Fig. 4.

RB loss results in aberrant neuronal migration. (A) Representative wild-type and KO organoids at 28 DIV sectioned and immunostained against Tuj1 and counterstained with DAPI. Arrowheads in the wild-type and KO organoid show Tuj1⁺ cells in or around the tubular region. An arrow in the KO organoid shows ectopic Tuj1⁺ cells outside the tubular region. Inset in the KO organoid shows an enlarged image of Tuj1⁺ cells with corresponding nuclei outside the tubular region. The graphs show the percentage of Tuj1-stained area in or around and outside the tubular region. (B) Representative wild-type and KO organoids at 28 DIV sectioned and immunostained against Vimentin and counterstained with DAPI. The graphs show the percentage of Vimentin-stained area in or around and outside the tubular region. (C) Relative VLDLR mRNA levels measured by RT-qPCR. (D) Representative wild-type and KO teratomas sectioned and immunostained against Tuj1 and GFAP, and counterstained with DAPI. Arrowheads in the wild-type and KO teratoma show Tuj1⁺ cells in or around the tubular region. An arrow in the KO teratoma shows ectopic Tuj1⁺ cells outside the tubular region. Inset in the KO teratoma shows an enlarged image of Tuj1⁺ cells with corresponding nuclei outside the tubular region. (E) A model of the role of RB during human brain development. In wild-type cells, RB regulates entry into S-phase. However, loss of RB promotes an accumulation of cells in S-phase and an increase in cell death that is associated with upregulation of CCNA2 and BAX. RB deficiency also leads to aberrant neuronal migration associated with an increase in expression of VLDLR. The results in A-D are mean±s.e.m. of three to five organoids from two to four independent experiments (*P<0.05, paired Student's t-test, n.s., not significant). Scale bars: 50 µm; 25 µm in insets.