Hypothalamic tanycytes, a radial glial-like ependymal cell population that expresses numerous genes selectively enriched in embryonic hypothalamic progenitors and adult neural stem cells, have recently been observed to serve as a source of adult-born neurons in the mammalian brain. The genetic mechanisms that regulate the specification and maintenance of tanycyte identity are unknown, but are critical for understanding how these cells can act as adult neural progenitor cells. We observe that LIM (Lin-11, Isl-1, Mec-3)-homeodomain gene Lhx2 is selectively expressed in hypothalamic progenitor cells and tanycytes. To test the function of Lhx2 in tanycyte development, we used an intersectional genetic strategy to conditionally delete Lhx2 in posteroventral hypothalamic neuroepithelium, both embryonically and postnatally. We observed that tanycyte development was severely disrupted when Lhx2 function was ablated during embryonic development. Lhx2-deficient tanycytes lost expression of tanycyte-specific genes, such as Rax, while also displaying ectopic expression of genes specific to cuboid ependymal cells, such as Rarres2. Ultrastructural analysis revealed that mutant tanycytes exhibited a hybrid identity, retaining radial morphology while becoming multiciliated. In contrast, postnatal loss of function of Lhx2 resulted only in loss of expression of tanycyte-specific genes. Using chromatin immunoprecipitation, we further showed that Lhx2 directly regulated expression of Rax, an essential homeodomain factor for tanycyte development. This study identifies Lhx2 as a key intrinsic regulator of tanycyte differentiation, sustaining Rax-dependent activation of tanycyte-specific genes while also inhibiting expression of ependymal cell-specific genes. These findings provide key insights into the transcriptional regulatory network specifying this still poorly characterized cell type.

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversity not captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, but not mouse, neocortex, and inhibiting HTR2A receptors in human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmitter signaling during neurogenesis in the developing human neocortex and highlight evolutionarily divergent mechanisms of physiological signaling.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. Although it has been shown that cells can traffic metabolic enzymes via EVs, much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Our metabolomics and functional analyses both revealed that EVs harbor L-asparaginase activity, catalyzed by the enzyme asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC EVs traffic Asrgl1. Our results demonstrate, for the first time, that NSC EVs function as independent metabolic units that are able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer's disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1(M146L)/APP(751SL) mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin-cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aβ oligomers were identified, the presence of A11-immunopositive Aβ plaques also suggested a direct role of plaque-associated Aβ oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages.

The existence of endogenous neural progenitors in the nigrostriatal system could represent a powerful tool for restorative therapies in Parkinson's disease. Sox-2 is a transcription factor expressed in pluripotent and adult stem cells, including neural progenitors. In the adult brain Sox-2 is expressed in the neurogenic niches. There is also widespread expression of Sox-2 in other brain regions, although the neurogenic potential outside the niches is uncertain. Here, we analyzed the presence of Sox-2(+) cells in the adult primate (Macaca fascicularis) brain in naïve animals (N = 3) and in animals exposed to systemic administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine to render them parkinsonian (N = 8). Animals received bromodeoxyuridine (100 mg/kg once a day during five consecutive days) to label proliferating cells and their progeny. Using confocal and electron microscopy we analyzed the Sox-2(+) cell population in the nigrostriatal system and investigated changes in the number, proliferation and neurogenic potential of Sox-2(+) cells, in control conditions and at two time points after MPTP administration. We found Sox-2(+) cells with self-renewal capacity in both the striatum and the substantia nigra. Importantly, only in the striatum Sox-2(+) was expressed in some calretinin(+) neurons. MPTP administration led to an increase in the proliferation of striatal Sox-2(+) cells and to an acute, concomitant decrease in the percentage of Sox-2(+)/calretinin(+) neurons, which recovered by 18 months. Given their potential capacity to differentiate into neurons and their responsiveness to dopamine neurotoxic insults, striatal Sox-2(+) cells represent good candidates to harness endogenous repair mechanisms for regenerative approaches in Parkinson's disease.

Neurogenesis decreases during aging and following cranial radiotherapy, causing a progressive cognitive decline that is currently untreatable. However, functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover, we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures, irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-β/Smad3 signalling. Strikingly, the blockade of TGF-β signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice, prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging.

Two neurogenic regions have been described in the adult brain, the lateral ventricle subventricular zone and the dentate gyrus subgranular zone. It has been suggested that neural stem cells also line the central canal of the adult spinal cord. Using transmission and scanning electron microscopy and immunostaining, we describe here the organization and cell types of the central canal epithelium in adult mice. The identity of dividing cells was determined by 3D ultrastructural reconstructions of [(3) H]thymidine-labeled cells and confocal analysis of bromodeoxyuridine labeling. The most common cell type lining the central canal had two long motile (9+2) cilia and was vimentin+, CD24+, FoxJ1+, Sox2+, and CD133+, but nestin- and glial fibrillary acidic protein (GFAP)-. These biciliated ependymal cells of the central canal (Ecc) resembled E2 cells of the lateral ventricles, but their basal bodies were different from those of E2 or E1 cells. Interestingly, we frequently found Ecc cells with two nuclei and four cilia, suggesting they are formed by incomplete cytokinesis or cell fusion. GFAP+ astrocytes with a single cilium and an orthogonally oriented centriole were also observed. The majority of dividing cells corresponded to biciliated Ecc cells. Central canal proliferation was most common during the active period of spinal cord growth. Pairs of labeled Ecc cells were observed within the central canal in adult mice 2.5 weeks post labeling. Our work suggests that the vast majority of postnatal dividing cells in the central canal are Ecc cells and their proliferation is associated with the growth of the spinal cord.

Recent studies have indicated oligodendroglial-vascular crosstalk during brain development, but the underlying mechanisms are incompletely understood. We report that oligodendrocyte precursor cells (OPCs) contact sprouting endothelial tip cells in mouse, ferret, and human neonatal white matter. Using transgenic mice, we show that increased or decreased OPC density results in cognate changes in white matter vascular investment. Hypoxia induced increases in OPC numbers, vessel density and endothelial cell expression of the Wnt pathway targets Apcdd1 and Axin2 in white matter, suggesting paracrine OPC-endothelial signaling. Conditional knockout of OPC Wntless resulted in diminished white matter vascular growth in normoxia, whereas loss of Wnt7a/b function blunted the angiogenic response to hypoxia, resulting in severe white matter damage. These findings indicate that OPC-endothelial cell interactions regulate neonatal white matter vascular development in a Wnt-dependent manner and further suggest this mechanism is important in attenuating hypoxic injury.

Neuroanatomists have long speculated that expanded primate brains contain an increased morphological diversity of inhibitory neurons (INs)1, and recent studies have identified primate-specific neuronal populations at the molecular level2. However, we know little about the developmental mechanisms that specify evolutionarily novel cell types in the brain. Here, we reconstruct gene expression trajectories specifying INs generated throughout the neurogenic period in macaques and mice by analysing the transcriptomes of 250,181 cells. We find that the initial classes of INs generated prenatally are largely conserved among mammals. Nonetheless, we identify two contrasting developmental mechanisms for specifying evolutionarily novel cell types during prenatal development. First, we show that recently identified primate-specific TAC3 striatal INs are specified by a unique transcriptional programme in progenitors followed by induction of a distinct suite of neuropeptides and neurotransmitter receptors in new-born neurons. Second, we find that multiple classes of transcriptionally conserved olfactory bulb (OB)-bound precursors are redirected to expanded primate white matter and striatum. These classes include a novel peristriatal class of striatum laureatum neurons that resemble dopaminergic periglomerular cells of the OB. We propose an evolutionary model in which conserved initial classes of neurons supplying the smaller primate OB are reused in the enlarged striatum and cortex. Together, our results provide a unified developmental taxonomy of initial classes of mammalian INs and reveal multiple developmental mechanisms for neural cell type evolution.

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and superresolved imaging, we identify a module that we term DISCO (distal centriole complex). The DISCO components CEP90, MNR, and OFD1 underlie human ciliopathies. This complex localizes to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR do not generate cilia, fail to assemble distal appendages, and do not transduce Hedgehog signals. Disrupting the satellite pools does not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruits the most proximal known distal appendage component, CEP83, to root distal appendage formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricts centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies.

The temporal lobe of the human brain contains the entorhinal cortex (EC). This region of the brain is a highly interconnected integrative hub for sensory and spatial information; it also has a key role in episodic memory formation and is the main source of cortical hippocampal inputs1-4. The human EC continues to develop during childhood5, but neurogenesis and neuronal migration to the EC are widely considered to be complete by birth. Here we show that the human temporal lobe contains many young neurons migrating into the postnatal EC and adjacent regions, with a large tangential stream persisting until the age of around one year and radial dispersal continuing until around two to three years of age. By contrast, we found no equivalent postnatal migration in rhesus macaques (Macaca mulatta). Immunostaining and single-nucleus RNA sequencing of ganglionic eminence germinal zones, the EC stream and the postnatal EC revealed that most migrating cells in the EC stream are derived from the caudal ganglionic eminence and become LAMP5+RELN+ inhibitory interneurons. These late-arriving interneurons could continue to shape the processing of sensory and spatial information well into postnatal life, when children are actively interacting with their environment. The EC is one of the first regions of the brain to be affected in Alzheimer's disease, and previous work has linked cognitive decline to the loss of LAMP5+RELN+ cells6,7. Our investigation reveals that many of these cells arrive in the EC through a major postnatal migratory stream in early childhood.

Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AAD(dim) fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information.

The adult subventricular zone (SVZ) of the lateral ventricle contains neural stem cells. In rodents, these cells generate neuroblasts that migrate as chains toward the olfactory bulb along the rostral migratory stream (RMS). The neural-stem-cell niche at the ventricular wall is conserved in various animal species, including primates. However, it is unclear how the SVZ and RMS organization in nonhuman primates relates to that of rodents and humans. Here we studied the SVZ and RMS of the adult and neonatal common marmoset (Callithrix jacchus), a New World primate used widely in neuroscience, by electron microscopy, and immunohistochemical detection of cell-type-specific markers. The marmoset SVZ contained cells similar to type B, C, and A cells of the rodent SVZ in their marker expression and morphology. The adult marmoset SVZ had a three-layer organization, as in the human brain, with ependymal, hypocellular, and astrocyte-ribbon layers. However, the hypocellular layer was very thin or absent in the adult-anterior and neonatal SVZ. Anti-PSA-NCAM staining of the anterior SVZ in whole-mount ventricular wall preparations of adult marmosets revealed an extensive network of elongated cell aggregates similar to the neuroblast chains in rodents. Time-lapse recordings of marmoset SVZ explants cultured in Matrigel showed the neuroblasts migrating in chains, like rodent type A cells. These results suggest that some features of neurogenesis and neuronal migration in the SVZ are common to marmosets, humans, and rodents. This basic description of the adult and neonatal marmoset SVZ will be useful for future studies on adult neurogenesis in primates.

Recent publications have shown that the lateral wall of the lateral ventricles in the Macaca fascicularis brain, in particular the subventricular zone (SVZ), contains neural stem cells throughout adulthood that migrate through a migratory pathway (RMS) to the olfactory bulb (OB). To date, a detailed and systematic cytoarchitectural and ultrastructural study of the monkey SVZ and RMS has not been done. We found that the organization of the SVZ was similar to that of humans, with the ependymal layer surrounding the lateral ventricles, a hypocellular GAP layer formed by astrocytic and ependymal expansions, and the astrocyte ribbon, composed of astrocytic bodies. We found no cells corresponding to the type C proliferating precursor of the rodent brain. Instead, proliferating cells, expressed as Ki-67 immunoreactivity, were predominantly young neurons concentrated in the anterior regions, and occasional astrocytes of the ribbon. We observed displaced ependymal cells of still unknown significance. New neurons tended to organize in chain-like structures, which were surrounded by astrocytes. This pattern was highly reminiscent of that observed in rodent RMS, but not in humans. These chains spread from the frontal SVZ along a GAP-like layer, uniquely composed of astrocytic expansions, to the olfactory bulb (OB). The number of neuronal chains and the number of chain-forming cells decreased gradually upon reaching the OB. The purpose of this work is to provide a reference for future studies in the field of adult neurogenesis that may lead to an understanding of the fate and functionality of newborn neurons in primates, and ultimately in humans.

The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinico-pathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs.

The human amygdala grows during childhood, and its abnormal development is linked to mood disorders. The primate amygdala contains a large population of immature neurons in the paralaminar nuclei (PL), suggesting protracted development and possibly neurogenesis. Here we studied human PL development from embryonic stages to adulthood. The PL develops next to the caudal ganglionic eminence, which generates inhibitory interneurons, yet most PL neurons express excitatory markers. In children, most PL cells are immature (DCX+PSA-NCAM+), and during adolescence many transition into mature (TBR1+VGLUT2+) neurons. Immature PL neurons persist into old age, yet local progenitor proliferation sharply decreases in infants. Using single nuclei RNA sequencing, we identify the transcriptional profile of immature excitatory neurons in the human amygdala between 4-15 years. We conclude that the human PL contains excitatory neurons that remain immature for decades, a possible substrate for persistent plasticity at the interface of the hippocampus and amygdala.

Ependymal cells are radial glia-derived multiciliated cells lining the lateral ventricles of the brain and spinal cord. Correct development and coordinated cilia beating is essential for proper cerebrospinal fluid (CSF) flow and neurogenesis modulation. Dysfunctions of ependymal cells were associated with transcription factor deregulation. Here we provide evidence that the transcriptional regulator ID4 is involved in ependymal cell development and maturation. We observed that Id4-deficient mice display altered ventricular cell cytoarchitecture, decreased ependymal cell number and enlarged ventricles. In addition, absence of ID4 during embryonic development resulted in decreased ependymal cell number and delayed maturation. Our findings open the way for a potential role of ID4 in ependymal cell development and motor cilia function.

Astrocytes play essential roles in the developing nervous system, including supporting synapse function. These astrocyte support functions emerge coincident with brain maturation and may be tailored in a region-specific manner. For example, gray matter astrocytes have elaborate synapse-associated processes and are morphologically and molecularly distinct from white matter astrocytes. This raises the question of whether there are unique environmental cues that promote gray matter astrocyte identity and synaptogenic function. We previously identified adrenergic receptors as preferentially enriched in developing gray versus white matter astrocytes, suggesting that noradrenergic signaling could be a cue that promotes the functional maturation of gray matter astrocytes. We first characterized noradrenergic projections during postnatal brain development in mouse and human, finding that process density was higher in the gray matter and increased concurrently with astrocyte maturation. RNA sequencing revealed that astrocytes in both species expressed α- and β-adrenergic receptors. We found that stimulation of β-adrenergic receptors increased primary branching of rodent astrocytes in vitro Conversely, astrocyte-conditional knockout of the β1-adrenergic receptor reduced the size of gray matter astrocytes and led to dysregulated sensorimotor integration in female mice. These studies suggest that adrenergic signaling to developing astrocytes impacts their morphology and has implications for adult behavior, particularly in female animals. More broadly, they demonstrate a mechanism through which environmental cues impact astrocyte development. Given the key roles of norepinephrine in brain states, such as arousal, stress, and learning, these findings could prompt further inquiry into how developmental stressors impact astrocyte development and adult brain function.SIGNIFICANCE STATEMENT This study demonstrates a role for noradrenergic signaling in the development of gray matter astrocytes. We provide new evidence that the β1-adrenergic receptor is robustly expressed by both mouse and human astrocytes, and that conditional KO of the β1-adrenergic receptor from female mouse astrocytes impairs gray matter astrocyte maturation. Moreover, female conditional KO mice exhibit behavioral deficits in two paradigms that test sensorimotor function. Given the emerging interest in moving beyond RNA sequencing to probe specific pathways that underlie astrocyte heterogeneity, this study provides a foundation for future investigation into the effect of noradrenergic signaling on astrocyte functions in conditions where noradrenergic signaling is altered, such as stress, arousal, and learning.

Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.

In the brain of adult mammals, neuronal precursors are generated in the subventricular zone in the lateral wall of the lateral ventricles and migrate into the olfactory bulbs (OBs) through a well-studied route called the rostral migratory stream (RMS). Recent studies have revealed that a comparable neural stem cell niche is widely conserved at the ventricular wall of adult vertebrates. However, little is known about the migration route of neuronal precursors in nonmammalian adult brains. Here, we show that, in the adult zebrafish, a cluster of neuronal precursors generated in the telencephalic ventricular zone migrates into the OB via a route equivalent to the mammalian RMS. Unlike the mammalian RMS, these neuronal precursors are not surrounded by glial tubes, although radial glial cells with a single cilium lined the telencephalic ventricular wall, much as in embryonic and neonatal mammals. To observe the migrating neuronal precursors in living brain tissue, we established a brain hemisphere culture using a zebrafish line carrying a GFP transgene driven by the neurogenin1 (ngn1) promoter. In these fish, GFP was observed in the neuronal precursors migrating in the RMS, some of which were aligned with blood vessels. Numerous ngn1:gfp-positive cells were observed migrating tangentially in the RMS-like route medial to the OB. Taken together, our results suggest that the RMS in the adult zebrafish telencephalon is a functional migratory pathway. This is the first evidence for the tangential migration of neuronal precursors in a nonmammalian adult telencephalon.