BMP7 expression in mammalian cortical radial glial cells increases the length of the neurogenic period

Abstract

The seat of human intelligence is the human cerebral cortex, which is responsible for our exceptional cognitive abilities. Identifying principles that lead to the development of the large-sized human cerebral cortex will shed light on what makes the human brain and species so special. The remarkable increase in the number of human cortical pyramidal neurons and the size of the human cerebral cortex is mainly because human cortical radial glial cells, primary neural stem cells in the cortex, generate cortical pyramidal neurons for more than 130 days, whereas the same process takes only about 7 days in mice. The molecular mechanisms underlying this difference are largely unknown. Here, we found that bone morphogenic protein 7 (BMP7) is expressed by increasing the number of cortical radial glial cells during mammalian evolution (mouse, ferret, monkey, and human). BMP7 expression in cortical radial glial cells promotes neurogenesis, inhibits gliogenesis, and thereby increases the length of the neurogenic period, whereas Sonic Hedgehog (SHH) signaling promotes cortical gliogenesis. We demonstrate that BMP7 signaling and SHH signaling mutually inhibit each other through regulation of GLI3 repressor formation. We propose that BMP7 drives the evolutionary expansion of the mammalian cortex by increasing the length of the neurogenic period.

Keywords: BMP7; SHH; cortical evolution; cortical gliogenesis; cortical neurogenesis; radial glia.

Figure 1.

Human cortical oRG cells and tRG cells exhibit distinct transcriptional signatures. (A–D) scRNA-Seq analysis of molecular profiles of human cortical cells at GW22, GW23, and GW26. tSNE plot showed EOMES (TBR2)-, NEUROG2-, and PPP1R17-expressing PyN-IPCs, suggesting that cortical neurogenesis extends to GW26. (E) Heat map of selected differentially expressed genes for oRG cells versus tRG cells. Note that HOPX and FAM107A are enriched in oRG cells. (F) Selected GO terms implicating oRG cells are neurogenic while tRG cells are gliogenic. (G) Schematic summarizing mouse and human cortical RG cell lineage progression. Note that the human OSVZ is a duplicated neurogenic zone, with a greatly prolonged period of cortical neurogenesis. Thus, the length of the cortical neurogenic period is more than 130 days (GW8–GW26) in humans.

Figure 2.

BMP7 is expressed by more and more cortical RG cells during mammalian development and evolution. (A–C) In situ hybridization shows that Bmp7 mRNA is expressed in the mouse medial cortex, but not in the dorsal cortex at E13.0 and E16.0. (D) Maps of BMP7-expressing cells in the human cortex at GW8. The original picture of BMP7 mRNA in situ hybridization on human GW8 cortical section is from previous studies (Abu-Khalil et al., 2004). (E–H) RNAscope in situ hybridization shows BMP7 expression in germinal zones of the human cortex and caudal ganglionic eminence (CGE) at GW18. Note BMP7 is also expressed in meninges. (I) Comparative scRNA-Seq analyses reveal an increase in the percentage of cortical RG cells that expressed BMP7 in the E15 mouse cortex, E39 ferret dorsal cortex, E78 rhesus monkey visual cortex, and GW14 human prefrontal cortex. (J) Increasing the percentage of human cortical RG cells that expressed BMP7 with increasing gestational age. We define a BMP7-expressing cell that expressed at least one UMI count. To compare UMI counts across samples, we took into account differences in sequencing depth between the samples according to the normalization procedure provided by 10× genomic. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence.

Figure 3.

BMP7-SMAD signaling inhibits EGFR expression. (A and B) IUE of vectors that express either GFP (control) or Bmp7-GFP in E14.5 WT mouse cortex. At E17.5, EGFR expression was significantly downregulated and pSMAD1/5/9 expression was strongly upregulated in the Bmp7-IUE cortex (arrows). (C) In the hGFAP-Cre;Smad4^F/F cortex, Bmp7-IUE showed less EGFR inhibition. (D) Quantification of numbers of EGFR-expressing cells in the cortex. (E) Bmp7 was overexpressed in the E14.5 or E15.5 cortex using IUE. The cortical GLI3R to GLI3FL ratio was significantly increased 48 h later (see Western blot in Fig. S8).

Figure 4.

Constitutively active SmoM2 inhibits BMP7 signaling and promotes cortical gliogenesis. (A–H) The expression of EGFR, OLIG2, and ASCL1 was increased, whereas the expression of HOPX, ID3, EOMES (TBR2), pSMAD1/5/9, and Bmp7 was decreased in the medial and/or dorsal cortex of hGFAP-Cre;SmoM2 mice compared to control mice at E16 (arrows).

Figure 5.

SHH-SMO signaling is required for cortical gliogenesis. (A) The expression of EGFR and OLIG2 was severely downregulated in the E17.5 cortex of hGFAP-Cre;Smo^F/F mice (Smo-cko). (B and C) Overexpression of Nog or dnPKA largely rescued the downregulation of EGFR and OLIG2 (arrows). (D and E) Quantification of numbers of cortical EGFR- and OLIG2-expressing cells. (F) Bmp7 mRNA expression was upregulated in the E17.5 cortex of hGFAP-Cre;Smo^F/F mice, but dnPKA-IUE blocked Bmp7 upregulation.

Figure 6.

Loss of SHH-SMO signaling in mouse cortical RG cells lengthens their neurogenic period. (A–C) Schematic of the workflow of scRNA-Seq analysis. CellTrace Yellow was injected into the lateral ventricle of Smo^F/F or hGFAP-Cre;Smo^F/F mice at E17.0. The cortex was collected at E18.0 for cell sorting and scRNA-Seq analysis. (D) Loss of Smo function in cortical RG cells resulted in significantly downregulation of Smo, Gli1, Ptch1, and upregulation of Bmp7, Id3, and Hopx, and inhibition of cell proliferation (downregulation of Cdk6 and Ccnd1, and upregulation of Cdkn1a and Trp53). A subset of WNT and BMP response neurogenic genes (Wnt7b, Axin2, Lef1, Dmrta2, Lhx2, and Emx2) was upregulated in cortical RG cells. (E and F) Numbers of EOMES-expressing cells were decreased in the E18 and P0 cortex, whereas increased in the P4 and P6 cortex of hGFAP-Cre;Smo^F/F mice, compared to Smo^F/F (control) mice, indicating that the length of the cortical neurogenic period is increased. Ctx, cortex; LV, lateral ventricle; Str, striatum.

Figure 7.

BMP7 and GLI3R coordinately protect cortical neurogenesis in human brain. Diagram showing that BMP7/GLI3R and SHH signaling mutually inhibit each other. BMP7 and GLI3R coordinately protect the genetic program of cortical neurogenesis and inhibit cortical gliogenesis. In contrast, SHH signaling promotes cortical gliogenesis and opposes BMP7/GLI3R functions during cortical development.