Literature context: RRID:AB_400326 Rat monoclonal anti-BrdU/CIdU A
Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically.
Literature context: 47580; Clone B44; Lot: 6042756; RRID:AB_400326 Chemicals, Peptides, and Recomb
Faithful DNA replication is challenged by stalling of replication forks during S phase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed S phase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout S phase. Moreover, cells that re-enter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged S phase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout S phase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events.
Literature context: on Dickinson Cat#347580; RRID:AB_400326 Rabbit polyclonal anti-POLE4 Th
DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.
Literature context: 347580; RRID:AB_400326 Anti-goat Cy5.5 Abcam ab6951; R
Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer.
Literature context: # 347580, RRID:AB_400326), Bcl-xL (
The bcl-2 family of survival and death promoting proteins play a key role in regulating cell numbers during nervous system development. Bcl-xL, an anti-apoptotic bcl-2 family member is highly expressed in the developing nervous system. However; the early embryonic lethality of the bcl-x germline null mouse precluded an investigation into its role in nervous system development. To identify the role of bcl-x in spinal cord neurogenesis, we generated a central nervous system-specific bcl-x conditional knockout (BKO) mouse. Apoptotic cell death in the BKO embryo was initially detected at embryonic day 11 (E11) in the ventrolateral aspect of the spinal cord corresponding to the location of motor neurons. Apoptosis reached its peak at E13 having spread across the ventral and into the dorsal spinal cord. By E18, the wave of apoptosis had passed and only a few apoptotic cells were observed. The duration and direction of spread of apoptosis across the spinal cord is consistent with the spatial and temporal sequence of neuronal differentiation. Motor neurons, the first neurons to become post mitotic in the spinal cord, were also the first apoptotic cells. As neurogenesis spread across the spinal cord, later born neuronal populations such as Lim2+ interneurons were also affected. The onset of apoptosis occurred in cells that had exited the cell cycle within the previous 24h and initiated neural differentiation as demonstrated by BrdU birthdating and βIII tubulin immunohistochemistry. This data demonstrates that spinal cord neurons become Bcl-xL dependent at an early post mitotic stage in developmental neurogenesis.
Literature context: body IdU BD Biosciences 347580, RRID:AB_400326 Antibody CldU Novus Biologicals
Classically, p53 tumor suppressor acts in transcription, apoptosis, and cell cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks that drive genomic instability, which isgenetically separable from transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53-mediated suppression of replication genome instability.
Literature context: U Becton Dickinson Cat# 347580; RRID:AB_400326 Rat anti-BrdU/CldU Abcam Cat# a
DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.
Literature context: t#347580; RRID:AB_400326 Rat monocl
RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.
Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment.
Literature context: t#347580; RRID:AB_400326 Caspase-3Â
The concerted production of neurons and glia by neural stem cells (NSCs) is essential for neural circuit assembly. In the developing cerebral cortex, radial glia progenitors (RGPs) generate nearly all neocortical neurons and certain glia lineages. RGP proliferation behavior shows a high degree of non-stochasticity, thus a deterministic characteristic of neuron and glia production. However, the cellular and molecular mechanisms controlling RGP behavior and proliferation dynamics in neurogenesis and glia generation remain unknown. By using mosaic analysis with double markers (MADM)-based genetic paradigms enabling the sparse and global knockout with unprecedented single-cell resolution, we identified Lgl1 as a critical regulatory component. We uncover Lgl1-dependent tissue-wide community effects required for embryonic cortical neurogenesis and novel cell-autonomous Lgl1 functions controlling RGP-mediated glia genesis and postnatal NSC behavior. These results suggest that NSC-mediated neuron and glia production is tightly regulated through the concerted interplay of sequential Lgl1-dependent global and cell intrinsic mechanisms.
Literature context: # 347580, RRID:AB_400326 Rabbit pol
In many vertebrates, postnatally generated neurons often migrate long distances to reach their final destination, where they help shape local circuit activity. Concerted action of extrinsic stimuli is required to regulate long-distance migration. Some migratory principles are evolutionarily conserved, whereas others are species and cell type specific. Here we identified a serotonergic mechanism that governs migration of postnatally generated neurons in the mouse brain. Serotonergic axons originating from the raphe nuclei exhibit a conspicuous alignment with subventricular zone-derived neuroblasts. Optogenetic axonal activation provides functional evidence for serotonergic modulation of neuroblast migration. Furthermore, we show that the underlying mechanism involves serotonin receptor 3A (5HT3A)-mediated calcium influx. Thus, 5HT3A receptor deletion in neuroblasts impaired speed and directionality of migration and abolished calcium spikes. We speculate that serotonergic modulation of postnatally generated neuroblast migration is evolutionarily conserved as indicated by the presence of serotonergic axons in migratory paths in other vertebrates.
Literature context: sduction LaboratoriesCat# 610958Mouse anti-BrdU Clone B44BD Transduction LaboratoriesCat# 347580Rat monoclonal anti BrdU Clone B
Cells exposed to hypoxia experience replication stress but do not accumulate DNA damage, suggesting sustained DNA replication. Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). However, oxygen is an essential cofactor for mammalian RNR (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here, we show that the RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favored over RRM1/RRM2 in order to preserve ongoing replication and avoid the accumulation of DNA damage. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and identified responsible residues in RRM2B. The importance of RRM2B in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance. Our data provide mechanistic insight into RNR biology, highlighting RRM2B as a hypoxic-specific, anti-cancer therapeutic target.
Literature context: U [1:300, RRID:AB_400326, BD Biosci
Vestibular hair cells in the inner ear encode head movements and mediate the sense of balance. These cells undergo cell death and replacement (turnover) throughout life in non-mammalian vertebrates. However, there is no definitive evidence that this process occurs in mammals. We used fate-mapping and other methods to demonstrate that utricular type II vestibular hair cells undergo turnover in adult mice under normal conditions. We found that supporting cells phagocytose both type I and II hair cells. Plp1-CreERT2-expressing supporting cells replace type II hair cells. Type I hair cells are not restored by Plp1-CreERT2-expressing supporting cells or by Atoh1-CreERTM-expressing type II hair cells. Destruction of hair cells causes supporting cells to generate 6 times as many type II hair cells compared to normal conditions. These findings expand our understanding of sensorineural plasticity in adult vestibular organs and further elucidate the roles that supporting cells serve during homeostasis and after injury.
Literature context: akes, NJ, RRID:AB_400326). Image ac
The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1. The ORC1 or ORC2-depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.
Literature context: ickinson, RRID:AB_400326; 4Â°C, over
Valproic acid (VPA), a widely used antiepileptic drug, is an inhibitor of histone deacetylases, which epigenetically modify cell proliferation/differentiation in developing tissues. A series of recent clinical studies in humans reported that VPA exposure in utero impaired histogenesis and the development of the central nervous system, leading to increased risks of congenital malformation and the impairment of higher brain functions in children. In the present study conducted in mice, we report that VPA exposure in utero (1) increases the amount of acetylated histone proteins, (2) alters the expression of G1-phase regulatory proteins, (3) inhibits the cell cycle exit of neural progenitor cells during the early stage of neocortical histogenesis, and (4) increases the production of projection neurons distributed in the superficial neocortical layers in embryonic brains. Together, our findings show that VPA exposure in utero alters proliferation/differentiation characteristics of neural progenitor cells and hence leads to the neocortical dysgenesis. SIGNIFICANCE STATEMENT: This study provides new insight into the mechanisms of how an altered in utero environment, such as drug exposure, affects the generation of neurons prenatally. The antiepileptic drug valproic acid (VPA) is a good target molecule as in utero exposure to VPA has been repeatedly reported to increase the risk of nervous system malformations and to impair higher brain functions in children. We show that VPA decreases the probability of differentiation of the neural progenitor cells (NPCs) in mice, resulting in an abnormally increased number of projection neurons in the superficial layers of the neocortex. Further, we suggest that histone deacetylase inhibition by VPA may be involved in the dysregulation of proliferation/differentiation characteristics of NPCs.
We found that the secreted protein periostin (Postn) is highly induced after partial pancreatectomy in regenerating areas containing mesenchymal stroma and tubular complexes. Importantly, after partial pancreatectomy, Postn-deficient mice exhibit impaired mesenchymal formation and reduced regeneration specifically within the pancreatic β-cell compartment. Furthermore, Postn-deficient mice demonstrate an increased sensitivity to streptozotocin. Notably, injection of Postn directly into the pancreas stimulated proliferation of vimentin-expressing cells within 24 hours, and by 3 days, a mesenchymal stroma was present containing proliferating duct-like cells expressing the progenitor markers Ngn3 and Pdx1. Intraperitoneal injection of Postn resulted in increased numbers of islets and long-term glucoregulatory benefits with no adverse effects found in other tissues. Delivery of Postn throughout the pancreas via the common bile duct resulted in increased numbers of small insulin-expressing clusters and a significant improvement in glucose tolerance. Therefore, Postn is novel molecule capable of potentiating pancreatic β-cell regeneration.
Literature context: # 347580, RRID:AB_400326, Clone B44
Proliferation of stem/progenitor cells during development provides for the generation of mature cell types in the CNS. While adult brain proliferation is highly restricted in the mammals, it is widespread in teleosts. The extent of adult neural proliferation in the weakly electric fish, Gymnotus omarorum has not yet been described. To address this, we used double thymidine analog pulse-chase labeling of proliferating cells to identify brain proliferation zones, characterize their cellular composition, and analyze the fate of newborn cells in adult G. omarorum. Short thymidine analog chase periods revealed the ubiquitous distribution of adult brain proliferation, similar to other teleosts, particularly Apteronotus leptorhynchus. Proliferating cells were abundant at the ventricular-subventricular lining of the ventricular-cisternal system, adjacent to the telencephalic subpallium, the diencephalic preoptic region and hypothalamus, and the mesencephalic tectum opticum and torus semicircularis. Extraventricular proliferation zones, located distant from the ventricular-cisternal system surface, were found in all divisions of the rombencephalic cerebellum. We also report a new adult proliferation zone at the caudal-lateral border of the electrosensory lateral line lobe. All proliferation zones showed a heterogeneous cellular composition. The use of short (24 h) and long (30 day) chase periods revealed abundant fast cycling cells (potentially intermediate amplifiers), sparse slow cycling (potentially stem) cells, cells that appear to have entered a quiescent state, and cells that might correspond to migrating newborn neural cells. Their abundance and migration distance differed among proliferation zones: greater numbers and longer range and/or pace of migrating cells were associated with subpallial and cerebellar proliferation zones.
Literature context: , 347580, RRID:AB_400326), and chic
Math1 is the defining molecule of the cerebellar rhombic lip and Pax6 is downstream in the Math1 pathway. In the present study, we discover that Wntless (Wls) is a novel molecular marker of the cells in the interior face of the rhombic lip throughout normal mouse cerebellar development. Wls expression is found complementary to the expression of Math1 and Pax6, which are localized to the exterior face of the rhombic lip. To determine the interaction between these molecules, we examine the loss-of-Math1 or loss-of-Pax6 in the cerebellum, i.e., the Math1-null and Pax6-null (Sey) mutant cerebella. The presence of Wls-positive cells in the Math1-null rhombic lip indicates that Wls expression is independent of Math1. In the Sey mutant cerebellum, there is an expansion of Wls-expressing cells into regions that are normally colonized by Pax6-expressing cells. The ectopic expression of Wls in the Pax6-null cerebellum suggests a negative interaction between Wls-expressing cells and Pax6-positive cells. These findings suggest that the rhombic lip is dynamically patterned by the expression of Wls, Math1, and Pax6. We also examine five rhombic lip cell markers (Wls, Math1, Pax6, Lmx1a, and Tbr2) to identify four molecularly distinct compartments in the rhombic lip during cerebellar development. The existence of spatial compartmentation in the rhombic lip and the interplay between Wls, Math1, and Pax6 in the rhombic lip provides novel views of early cerebellar development.
Paternal care is rare among mammals, occurring in ≈6% of species. California mice (Peromyscus californicus) are unusual; fathers participate extensively in raising their young and display the same components of parental care as mothers, with the exception of nursing. Parenting is a complex experience, having stressful and enriching aspects. The hippocampus is sensitive to experience and responds to both stress and environmental enrichment with changes in structure and function. In rats, where females care exclusively for offspring, parenting is associated with suppressed hippocampal adult neurogenesis. Since this effect has been causally linked to lactation, it is unlikely that fathers would show a similar change. To investigate this issue, we examined adult neurogenesis in the hippocampus of California mouse fathers compared to males without pups and observed reduced adult neurogenesis. Similar effects were found in California mouse mothers. Next, we investigated whether behaviors linked to the hippocampus, namely, object recognition and novelty-suppressed feeding, were altered in fathers, and observed no substantial changes. During caregiving, suppressed adult neurogenesis does not appear to be related to changes in behaviors associated with the hippocampus, although it is possible that there are other effects on hippocampal function.
The anatomy of the mammalian thalamus is characterized by nuclei, which can be readily identified in postnatal animals. However, the molecular mechanisms that guide specification and differentiation of neurons in specific thalamic nuclei are still largely unknown, and few molecular markers are available for most of these thalamic subregions at early stages of development. We therefore searched for patterned gene expression restricted to specific mouse thalamic regions by in situ hybridization during the onset of thalamic neurogenesis (embryonic [E] days E10.5-E12.5). To obtain correct regional information, we used Shh as a landmark and compared spatial relationships with the zona limitans intrathalamica (Zli), the border of the p2 and p3 compartments of the diencephalon. We identified genes that are expressed specifically in the ventricular zone of the thalamic neuroepithelium and also identified a number of genes that already exhibited regional identity at E12.5. Although many genes expressed in the mantle regions of the thalamus at E12.5 showed regionally restricted patterns, none of these clearly corresponded to individual thalamic nuclei. We next examined gene expression at E15.5, when thalamocortical axons (TCAs) project from distinct regions of the thalamus and reach their targets in the cerebral cortex. Regionally restricted patterns of gene expression were again seen for many genes, but some regionally bounded expression patterns in the early postnatal thalamus had shifted substantially by E15.5. These findings reveal that nucleogenesis in the developing thalamus is associated with selective and complex changes in gene expression and provide a list of genes that may actively regulate the development of thalamic nuclei.
The amygdala is located in the caudal part of the ventral telencephalon. It is composed of many subdivisions and is involved in the control of emotion. It is important to know the mechanisms of amygdalar development in order to analyze the pathogenesis of emotional disorders, but they are still not adequately understood. In the present study the migration, differentiation, and distribution of amygdalar neurons in the mouse embryo were investigated by means of in utero electroporation. Ventricular zone cells in restricted regions, that is, the caudal ganglionic eminence (CGE), the ventral pallium, the lateral pallium, and the diencephalon, were labeled with an expression vector of the enhanced green fluorescent protein (EGFP) gene. Labeling at embryonic day (E)10 revealed that the central nucleus originates from the neuroepithelium in the ganglionic eminence and the labeling at E11 and E12 revealed that the basolateral complex originates from the neuroepithelium of the ventral and lateral pallia. The introduction of the EGFP gene into the neuroepithelium of the third ventricle at E11 showed that the medial nucleus originates, at least in part, from the neuroepithelium of the diencephalon and migrates over the diencephalo-telencephalic boundary. The radial glial arrangement corresponded well with the initial migration of amygdalar neurons, and the radial processes later formed the boundary demarcating the basolateral complex. These findings indicate that the neurons originating from the temporally and spatially restricted neuroepithelium in both the telencephalon and diencephalon migrate and differentiate to form the mosaic of amygdalar subdivisions.
Debilitating hearing and balance deficits often arise through damage to the inner ear's hair cells. For humans and other mammals, such deficits are permanent, but nonmammalian vertebrates can quickly recover hearing and balance through their innate capacity to regenerate hair cells. The biological basis for this difference has remained unknown, but recent investigations in wounded balance epithelia have shown that proliferation follows cellular spreading at sites of injury. As mammalian ears mature during the first weeks after birth, the capacity for spreading and proliferation declines sharply. In seeking the basis for those declines, we investigated the circumferential bands of F-actin that bracket the apical junctions between supporting cells in the gravity-sensitive utricle. We found that those bands grow much thicker as mice and humans mature postnatally, whereas their counterparts in chickens remain thin from hatching through adulthood. When we cultured utricular epithelia from chickens, we found that cellular spreading and proliferation both continued at high levels, even in the epithelia from adults. In contrast, the substantial reinforcement of the circumferential F-actin bands in mammals coincides with the steep declines in cell spreading and production established in earlier experiments. We propose that the presence of thin F-actin bands at the junctions between avian supporting cells may contribute to the lifelong persistence of their capacity for shape change, cell proliferation, and hair cell replacement and that the postnatal reinforcement of the F-actin bands in maturing humans and other mammals may have an important role in limiting hair cell regeneration.
The alpha(2A)-adrenoceptor (AR) subtype, a G protein-coupled receptor located both pre- and postsynaptically, mediates adrenaline/noradrenaline functions. The present study aimed to determine the alpha(2A)-AR distribution in the adult zebrafish (Danio rerio) brain by means of immunocytochemistry. Detailed mapping showed labeling of alpha(2A)-ARs, in neuropil, neuronal somata and fibers, glial processes, and blood vessels. A high density of alpha(2A)-AR immunoreactivity was found in the ventral telencephalic area, preoptic, pretectal, hypothalamic areas, torus semicircularis, oculomotor nucleus (NIII), locus coreruleus (LC), medial raphe, medial octavolateralis nucleus (MON), magnocellular octaval nucleus (MaON), reticular formation (SRF, IMRF, IRF), rhombencephalic nerves and roots (DV, V, VII, VIII, X), and cerebellar Purkinje cell layer. Moderate levels of alpha(2A)-ARs were observed in the medial and central zone nuclei of dorsal telencephalic area, in the periventricular gray zone of optic tectum, in the dorsomedial part of optic tectum layers, and in the molecular and granular layers of all cerebellum subdivisions. Glial processes were found to express alpha(2A)-ARs in rhombencephalon, intermingled with neuronal fibers. Medium-sized neurons were labeled in telencephalic, diencephalic, and mesencephlic areas, whereas densely labeled large neurons were found in rhombencephalon, locus coeruleus, reticular formation, oculomotor area, medial octavolateralis and magnocellular octaval nuclei, and Purkinje cell somata. The functional role of alpha(2A)-ARs on neurons and glial processes is not known at present; however, their strong relation to the ventricular system, somatosensory nuclei, and nerves supports a possible regulatory role of alpha(2A)-ARs in autonomic functions, nerve output, and sensory integration in adult zebrafish brain.
It is well established that neurogenesis in the dentate gyrus slows with aging, but it is unclear whether this change is due to slowing of the cell cycle, as occurs during development, or to loss of precursor cells. In the current study, we find that the cell cycle time of granule cell precursors in middle-aged male rats is not significantly different from that in young adults. The size of the precursor pool, however, was 3-4 times smaller in the middle-aged rats, as determined using both cumulative bromodeoxyuridine (BrdU) labeling as well as labeling with the endogenous marker of cell proliferation, proliferating cell nuclear antigen (PCNA). Loss of precursor cells was much greater in the granule cell layer than in the hilus, suggesting that dividing cells in the hilus belong to a distinct population, most likely glial progenitors, that are less affected by aging than neuronal precursors. BrdU-labeled precursor cells and young neurons, labeled with doublecortin, appeared to be lost equally from rostral and caudal, as well as suprapyramidal and infrapyramidal, subregions of the granule cell layer. However, doublecortin staining did show large parts of the caudal granule cell layer with few if any young neurons at both ages. Taken together, these findings indicate that precursor cells are not distributed evenly within the dentate gyrus in adulthood but that precursors are lost from throughout the dentate gyrus in old age with no concomitant change in the cell cycle time.
Pioneering work indicates that the final position of neurons in specific layers of the mammalian cerebral cortex is determined primarily by birthdate. Glutamatergic projection neurons are born in the cortical proliferative zones of the dorsal telencephalon, and follow an "inside-out" neurogenesis gradient: later-born cohorts migrate radially past earlier-born neurons to populate more superficial layers. GABAergic interneurons, the major source of cortical inhibition, comprise a heterogeneous population and are produced in proliferative zones of the ventral telencephalon. Mechanisms by which interneuron subclasses find appropriate layer-specific cortical addresses remain largely unexplored. Major cortical interneuron subclasses can be identified based on expression of distinct calcium-binding proteins including parvalbumin, calretinin, or calbindin. We determined whether cortical layer-patterning of interneurons is dependent on phenotype. Parvalbumin-positive interneurons populate cortical layers with an inside-out gradient, and birthdate is isochronous to projection neurons in the same layers. In contrast, another major GABAergic subtype, labeled using calretinin, populates the cerebral cortex using an opposite "outside-in" gradient, heterochronous to neighboring neurons. In addition to birthdate, phenotype is also a determinant of cortical patterning. Discovery of a cortical subpopulation that does not follow the well-established inside-out gradient has important implications for mechanisms of layer formation in the cerebral cortex.
NeuroD is a basic helix-loop-helix (bHLH) transcription factor critical for determining neuronal cell fate and regulating withdrawal from the cell cycle. We showed previously that, in goldfish, neuroD is expressed in the rod photoreceptor lineage, and we inferred that neuroD is also expressed in a subset of amacrine cells and nascent cone photoreceptors. Here we extended that study by examining the temporal and spatial expression pattern of neuroD in the embryonic and larval zebrafish and by identifying the cell types that express this gene. NeuroD expression in the developing zebrafish retina is dynamic, spanning early retinogenesis and the maturation of cone photoreceptors. In early retinogenesis neuroD expression expands from a small patch in the ventronasal retina, through the remaining retinal neuroepithelium. As retinogenesis progresses, neuroD expression becomes restricted to amacrine cells, immature cones, and cells of rod and cone lineages. This expression achieves an adult pattern by 96 hours postfertilization (hpf), whereupon the temporal pattern of neuroD expression in central retina is spatially recapitulated at the germinative margin. The cellular pattern of expression suggests that neuroD regulates aspects of rod and cone genesis, but through separate cellular lineages. Furthermore, neuroD is coexpressed with the cone-rod-homeobox transcription factor (Crx) in putative cone progenitors and nascent cone photoreceptors, suggesting that, in the zebrafish retina, as in other vertebrate retinas, similar genetic cascades regulate photoreceptor genesis and maturation.
Adult neurogenesis, the generation of new neurons from adult precursor cells, occurs in the brains of a phylogenetically diverse array of animals. In the higher (amniotic) vertebrates, these precursor cells are glial cells that reside within specialized regions, known as neurogenic niches, the elements of which both support and regulate neurogenesis. The in vivo identity and location of the precursor cells responsible for adult neurogenesis in nonvertebrate taxa, however, remain largely unknown. Among the invertebrates, adult neurogenesis has been particularly well characterized in freshwater crayfish (Arthropoda, Crustacea), although the identity of the precursor cells sustaining continuous neuronal proliferation in these animals has yet to be established. Here we provide evidence suggesting that, as in the higher vertebrates, the precursor cells maintaining adult neurogenesis in the crayfish Procambarus clarkii are glial cells. These precursor cells reside within a specialized region, or niche, on the ventral surface of the brain, and their progeny migrate from this niche along glial fibers and then proliferate to form new neurons in the central olfactory pathway. The niche in which these precursor cells reside has many features in common with the neurogenic niches of higher vertebrates. These commonalities include: glial cells functioning as both precursor and support cells, directed migration, close association with the brain vasculature, and specialized basal laminae. The cellular machinery maintaining adult neurogenesis appears, therefore, to be shared by widely disparate taxa. These extensive structural and functional parallels suggest a common strategy for the generation of new neurons in adult brains.
Hair cells of the inner ear are damaged by intense noise, aging, and aminoglycoside antibiotics. Gentamicin causes oxidative damage to hair cells, inducing apoptosis. In mammals, hair cell loss results in a permanent deficit in hearing and balance. In contrast, avians can regenerate lost hair cells to restore auditory and vestibular function. This study examined the changes of myosin VI and myosin VIIa, two unconventional myosins that are critical for normal hair cell formation and function, during hair cell death and regeneration. During the late stages of apoptosis, damaged hair cells are ejected from the sensory epithelium. There was a 4-5-fold increase in the labeling intensity of both myosins and a redistribution of myosin VI into the stereocilia bundle, concurrent with ejection. Two separate mechanisms were observed during hair cell regeneration. Proliferating supporting cells began DNA synthesis 60 hours after gentamicin treatment and peaked at 72 hours postgentamicin treatment. Some of these mitotically produced cells began to differentiate into hair cells at 108 hours after gentamicin (36 hours after bromodeoxyuridine (BrdU) administration), as demonstrated by the colabeling of myosin VI and BrdU. Myosin VIIa was not expressed in the new hair cells until 120 hours after gentamicin. Moreover, a population of supporting cells expressed myosin VI at 78 hours after gentamicin treatment and myosin VIIa at 90 hours. These cells did not label for BrdU and differentiated far too early to be of mitotic origin, suggesting they arose by direct transdifferentiation of supporting cells into hair cells.
Radix Angelicae Sinensis (RAS) decoction can markedly inhibit acute and chronic inflammation caused by various phlogistic agents. Similar effects are equally seen in adrenalectomized rats. RAS can also suppress the biosynthesis or release of prostaglandin E2 in inflamed tissues induced by carrageenan, as well as significantly decrease the hemolytic activity of complement bypass, but shows no effect on the inflammation caused by histamine.