BRN2 as a key gene drives the early primate telencephalon development - PubMed (original) (raw)

. 2022 Mar 4;8(9):eabl7263.

doi: 10.1126/sciadv.abl7263. Epub 2022 Mar 4.

Yicheng Guo 3, Chu Chu 1, Dahai Liu 4, Kui Duan 1 2, Yu Yin 1 2, Chenyang Si 1 2, Yu Kang 1 2, Junjun Yao 1, Xuewei Du 1, Junliang Li 1, Shumei Zhao 1, Zongyong Ai 1, Qingyuan Zhu 1, Weizhi Ji 1 2, Yuyu Niu 1 2, Tianqing Li 1 2

Affiliations

BRN2 as a key gene drives the early primate telencephalon development

Xiaoqing Zhu et al. Sci Adv. 2022.

Abstract

Evolutionary mutations in primate-specific genes drove primate cortex expansion. However, whether conserved genes with previously unidentified functions also play a key role in primate brain expansion remains unknown. Here, we focus on BRN2 (POU3F2), a gene encoding a neural transcription factor commonly expressed in both primates and mice. Compared to the limited effects on mouse brain development, BRN2 biallelic knockout in cynomolgus monkeys (Macaca fascicularis) is lethal before midgestation. Histology analysis and single-cell transcriptome show that BRN2 deficiency decreases RGC expansion, induces precocious differentiation, and alters the trajectory of neurogenesis in the telencephalon. BRN2, serving as an upstream factor, controls specification and differentiation of ganglionic eminences. In addition, we identified the conserved function of BRN2 in cynomolgus monkeys to human RGCs. BRN2 may function by directly regulating SOX2 and STAT3 and maintaining HOPX. Our findings reveal a previously unknown mechanism that BRN2, a conserved gene, drives early primate telencephalon development by gaining novel mechanistic functions.

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Figures

Fig. 1.

Fig. 1.. The spatiotemporal distribution of BRN2 coordinates with RGC expansion in the developing cynomolgus monkey cortex.

(A and B) Dynamic expressions of PAX6 and BRN2 during the monkey cortical expansion through brain coronal sections of the primary visual cortex. BRN2 was initially expressed in NEPCs at embryonic day 30 (E30) (A) and gradually extended along the VZ spread to SVZ at E49 (B). Cortical layers were identified by staining of 4′,6-diamidino-2-phenylindole (DAPI) and PAX6. (C) Immunohistochemical analyses for Brn2 and Pax6 were performed in E9.5 (top) and E12.5 (bottom) mouse neocortices. (D) Images showing the expression patterns of BRN2 in E40 monkey coronal brain sections of the primary visual cortex. (E) Double staining of BRN2 and PAX6 in the E40 VZ and SVZ of BRN2-expressing (BRN2+) or BRN2-absent (BRN2−) zones, respectively. An obvious SVZ was formed in the BRN2+ zone but not in the BRN2− zone. (F) Quantification of BRN2 and PAX6 expression in the BRN2+ and BRN2− zones, respectively. Data are presented as means ± SEM (n = 3 slices); >300 cells per group were counted. (G) Expressions of p-VIMENTIN (PVI) and KI67 in the E40 VZ and SVZ of BRN2+ or BRN2− zones, respectively. The quantification data were only from the cells in the apical regions where cell division occurs. Data are presented as means ± SEM, n = 3 slices; >400 cells per group were counted. Arrowheads indicate positive cells. (a to d) Areas in rectangles are shown with higher magnification. (H) The left panel is a cortical graph at E40, and the right panel is the schematic diagram for measuring the thicknesses of whole-cortex and BRN2-expressing cells in monkey fetal brain. (I) Coordinating BRN2 distribution with the thicknesses of the cortex in E40 monkey neocortex, relative to (H). Start and lateral sites in (H) correspond to those in (I). Blue, DAPI, nuclear staining. Scale bars, 100 μm.

Fig. 2.

Fig. 2.. Generation of _BRN2_-knockout (_BRN2_−/−) monkeys using a CRISPR-Cas9 system.

(A) Workflow of generation and analysis of _BRN2_-KO monkeys. RNP, gRNA-Cas9 nuclease complex. (B) Schematic diagram of target gene editing. Triangles with different colors show the different locations where the sgRNAs target. Two pairs of sgRNA (A+B or A+C) in principle induced type I [with about 1091–base pair (bp) deletion] and type II (with near-488-bp deletion) gene mutations, respectively. (C) Detailed information of all fetuses in this study. Two fetuses (080460 and 081012) were miscarried during gestation, and we failed to collect their tissues for further analysis. (D) Representative images of wild-type (WT; BRN2+/+) and _BRN2_-knockout (KO, _BRN2_−/−) fetus at E29, E36, and E49. Inserts are magnifications of monkey brains, showing abnormal cerebrovascular development in the _BRN2_−/− E36 and E49 fetuses. Scale bars, 3.7 mm. NA, non-examination. (E) Agarose gel electrophoresis of the PCR products from all knockout monkey samples. BRN2+/+ sample (123044-1#) is shown as a negative control of the target gene editing. P, placenta; B, brain cells. (F) Immunostainings of BRN2 and SOX1 in the BRN2+/+ and _BRN2_−/− monkey fetal cortex, confirming BRN2 deletion in the _BRN2_−/− cortex. Blue, DAPI, nuclear staining. Scale bars, 100 μm. IHC, immunohistochemistry.

Fig. 3.

Fig. 3.. BRN2 deletion results in abnormal development of monkey cortex.

(A to D) The coronal sections from the same region of primary somatosensory cortices were used to perform comparison analysis between E49 BRN2+/+ and _BRN2_−/− telencephalon (see Materials and Methods). (A) SOX2 and PAX6 expressions in the _BRN2_−/− and BRN2+/+ cortex, respectively. (B and B′) Representative staining images (B) and quantification (B′) of PVI, KI67, and DAPI in _BRN2_−/− and BRN2+/+ cortical cells. DAPI staining was used to identify cell division orientation. Quantification data of division orientations of RGCs are presented as means ± SEM (n = 6 representative images; >100 cells per group were counted, *P < 0.01). (C) Representative staining images of TUJ1 and SOX1 in _BRN2_−/− and _BRN2_+/+ cortical cells, respectively. (D and D′) HOPX and SOX1 expressions in the _BRN2_−/− and _BRN2_+/+ cortex, respectively. Right images are higher magnifications of left images. (D′) Quantification of HOPX cell migration into the SVZ region from VZ region (means ± SEM, _n_ = 4 representative images; >500 cells per group were counted, *P < 0.01). Scale bars, 100 μm. (E and F) Visualization of major classes of neural cells from BRN2+/+ and _BRN2_−/− single cells across three different stages by Uniform Manifold Approximation and Projection (UMAP) analysis. (G) Plots of classical markers representing RGC (SOX2), intermediate progenitor (IP) (EOMES), and excitatory neuron (ExN) (STMN2), respectively. (H) Proportion dynamic of each cell type over cortical development. (I) Developmental trajectory of cortical cells across three different stages. RG-IP-N (RGC-IP-to-neuron) is the indirect way, while RG-N (RGC-to-neuron) is the direct way. The dotted lines show developmental trajectories of RGCs. The arrowheads show the directions of these trajectories. (J) DEGs between the _BRN2_−/− and BRN2+/+ cortex (see table S3). Representative transcription factors (TFs), gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways are shown. Blue, DAPI, nuclear staining.

Fig. 4.

Fig. 4.. BRN2 loss produced abnormities of monkey interneuron development.

(A) BRN2 and COUP-TFII staining showed the wide distribution of BRN2 in the telencephalic ganglionic eminences (GEs). PSB, pallial-subpallial boundary. (B) NKX2-1 and COUP-TFII expression in BRN2+/+ and _BRN2_−/− GEs, showing that COUP-TFII was specifically expressed in BRN2+/+ CGE/LGE, whereas it was obviously activated in the whole _BRN2_−/− GEs (including both LGE/CGE and MGE). The sections of BRN2+/+ and _BRN2_−/− were from equivalent coronal levels (details are in Materials and Methods). (C) Visualization of major classes of interneuron and interneuron progenitor (iNP) by UMAP analysis. (D) Developmental trajectory of interneurons and iNPs showing that BRN2 loss changed interneuron development trajectory. Cells in cluster 3 were markedly decreased in _BRN2_−/− monkeys. (E) DEGs between BRN2+/+ and _BRN2_−/− interneurons and iNPs (table S4). Representative transcription factors (TFs), GO terms, and KEGG pathways are shown. (F and G) The sections of BRN2+/+ and BRN2−/− telencephalon were from equivalent coronal levels. (F and G) Representative staining images of HOPX and SOX1 in BRN2+/+ (F) and _BRN2_−/− (G) GEs. F1 and G1 are magnifications of the squares in (F) and (G). (H) PAX6 and SOX2 expression in BRN2+/+ and _BRN2_−/− GEs. (I) SST and NKX2-1 expression in BRN2+/+ and _BRN2_−/− GEs, respectively. (J) CALRETININ (CR) expression in BRN2+/+ and _BRN2_−/− telencephalon. Arrows indicate the migration orientations of CR interneurons. All images of immunofluorescence were from coronal sections spanning the rostral-caudal extent of the telencephalon. Scale bars, 500 μm. Blue, DAPI, nuclear staining.

Fig. 5.

Fig. 5.. BRN2 function on monkey RGCs is conserved for human RGCs.

(A) Schematic of generating _BRN2_-knockout (_BRN2_−/−) human embryonic stem cells (ESCs) by CRISPR-Cas9 gene editing. (B) Generation of cortical organoids using human wild-type (BRN2+/+) and _BRN2_−/− ESCs, respectively, showing that _BRN2_−/− ESCs lost the ability to generate cortical organoids. The top panel shows the diagram of cortical organoid generation. (C) Expression of representative neuroepithelium markers in cortical organoids from BRN2+/+ and _BRN2_−/− ESCs. (D) Comparison of proliferation markers KI67 and PVI in BRN2+/+ or _BRN2_−/− cortical organoids. DAPI staining was used to identify cell division orientation. (E) Quantifications of cell division orientation in BRN2+/+ and _BRN2_−/− cells of cortical organoids (means ± SEM, n = 4 independent experiments; >200 cells per group were counted), respectively. (F) Comparison of CUX1 (an upper-layer neuron marker) and FOXP2 (a deep-layer neuron marker) expressions between BRN2+/+ and _BRN2_−/− cortical organoids, respectively. (G) Quantification of SOX2-, PAX6-, CUX1-, and FOXP2-positive cells in BRN2+/+ and _BRN2_−/− cortical organoids, respectively. Data are presented as means ± SEM, n = 3 slices; >300 cells per group were counted. *P < 0.05. Scale bars, 100 μm.

Fig. 6.

Fig. 6.. The regulatory mechanisms of BRN2 on primate telencephalon development.

(A and B) Comparison of BRN1 expressions between BRN2+/+ and _BRN2_−/− human ESC–derived cortical organoids (A) and between BRN2+/+ and _BRN2_−/− monkey cortical cells (B). Data are presented as means ± SEM (n = 3). NS, no significance, P > 0.05. (C) Gene plots show densities of BRN2, polymerase II (Pol II), and control (IgG) at the SOX2 promoter in human developing cortex, indicating that BRN2 binds to the promoter region of SOX2. (D) Gene plots show densities of BRN2, Pol II, and control (IgG) at the STAT3 promoter in human developing cortex, indicating that BRN2 regulates STAT3 expression by directly binding to the promoter region of STAT3. (E) Comparison of STAT3 expressions in the BRN2+/+ and _BRN2_−/− cortex, which exhibited a significant down-regulation in the _BRN2_−/− cortex. **P < 0.01. (F) Summary of BRN2 regulatory mechanisms on monkey telencephalon development. aRGC, apical RGC. The thickness and fineness of the line represent the strong and weak activity, respectively. Arrows indicate the activation, and trident lines indicate the repression.

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