Extended Data Fig. 9: Lower levels of AKNA in differentiating neurons mediate non-centrosomal microtubule polymerization and allow migration into the cortical plate.
From: The centrosome protein AKNA regulates neurogenesis via microtubule organization
a, b, Primary E14 cortical cells were sorted for PROMININ 1 (CD133) to isolate NSCs or for PSA-NCAM to isolate neurons as indicated in the panels. PROM1+ cells express the NSC marker nestin, whereas PSA-NCAM+ cells express the neuronal marker TUBB3, showing the specificity of the FACS (a) (n = 2 independent experiments). Microtubule regrowth assay in purified NSCs shows centrosomal microtubule polymerization (b, top), and purified neurons show largely non-centrosomal microtubule polymerization patterns (b, bottom) (n = 2 independent experiments). c, Micrographs showing co-electroporation of a CAG–dsRED and DCX–GFP at E13 and analysis at E15. Note the onset of Dcx–GFP expression (c′′) only in the SVZ and cortical plate of the developing cortex, whereas DsRED+ cells are also found in the ventricular zone (c, c′) (n = 3 embryos). d, e, Micrographs showing electroporated cells (GFP+) in control (Dcx-GFP) (d) and Akna overexpressing (Dcx-Akna) (e) conditions (n = 3 embryos each conditions). Note that many AKNA overexpression cells accumulate in the SVZ, and are unable to migrate into the cortical plate. f, Micrographs of AKNA overexpression cells retained in the SVZ co-stained for TBR1, CTIP2, AND CUX1, which label neurons of different layer identity (n = 3 embryos). Note that the neuronal populations accumulating below the cortical plate upon Dcx-driven Akna expression contain neurons that are positive for each of these neuronal identities. g, Micrographs showing that electroporated cells (GFP+) after control and Dcx-driven miRNA mediated downregulation of AKNA enter the cortical plate equally well (n = 3 embryos). h, Line graph illustrating the distribution of GFP+ cells after IUE for Dcx-miRNA(neg), and Dcx-Akna miRNA no. 4. Mean ± s.e.m. as a transparent band (Dcx-miRNA(neg), n = 4; Dcx-Akna miRNA no. 4, n = 3 embryos). Scale bars, 5 μm (a, b), 50 μm (c–g).
