Iulian Ilieş

Adult stem cells in the knifefish cerebellum

Adult neurogenesis has been described in dozens of brain regions in teleost fish, with the largest number of new neurons being generated in the cerebellum. Here, we characterized the cerebellar neural stem/progenitor cells (NSPCs) in the brown ghost knifefish (Apteronotus leptorhynchus), an established model system of adult neurogenesis. The majority of the new cerebellar cells arise from neurogenic niches located medially, at the interface of the dorsal/ventral molecular layers and the granular layer. NSPCs within these niches give rise to transit‐amplifying progenitors which populate the molecular layer, where they continue to proliferate during their migration toward target areas in the granular layer. At any given time, the majority of proliferating cells are located in the molecular layer. Immunohistochemical staining revealed that the stem cell markers Sox2, Meis1/2/3, Islet1, and, to a lesser extent, Pax6, are widely expressed in all regions of the adult cerebellum. A large subpopulation of these NSPCs coexpress S100, GFAP, and/or vimentin, indicating astrocytic identity. This is further supported by the specific effect of the gliotoxin l‐methionine sulfoximine, which leads to a targeted decrease in the number of GFAP+ cells that coexpress Sox2 or the proliferation marker PCNA. Pulse‐chase analysis of the label size associated with new cells after administration of 5‐bromo‐2′‐deoxyuridine demonstrated that, on average, two additional cell divisions occur after completion of the initial mitotic cycle. Overall numbers of NSPCs in the cerebellum niches increase consistently over time, presumably in parallel with the continuous growth of the brain.

Neuroanatomical and Morphological Trait Clusters in the Ant Genus Pheidole: Evidence for Modularity and Integration in Brain Structure

A central question in brain evolution concerns how selection has structured neuromorphological variation to generate adaptive behavior. In social insects, brain structures differ between reproductive and sterile castes, and worker behavioral specializations related to morphology, age, and ecology are associated with intra- and interspecific variation in investment in functionally different brain compartments. Workers in the hyperdiverse ant genus Pheidole are morphologically and behaviorally differentiated into minor and major subcastes that exhibit distinct species-typical patterns of brain compartment size variation. We examined integration and modularity in brain organization and its developmental patterning in three ecotypical Pheidole species by analyzing intra- and interspecific morphological and neuroanatomical covariation. Our results identified two trait clusters, the first involving olfaction and social information processing and the second composed of brain regions regulating nonolfactory sensorimotor functions. Patterns of size covariation between brain compartments within subcastes were consistent with levels of behavioral differentiation between minor and major workers. Globally, brains of mature workers were more heterogeneous than brains of newly eclosed workers, suggesting diversified developmental trajectories underscore species- and subcaste-typical brain organization. Variation in brain structure associated with the striking worker polyphenism in our sample of Pheidole appears to originate from initially differentiated brain templates that further diverge through species- and subcaste-specific processes of maturation and behavioral development.

Age-related changes in stem cell dynamics, neurogenesis, apoptosis, and gliosis in the adult brain: a novel teleost fish model of negligible senescence.

Adult neurogenesis, the generation of new neurons in the adult central nervous system, is a reported feature of all examined vertebrate species. However, a dramatic decline in the rates of cell proliferation and neuronal differentiation occurs in mammals, typically starting near the onset of sexual maturation. In the present study, we examined possible age‐related changes associated with adult neurogenesis in the brain of brown ghost knifefish (Apteronotus leptorhynchus), a teleost fish distinguished by its enormous neurogenic potential. Contrary to the well‐established alterations in the mammalian brain during aging, in the brain of this teleostean species we could not find evidence for any significant age‐related decline in the absolute levels of stem/progenitor cell proliferation, neuronal and glial differentiation, or long‐term survival of newly generated cells. Moreover, there was no indication that the amount of glial fibrillary acidic protein or the number of apoptotic cells in the brain was altered significantly over the course of adult life. We hypothesize that this first demonstration of negligible cellular senescence in the vertebrate brain is related to the continued growth of this species and to the lack of reproductive senescence during adulthood. The establishment of the adult brain of this species as a novel model of negligible senescence provides new opportunities for the advancement of our understanding of the biology of aging and the fundamental mechanisms that underlie senescence in the brain.

Quantitative analysis reveals dominance of gliogenesis over neurogenesis in an adult brainstem oscillator

Neural stem/progenitor cells in the neurogenic niches of the adult brain are widely assumed to give rise predominantly to neurons, rather than glia. Here, we performed a quantitative analysis of the resident neural progenitors and their progeny in the adult pacemaker nucleus (Pn) of the weakly electric fish Apteronotus leptorhynchus. Approximately 15% of all cells in this brainstem nucleus are radial glia‐like neural stem/progenitor cells. They are distributed uniformly within the tissue and are characterized by the expression of Sox2 and Meis 1/2/3. Approximately 2–3% of them are mitotically active, as indicated by expression of proliferating cell nuclear antigen. Labeling of proliferating cells with a single pulse of BrdU, followed by chases of up to 100 days, revealed that new cells are generated uniformly throughout the nucleus and do not undergo substantial migration. New cells differentiate into S100+ astrocytes and Hu C/D+ small interneurons at a ratio of 4:1, reflecting the proportions of the total glia and neurons in this brain region. The continuous addition of new cells leads to a diffuse growth of the Pn, which doubles in volume and total cell number over the first 2 years following sexual maturation of the fish. However, the number of pacemaker and relay cells, which constitute the oscillatory neural network, remains constant throughout adult life. We hypothesize that the dominance of gliogenesis is an adaptation to the high‐frequency firing of the oscillatory neurons in this nucleus.

Proteome analysis reveals protein candidates involved in early stages of brain regeneration of teleost fish

Exploration of the molecular dynamics underlying regeneration in the central nervous system of regeneration-competent organisms has received little attention thus far. By combining a cerebellar lesion paradigm with differential proteome analysis at a post-lesion survival time of 30 min, we screened for protein candidates involved in the early stages of regeneration in the cerebellum of such an organism, the teleost fish Apteronotus leptorhynchus. Out of 769 protein spots, the intensity of 26 spots was significantly increased by a factor of at least 1.5 in the lesioned hemisphere, relative to the intact hemisphere. The intensity of 9 protein spots was significantly reduced by a factor of at least 1.5. The proteins associated with 15 of the spots were identified by peptide mass fingerprinting and/or tandem mass spectrometry, resulting in the identification of a total of 11 proteins. Proteins whose abundance was significantly increased include: erythrocyte membrane protein 4.1N, fibrinogen gamma polypeptide, fructose-biphosphate aldolase C, alpha-internexin neuronal intermediate filament protein, major histocompatibility complex class I heavy chain, 26S proteasome non-ATPase regulatory subunit 8, tubulin alpha-1C chain, and ubiquitin-specific protease 5. Proteins with significantly decreased levels of abundance include: brain glycogen phosphorylase, neuron-specific calcium-binding protein hippocalcin, and spectrin alpha 2. We hypothesize that these proteins are involved in energy metabolism, blood clotting, electron transfer in oxidative reactions, cytoskeleton degradation, apoptotic cell death, synaptic plasticity, axonal regeneration, and promotion of mitotic activity.

Large-scale identification of proteins involved in the development of a sexually dimorphic behavior

Sexually dimorphic behaviors develop under the influence of sex steroids, which induce reversible changes in the underlying neural network of the brain. However, little is known about the proteins that mediate these activational effects of sex steroids. Here, we used a proteomics approach for large-scale identification of proteins involved in the development of a sexually dimorphic behavior, the electric organ discharge of brown ghost knifefish, Apteronotus leptorhynchus. In this weakly electric fish, the discharge frequency is controlled by the medullary pacemaker nucleus and is higher in males than in females. After lowering the discharge frequency by chronic administration of β-estradiol, 2-dimensional difference gel electrophoresis revealed 62 proteins spots in tissue samples from the pacemaker nucleus that exhibited significant changes in abundance of >1.5-fold. The 20 identified protein spots indicated, among others, a potential involvement of astrocytes in the establishment of the behavioral dimorphism. Indeed, immunohistochemical analysis demonstrated higher expression of the astrocytic marker protein GFAP and increased gap-junction coupling between astrocytes in females compared with males. We hypothesize that changes in the size of the glial syncytium, glial coupling, and/or number of glia-specific potassium channels lead to alterations in the firing frequency of the pacemaker nucleus via a mechanism mediating the uptake of extracellular potassium ions from the extracellular space.

Determination of relative age using growth increments of scales as a minimally invasive method in the tropical freshwater Apteronotus leptorhynchus

This study describes a method for the determination of relative age in a tropical teleost, the brown ghost knifefish Apteronotus leptorhynchus. This method is based on identification of the maximum number of scale circuli, which is thought to be associated with the oldest scales, and thus to be the most indicative of the age of a given fish. Relative age can be inferred by relating differences in maximum circulus counts to the average rate of circulus addition, which was estimated at 34 circuli per year in adult fish through oxytetracycline marking. This method shows high inter-investigator reliability and has a limited effect on fish because of the low number of scales required in order to determine the maximum number of circuli with a sufficiently high confidence level. Analysis of the frequency distribution of the circulus counts revealed periodic patterns that are similar among fish, presumably reflecting the environmental life history of the individuals. Regression analysis and comparison of addition rates showed that scale circulus counts and otolith ring counts are equivalent approaches for age estimation, but scale analysis is superior because of its limited invasiveness and the lower demand in terms of technical skills and expensive instrumentation.

Matrix metalloproteinase-2 and -9 in the cerebellum of teleost fish: Functional implications for adult neurogenesis

Matrix metalloproteinases (MMPs) are a family of highly conserved zinc-dependent proteases involved in both development and pathogenesis. The present study examines the role of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in adult neurogenesis, using the corpus cerebelli, a subdivision of the cerebellum, of knifefish (Apteronotus leptorhynchus) as a model system. Transcripts of five isoforms of these gelatinases were identified in the central nervous system of this species. Sequence similarity analysis and homology modeling indicated that functionally and structurally critical elements were highly conserved in knifefish gelatinases. Immunohistochemical staining revealed a differential distribution of MMP-2 and MMP-9 at both the cellular and subcellular level. MMP-2 expression was found mainly in Sox2-immunopositive stem/progenitor cells, both quiescent and mitotically active; and was localized in both the cytoplasmic compartment and the nucleus. By contrast, MMP-9 immunoreactivity was absent in neurogenic niches and displayed a more homogenous distribution, with low to moderate intensity levels, in the molecular and granular layers. MMP-9 expression appeared to be restricted to the extracellular space. In situ zymography indicated that gelatinase activity matched the cellular and subcellular distributions of the two MMPs. The observed patterns of gelatinase activity and expression support the hypothesis that MMP-2 is primarily involved in regulation of the activity of stem/progenitor cells that give rise to new granule neurons, whereas MMP-9 facilitates migration of the progeny of these cells by proteolysis of extracellular matrix proteins.

Mature B cells accelerate wound healing after acute and chronic diabetic skin lesions

Chronic wounds affect 12–15% of patients with diabetes and are associated with a drastic decrease in their quality of life. Here, we demonstrate that purified mature naive B220+/CD19+/IgM+/IgD+ B cells improve healing of acute and diabetic murine wounds after a single topical application. B cell treatment significantly accelerated acute wound closure by 2–3 days in wild‐type mice and 5–6 days in obese diabetic mice. The treatment led to full closure in 43% of chronic diabetic wounds, as compared to only 5% in saline‐treated controls. Applying equivalent numbers of T cells or disrupted B cells failed to reproduce these effects, indicating that live B cells mediated pro‐healing responses. Topically applied B cell treatment was associated with significantly reduced scar size, increased collagen deposition and maturation, enhanced angiogenesis, and increased nerve growth into and under the healing wound. β‐III tubulin+ nerve endings in scars of wounds treated acutely with B cells showed increased relative expression of growth‐associated protein 43. The improved healing associated with B cell treatment was supported by significantly increased fibroblast proliferation and decreased apoptosis in the wound bed and edges, altered kinetics of neutrophil infiltration, as well as an increase in TGF‐β and a significant reduction in MMP2 expression in wound granulation tissue. Our findings indicate that the timeline and efficacy of wound healing can be experimentally manipulated through the direct application of mature, naive B cells, which effectively modify the balance of mature immune cell populations within the wound microenvironment and accelerate the healing process.

Additive neurogenesis supported by multiple stem cell populations mediates adult spinal cord development: A spatiotemporal statistical mapping analysis in a teleost model of indeterminate growth

The knifefish Apteronotus leptorhynchus exhibits indeterminate growth throughout adulthood. This phenomenon extends to the spinal cord, presumably through the continuous addition of new neurons and glial cells. However, little is known about the developmental dynamics of cells added during adult growth. The present work characterizes the structural and functional development of the adult spinal cord in this model organism through a comprehensive quantitative analysis of the spatial and temporal dynamics of new cells at various developmental stages. This analysis, based on a novel statistical mapping approach, revealed within the adult spinal cord a wide distribution of both mitotically active and quiescent Sox2‐expressing stem/progenitor cells (SPCs). While such cells are particularly concentrated within the ependymal layer near the central canal, the majority of them reside in the parenchyma, resembling the distribution of SPCs observed in the mammalian spinal cord. The active SPCs in the adult knifefish spinal cord give rise to transit amplifying progenitor cells that undergo a few additional mitotic divisions before developing into Hu C/D+ neurons and S100+ glial cells. There is no evidence of long‐distance migration of the newborn cells. The persistence of cell proliferation and differentiation, combined with low levels of apoptosis, leads to a continuous addition of cells to the existing tissue. Newly generated neurons have functional and behavioral relevance, as indicated by the integration of axons of new electromotor neurons into the electric organ of these weakly electric fish. This results in a gradual increase in the amplitude of the electric organ discharge during adult development.