Estrogens regulate early embryonic development of the olfactory sensory system via estrogen-responsive glia

ABSTRACT Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour.

Cellular development and evolution of the mammalian cerebellum

The expansion of the neocortex, one of the hallmarks of mammalian evolution, was accompanied by an increase in the number of cerebellar neurons. However, little is known about the evolution of the cellular programs underlying cerebellum development in mammals. In this study, we generated single-nucleus RNA-sequencing data for ~400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse, and the marsupial opossum. Our cross-species analyses revealed that the cellular composition and differentiation dynamics throughout cerebellum development are largely conserved, except for human Purkinje cells. Global transcriptome profiles, conserved cell state markers, and gene expression trajectories across neuronal differentiation show that the cerebellar cell type-defining programs have been overall preserved for at least 160 million years. However, we also discovered differences. We identified 3,586 genes that either gained or lost expression in cerebellar cells in one of the species, and 541 genes that evolved new expression trajectories during neuronal differentiation. The potential functional relevance of these cross-species differences is highlighted by the diverged expression patterns of several human disease-associated genes. Altogether, our study reveals shared and lineage-specific programs governing the cellular development of the mammalian cerebellum, and expands our understanding of the evolution of mammalian organ development.

Dinophiliformia early neurogenesis suggests the evolution of conservative neural structures across the Annelida phylogenetic tree

Despite the increasing data concerning the structure of the adult nervous system in various Lophotrochozoa groups, the early events during the neurogenesis of rare and unique groups need clarification. Annelida are a diverse clade of Lophotrochozoa, and their representatives demonstrate a variety of body plans, lifestyles, and life cycles. Comparative data about the early development are available for Errantia, Sedentaria, Sipuncula, and Palaeoannelida; however, our knowledge of Dinophiliformia is currently scarce. Representatives of Dinophiliformia are small interstitial worms combining unique morphological features of different Lophotrochozoan taxa and expressing paedomorphic traits. We describe in detail the early neurogenesis of two related species: Dimorphilus gyrociliatus and Dinophilus vorticoides, from the appearance of first nerve cells until the formation of an adult body plan. In both species, the first cells were detected at the anterior and posterior regions at the early trochophore stage and demonstrated positive reactions with pan-neuronal marker anti-acetylated tubulin only. Long fibers of early cells grow towards each other and form longitudinal bundles along which differentiating neurons later appear and send their processes. We propose that these early cells serve as pioneer neurons, forming a layout of the adult nervous system. The early anterior cell of D. vorticoides is transient and present during the short embryonic period, while early anterior and posterior cells in D. gyrociliatus are maintained throughout the whole lifespan of the species. During development, the growing processes of early cells form compact brain neuropile, paired ventral and lateral longitudinal bundles; unpaired medial longitudinal bundle; and commissures in the ventral hyposphere. Specific 5-HT- and FMRFa-immunopositive neurons differentiate adjacent to the ventral bundles and brain neuropile in the middle trochophore and late trochophore stages, i.e. after the main structures of the nervous system have already been established. Processes of 5-HT- and FMRFa-positive cells constitute a small proportion of the tubulin-immunopositive brain neuropile, ventral cords, and commissures in all developmental stages. No 5-HT- and FMRFa-positive cells similar to apical sensory cells of other Lophotrochozoa were detected. We conclude that: (i) like in Errantia and Sedentaria, Dinophiliformia neurogenesis starts from the peripheral cells, whose processes prefigure the forming adult nervous system, (ii) Dinophiliformia early cells are negative to 5-HT and FMRFa antibodies like Sedentaria pioneer cells.

Dinophiliformia early neurogenesis suggests the evolution of conservative neural structures across the Annelida phylogenetic tree

Despite the increasing data concerning the structure of the adult nervous system in various Lophotrochozoa groups, the early events during the neurogenesis of rare and unique groups need clarification. Annelida are a diverse clade of Lophotrochozoa, and their representatives demonstrate a variety of body plans, lifestyles, and life cycles. Comparative data about the early development are available for Errantia, Sedentaria, Sipuncula and Palaeoannelida; however, our knowledge of Dinophiliformia is currently scarce. Representatives of Dinophiliformia are small interstitial worms combining unique morphological features of different Lophotrochozoan taxa and expressing paedomorphic traits. We describe in detail the early neurogenesis of two related species: Dimorphilus gyrociliatus and Dinophilus vorticoides, from the appearance of first nerve cells until the formation of an adult body plan. In both species, the first cells were detected at the anterior and posterior regions at the early trochophore stage and demonstrated positive reactions with pan-neuronal marker anti-acetylated tubulin only. Long fibers of early cells grow towards each other and form longitudinal bundles along which differentiating neurons later appear and send their processes. We propose that these early cells serve as pioneer neurons, forming a layout of the adult nervous system. The early anterior cell of D. vorticoides is transient and present during the short embryonic period, while early anterior and posterior cells in D. gyrociliatus are maintained throughout the whole lifespan of the species. During development, the growing processes of early cells form compact brain neuropile, paired ventral and lateral longitudinal bundles; unpaired medial longitudinal bundle; and commissures in the ventral hyposphere. Specific 5-HT- and FMRFa-immunopositive neurons differentiate adjacent to the ventral bundles and brain neuropile in the middle trochophore and late trochophore stages, i.e. after the main structures of the nervous system have already been established. Processes of 5-HT- and FMRFa-positive cells constitute a small proportion of the tubulin-immunopositive brain neuropile, ventral cords, and commissures in all developmental stages. No 5-HT- and FMRFa-positive cells similar to apical sensory cells of other Lophotrochozoa were detected. We conclude that: (i) like in Errantia and Sedentaria, Dinophiliformia neurogenesis starts from the peripheral cells, whose processes prefigure the forming adult nervous system, (ii) Dinophiliformia early cells are negative to 5-HT and FMRFa antibodies like Sedentaria pioneer cells.

Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism

Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.

Integrative genomic analysis of early neurogenesis reveals a temporal genetic program for differentiation and specification of preplate and Cajal-Retzius neurons

Neurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early-born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. Genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays in vitro and in vivo. In addition, at E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneities in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron differentiation.

Cortical Organoids Model Early Brain Development Disrupted by 16p11.2 Copy Number Variants in Autism

Reciprocal deletion and duplication of 16p11.2 region is the most common copy number variation (CNV) associated with Autism Spectrum Disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV has mirror-opposite effect on neuronal maturation, proliferation, and synapse number, in concordance with its effect on brain growth in humans. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with excess of neurons and depletion of neural progenitors observed in deletions, and mirror phenotypes in duplications. Transcriptomic and proteomic profiling revealed multiple dysregulated pathways, including defects in neuron migration. Inhibition of activity of the small GTPase RhoA rescued migration deficits. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.