FGF dependent regulation of Zfhx1b gene expression promotes the formation of definitive neural stem cells in the mouse anterior neurectoderm

<p>Abstract</p> <p>Background</p> <p>Mouse definitive neural stem cells (NSCs) are derived from a population of LIF-responsive primitive neural stem cells (pNSCs) within the neurectoderm, yet details on the early signaling and transcriptional mechanisms that control this lineage transition are lacking. Here we tested whether FGF and Wnt signaling pathways can regulate <it>Zfhx1b </it>expression to control early neural stem cell development.</p> <p>Results</p> <p>By microinjecting FGF8b into the pro-amniotic cavity <it>ex vivo </it>at 7.0 days post-coitum (dpc) and culturing whole embryos, we demonstrate that neurectoderm-specific gene expression (for example, <it>Sox2</it>, <it>Nestin</it>, <it>Zfhx1b</it>) is increased, whereas Wnt3a represses neurectoderm gene expression. To determine whether FGF signaling also mediates the lineage transition from a pNSC to a NSC, 7.0-dpc embryos were microinjected with either FGF8b or inhibitors of the FGF receptor-MAP kinase signaling pathway <it>ex vivo</it>, cultured as whole embryos to approximately 8.5 dpc and assayed for clonal NSC colony formation. We show that pre-activation of FGF signaling in the anterior neurectoderm causes an increase in the number of colony forming NSCs derived later from the anterior neural plate, whereas inhibition of FGF signaling significantly reduces the number of NSC colonies. Interestingly, inhibition of FGF signaling causes the persistence of LIF-responsive pNSCs within the anterior neural plate and over-expression of <it>Zfhx1b </it>in these cells is sufficient to rescue the transition from a LIF-responsive pNSC to an FGF-responsive NSC.</p> <p>Conclusion</p> <p>Our data suggest that definitive NSC fate specification in the mouse neurectoderm is facilitated by FGF activation of <it>Zfhx1b</it>.</p

Duplicate dmbx1 genes regulate progenitor cell cycle and differentiation during zebrafish midbrain and retinal development

Abstract Background The Dmbx1 gene is important for the development of the midbrain and hindbrain, and mouse gene targeting experiments reveal that this gene is required for mediating postnatal and adult feeding behaviours. A single Dmbx1 gene exists in terrestrial vertebrate genomes, while teleost genomes have at least two paralogs. We compared the loss of function of the zebrafish dmbx1a and dmbx1b genes in order to gain insight into the molecular mechanism by which dmbx1 regulates neurogenesis, and to begin to understand why these duplicate genes have been retained in the zebrafish genome. Results Using gene knockdown experiments we examined the function of the dmbx1 gene paralogs in zebrafish, dmbx1a and dmbx1b in regulating neurogenesis in the developing retina and midbrain. Dose-dependent loss of dmbx1a and dmbx1b function causes a significant reduction in growth of the midbrain and retina that is evident between 48-72 hpf. We show that this phenotype is not due to patterning defects or persistent cell death, but rather a deficit in progenitor cell cycle exit and differentiation. Analyses of the morphant retina or anterior hindbrain indicate that paralogous function is partially diverged since loss of dmbx1a is more severe than loss of dmbx1b. Molecular evolutionary analyses of the Dmbx1 genes suggest that while this gene family is conservative in its evolution, there was a dramatic change in selective constraint after the duplication event that gave rise to the dmbx1a and dmbx1b gene families in teleost fish, suggestive of positive selection. Interestingly, in contrast to zebrafish dmbx1a, over expression of the mouse Dmbx1 gene does not functionally compensate for the zebrafish dmbx1a knockdown phenotype, while over expression of the dmbx1b gene only partially compensates for the dmbx1a knockdown phenotype. Conclusion Our data suggest that both zebrafish dmbx1a and dmbx1b genes are retained in the fish genome due to their requirement during midbrain and retinal neurogenesis, although their function is partially diverged. At the cellular level, Dmbx1 regulates cell cycle exit and differentiation of progenitor cells. The unexpected observation of putative post-duplication positive selection of teleost Dmbx1 genes, especially dmbx1a, and the differences in functionality between the mouse and zebrafish genes suggests that the teleost Dmbx1 genes may have evolved a diverged function in the regulation of neurogenesis

Identification of a BMP inhibitor-responsive promoter module required for expression of the early neural gene zic1

AbstractExpression of the transcription factor zic1 at the onset of gastrulation is one of the earliest molecular indicators of neural fate determination in Xenopus. Inhibition of bone morphogenetic protein (BMP) signaling is critical for activation of zic1 expression and fundamental for establishing neural identity in both vertebrates and invertebrates. The mechanism by which interruption of BMP signaling activates neural-specific gene expression is not understood. Here, we report identification of a 215 bp genomic module that is both necessary and sufficient to activate Xenopus zic1 transcription upon interruption of BMP signaling. Transgenic analyses demonstrate that this BMP inhibitory response module (BIRM) is required for expression in the whole embryo. Multiple consensus binding sites for specific transcription factor families within the BIRM are required for its activity and some of these regions are phylogenetically conserved between orthologous vertebrate zic1 genes. These data suggest that interruption of BMP signaling facilitates neural determination via a complex mechanism, involving multiple regulatory factors that cooperate to control zic1 expression

Ectopic Expression of Neurogenin 2 Alone is Sufficient to Induce Differentiation of Embryonic Stem Cells into Mature Neurons

Recent studies show that combinations of defined key developmental transcription factors (TFs) can reprogram somatic cells to pluripotency or induce cell conversion of one somatic cell type to another. However, it is not clear if single genes can define a cell̀s identity and if the cell fate defining potential of TFs is also operative in pluripotent stem cells in vitro. Here, we show that ectopic expression of the neural TF Neurogenin2 (Ngn2) is sufficient to induce rapid and efficient differentiation of embryonic stem cells (ESCs) into mature glutamatergic neurons. Ngn2-induced neuronal differentiation did not require any additional external or internal factors and occurred even under pluripotency-promoting conditions. Differentiated cells displayed neuron-specific morphology, protein expression, and functional features, most importantly the generation of action potentials and contacts with hippocampal neurons. Gene expression analyses revealed that Ngn2-induced in vitro differentiation partially resembled neurogenesis in vivo, as it included specific activation of Ngn2 target genes and interaction partners. These findings demonstrate that a single gene is sufficient to determine cell fate decisions of uncommitted stem cells thus giving insights into the role of key developmental genes during lineage commitment. Furthermore, we present a promising tool to improve directed differentiation strategies for applications in both stem cell research and regenerative medicine

A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships(1,2). Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome(3), transcriptome(4) and proteome(5) data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes

Origin and diversification of neural stem cells during mammalian brain development

grantor: University of TorontoNeural stem cells are self-renewing precursor cells that have a fundamental role in generating cellular diversity in the developing mammalian brain. However, the establishment of this unique class of cells during the course of embryogenesis has previously not been characterized. In this thesis, I investigate mammalian neural stem cell ontogenesis using mouse as a model system. First, evidence is presented suggesting that neural stem cell formation is preceded by a primitive neural stem cell stage before the onset of neurogenesis. Primitive neural stem cells display distinct growth factor requirements for the production of progenitor cells and have a broad range of neural and non-neural lineage potential. The transition from primitive neural stem cell to definitive neural stem cell is correlated with an alteration in growth factor dependence and a restriction in multilineage potential. Acquisition of primitive neural stem cell identity is negatively regulated by TGFß signaling proteins, which act to inhibit neural differentiation. Second, results from experiments performed after the onset of neurogenesis reveal that the entire germinal zone is initially composed of a small population of neural stem cells that are critically dependent upon FGF as a stimulus for generating progenitor cells. As neurogenesis proceeds, the FGF-responsive neural stem cell population expands and also gives rise to a distinct EGF-responsive neural stem cell population. A heterogeneous population of FGF- and EGF-responsive neural stem cells (both with self-renewal and multilineage potential) persists in germinal zone compartments throughout the brain. Finally, experiments show that during neurogenesis, neural stem cells and their early progenitors isolated from distinct brain compartments maintain a region-specific pattern of regulatory gene expression in the absence of their in vivo environment. However, this regional specification may not be irreversible and can be altered by local inductive cues. Overall, the results of this research provide the first description of mammalian neural stem cell ontogenesis. An ontogenetic model for the origin and diversification of mammalian neural stem cells is discussed in the context of brain development and adult neural homeostasis. The thesis concludes with an attempt to consolidate what is currently known about stem cell ontogenesis and behavior in various tissues and diverse organisms in order to elucidate the role of stem cells during metazoan evolution.Ph.D

Retinal Stem Cell ‘Retirement Plans’: Growth, Regulation and Species Adaptations in the Retinal Ciliary Marginal Zone

The vertebrate retina develops from a specified group of precursor cells that adopt distinct identities and generate lineages of either the neural retina, retinal pigmented epithelium, or ciliary body. In some species, including teleost fish and amphibians, proliferative cells with stem-cell-like properties capable of continuously supplying new retinal cells post-embryonically have been characterized and extensively studied. This region, termed the ciliary or circumferential marginal zone (CMZ), possibly represents a conserved retinal stem cell niche. In this review, we highlight the research characterizing similar CMZ-like regions, or stem-like cells located at the peripheral margin, across multiple different species. We discuss the proliferative parameters, multipotency and growth mechanisms of these cells to understand how they behave in vivo and how different molecular factors and signalling networks converge at the CMZ niche to regulate their activity. The evidence suggests that the mature retina may have a conserved propensity for homeostatic growth and plasticity and that dysfunction in the regulation of CMZ activity may partially account for dystrophic eye growth diseases such as myopia and hyperopia. A better understanding of the properties of CMZ cells will enable important insight into how an endogenous generative tissue compartment can adapt to altered retinal physiology and potentially even restore vision loss caused by retinal degenerative conditions

Neural stem cell heterogeneity

International audienceThe ‘identity’ of the neural stem cell (NSC) in the adult mammalian brain has captivated the interest and imagination of scientists since the seminal findings in the 1990s revealing the existence of a unique subset of glial-like cells that fulfill the stem cell criteria of self-renewal and multipotentality. That work was followed by over a decade of unprecedented insight, where the morphological and molecular profiles of these NSCs, their regulation by the tissue microenvironment (or niche) they inhabit, and their potential functional role in mediating behavior, were revealed. These striking revelations about the nature of NSCs have led to a broader interest from the neurobiology community to address the extent to which the properties of NSCs identified in the mammalian (primarily rodent) brain have been conserved in vertebrate evolution. In the last 5 years or so, research in non-mammalian vertebrates has shown that, while some NSC properties appear to be conserved, there are intriguing disparities that have emerged in the data. These include: distinct regenerative properties that are not apparent in mammalian NSCs; distinct attributes of the stem cell niche that do not align with observations from the two most studied neurogenic niches in rodents – the forebrain subventricular zone (SVZ) and the hippocampal subgranular zone (SGZ); and even the existence of different types of adult NSCs with contrasting morphologies, molecular profiles, and perhaps functional roles in the brain. We believe that by bringing together a group of leading neurobiologists in the field to contribute to a Special Issue on the heterogeneous nature of adult vertebrate NSCs, we could enrich our contemporary perspective on the properties of adult NSCs and how adaptions in different species have diversified these properties. Another important impact would be in the area of regenerative medicine. A common desire among clinician-scientists is to harness the regenerative power of NSCs in animal models and apply this to human neural regeneration. The authors contributing to this Special Issue have, indeed, fulfilled these objectives. It is widely believed that NSCs transit through active and quiescent states throughout life, responding to the demands of tissue development or adult neurogenic plasticity. We learn from Adams and Morshead that there may be at least two subpopulations of mammalian NSCs, primitive and definitive, that are lineage related and have what appear to be distinct roles in maintaining neurogenesis in the brain and responding to damage. The notion that NSCs exist as distinct sub-populations raises the possibility that rather than different proliferative states of the same cell, it may be that different subsets of very slow or relatively fast dividing NSCs exist. Bardella, Al-Shammari, Soares and colleagues explain how the SVZ exhibits constitutive semi-activated inflammatory regulation unlike surrounding brain tissue. This has led to the notion that inflammatory responses to damage within the SVZ niche may be different relative to the rest of the brain and may also underlie the susceptibility of SVZ tumorigenesis. The identity of the tumor initiating cells in subependymomas or gliomas remains elusive, but evidence suggests that different NSCs or their progeny could serve as cancer stem cells that form tumors with different degrees of growth and invasiveness. Yoo and Blackshaw inform us that the postnatal mouse hypothalamus harbors various subpopulations of tanycytes, which exhibit radial-glial like NSC characteristics similar to the NSCs found in the SVZ, or Müller cells in the retina, and can give rise to neurons in vivo. The authors explain how these distinct tanycyte sub-populations might contribute to the process of neurogenesis that, in turn, regulates energy homeostasis, body temperature, and reproductive behavior. Becker and colleagues compare the cellular and molecular responses of the spinal cord ventricular zone (VZ) between anamniotes and mammals in order to derive general principles of regenerative neurogenesis. One example involves the ependymo-radial glial (ERG) cell in the zebrafish spinal cord. The ERG cells not only serve to promote axon growth of neurons, but also serve as a multipotent NSC population that can regenerate spinal cord tissue in response to damage. Mouse spinal cord ependymal zone cells with characteristics of NSCs have been identified and these cells increase in proliferation in response to injury, but unlike their properties in vitro, these cells are biased to generate glial cells (primarily astrocytes) in vivo. The authors offer intriguing insight into the differences between the human spinal cord and that of regenerative species like zebrafish that might explain why cells with NSC characteristics fail to mount an in vivo regenerative response. Joven and Simon reveal how homeostatic neurogenesis in the postembryonic brain varies in different species of salamander, some of which have little to no neurogenesis in older stages of life. Despite this variation, most species are capable of mounting a regenerative response in the CNS mediated by the proliferation of brain ependymoglial cells or spinal cord ependymal cells. Interestingly, the presence of reactive oxygen species at sites of injury is critical for this regenerative response, which seems to correlate with some animals adapting to variable oxygen levels in their natural habitat. It is possible that such “local” adaptation of cells with NSC properties in some vertebrate species could provide new insight into ways of manipulating NSCs in others. Like in the mammalian forebrain, there may be at least two distinct types of ependymoglial NSCs that are either relatively quiescent (GFAP+ and Notch+) or relatively active (GFAP+ and Notch-) in the salamander brain. Lindsey and colleagues highlight how, in zebrafish, heterogeneity of NSCs may correlate with varying capacities for brain growth, plasticity and regeneration. While active and quiescent radial glial like NSCs exist in fish as they do in mammals, a distinct type of NSC also exists in fish with neuroepithelial like properties. Through development, different brain regions contain biased proportions of different NSC subtypes with the potential to modulate the rates of neurogenesis in a region-specific manner in response to distinct sensory cues. Moreover, in response to injury there are differential responses of neuroepithelial like and radial glial like NSCs in distinct brain regions. Interestingly, an inflammatory niche may regulate reactive neurogenesis from quiescent radial glial like NSCs in zebrafish, reminiscent of the SVZ in mice. Some common themes have emerged from these reviews: (1) NSCs exist assubpopulations often with different molecular profiles, morphologies, proliferative characteristics and regenerative potentials; (2) in most instances, the lineage relationship of these distinct types of NSCs and the reasons for their biased distributions in the brain remain unresolved; (3) NSC niches dictate behavior of NSCs in a region-specific manner; (4) adaptations in the niche suggest that inflammation and reactive oxygen species, which are normally thought to limit regeneration, may actually promote neurogenesis in specific circumstances; (5) a common future goal is to use single cell transcriptomic approaches to resolve identities and lineages within neurogenic niches. Overall, this Special Issue identifies important areas of future research that will bring us closer to understanding the mechanistic basis of the development and maintenance of NSC heterogeneity that could be used to better manipulate specific human NSCs for therapeutic innovation. [no pdf