Plag1 regulates neuronal gene expression and neuronal differentiation of neocortical neural progenitor cells

Abstract

Neural progenitor cells (NPCs, also known as radial glial progenitors) produce neurons and then glial cells such as astrocytes during development of the mouse neocortex. Given that this sequential generation of neural cells is critical for proper brain formation, the neurogenic potential of NPCs must be precisely controlled. Here, we show that the transcription factor Plag1 plays an important role in the regulation of neurogenic potential in mouse neocortical NPCs. We found that Hmga2, a key neurogenic factor in neocortical NPCs, induces expression of the Plag1 gene. Analysis of the effects of over‐expression or knockdown of Plag1 indicated that Plag1 promotes the production of neurons at the expense of astrocyte production in embryonic neocortical cultures. Furthermore, over‐expression of Plag1 promoted and knockdown of Plag1 suppressed neuronal differentiation of neocortical NPCs in vivo. Transcriptomic analysis showed that Plag1 increases the expression of a set of neuronal genes in NPCs. Our results thus identify Plag1 as a regulator of neuronal gene expression and neuronal differentiation in NPCs of the developing mouse neocortex.

HMGA Genes and Proteins in Development and Evolution

“…On the contrary, depletion of Plag1 in NPCs at E11.5 + 3DIV increases astrocytes. Consistent with these results, in vivo experiments confirm the observations made on cultured NPCs: in utero electroporation experiments show that Plag1 overexpression promotes neuronal differentiation in NPCs, while interference on Plag1 function does the opposite [126]. By transcriptomic analysis, a set of 46 genes, most of which involved in neuronal development, was downregulated in vivo by interfering with Plag1 function, while 1 gene was found upregulated; for many of them, a PLAG1 binding motif was found in the surroundings of their transcription start site [126].…”

“…Similar to Hmga2, Igf2bp2 is initially expressed at high levels in cortical NPCs in the early neurogenic phase, and then downregulated in late NPCs in the gliogenic phase. Igf2bp2 expression is increased by Hmga2 overexpression in NPCs, while depletion of Hmga2 reduces Igf2bp2 levels of expression [76,126]. Igf2bp2 overexpression in late NPCs (E17.5 + 3DIV) prevents their differentiation into astrocytes, instead favoring a neurogenic potential [76]; on the other hand, depletion of Igf2bp2 in early NPCs (E11.5 + 3DIV) favors gliogenesis and impairs neurogenesis, without any variation in proliferation or in cell death [76].…”

“…PLAG1 is a zinc-finger transcription factor, expressed in various tissues including the telencephalic VZ of the mouse, where it is required for normal development of the neocortex [127,128]. Again, similar to Hmga2, the expression of Plag1 in NPCs declines as they proceed along development [126]; moreover, HMGA2 binds directly to the promoter of Plag1, whose expression is upregulated or downregulated, respectively, by gain-or loss-of-function of Hmga2 in NPCs [126]. In vitro experiments on early stage (E11.5 + 3DIV) NPCs overexpressing Plag1 show that these cells produce a larger proportion of neurons compared to controls, while similar overexpression in a later astrogenetic phase (E11.5 + 9DIV) causes cells to rescue their neurogenetic potential and produce larger proportion of neurons and fewer astrocytes.…”

A simple method for gene expression in endo- and ectodermal cells in mouse embryos before neural tube closure

“…The 528 amount of each target mRNA was normalized by that of Actb mRNA. Primer sequences 529 were described previously (Sakai et al, 2019). 530…”

“…Ring1B target genes in NPCs in vivo: Ring1A/B KO/Ring1A KO ≥ 3.0, 562 expression in control (Ring1A KO) ≥ 1.0 RPKM. 563 564 ChIP-seq analysis 565ChIP analysis was performed as previously described(Sakai et al, 2019) with 566 antibodies to Hmga2 (Cell Signaling) and to H3K27me3 (MBL). Two million cells 567 directly isolated from the neocortex of embryos at E11.0 were used for ChIP-seq of 568 Hmga2, and 100,000 NPCs isolated as CD133 high cells by FACS from the neocortex of 569 embryos at E11.0 were used for ChIP-seq of H3K27me3.…”