Ages: A–a: E14, B–b: P21, C: E14, D–d: P21, E: P21.