Germination Niche Research Articles

Neural precursor cells in the ischemic brain - integration, cellular crosstalk, and consequences for stroke recovery.

After an ischemic stroke, neural precursor cells (NPCs) proliferate within major germinal niches of the brain. Endogenous NPCs subsequently migrate toward the ischemic lesion where they promote tissue remodeling and neural repair. Unfortunately, this restorative process is generally insufficient and thus unable to support a full recovery of lost neurological functions. Supported by solid experimental and preclinical data, the transplantation of exogenous NPCs has emerged as a potential tool for stroke treatment. Transplanted NPCs are thought to act mainly via trophic and immune modulatory effects, thereby complementing the restorative responses initially executed by the endogenous NPC population. Recent studies have attempted to elucidate how the therapeutic properties of transplanted NPCs vary depending on the route of transplantation. Systemic NPC delivery leads to potent immune modulatory actions, which prevent secondary neuronal degeneration, reduces glial scar formation, diminishes oxidative stress and stabilizes blood–brain barrier integrity. On the contrary, local stem cell delivery allows for the accumulation of large numbers of transplanted NPCs in the brain, thus achieving high levels of locally available tissue trophic factors, which may better induce a strong endogenous NPC proliferative response. Herein we describe the diverse capabilities of exogenous (systemically vs. locally transplanted) NPCs in enhancing the endogenous neurogenic response after stroke, and how the route of transplantation may affect migration, survival, bystander effects and integration of the cellular graft. It is the authors’ claim that understanding these aspects will be of pivotal importance in discerning how transplanted NPCs exert their therapeutic effects in stroke.

Primary cilia are required in a unique subpopulation of neural progenitors.

The apical domain of embryonic (radial glia) and adult (B1 cells) neural stem cells (NSCs) contains a primary cilium. This organelle has been suggested to function as an antenna for the detection of morphogens or growth factors. In particular, primary cilia are essential for Hedgehog (Hh) signaling, which plays key roles in brain development. Their unique location facing the ventricular lumen suggests that primary cilia in NSCs could play an important role in reception of signals within the cerebrospinal fluid. Surprisingly, ablation of primary cilia using conditional alleles for genes essential for intraflagellar transport [kinesin family member 3A (Kif3a) and intraflagellar transport 88 (Ift88)] and Cre drivers that are activated at early [Nestin; embryonic day 10.5 (E10.5)] and late [human glial fibrillary acidic protein (hGFAP); E13.5] stages of mouse neural development resulted in no apparent developmental defects. Neurogenesis in the ventricular-subventricular zone (V-SVZ) shortly after birth was also largely unaffected, except for a restricted ventral domain previously known to be regulated by Hh signaling. However, Kif3a and Ift88 genetic ablation also disrupts ependymal cilia, resulting in hydrocephalus by postnatal day 4. To directly study the role of B1 cells' primary cilia without the confounding effects of hydrocephalus, we stereotaxically targeted elimination of Kif3a from a subpopulation of radial glia, which resulted in ablation of primary cilia in a subset of B1 cells. Again, this experiment resulted in decreased neurogenesis only in the ventral V-SVZ. Primary cilia ablation led to disruption of Hh signaling in this subdomain. We conclude that primary cilia are required in a specific Hh-regulated subregion of the postnatal V-SVZ.

Molecular targets of chromatin repressive mark H3K9me3 in primate progenitor cells within adult neurogenic niches

Histone 3 Lysine 9 (H3K9) methylation is known to be associated with pericentric heterochromatin and important in genomic stability. In this study, we show that trimethylation at H3K9 (H3K9me3) is enriched in an adult neural stem cell niche- the subventricular zone (SVZ) on the walls of the lateral ventricle in both rodent and non-human primate baboon brain. Previous studies have shown that there is significant correlation between baboon and human regarding genomic similarity and brain structure, suggesting that findings in baboon are relevant to human. To understand the function of H3K9me3 in this adult neurogenic niche, we performed genome-wide analyses using ChIP-Seq (chromatin immunoprecipitation and deep-sequencing) and RNA-Seq for in vivo SVZ cells purified from baboon brain. Through integrated analyses of ChIP-Seq and RNA-Seq, we found that H3K9me3-enriched genes associated with cellular maintenance, post-transcriptional and translational modifications, signaling pathways, and DNA replication are expressed, while genes involved in axon/neuron, hepatic stellate cell, or immune-response activation are not expressed. As neurogenesis progresses in the adult SVZ, cell fate restriction is essential to direct proper lineage commitment. Our findings highlight that H3K9me3 repression in undifferentiated SVZ cells is engaged in the maintenance of cell type integrity, implicating a role for H3K9me3 as an epigenetic mechanism to control cell fate transition within this adult germinal niche.

Germination niche breadth differs in two co-occurring palms of the Atlantic Rainforest

Niche germination breadth may determine species occurrence under distinct environmental conditions. We choose Euterpe edulis and Lytocaryum weddellianum to evaluate germination niche breadth under light or dark at water potential of 0MPa, and in either decreasing water potentials (−0.4 and −0.8MPa) or flooded. In contrast to L. weddellianum, E. edulis showed high seed germination at both light conditions and expressive seed germination at low water potentials and flooding. E. edulis had wide seed germination niche breadth and seed addition of the same geographic region might be used for the reintroduction of populations of this threatened palm in different types of habitats of the Atlantic Rainforest. L. weddellianum, however, is unlikely to be able to germinate under altered conditions due to narrower germination niches and might be more vulnerable to extinction if the restricted germination cues no longer occur. Thus, ex situ initiatives might be used for the species conservation.

Predicting the impact of increasing temperatures on seed germination among populations of Western AustralianBanksia(Proteaceae)

AbstractTemperature is a significant factor influencing seed germination and for many species temperature-mediated germination cues are vital for plant persistence. Rising temperatures forecast as a result of anthropogenic climate change may have a substantial influence on the population and range dynamics of plant species. Here, we report on the thermal constraints on seed germination in natural populations of four congenericBanksiaspecies collected from a longitudinal climate gradient in Western Australia. We investigated whether germination niche: (1) varied between species; (2) varied among populations of each species; and (3) varied in a consistent manner reflecting the climatic gradients of seed origin. We hypothesized that species would differ and that populations from warmer sites would have a broader temperature window for germination than populations from cooler sites. Species differed in the breadth of their germination niche, but temperatures that stimulated the most rapid and complete germination were similar across all species. A sharp reduction in germination percentage occurred above the optimum temperature, which coincided with significant delays in germination relative to the optimum. The temperatures causing these declines varied among populations. Across the species, there was a significant correlation between optimum germination temperature and mean annual temperature at seed source; however, there was no relationship at the population level for individual species. These data provide insight into the vulnerability ofBanksiaspecies to climate change, with those populations that require lower temperatures for germination, or have narrower optimal ranges for germination, likely to be most vulnerable to a warming climate.

Germination niche of the permanent wetland specialist, Parnassia grandifolia DC

AbstractTemperate wetland species often require light and warm temperatures for seed germination. However, recent studies indicate that species which specialize on permanently saturated wetlands that are maintained by groundwater discharge (fens, seeps and mountain springs), rather than wetlands with surface-water-driven hydrologic regimes, diverge from the typical wetland germination niche by germinating at cool temperatures and lacking photoblastic seeds. We conducted laboratory experiments that manipulated stratification conditions (non-stratified versus cold stratification in light and darkness), thermal regime (15/6, 25/15 and 35/20°C), and light (14 h photoperiod versus continuous darkness) to test whether seeds of the North American calcareous fen specialist Parnassia grandifolia diverged from the typical temperate wetland germination niche. After 30 d, fresh seeds were conditionally dormant and could only germinate to high percentages in light at 25/15°C. During 16 weeks of incubation, non-stratified seeds germinated to low percentages ( < 40%) at all thermal regimes in darkness. In contrast, cold-stratified seeds germinated to high percentages in both light and darkness at all thermal regimes, although germination was incomplete (no cotyledon emergence) at 35/20°C. Further, seeds did not require light during cold stratification to germinate to high percentages when incubated in light or darkness. Thus, seeds diverged from the typical temperate wetland germination syndrome in lacking a light and warm temperature requirement for germination. Our results reinforce previous work from European fens and Mediterranean wetlands. This indicates that multiple germination strategies are found in fen wetlands that are maintained by the continuous or near-continuous discharge of cool groundwater.

Increasing temperatures can improve seedling establishment in arid-adapted savanna trees.

Plant species are shifting their ranges in response to global climate change, thus intensifying the need to predict such changes accurately. As the environmental requirements controlling plant distribution act differently at each developmental stage, there is a need to acquire a demographic-specific understanding of the factors which determine these distributions. Here we investigated the germination niche of two common savanna species Acacia nigrescens and Colophospermum mopane, with the aims to disentangle the direct and indirect effects of temperature on seed germination and establishment and to explore the impact of higher temperatures on the establishment success of savanna trees. Under laboratory conditions, we used thermal gradient plates to determine the thermal germination niche of both species, and a water stress experiment was conducted on C. mopane to account for water-temperature interactions. Using these data we parameterised a soil-moisture model to determine germination and establishment success under field conditions at current and future temperatures (+4°C). Based on this model, higher future temperatures will not limit germination directly, but they will reduce the number of germination events by reducing the time window of suitable available soil water. Conversely, warmer conditions will accelerate the rate of radicle extension and increase the frequency of seedling establishment events. An additional advantage of higher temperatures is that fewer seeds will germinate, resulting in slower seed bank depletion when successful seedling establishment events do occur.

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

Neonatal pial surface electroporation.

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

Are seed germination and ecological breadth associated? Testing the regeneration niche hypothesis with bromeliads in a heterogeneous neotropical montane vegetation

The role of seed germination in contributing to species ecological breadth and geographic distribution is still a matter of debate. Here, we attempted to relate seed germination requirements with ecological breadth in 12 bromeliad species from heterogeneous montane vegetation in southeastern Brazil. Seeds were set to germinate under both light and dark conditions at a broad range of temperatures to determine the breadth of the germination niche. We ran a RLQ analysis based on the matrices of species occurrence, environmental parameters and germination traits and found a significant association between germination traits, and the characteristics of sites where adult plants occur. The variation of germination responses to environmental factors was not random with habitat-generalist plants having broader germination niches and habitat-specialist plants having narrower germination niches. The RLQ analysis showed that substrate moisture and light environment were the most important factors correlated with germination traits. Phylogenetic niche conservatism appears to play a role in the patterns found here, especially in the Tillandsioideae. There is an association between the regeneration niche of a species and its ecological range, and this also provides support for the idea that the regeneration niche may help assemble plant species into heterogeneous, species-rich communities.

Neonatal Peripheral Immune Challenge Activates Microglia and Inhibits Neurogenesis in the Developing Murine Hippocampus

The early postnatal period represents an important window in rodent hippocampal development with peak hilar neurogenesis and widespread microgliogenesis occurring in the first week of life. Inflammation occurring during this period may negatively influence development, potentially facilitating or increasing susceptibility to later-life pathology. We administered the Gram-negative bacterial coat protein lipopolysaccharide (LPS) systemically at postnatal day 5 (1 mg/kg i.p.) and assessed potential effects on microgliogenesis, inflammation and neurogenesis in the developing hippocampus. LPS administration led to an acute but transient increase in absolute number and density of ionized calcium-binding adaptor molecule 1-immunoreactive microglia, a change attributable to increased proliferation of central nervous system-resident microglia/microglial precursor cells but not infiltration of peripheral monocyte-derived macrophages. qRT-PCR analysis of hippocampal gene expression showed these LPS-mediated changes to be associated with persistent dysregulation of genes associated with both M1 and M2 microglial phenotypes, indicating prolonged alteration in hippocampal inflammatory status. Further, analysis of progenitor cell regulation in the hippocampal subgranular zone revealed a transient inhibition of the neuronal differentiation pathway up to 2 weeks after LPS administration, a change occurring specifically through effects on type 3 neural progenitor cells and independently of altered cell proliferation or survival of newly born cells. Together, our results show that systemic inflammation occurring during the early neonatal period is sufficient to alter inflammatory status and dysregulate the ongoing process of neurogenesis in the developing hippocampal germinal niche.

Increased Precursor Cell Proliferation after Deep Brain Stimulation for Parkinson's Disease: A Human Study

ObjectiveDeep brain stimulation (DBS) has been used for more than a decade to treat Parkinson's disease (PD); however, its mechanism of action remains unknown. Given the close proximity of the electrode trajectory to areas of the brain known as the “germinal niches,” we sought to explore the possibility that DBS influences neural stem cell proliferation locally, as well as more distantly.MethodsWe studied the brains of a total of 12 idiopathic Parkinson's disease patients that were treated with DBS (the electrode placement occurred 0.5–6 years before death), and who subsequently died of unrelated illnesses. These were compared to the brains of 10 control individuals without CNS disease, and those of 5 PD patients with no DBS.ResultsImmunohistochemical analyses of the subventricular zone (SVZ) of the lateral ventricles, the third ventricle lining, and the tissue surrounding the DBS lead revealed significantly greater numbers of proliferating cells expressing markers of the cell cycle, plasticity, and neural precursor cells in PD-DBS tissue compared with both normal brain tissue and tissue from PD patients not treated with DBS. The level of cell proliferation in the SVZ in PD-DBS brains was 2–6 fold greater than that in normal and untreated PD brains.ConclusionsOur data suggest that DBS is capable of increasing cellular plasticity in the brain, and we hypothesize that it may have more widespread effects beyond the electrode location. It is unclear whether these effects of DBS have any symptomatic or other beneficial influences on PD.

Neurogenesis or non-neurogenesis: that is the question

Neural stem/precursor cells (NPCs) that reside within germinal niches of the adult CNS have more complex roles than previously expected. In addition to their well-documented neurogenic functions, emerging evidence indicates that NPCs exert non-neurogenic functions that contribute to the regulation and preservation of tissue homeostasis under both physiological and pathological conditions. In this issue of the JCI, Mohammad et al. found that DCs efficiently patrol the CNS only when the germinal niche of the subventricular zone functions properly. Indeed, DCs traveled from the ventricles along the rostral migratory stream to the olfactory bulb (a cervical lymph node access point) to dampen anti-CNS immune responses. The authors' findings further support a non-neurogenic role for NPCs in maintaining tissue homeostasis and promoting tissue protection in the adult brain.

Axonal Control of the Adult Neural Stem Cell Niche

The ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSCs) in the walls of the lateral ventricles of the adult brain. How the adult brain's neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that activation of these receptors in B1 cells induced small inward currents. Intraventricular infusion of 5HT2C agonist or antagonist increased or decreased V-SVZ proliferation, respectively. These results indicate that supraependymal 5HT axons directly interact with NSCs to regulate neurogenesis via 5HT2C.

Conditional ablation of Tbr2 results in abnormal development of the olfactory bulbs and subventricular zone‐rostral migratory stream

Development of the olfactory bulb (OB) is a complex process that requires contributions from several progenitor cell niches to generate neuronal diversity. Previous studies showed that Tbr2 is expressed during the generation of glutamatergic OB neurons in rodents. However, relatively little is known about the role of Tbr2 in the developing OB or in the subventricular zone-rostral migratory stream (SVZ-RMS) germinal niche that gives rise to many OB neurons. Here, we use conditional gene ablation strategies to knockout Tbr2 during embryonic mouse olfactory bulb morphogenesis, as well as during perinatal and adult neurogenesis from the SVZ-RMS niche, and describe the resulting phenotypes. We find that Tbr2 is important for the generation of mitral cells in the OB, and that the olfactory bulbs themselves are hypoplastic and disorganized in Tbr2 mutant mice. Furthermore, we show that the SVZ-RMS niche is expanded and disordered following loss of Tbr2, which leads to ectopic accumulation of neuroblasts in the RMS. Lastly, we show that adult glutamatergic neurogenesis from the SVZ is impaired by loss of Tbr2. Tbr2 is essential for proper morphogenesis of the OB and SVZ-RMS, and is important for the generation of multiple lineages of glutamatergic olfactory bulb neurons.

Cerebrospinal fluid derived from progressive multiple sclerosis patients promotes neuronal and oligodendroglial differentiation of human neural precursor cells in vitro

In the adult CNS, tissue-specific germinal niches, such as the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus, contain multipotent neural precursor cells (NPCs) with the capacity to self-renew and differentiate into functional brain cells (i.e. neurons, astrocytes or oligodendrocytes). Due to their intrinsic plasticity, NPCs can be considered an essential part of the cellular mechanism(s) by which the CNS tries to repair itself after an injury. In inflammatory CNS disorders, such as multiple sclerosis (MS), neurogenesis and gliogenesis occur as part of an ‘intrinsic’ self-repair process. However, full and long-lasting repair in progressive MS is not achieved.Recent data suggest that endogenous NPCs, while trying to repair the damaged CNS in MS, may become the target of the disease itself. It is possible that factors produced during MS, like CNS-infiltrating blood-borne inflammatory mononuclear cells, reactive CNS-resident cells, and humoral mediators, can alter the physiological properties of NPCs, ultimately impairing their ability to promote neural regeneration.Here, we investigate the effect of cerebrospinal fluid (CSF) derived from primary progressive (PPMS) and secondary progressive (SPMS) MS patients (CSF–MS) on the survival, proliferation, and differentiation of commercially available human embryonic-derived NPCs named ENStem-A. We found that PPMS derived CSF markedly reduced the proliferation of ENStem-A and increased their differentiation toward neuronal and oligodendroglial cells, compared to control CSF. Similar but less striking results were seen when ENstem-A were treated with SPMS derived CSF. Our findings suggest that in both SPMS and PPMS the CNS milieu, as determined by extrapolation from CSF findings, may stimulate the endogenous pool of NPCs to differentiate into neurons and oligodendrocytes.

Abstract 366: Downregulation of THBS1 is a critical step in glioblastoma angiogenesis.

Abstract One of the hallmarks of Glioblastoma multiforme (GBM) is its abundant vasculature. These blood vessels support the tumor in a number of ways, including providing a niche for glioblastoma cancer stem cells. GBM tumors drive the formation of their vasculature and associated germinal niches through reciprocal inductive interactions between GBM cells and endothelial cells. Targeting the primary drivers of angiogenesis, VEGF and bFGF, in anti-angiogenic cancer treatments has value but is not curative. We hypothesize that identification of additional angiogenic molecules and pathways will provide new avenues for treatment. We used microarrays to perform global gene expression profiling to identify genes that are increased or decreased in expression as a consequence of functional interactions between GBM and brain microvascular endothelial cells. We identified several regulators of angiogenesis whose expression is modulated by this cell-cell interaction, but were particularly interested in the downregulation of Thrombospondin-1 (THBS1) and its associated receptors. THBS1 is a powerful anti-angiogenic protein and has previously been implicated in cancer, but its precise mechanism in glioblastoma angiogenesis is not yet known. A number of in-vitro experiments validated the importance of THBS1 in the interaction between GBM and endothelial cells. In-vivo mouse xenograft experiments showed that overexpression of THBS1 strongly inhibited tumor formation. We believe these results reveal the importance of factors other than VEGF in glioblastoma angiogenesis and provide a rationale to evaluate combinatorial targeted anti-angiogenic therapy for GBM. Citation Format: Michael D. Brooks, Erin Jackson, David Piwnica-Worms, Robi D. Mitra, Joshua B. Rubin. Downregulation of THBS1 is a critical step in glioblastoma angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 366. doi:10.1158/1538-7445.AM2013-366

Characterising the germination niche of two common savanna trees

Characterising the germination niche of two common savanna trees

The temperature dimension of the seed germination niche in fen wetlands

The seed germination niche partly determines adaptation, ecological breadth and geographic range in plant species. In temperate wetlands, environmental temperature is the chief regulator of germination timing, but the ecological significance of high and low temperatures during dormancy break and germination is still poorly understood. Our aim was to characterize the temperature dimension of the germination niche in mountain base-rich fens, determining (1) the effect of different temperatures on dormancy break and germination, and (2) whether different germination strategies may be identified at the species level. We conducted laboratory germination experiments with seeds of 15 species from these habitats, collected in 18 fen sites in the Cantabrian Mountains (Spain) for two consecutive years. In all the species, the seeds were totally or conditionally dormant at dispersal and stratification produced a significant increase of germination. In most cases, there was not an obligatory requirement for cold temperatures during dormancy break, since warm stratification promoted germination as well. Although the optimal germination thermoperiod was generally high (30/20 °C), most species could also germinate at lower temperatures after cold-stratification. We also identified a group of species associated to cold-water springs that germinated only at low temperatures. Our results demonstrate that dormancy break in mountain base-rich fens does not obligatorily depend on cold temperatures during overwintering. Furthermore, germination at cool temperatures may be more widespread in wetland habitats than previously thought. The existence of two distinctive germination strategies, ‘warm’ and ‘cool’, can potentially give rise to divergent species responses to climate change.

The role of EGFR and ErbB family related proteins in the oligodendrocyte specification in germinal niches of the adult mammalian brain

In the adult brain, multipotent progenitor cells have been identified in three areas: the ventricular-subventricular zone (VZ-SVZ), adjacent to the striatal wall of the lateral ventricles, the subgranular zone (SGZ), located at the dentate gyrus of the hippocampus and the subcallosal zone (SCZ), located between the corpus callosum and the CA1 and CA2 regions of the hippocampus. The neural progenitor cells of these regions express the epidermal growth factor receptor (EGFR, ErbB-1 or HER1). EGF, the most important ligand for the EGFR, is a potent mitogenic agent that stimulates proliferation, survival, migration and differentiation into the oligodendrocyte lineage. Other ErbB receptors also activate several intracellular pathways for oligodendrocyte specification, migration and survival. However, the specific downstream pathways related to oligodendrogenesis and the hierarchic interaction among intracellular signaling cascades is not well-known. We summarize the current data regarding the role of EGFR and ErbB family signaling on neural stem cells and the downstream cascades involved in oligodendrogenesis in the neurogenic niches of the adult brain. Understanding the mechanisms that regulate proliferation, differentiation, migration of oligodendrocytes and myelination is of critical importance for the field of neurobiology and constitutes a crucial step in the design of stem-cell-based therapies for demyelinating diseases.