NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients

Abstract The relationships between impaired cortical development and consequent malformations in neurodevelopmental disorders, as well as the genes implicated in these processes, are not fully elucidated to date. In this study, we report six novel cases of patients affected by BBSOAS (Boonstra‐Bosch‐Schaff optic atrophy syndrome), a newly emerging rare neurodevelopmental disorder, caused by loss‐of‐function mutations of the transcriptional regulator NR2F1. Young patients with NR2F1 haploinsufficiency display mild to moderate intellectual disability and show reproducible polymicrogyria‐like brain malformations in the parietal and occipital cortex. Using a recently established BBSOAS mouse model, we found that Nr2f1 regionally controls long‐term self‐renewal of neural progenitor cells via modulation of cell cycle genes and key cortical development master genes, such as Pax6. In the human fetal cortex, distinct NR2F1 expression levels encompass gyri and sulci and correlate with local degrees of neurogenic activity. In addition, reduced NR2F1 levels in cerebral organoids affect neurogenesis and PAX6 expression. We propose NR2F1 as an area‐specific regulator of mouse and human brain morphology and a novel causative gene of abnormal gyrification.

The EMBO Journal Michele Bertacchi

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The EMBO Journal e104163 | 2020 ª 2020 The Authors Figure EV1. MRI features of six novel BBSOAS patients and NR2F1 expression along the L-M and A-P axes.
A Two-year-old female patient with thinning of the optic chiasm (left image) and optic nerves (right image). B Four-year-old male patient showing prominent occipital convolutions (left) together with thinning of the posterior half of corpus callosum (right). C Six-year-old female patient with thinning of the posterior half of corpus callosum (left) and of the optic chiasm (right). D Three-year-old female patient with small asymmetry of the occipital cortex (left), thinning of the posterior half of corpus callosum (right), and normal thickness of optic nerves (not shown). E Seven-year-old female patient with thinning of the optic chiasm (left) and mild thinning of posterior corpus callosum (right). F Twelve-year-old male patient showing mild thinning of the optic nerves (left) and of the posterior half of corpus callosum (right). G-G‴ Low-magnification DAPI staining and schematic representation of a GW11 coronal section (G) displaying the position of high-magnification views in (G 0 -G‴) and in Fig 1H and H 0 . Colors in (G) display lateral, dorsal, and medial areas taken for NR2F1 pixel intensity measurements at GW11 (see Fig 1I). Representative images of NR2F1 (red) immunofluorescence (IF) at different levels along the latero-medial (L-M) extent of the cortex, as shown in (G 0 -G″). H Low-magnification DAPI staining of a GW14 sagittal section displaying the position of high-magnification images showed in (I-I″, K-K″) and in Fig 1J-L 0 , N, N 0 and P-P″. I, J NR2F1 (red) IF at GW14 depicting increasing expression levels in the posterior-most cortex (I″), as quantified in (J). n ≥ 4 sections from n = 1 fetal brain. K-L″ TBR2 (green) and SOX2 (red) IF of a GW14 primary convolution (detail taken from H). The average number of different NP classes in the fissure (K 0 ) and convoluted region (K″) is quantified in (L-L″). The convoluted region is populated by a higher number of both apical and basal progenitors. n ≥ 4 sections from n = 1 fetal brain. M Schematic representation of human NR2F1 expression levels correlated with distinct cytoarchitectures along cortical convolutions. Expression of NR2F1 displays local modules of low versus medium/high expression levels in sulci and gyri, respectively. Medium/high NR2F1 expression in gyri is associated with high basal RG numbers and high neurogenic activity promoting radial and tangential expansion, whereas low expression is correlated with a small progenitor pool and low neurogenic potential.

Michele Bertacchi
The The The EMBO Journal e104163 | 2020 ª 2020 The Authors Figure EV4. Delayed neurogenesis and posterior cortical thickening in Nr2f1-deficient mice.
Differentiating cells (EdU + Satb2 + Ki67 À ) can be recognized as they migrate toward the CP. D Short-term EdU injection as in (A), after re-normalization on the total number of EdU + cells, to take account of the increased size of progenitor pool in mutant brains compared to WT. n ≥ 4 sections from n = 2 brains. E Average number of EdU + cells in the LP of control (blue columns) and mutant (orange columns) embryos at P0 upon EdU injection at the indicated stages. Mutant cortices are characterized by a higher density of differentiating neurons at mid-late corticogenesis. n ≥ 4 sections from n = 2 brains. F-F″ Ctip2 (green), Tbr1 (blue), and EdU (red; injected at E12.5) triple IF of P0 WT (F) and KO (F 0 ) brains showing a high number of E12.5-born Tbr1 + EdU + neurons in lower layers of mutant cortices. Graph in (F″) quantifies the average number of EdU + neurons expressing distinct laminar markers (Tbr1 and Ctip2) in WT and KO embryos injected with EdU at E12.5. n ≥ 4 sections from n = 2 brains. G-G″ Cux1 (green) and EdU (red; injected at E15. Data information: Nuclei (blue) were stained with DAPI. In (C, F-G), the number of positive cells was quantified in 100 lm-width boxes, randomly placed across the LP. In graphs, data are represented as means AE SEM. Two-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).