Embryonic Origin of Postnatal Neural Stem Cells.

Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes.

Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here, we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmunoelectron microscopy showed that these corresponded to MVBs. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to ß-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal-transduction pathway.

Myb promotes centriole amplification and later steps of the multiciliogenesis program.

The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.

Basin scale sources of siltation in a contaminated hydropower reservoir.

Siltation and the loss of hydropower reservoir capacity is a global challenge with a predicted 26 % loss of storage at the global scale by 2050. Like in many other Latin American contexts, soil erosion constitutes one of the most significant water pollution problems in Chile with serious siltation consequences downstream. Identifying the sources and drivers affecting hydropower siltation and water pollution is a critical need to inform adaptation and mitigation strategies especially in the context of changing climate regimes e.g. rainfall patterns. We investigated, at basin scale, the main sources of sediments delivered to one of the largest hydropower reservoirs in South America using a spatio-temporal geochemical fingerprinting approach. Mining activities contributed equivalent to 9 % of total recent sediment deposited in the hydropower lake with notable concentrations of sediment-associated pollutants e.g. Cu and Mo in bed sediment between the mine tributary and the reservoir sediment column. Agricultural sources represented ca. 60 % of sediment input wherein livestock production and agriculture promoted the input of phosphorus to the lake. Evaluation of the lake sediment column against the tributary network showed that the tributary associated with both dominant anthropogenic activities (mining and agriculture) contributed substantially more sediment, but sources varied through time: mining activities have reduced in proportional contribution since dam construction and proportional inputs from agriculture have increased in recent years, mainly promoted by recent conversion of steep lands from native vegetation to agriculture. Siltation of major hydropower basins presents a global challenge exemplified by the Rapel basin. The specific challenges faced here highlight the urgent need for co-design of evidence-led, context-specific solutions that address the interplay of drivers both within and without the basin and its communities, enhancing the social acceptability of sediment management strategies to support the sustainability of clean, hydropower energy production.

Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE.

Integrating positional information at the level of Smad1/5/8.

The intensity of the BMP signal is determined by cell surface receptors that phosphorylate Smad1/5/8 at the C-terminus. In addition to this BMP-activated phosphorylation, recent studies have shown that sequential phosphorylations by MAPK and GSK3 kinases can negatively regulate the activity of the pSmad1Cter signal. These phosphorylations in the linker region cause Smad1 to be transported to the centrosomal region, polyubiquitinylated and degraded by the proteasomal machinery. In Xenopus embryos, Wnt signals, which regulate GSK3, induce ectoderm to adopt an epidermal fate, and this Wnt effect requires an active BMP-Smad1/5/8 signaling pathway. These findings have profound implications for understanding how dorsal-ventral and anterior-posterior patterning are seamlessly integrated in the early embryonic morphogenetic field.

Asymmetric mitosis: Unequal segregation of proteins destined for degradation.

Mitotic cell division ensures that two daughter somatic cells inherit identical genetic material. Previous work has shown that signaling by the Smad1 transcription factor is terminated by polyubiquitinylation and proteasomal degradation after essential phosphorylations by MAPK and glycogen synthase kinase 3 (GSK3). Here, we show that, unexpectedly, proteins specifically targeted for proteasomal degradation are inherited preferentially by one mitotic daughter during somatic cell division. Experiments with dividing human embryonic stem cells and other mammalian cultured cell lines demonstrated that in many supposedly equal mitoses the segregation of proteins destined for degradation (Smad1 phosphorylated by MAPK and GSK3, phospho-beta-catenin, and total polyubiquitinylated proteins) was asymmetric. Transport of pSmad1 targeted for degradation to the centrosome required functional microtubules. In vivo, an antibody specific for Mad phosphorylated by MAPK showed that this antigen was associated preferentially with one of the two centrosomes in Drosophila embryos at cellular blastoderm stage. We propose that this remarkable cellular property may be explained by the asymmetric inheritance of peripheral centrosomal proteins when centrioles separate and migrate to opposite poles of the cell, so that one mitotic daughter remains pristine. We conclude that many mitotic divisions are unequal, unlike what was previously thought.

Development of Ependymal and Postnatal Neural Stem Cells and Their Origin from a Common Embryonic Progenitor.

The adult mouse brain contains an extensive neurogenic niche in the lateral walls of the lateral ventricles. This epithelium, which has a unique pinwheel organization, contains multiciliated ependymal (E1) cells and neural stem cells (B1). This postnatal germinal epithelium develops from the embryonic ventricular zone, but the lineage relationship between E1 and B1 cells remains unknown. Distinct subpopulations of radial glia (RG) cells in late embryonic and early postnatal development either expand their apical domain >11-fold to form E1 cells or retain small apical domains that coalesce into the centers of pinwheels to form B1 cells. Using independent methods of lineage tracing, we show that individual RG cells can give rise to clones containing E1 and B1 cells. This study reveals key developmental steps in the formation of the postnatal germinal niche and the shared cellular origin of E1 and B1 cells.

First use of a compound-specific stable isotope (CSSI) technique to trace sediment transport in upland forest catchments of Chile.

Land degradation is a problem affecting the sustainability of commercial forest plantations. The identification of critical areas prone to erosion can assist this activity to better target soil conservation efforts. Here we present the first use of the carbon-13 signatures of fatty acids (C14 to C24) in soil samples for spatial and temporal tracing of sediment transport in river bodies of upland commercial forest catchments in Chile. This compound-specific stable isotope (CSSI) technique was tested as a fingerprinting approach to determine the degree of soil erosion in pre-harvested forest catchments with surface areas ranging from 12 to 40ha. For soil apportionment a mixing model based on a Bayesian inference framework was used (CSSIAR v.2.0). Approximately four potential sediment sources were used for the calculations of all of the selected catchments. Unpaved forestry roads were shown to be the main source of sediment deposited at the outlet of the catchments (30-75%). Furthermore, sampling along the stream channel demonstrated that sediments were mainly comprised of sediment coming from the unpaved roads in the upper part of the catchments (74-98%). From this it was possible to identify the location and type of primary land use contributing to the sediment delivered at the outlet of the catchments. The derived information will allow management to focus efforts to control or mitigate soil erosion by improving the runoff features of the forest roads. The use of this CSSI technique has a high potential to help forestry managers and decision makers to evaluate and mitigate sources of soil erosion in upland forest catchments. It is important to highlight that this technique can also be a good complement to other soil erosion assessment and geological fingerprinting techniques, especially when attempting to quantify (sediment loads) and differentiate which type of land use most contributes to sediment accumulation.

Identification of proliferative progenitors associated with prominent postnatal growth of the pons.

The pons controls crucial sensorimotor and autonomic functions. In humans, it grows sixfold postnatally and is a site of paediatric gliomas; however, the mechanisms of pontine growth remain poorly understood. We show that the murine pons quadruples in volume postnatally; growth is fastest during postnatal days 0-4 (P0-P4), preceding most myelination. We identify three postnatal proliferative compartments: ventricular, midline and parenchymal. We find no evidence of postnatal neurogenesis in the pons, but each progenitor compartment produces new astroglia and oligodendroglia; the latter expand 10- to 18-fold postnatally, and are derived mostly from the parenchyma. Nearly all parenchymal progenitors at P4 are Sox2(+)Olig2(+), but by P8 a Sox2(-) subpopulation emerges, suggesting a lineage progression from Sox2(+) 'early' to Sox2(-) 'late' oligodendrocyte progenitor. Fate mapping reveals that >90% of adult oligodendrocytes derive from P2-P3 Sox2(+) progenitors. These results demonstrate the importance of postnatal Sox2(+)Olig2(+) progenitors in pontine growth and oligodendrogenesis.

Source identification, apportionment and toxicity of indoor and outdoor PM2.5 airborne particulates in a region characterised by wood burning.

The occurrence of airborne particulate matter has been flagged as "of concern" in several megacities, especially in Asia. Selected Chilean regions have similar problems as wood burning is the major source of heating in homes. This concern has led to mitigation measures restricting the burning of wood at periods when the particulate matter smaller than 2.5 µm (PM2.5) concentrations are predicted to be high. This work investigates the linkage between indoor and outdoor particle concentrations, determines their source through the polyaromatic hydrocarbon (PAH) signature and investigates the efficacy of the current management practice of burning restrictions. The PM2.5 fraction was collected at 12 different properties with coincident indoor and outdoor sampling using a low-volume active sampler for 24 hours. Indoor concentrations of PM2.5 ranged from 6 to 194 µg m(-3) with a mean of 72 µg m(-3) and corresponding outdoor concentrations ranged from 5 to 367 µg m(-3) with a mean of 85 µg m(-3) over the winter periods of 2014 and 2015; the Chilean national permitted maximum in outdoor air is 50 µg m(-3) in 24 hours. Higher concentrations were measured when the outdoor air temperature was lower. The PAHs were analysed on the PM2.5 fraction; the indoor concentrations ranged from 2 to 291 ng m(-3) with a mean of 51 ng m(-3) compared to an outdoor concentration between 3 and 365 ng m(-3) with a mean of 71 ng m(-3). Multivariate statistical analysis of the PAH profiles using principal components analysis (PCA) and polytopic vector analysis (PVA) identified wood burning, static and mobile diesel emissions and kerosene combustion as the major contributors to the particulate matter. When converted to toxicity equivalents (BaP-TEQ), the highest toxicity arising from PAHs in the indoor air was associated with a property that used a "leaky" combined wood stove and heater and also used a wood-fired brazier for local heating. In outdoor air, there was a relationship between the housing density and the BaP-TEQ, such that denser housing had higher BaP-TEQ values. The restrictions in wood burning on selected days may have had a measureable effect on the PM2.5 concentrations in that region but the effects were small and only present for the day of the restriction.

A Dorsal SHH-Dependent Domain in the V-SVZ Produces Large Numbers of Oligodendroglial Lineage Cells in the Postnatal Brain.

Neural stem cells in different locations of the postnatal mouse ventricular-subventricular zone (V-SVZ) generate different subtypes of olfactory bulb (OB) interneurons. High Sonic hedgehog (SHH) signaling in the ventral V-SVZ regulates the production of specific subtypes of neurons destined for the OB. Here we found a transient territory of high SHH signaling in the dorsal V-SVZ beneath the corpus callosum (CC). Using intersectional lineage tracing in neonates to label dorsal radial glial cells (RGCs) expressing the SHH target gene Gli1, we demonstrate that this region produces many CC cells in the oligodendroglial lineage and specific subtypes of neurons in the OB. The number of oligodendroglial cells generated correlated with the levels of SHH signaling. This work identifies a dorsal domain of SHH signaling, which is an important source of oligodendroglial cells for the postnatal mammalian forebrain.

Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons.

The mammalian neocortex is composed of two major neuronal cell types with distinct origins: excitatory pyramidal neurons and inhibitory interneurons, generated in dorsal and ventral progenitor zones of the embryonic telencephalon, respectively. Thus, inhibitory neurons migrate relatively long distances to reach their destination in the developing forebrain. The role of lineage in the organization and circuitry of interneurons is still not well understood. Utilizing a combination of genetics, retroviral fate mapping, and lineage-specific retroviral barcode labeling, we find that clonally related interneurons can be widely dispersed while unrelated interneurons can be closely clustered. These data suggest that migratory mechanisms related to the clustering of interneurons occur largely independent of their clonal origin.

Astrocyte development and heterogeneity.

Astrocytes have many roles within the brain parenchyma, and a subpopulation restricted to germinal niches functions as neural stem cells (NSCs) that produce various types of neuronal progeny in relation to spatiotemporal factors. A growing body of evidence supports the concept of morphological and molecular differences between astrocytes in different brain regions, which might relate to their derivation from regionally patterned radial glia. Indeed, the notion that astrocytes are molecularly and functionally heterogeneous could help explain how the central nervous system (CNS) retains embryonic positional information into adulthood. Here, we discuss recent evidence for regionally encoded functions of astrocytes in the developing and adult CNS to provide an integrated concept of the origin and possible function of astrocyte heterogeneity. We focus on the regionalization of NSCs in the ventricular-subventricular zone (V-SVZ) of the adult mammalian brain and emerging evidence for a segmental organization of astrocytes in the developing spinal cord and forebrain. We propose that astrocytes' diversity will provide fundamental clues to understand regional brain organization and function.

Adult neural stem cells in distinct microdomains generate previously unknown interneuron types.

Throughout life, neural stem cells (NSCs) in different domains of the ventricular-subventricular zone (V-SVZ) of the adult rodent brain generate several subtypes of interneurons that regulate the function of the olfactory bulb. The full extent of diversity among adult NSCs and their progeny is not known. Here, we report the generation of at least four previously unknown olfactory bulb interneuron subtypes that are produced in finely patterned progenitor domains in the anterior ventral V-SVZ of both the neonatal and adult mouse brain. Progenitors of these interneurons are responsive to sonic hedgehog and are organized into microdomains that correlate with the expression domains of the Nkx6.2 and Zic family of transcription factors. This work reveals an unexpected degree of complexity in the specification and patterning of NSCs in the postnatal mouse brain.

Adult neural stem cells bridge their niche.

Major developments in the neural stem cell (NSC) field in recent years provide new insights into the nature of the NSC niche. In this perspective, we integrate recent anatomical data on the organization of the two main neurogenic niches in the adult brain, the ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ), with signaling pathways that control the behavior of NSCs. NSCs in the adult brain stretch into physiologically distinct compartments of their niche. We propose how adult NSCs' morphology may allow these cells to integrate multiple signaling pathways arising from unique locations of their niche.

Regional astrocyte allocation regulates CNS synaptogenesis and repair.

Astrocytes, the most abundant cell population in the central nervous system (CNS), are essential for normal neurological function. We show that astrocytes are allocated to spatial domains in mouse spinal cord and brain in accordance with their embryonic sites of origin in the ventricular zone. These domains remain stable throughout life without evidence of secondary tangential migration, even after acute CNS injury. Domain-specific depletion of astrocytes in ventral spinal cord resulted in abnormal motor neuron synaptogenesis, which was not rescued by immigration of astrocytes from adjoining regions. Our findings demonstrate that region-restricted astrocyte allocation is a general CNS phenomenon and reveal intrinsic limitations of the astroglial response to injury.

Mad is required for wingless signaling in wing development and segment patterning in Drosophila.

A key question in developmental biology is how growth factor signals are integrated to generate pattern. In this study we investigated the integration of the Drosophila BMP and Wingless/GSK3 signaling pathways via phosphorylations of the transcription factor Mad. Wingless was found to regulate the phosphorylation of Mad by GSK3 in vivo. In epistatic experiments, the effects of Wingless on wing disc molecular markers (senseless, distalless and vestigial) were suppressed by depletion of Mad with RNAi. Wingless overexpression phenotypes, such as formation of ectopic wing margins, were induced by Mad GSK3 phosphorylation-resistant mutant protein. Unexpectedly, we found that Mad phosphorylation by GSK3 and MAPK occurred in segmental patterns. Mad depletion or overexpression produced Wingless-like embryonic segmentation phenotypes. In Xenopus embryos, segmental border formation was disrupted by Smad8 depletion. The results show that Mad is required for Wingless signaling and for the integration of gradients of positional information.

Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal.

BMP receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1(Cter) signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1(GSK3) antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.

xBtg-x regulates Wnt/beta-Catenin signaling during early Xenopus development.

In Xenopus, two signaling systems, maternal beta-Catenin and Nodal-related, are required for induction of the Spemann organizer and establishment of the body plan. By screening cDNA macroarrays for genes activated by these two signaling pathways, we identified Xenopus xBtg-x, a novel member of the Btg/Tob gene family of antiproliferative proteins. We show that xBtg-x is expressed in the dorsal mesendoderm (Spemann organizer tissue) of gastrula stage embryos and that its expression is regulated by both beta-Catenin and Nodal-related signals. Microinjection of synthetic xBtg-x mRNA into Xenopus embryos induced axis duplication and completely rescued the ventralizing effects of UV irradiation through the activation of the canonical Wnt/beta-Catenin signaling pathway. Interestingly, xBtg-x stimulated beta-Catenin-dependent transcription without affecting the stability of beta-Catenin protein. These data suggest that xBtg-x is a novel component of the Wnt/beta-Catenin signaling pathway regulating early embryonic patterning.