Intermediate progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants

ABSTRACT Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated owing to lack of appropriate genetic tools. Here, we report a Split GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, through their dynamic control of the Notch ligand Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems.

Regulation of Neural Stem Cell Competency and Commitment during Indirect Neurogenesis

Indirect neurogenesis, during which neural stem cells generate neurons through intermediate progenitors, drives the evolution of lissencephalic brains to gyrencephalic brains. The mechanisms that specify intermediate progenitor identity and that regulate stem cell competency to generate intermediate progenitors remain poorly understood despite their roles in indirect neurogenesis. Well-characterized lineage hierarchy and available powerful genetic tools for manipulating gene functions make fruit fly neural stem cell (neuroblast) lineages an excellent in vivo paradigm for investigating the mechanisms that regulate neurogenesis. Type II neuroblasts in fly larval brains repeatedly undergo asymmetric divisions to generate intermediate neural progenitors (INPs) that undergo limited proliferation to increase the number of neurons generated per stem cell division. Here, we review key regulatory genes and the mechanisms by which they promote the specification and generation of INPs, safeguarding the indirect generation of neurons during fly larval brain neurogenesis. Homologs of these regulators of INPs have been shown to play important roles in regulating brain development in vertebrates. Insight into the precise regulation of intermediate progenitors will likely improve our understanding of the control of indirect neurogenesis during brain development and brain evolution.

Young transposable elements rewired gene regulatory networks in human and chimpanzee hippocampal intermediate progenitors

The hippocampus is associated with essential brain functions such as learning and memory. Human hippocampal volume is significantly greater than expected when compared to non-human apes, suggesting a recent expansion. Intermediate progenitors, which are able to undergo multiple rounds of proliferative division before a final neurogenic division, may have played a role in the evolutionary hippocampal expansion. To investigate the evolution of gene regulatory networks underpinning hippocampal neurogenesis in apes, we leveraged the differentiation of human and chimpanzee induced Pluripotent Stem Cells into TBR2-positive hippocampal intermediate progenitors (hpIPCs). We find that the gene networks active in hpIPCs are significantly different between humans and chimpanzees, with ~2,500 genes differentially expressed. We demonstrate that species-specific transposon-derived enhancers contribute to these transcriptomic differences. Young transposons, predominantly Endogenous Retroviruses (ERVs) and SINE-Vntr-Alus (SVAs), were co-opted as enhancers in a species-specific manner. Human-specific SVAs provided substrates for thousands of novel TBR2 binding sites, and CRISPR-mediated repression of these SVAs attenuates the expression of ~25% of the genes that are upregulated in human intermediate progenitors relative to the same cell population in the chimpanzee.

An epigenetic circuit controls neurogenic programs during neocortex development

ABSTRACT The production and expansion of intermediate progenitors (IPs) are essential for neocortical neurogenesis during development and over evolution. Here, we have characterized an epigenetic circuit that precisely controls neurogenic programs, particularly properties of IPs, during neocortical development. The circuit comprises a long non-coding RNA (LncBAR) and the BAF (SWI/SNF) chromatin-remodeling complex, which transcriptionally maintains the expression of Zbtb20. LncBAR knockout neocortex contains more deep-layer but fewer upper-layer projection neurons. Intriguingly, loss of LncBAR promotes IP production, but paradoxically prolongs the duration of the cell cycle of IPs during mid-later neocortical neurogenesis. Moreover, in LncBAR knockout mice, depletion of the neural progenitor pool at embryonic stage results in fewer adult neural progenitor cells in the subventricular zone of lateral ventricles, leading to a failure in adult neurogenesis to replenish the olfactory bulb. LncBAR binds to BRG1, the core enzymatic component of the BAF chromatin-remodeling complex. LncBAR depletion enhances association of BRG1 with the genomic locus of, and suppresses the expression of, Zbtb20, a transcription factor gene known to regulate both embryonic and adult neurogenesis. ZBTB20 overexpression in LncBAR-knockout neural precursors reverses compromised cell cycle progressions of IPs.

Extracellular LGALS3BP regulates neural progenitor position and relates to human cortical complexity

Basal progenitors (BPs), including intermediate progenitors and basal radial glia, are generated from apical radial glia and are enriched in gyrencephalic species like humans, contributing to neuronal expansion. Shortly after generation, BPs delaminate towards the subventricular zone, where they further proliferate before differentiation. Gene expression alterations involved in BP delamination and function in humans are poorly understood. Here, we study the role of LGALS3BP, so far known as a cancer biomarker, which is a secreted protein enriched in human neural progenitors (NPCs). We show that individuals with LGALS3BP de novo variants exhibit altered local gyrification, sulcal depth, surface area and thickness in their cortex. Additionally, using cerebral organoids, human fetal tissues and mice, we show that LGALS3BP regulates the position of NPCs. Single-cell RNA-sequencing and proteomics reveal that LGALS3BP-mediated mechanisms involve the extracellular matrix in NPCs’ anchoring and migration within the human brain. We propose that its temporal expression influences NPCs’ delamination, corticogenesis and gyrification extrinsically.

CNOT3 interacts with the Aurora B and MAPK/ERK kinases to promote survival of differentiating mesendodermal progenitor cells

Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B, which requires sequences in the NOT box domain of CNOT3, and regulates MAPK/ERK signalling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse ES cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of cells with ectodermal characteristics. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4 and Wnt3 due to reduced survival and proliferation of differentiating mesendoderm cells. We also show that the double mutation alters the balance of interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a potential adaptor function for CNOT3 that regulates the Ras/MEK/ERK pathway during embryogenesis. [Media: see text]

Developmental Origins of Human Cortical Oligodendrocytes and Astrocytes

Human cortical radial glial cells are primary neural stem cells that give rise to cortical glutaminergic projection pyramidal neurons, glial cells (oligodendrocytes and astrocytes) and olfactory bulb GABAergic interneurons. One of prominent features of the human cortex is enriched with glial cells, but there are major gaps in understanding how these glial cells are generated. Herein, by integrating analysis of published human cortical single-cell RNA-Seq datasets with our immunohistochemistical analyses, we show that around gestational week 18, EGFR-expressing human cortical truncated radial glial cells (tRGs) give rise to basal multipotent intermediate progenitors (bMIPCs) that express EGFR, ASCL1, OLIG2 and OLIG1. These bMIPCs undergo several rounds of mitosis and generate cortical oligodendrocytes, astrocytes and olfactory bulb interneurons. We also characterized molecular features of the cortical tRG. Integration of our findings suggests a general picture of the lineage progression of cortical radial glial cells, a fundamental process of the developing human cerebral cortex.

Cell-Type-Specific Gene Expression in Developing Mouse Neocortex: Intermediate Progenitors Implicated in Axon Development

Cerebral cortex projection neurons (PNs) are generated from intermediate progenitors (IPs), which are in turn derived from radial glial progenitors (RGPs). To investigate developmental processes in IPs, we profiled IP transcriptomes in embryonic mouse neocortex, using transgenic Tbr2-GFP mice, cell sorting, and microarrays. These data were used in combination with in situ hybridization to ascertain gene sets specific for IPs, RGPs, PNs, interneurons, and other neural and non-neural cell types. RGP-selective transcripts (n = 419) included molecules for Notch receptor signaling, proliferation, neural stem cell identity, apical junctions, necroptosis, hippo pathway, and NF-κB pathway. RGPs also expressed specific genes for critical interactions with meningeal and vascular cells. In contrast, IP-selective genes (n = 136) encoded molecules for activated Delta ligand presentation, epithelial-mesenchymal transition, core planar cell polarity (PCP), axon genesis, and intrinsic excitability. Interestingly, IPs expressed several “dependence receptors” (Unc5d, Dcc, Ntrk3, and Epha4) that induce apoptosis in the absence of ligand, suggesting a competitive mechanism for IPs and new PNs to detect key environmental cues or die. Overall, our results imply a novel role for IPs in the patterning of neuronal polarization, axon differentiation, and intrinsic excitability prior to mitosis. Significantly, IPs highly express Wnt-PCP, netrin, and semaphorin pathway molecules known to regulate axon polarization in other systems. In sum, IPs not only amplify neurogenesis quantitatively, but also molecularly “prime” new PNs for axogenesis, guidance, and excitability.

Characterization and Stage-Dependent Lineage Analysis of Intermediate Progenitors of Cortical GABAergic Interneurons

Intermediate progenitors of both excitatory and inhibitory neurons, which can replenish neurons in the adult brain, were recently identified. However, the generation of intermediate progenitors of GABAergic inhibitory neurons (IPGNs) has not been studied in detail. Here, we characterized the spatiotemporal distribution of IPGNs in mouse cerebral cortex. IPGNs generated neurons during both embryonic and postnatal stages, but the embryonic IPGNs were more proliferative. Our lineage tracing analyses showed that the embryonically proliferating IPGNs tended to localize to the superficial layers rather than the deep cortical layers at 3 weeks after birth. We also found that embryonic IPGNs derived from the medial and caudal ganglionic eminence (CGE) but more than half of the embryonic IPGNs were derived from the CGE and broadly distributed in the cerebral cortex. Taken together, our data indicate that the broadly located IPGNs during embryonic and postnatal stages exhibit a different proliferative property and layer distribution.

Distinct roles of Fto and Mettl3 in controlling development of the cerebral cortex through transcriptional and translational regulations

Proper development of the mammalian cerebral cortex relies on precise gene expression regulation, which is controlled by genetic, epigenetic, and epitranscriptomic factors. Here we generate RNA demethylase Fto and methyltransferase Mettl3 cortical-specific conditional knockout mice, and detect severe brain defects caused by Mettl3 deletion but not Fto knockout. Transcriptomic profiles using RNA sequencing indicate that knockout of Mettl3 causes a more dramatic alteration on gene transcription than that of Fto. Interestingly, we conduct ribosome profiling sequencing, and find that knockout of Mettl3 leads to a more severe disruption of translational regulation of mRNAs than deletion of Fto and results in altered translation of crucial genes in cortical radial glial cells and intermediate progenitors. Moreover, Mettl3 deletion causes elevated translation of a significant number of mRNAs, in particular major components in m6A methylation. Our findings indicate distinct functions of Mettl3 and Fto in brain development, and uncover a profound role of Mettl3 in regulating translation of major mRNAs that control proper cortical development.