Phagocytic activity of neuronal progenitors regulates adult neurogenesis

Whereas thousands of new neurons are generated daily during adult life, only a fraction of them survive and become part of neural circuits; the rest die, and their corpses are presumably cleared by resident phagocytes. How the dying neurons are removed and how such clearance influences neurogenesis are not well understood. Here, we identify an unexpected phagocytic role for the doublecortin (DCX)-positive neuronal progenitor cells during adult neurogenesis. Our in vivo and ex vivo studies demonstrate that DCX(+) cells comprise a significant phagocytic population within the neurogenic zones. Intracellular engulfment protein ELMO1, which promotes Rac activation downstream of phagocytic receptors, was required for phagocytosis by DCX(+) cells. Disruption of engulfment in vivo genetically (in Elmo1-null mice) or pharmacologically (in wild-type mice) led to reduced uptake by DCX(+) cells, accumulation of apoptotic nuclei in the neurogenic niches and impaired neurogenesis. Collectively, these findings indicate a paradigm wherein DCX(+) neuronal precursors also serve as phagocytes, and that their phagocytic activity critically contributes to neurogenesis in the adult brain.     

Signalling by FGF8 from the isthmus patterns anterior hindbrain and establishes the anterior limit of Hox gene expression

Current evidence suggests that the anterior segment of the vertebrate hindbrain, rhombomere 1, gives rise to the entire cerebellum. It is situated where two distinct developmental patterning mechanisms converge: graded signalling from an organising centre (the isthmus) located at the midbrain/hindbrain boundary confronts segmentation of the hindbrain. The unique developmental fate of rhombomere 1 is reflected by it being the only hindbrain segment in which no Hox genes are expressed. In this study we show that ectopic FGF8 protein, a candidate for the isthmic organising activity, is able to induce and repress gene expression within the hindbrain in a manner appropriate to rhombomere 1. Using a heterotopic, heterospecific grafting strategy we demonstrate that rhombomere 1 is able to express Hox genes but that both isthmic tissue and FGF8 inhibit their expression. Inhibition of FGF8 function in vivo shows that it is responsible for defining the anterior limit of Hox gene expression within the developing brain and thereby specifies the extent of the rl territory. Previous studies have suggested that a retinoid morphogen gradient determines the axial limit of expression of individual Hox genes within the hindbrain. We propose a model whereby activation by retinoids is antagonised by inhibition by FGF8 in the anterior hindbrain to set aside the territory from which the cerebellum will develop.     

Leech neurogenesis. II. Mesodermal control of neuronal patterns

This paper reports analyses of the effects of eliminating mesoderm from one or both sides of embryos of the glossiphoniid leech Theromyzon rude on the differentiation and distribution of ectodermal cells, especially identified peripheral neurons and central 5-hydroxytryptamine (5-HT)-containing neurons arising from the bilateral pair of cell lines called the n bandlets (n-kinship cells). In mesoderm-deprived regions, no segmental hemiganglia formed, and identified neurons were not organized into recognizable patterns, although 5-HT neurons underwent neurochemical differentiation and grew axons. In unilaterally mesoderm-deprived embryos, segmental hemiganglia were formed in a midbody experimental zone, and cells that had abnormally crossed the ventral midline from the deprived side gave rise to identified neurons that were incorporated as supernumeraries into the normal organization of hemiganglia on the nondeprived side. In a posterior experimental zone, ganglionic morphology was disrupted on both sides. We conclude that precursor cells are committed to specific neuronal fates regardless of whether they occupy normal positions and that mesodermal tissues provide positional cues necessary for such precursor cells to find positions appropriate to their fates.     

Wnt1 regulates neurogenesis and mediates lateral inhibition of boundary cell specification in the zebrafish hindbrain

The formation of localised signalling centres is essential for patterning of a number of tissues during development. Previous work has revealed that a distinct population of boundary cells forms at the interface of segments in the vertebrate hindbrain, but the role of these cells is not known. We have investigated the function of the Wnt1 signalling molecule that is expressed by boundary and roof plate cells in the zebrafish hindbrain. Knockdown of wnt1 or of tcf3b, a mediator of Wnt signalling, leads to ectopic expression of boundary cell markers, rfng and foxb1.2, in non-boundary regions of the hindbrain. Ectopic boundary marker expression also occurs following knockdown of rfng, a modulator of Notch signalling required for wnt1 expression at hindbrain boundaries. We show that the boundary and roof plate expression of wnt1 each contribute to upregulation of proneural and delta gene expression and neurogenesis in non-boundary regions, which in turn blocks ectopic boundary marker expression. Boundary cells therefore play a key role in the regulation of cell differentiation in the zebrafish hindbrain. The network of genes underlying the regulation of neurogenesis and lateral inhibition of boundary cell formation by Wnt1 has a striking similarity to mechanisms at the dorsoventral boundary in the Drosophila wing imaginal disc.     

Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the rostral hindbrain

Little is known about how the generation of specific neuronal types at stereotypic positions within the hindbrain is linked to Hox gene-mediated patterning. Here, we show that during neurogenesis, Hox paralog group 2 genes control both anteroposterior (A-P) and dorsoventral (D-V) patterning. Hoxa2 and Hoxb2 differentially regulate, in a rhombomere-specific manner, the expression of several genes in broad D-V-restricted domains or narrower longitudinal columns of neuronal progenitors, immature neurons, and differentiating neuronal subtypes. Moreover, Hoxa2 and Hoxb2 can functionally synergize in controlling the development of ventral neuronal subtypes in rhombomere 3 (r3). Thus, in addition to their roles in A-P patterning, Hoxa2 and Hoxb2 have distinct and restricted functions along the D-V axis during neurogenesis, providing insights into how neuronal fates are assigned at stereotypic positions within the hindbrain.     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis.     

Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis. Received: 20 September 1997 / Accepted: 8 October 1997     

Signaling in adult neurogenesis: from stem cell niche to neuronal networks

The mechanisms that determine why neurogenesis is restricted to few regions of the adult brain in mammals, in contrast to its more widespread nature in other vertebrates such as zebrafish, remain to be fully understood. The local environment must provide key signals that instruct stem cell and neurogenic fate, because non-neurogenic progenitors can be instructed towards neurogenesis in this environment. Here, we discuss the recent progress in understanding key factors in the local stem cell niche of the adult mammalian brain, including surprising sources of new signals such as endothelial cells, complement factors and microglia. Moreover, new insights have been gained into how neuronal diversity is instructed in adult neurogenesis, prompting a new view of stem and progenitor cell heterogeneity in the adult mammalian brain.     

Macoilin, a conserved nervous system-specific ER membrane protein that regulates neuronal excitability

Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O₂ responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca²⁺ transients, at least in some neurons: in maco-1 mutants the O₂-sensing neuron PQR is unable to generate a Ca²⁺ response to a rise in O₂. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O₂, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca²⁺ channels, also fails to disrupt Ca²⁺ responses in the PQR cell body to O₂ stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca²⁺ channel α1 subunit, recapitulate the Ca²⁺ response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or traffic of ion channels or ion channel regulators.     

A novel approach to selectively target neuronal subpopulations reveals genetic pathways that regulate tangential migration in the vertebrate hindbrain

Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications.     

Connexin 36 expression regulates neuronal differentiation from neural progenitor cells

Background

Gap junction communication has been shown in glial and neuronal cells and it is thought they mediate inter- and intra-cellular communication. Connexin 36 (Cx36) is expressed extensively in the developing brain, with levels peaking at P14 after which its levels fall and its expression becomes entirely neuronal. These and other data have led to the hypothesis that Cx36 may direct neuronal coupling and neurogenesis during development.

Methodology/Principal Findings

To investigate Cx36 function we used a neurosphere model of neuronal cell development and developed lentiviral Cx36 knockdown and overexpression strategies. Cx36 knockdown was confirmed by western blotting, immunocytochemistry and functionally by fluorescence recovery after photobleaching (FRAP). We found that knockdown of Cx36 in neurosphere neuronal precursors significantly reduced neuronal coupling and the number of differentiated neurons. Correspondingly, the lentiviral mediated overexpression of Cx36 significantly increased the number of neurons derived from the transduced neurospheres. The number of oligodendrocytes was also significantly increased following transduction with Cx36 indicating they may support neuronal differentiation.

Conclusions/Significance

Our data suggests that astrocytic and neuronal differentiation during development are governed by mechanisms that include the differential expression of Cx36.     

'Multifrequency' location and clustering of sequence patterns from proteins

In previous work, we have shown that a set of characteristics,defined as (code frequency) pairs, can be derived from a proteinfamily by the use of a signal-processing method. This methodenables the location and extraction of sequence patterns bytaking into account each (code frequency) pair individually.In the present paper, we propose to extend this method in orderto detect and visualize patterns by taking into account severalpairs simultaneously. Two ‘multifrequency’ methodsare described. The first one is based on a rewriting of thesequences with new symbols which summarize the frequency information.The second method is based on a clustering of the patterns associatedwith each pair. Both methods lead to the definition of significantconsensus sequences. Some results obtained with calcium-bindingproteins and serine proteases are also discussed. Received on March 6, 1990; accepted on September 24, 1990     

Polysialic acid regulates the clustering, migration, and neuronal differentiation of progenitor cells in the adult hippocampus

Newborn cells of the adult dentate gyrus in the hippocampus are characterized by their abundant expression of polysialic acid (PSA), a carbohydrate attached to the neural cell adhesion molecule (NCAM). PSA+ newborn cells of the dentate gyrus form clusters with proliferating neural progenitor cells, migrate away from these clusters, and terminally differentiate. To identify the roles of PSA in the development of adult progenitors of the dentate gyrus, we injected endoneuraminidase N (endoN) into the hippocampus of adult rats to specifically cleave PSA from NCAM. Two days later, we administered the mitotic marker, 5-bromo-2'-deoxyuridine (BrdU). Three days after BrdU injection, BrdU+ cells were found inside and outside the clusters of newborn cells. In endoN-treated animals, the total number of BrdU+ cells was not changed but significantly more BrdU+ cells were present within clusters, suggesting that PSA normally facilitates the migration of progenitors away from the clusters. Seven days post-BrdU injection, endoN-treated animals had significantly more BrdU+ cells which were also positive for the mature neuronal nuclear marker NeuN compared with controls, indicating that the loss of PSA from progenitor cells increases neuronal differentiation. This report is the first demonstration that PSA is involved in controlling the spatio-temporal neuronal maturation of adult hippocampal progenitors in the normal brain. In vitro, the removal of PSA from adult-derived neural progenitors significantly enhanced neuronal differentiation, strengthening our in vivo findings and indicating that PSA removal on isolated progenitor cells, apart from a complex in vivo environment, induces neuronal maturation.     

rpm-1, a conserved neuronal gene that regulates targeting and synaptogenesis in C. elegans

Little is known of mechanisms regulating presynaptic differentiation. We identified rpm-1 in a screen for mutants with defects in patterning of a presynaptic marker at certain interneuronal synapses. The predicted RPM-1 protein contains zinc binding, RCC1, and other conserved motifs. In rpm-1 mutants, mechanosensory neurons fail to accumulate tagged vesicles, retract synaptic branches, and ectopically extend axons. Some motor neurons branch and overgrow; others show altered synaptic organization. Expression of RPM-1 in the presynaptic mechanosensory neurons is sufficient to rescue phenotypes in these cells. Certain rpm-1 phenotypes are temperature sensitive, revealing that RPM-1 function can be bypassed by maintaining mutants at the permissive temperature at stages commensurate with synapse formation in wild-type animals. These results indicate that RPM-1 functions cell autonomously during synaptogenesis to regulate neuronal morphology.     

Purification of axonin-1, a protein that is secreted from axons during neurogenesis.

Using selective metabolic labelling in a compartmental cell culture system two proteins, denoted axonin-1 and axonin-2, were found to be secreted by axons of dorsal root ganglia neurons from chicken embryos. Based on its characteristic coordinates and spot morphology in two-dimensional gel electrophoresis, axonin-1 was detected in the cerebrospinal fluid and the vitreous fluid, axonin-1 was purified 476-fold to homogeneity by a four-step chromatographic procedure. The identity of the purified protein as axonin-1 was confirmed by immunological methods. Axonin-1 is a glycoprotein that subdivides into at least 16 immunologically similar isoelectric variants; their molecular weight range extends from 132 to 140 kd and their pI range from 5.3 to 6.2. In the vitreous fluid of the embryo, axonin-1 could first be detected on the embryonic day 5 and highest concentrations were measured during the second half of embryonic life; in the vitreous fluid of the adult chicken, concentrations were approximately 20 times lower. The early onset of secretion and the time course of expression suggest a role for axonin-1 in the development of the nervous system.