Neural stem cells (NSCs) differentiate into neuron-fated intermediate progenitor cells (IPCs) via cell division. Although differentiation from NSCs to IPCs is a discrete process, recent transcriptome analyses identified a continuous transcriptional trajectory during this process, raising the question of how to reconcile these contradictory observations. In mouse NSCs, Hes1 expression oscillates, regulating the oscillatory expression of the proneural gene Neurog2, while Hes1 expression disappears in IPCs. Thus, the transition from Hes1 oscillation to suppression is involved in the differentiation of NSCs to IPCs. Here, we found that Neurog2 oscillations induce the accumulation of Tbr2, which suppresses Hes1 expression, generating an IPC-like gene expression state in NSCs. In the absence of Tbr2, Hes1 expression is up-regulated, decreasing the formation of IPCs. These results indicate that the Neurog2-Tbr2 axis forms a continuous transcriptional trajectory to an IPC-like neurogenic state in NSCs, which then differentiate into IPCs via cell division.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD\'s critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFβ signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFβ signaling in Gli1+ cell function, the receptor for TGFβ, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFβ signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFβ signaling reduced proliferation and increased apoptosis. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFβ controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFβ directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

Lipopolysaccharide (LPS) is a natural agonist of toll-like receptor 4 that serves a role in innate immunity. The current study evaluated the LPS-mediated regulation of neurogenesis in the subventricular zone (SVZ) progenitors, that is, the basal radial glia and intermediate progenitors (IPs), in ferrets. Ferret pups were subcutaneously injected with LPS (500 μg/g of body weight) on postnatal days (PDs) 6 and 7. Furthermore, 5-ethynyl-2\'-deoxyuridine (EdU) and 5-bromo-2\'-deoxyuridine (BrdU) were administered on PDs 5 and 7, respectively, to label the post-proliferative and proliferating cells in the inner SVZ (iSVZ) and outer SVZ (oSVZ). A significantly higher density of BrdU single-labeled proliferating cells was observed in the iSVZ of LPS-exposed ferrets than in controls but not in post-proliferative EdU single-labeled and EdU/BrdU double-labeled self-renewing cells. BrdU single-labeled cells exhibited a lower proportion of Tbr2 immunostaining in LPS-exposed ferrets (22.2%) than in controls (42.6%) and a higher proportion of Ctip2 immunostaining in LPS-exposed ferrets (22.2%) than in controls (8.6%). The present findings revealed that LPS modified the neurogenesis of SVZ progenitors. Neonatal LPS exposure facilitates the proliferation of SVZ progenitors, followed by the differentiation of Tbr2-expressing IPs into Ctip2-expressing immature neurons.

The hippocampus is associated with essential brain functions, such as learning and memory. Human hippocampal volume is significantly greater than expected compared with that of 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 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 (or EOMES)-positive hippocampal intermediate progenitor cells (hpIPCs). We found that the gene networks active in hpIPCs are significantly different between humans and chimpanzees, with ∼2500 genes being differentially expressed. We demonstrate that species-specific transposon-derived enhancers contribute to these transcriptomic differences. Young transposons, predominantly endogenous retroviruses 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 attenuated the expression of ∼25% of the genes that are upregulated in human intermediate progenitors relative to the same cell population in the chimpanzee.

We previously observed that developmental marginal zinc deficiency affects neurogenesis. Maternal phthalate exposure could disrupt fetal zinc homeostasis by triggering an acute phase response, causing maternal liver zinc retention that limits zinc availability to the fetus. Thus, we currently investigated whether exposure to di-2-ethylhexyl phthalate (DEHP) during gestation in rats alters fetal brain neurogenesis by impairing zinc homeostasis. Dams consumed an adequate (25 μg zinc/g diet) (C) or a marginal zinc deficient (MZD) (10 μg zinc/g diet) diet, without or with DEHP (300 mg/kg BW) (C + DEHP, MZD + DEHP) from embryonic day (E) 0 to E19. To evaluate neurogenesis we measured parameters of neural progenitor cells (NPC) proliferation and differentiation. Maternal exposure to DEHP and/or zinc deficiency lowered fetal brain cortical tissue (CT) zinc concentrations. Transcription factors involved in NPC proliferation (PAX6, SOX2, EMX1), differentiation (TBR2, TBR1) and mature neurons (NeuN) were lower in MZD, MZD + DEHP and C + DEHP than in C E19 brain CT, being the lowest in the MZD + DEHP group. VGLUT1 levels, a marker of glutamatergic neurons, showed a similar pattern. Levels of a marker of GABAergic neurons, GAD65, did not vary among groups. Phosphorylated ERK1/2 levels were reduced by both MZD and DEHP, and particularly in the MZD + DEHP group. MEHP-treated human neuroblastoma IMR-32 cells and E19 brains from DEHP-treated dams showed that the zinc-regulated phosphatase PP2A can be in part responsible for DEHP-mediated ERK1/2 downregulation and impaired neurogenesis. Overall, gestational exposure to DEHP caused secondary zinc deficiency and impaired neurogenesis. These harmful effects could have long-term consequences on the adult offspring brain structure and function.

The mammalian retina contains more than 40 retinal ganglion cell (RGC) subtypes based on their unique morphologies, functions, and molecular profiles. Among them, intrinsically photosensitive RGCs (ipRGCs) are the first specified RGC type emerging from a common retinal progenitor pool during development. Previous work has shown that T-box transcription factor T-brain 2 (Tbr2) is essential for the formation and maintenance of ipRGCs, and that Tbr2-expressing RGCs activate Opn4 expression upon native ipRGC ablation, suggesting that Tbr2+ RGCs contain a reservoir for ipRGCs. However, the identity of Tbr2+ RGCs has not been fully vetted. Here, using genetic sparse labeling and single cell recording, we showed that Tbr2-expressing retinal neurons include RGCs and a subset of GABAergic displaced amacrine cells (dACs). Most Tbr2+ RGCs are intrinsically photosensitive and morphologically resemble native ipRGCs with identical retinofugal projections. Tbr2+ RGCs also include a unique and rare Pou4f1-expressing OFF RGC subtype. Using a loss-of-function strategy, we have further demonstrated that Tbr2 is essential for the survival of these RGCs and dACs, as well as maintaining the expression of Opn4. These data set a strong foundation to study how Tbr2 regulates ipRGC development and survival, as well as the expression of molecular machinery regulating intrinsic photosensitivity.

Intermediate progenitor cells (IPCs) are neocortical neuronal precursors. Although IPCs play crucial roles in corticogenesis, their molecular features remain largely unknown. In this study, we aimed to characterize the molecular profile of IPCs. We isolated TBR2-positive (+) IPCs and TBR2-negative (-) cell populations in the developing mouse cortex. Comparative genome-wide gene expression analysis of TBR2+ IPCs versus TBR2- cells revealed differences in key factors involved in chromatid segregation, cell-cycle regulation, transcriptional regulation, and cell signaling. Notably, mutation of many IPC genes in human has led to intellectual disability and caused a wide range of cortical malformations, including microcephaly and agenesis of corpus callosum. Loss-of-function experiments in cortex-specific mutants of Esco2, one of the novel IPC genes, demonstrate its critical role in IPC maintenance, and substantiate the identification of a central genetic determinant of IPC biogenesis. Our data provide novel molecular characteristics of IPCs in the developing mouse cortex.

CHD8 haploinsufficiency causes autism and macrocephaly with high penetrance in the human population. Chd8 heterozygous mice exhibit relatively subtle brain overgrowth and little gene expression changes in the embryonic neocortex. The purpose of this study was to generate new, sub-haploinsufficient Chd8 mouse models to allow us to identify and study the functions of CHD8 during embryonic cortical development.
To examine the possibility that certain phenotypes may only appear at sub-heterozygous Chd8 levels in the mouse, we created an allelic series of Chd8-deficient mice to reduce CHD8 protein levels to approximately 35% (mild hypomorph), 10% (severe hypomorph) and 0% (neural-specific conditional knockout) of wildtype levels. We used RNA sequencing to compare transcriptional dysregulation, structural MRI and brain weight to investigate effects on brain size, and cell proliferation, differentiation and apoptosis markers in immunostaining assays to quantify changes in neural progenitor fate.
Mild Chd8 hypomorphs displayed significant postnatal lethality, with surviving animals exhibiting more pronounced brain hyperplasia than heterozygotes. Over 2000 genes were dysregulated in mild hypomorphs, including autism-associated neurodevelopmental and cell cycle genes. We identify increased proliferation of non-ventricular zone TBR2+ intermediate progenitors as one potential cause of brain hyperplasia in these mutants. Severe Chd8 hypomorphs displayed even greater transcriptional dysregulation, including evidence for p53 pathway upregulation. In contrast to mild hypomorphs, these mice displayed reduced brain size and increased apoptosis in the embryonic neocortex. Homozygous, conditional deletion of Chd8 in early neuronal progenitors resulted in pronounced brain hypoplasia, partly caused by p53 target gene derepression and apoptosis in the embryonic neocortex. Limitations Our findings identify an important role for the autism-associated factor CHD8 in controlling the proliferation of intermediate progenitors in the mouse neocortex. We propose that CHD8 has a similar function in human brain development, but studies on human cells are required to confirm this. Because many of our mouse mutants with reduced CHD8 function die shortly after birth, it is not possible to fully determine to what extent reduced CHD8 function results in autism-associated behaviours in mice.
Together, these findings identify important, dosage-sensitive functions for CHD8 in p53 pathway repression, neurodevelopmental gene expression and neural progenitor fate in the embryonic neocortex. We conclude that brain development is acutely sensitive to reduced CHD8 expression and that the varying sensitivities of different progenitor populations and cellular processes to CHD8 dosage result in non-linear effects on gene transcription and brain growth. Shaun Hurley, Conor Mohan and Philipp Suetterlin have contributed equally to this work.

Previous studies demonstrated specific expression of transcription factor Tbr2 in unipolar brush cells (UBCs) of the cerebellum during development and adulthood. To further study UBCs and the role of Tbr2 in their development we examined UBC morphology in transgenic mouse lines (reporter and lineage tracer) and also examined the effects of Tbr2 deficiency in Tbr2 (MGI: Eomes) conditional knock-out (cKO) mice. In Tbr2 reporter and lineage tracer cerebellum, UBCs exhibited more complex morphologies than previously reported including multiple dendrites, bifurcating dendrites, and up to four dendritic brushes. We propose that \"dendritic brush cells\" (DBCs) may be a more apt nomenclature. In Tbr2 cKO cerebellum, mature UBCs were completely absent. Migration of UBC precursors from rhombic lip to cerebellar cortex and other nuclei was impaired in Tbr2 cKO mice. Our results indicate that UBC migration and differentiation are sensitive to Tbr2 deficiency. To investigate whether UBCs develop similarly in humans as in rodents, we studied Tbr2 expression in mid-gestational human cerebellum. Remarkably, Tbr2+ UBC precursors migrate along the same pathways in humans as in rodent cerebellum and disperse to create the same \"fountain-like\" appearance characteristic of UBCs exiting the rhombic lip.