Kevin A. Burns

Hypoxia-ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain

Recent studies suggest that postmitotic neurons can reenter the cell cycle as a prelude to apoptosis after brain injury. However, most dying neurons do not pass the G1/S-phase checkpoint to resume DNA synthesis. The specific factors that trigger abortive DNA synthesis are not characterized. Here we show that the combination of hypoxia and ischemia induces adult rodent neurons to resume DNA synthesis as indicated by incorporation of bromodeoxyuridine (BrdU) and expression of G1/S-phase cell cycle transition markers. After hypoxia-ischemia, the majority of BrdU- and neuronal nuclei (NeuN)-immunoreactive cells are also terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-stained, suggesting that they undergo apoptosis. BrdU+ neurons, labeled shortly after hypoxia-ischemia, persist for >5 d but eventually disappear by 28 d. Before disappearing, these BrdU+/NeuN+/TUNEL+ neurons express the proliferating cell marker Ki67, lose the G1-phase cyclin-dependent kinase (CDK) inhibitors p16INK4 and p27Kip1 and show induction of the late G1/S-phase CDK2 activity and phosphorylation of the retinoblastoma protein. This contrasts to kainic acid excitotoxicity and traumatic brain injury, which produce TUNEL-positive neurons without evidence of DNA synthesis or G1/S-phase cell cycle transition. These findings suggest that hypoxia-ischemia triggers neurons to reenter the cell cycle and resume apoptosis-associated DNA synthesis in brain. Our data also suggest that the demonstration of neurogenesis after brain injury requires not only BrdU uptake and mature neuronal markers but also evidence showing absence of apoptotic markers. Manipulating the aberrant apoptosis-associated DNA synthesis that occurs with hypoxia-ischemia and perhaps neurodegenerative diseases could promote neuronal survival and neurogenesis.

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ‘fossil’ transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17–18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes

Xenbase (http://www.xenbase.org), the Xenopus frog model organism database, integrates a wide variety of data from this biomedical model genus. Two closely related species are represented: the allotetraploid Xenopus laevis that is widely used for microinjection and tissue explant-based protocols, and the diploid Xenopus tropicalis which is used for genetics and gene targeting. The two species are extremely similar and protocols, reagents and results from each species are often interchangeable. Xenbase imports, indexes, curates and manages data from both species; all of which are mapped via unique IDs and can be queried in either a species-specific or species agnostic manner. All our services have now migrated to a private cloud to achieve better performance and reliability. We have added new content, including providing full support for morpholino reagents, used to inhibit mRNA translation or splicing and binding to regulatory microRNAs. New genomes assembled by the JGI for both species and are displayed in Gbrowse and are also available for searches using BLAST. Researchers can easily navigate from genome content to gene page reports, literature, experimental reagents and many other features using hyperlinks. Xenbase has also greatly expanded image content for figures published in papers describing Xenopus research via PubMedCentral.

Rac1 Controls the Formation of Midline Commissures and the Competency of Tangential Migration in Ventral Telencephalic Neurons

Previous studies using dominant-mutant constructs have implicated Rac1 GTPase in neuritogenesis and neuronal migration. However, overexpression of dominant mutants generally blocks Rho–GTPase activity; thus, it may not reveal the specific or physiological functions of Rac1. To address this issue, we have applied a conditional gene-targeting strategy, using Foxg1–Cre mice to delete Rac1 in the ventricular zone (VZ) of telencephalon and Dlx5/6–Cre–IRES (internal ribosomal entry site)–EGFP (enhanced green fluorescent protein) (Dlx5/6–CIE) in the subventricular zone (SVZ) of ventral telencephalon, respectively. Surprisingly, the deletion of Rac1 in VZ progenitors did not prevent axonal outgrowth of telencephalic neurons. However, the anterior commissure was absent, and the corpus callosal as well as hippocampal commissural axons failed to cross the midline in Rac1/Foxg1–Cre knock-out embryos. The thalamocortical and corticothalamic axons also showed defasciculation or projection defects. These results suggest that Rac1 controls axon guidance rather than neuritogenesis. In addition, although Rac1/Foxg1–Cre knock-out embryos showed delayed radial migration of cortical projection neurons and severe impairment of tangential migration by the ventral telencephalon-derived interneurons, deletion of Rac1 in the SVZ by Dlx5/6–CIE mice produced no discernible defects in tangential migration. These contrasting effects of Rac1 deletion on tangential migration suggest that Rac1 is dispensable for cellular motility per se during neuronal migration. Together, these results underscore the challenge of deciphering the biological functions of Rac1, and Rho–GTPases in general, during mammalian brain development. Moreover, they indicate that Rac1 has a critical role in axon guidance and in acquisition of migratory competency during differentiation of the progenitors for the ventral telencephalon-derived interneurons.

Coordinated control of self-renewal and differentiation of neural stem cells by Myc and the p19ARF–p53 pathway

The modes of proliferation and differentiation of neural stem cells (NSCs) are coordinately controlled during development, but the underlying mechanisms remain largely unknown. In this study, we show that the protooncoprotein Myc and the tumor suppressor p19ARF regulate both NSC self-renewal and their neuronal and glial fate in a developmental stage–dependent manner. Early-stage NSCs have low p19ARF expression and retain a high self-renewal and neurogenic capacity, whereas late-stage NSCs with higher p19ARF expression possess a lower self-renewal capacity and predominantly generate glia. Overexpression of Myc or inactivation of p19ARF reverts the properties of late-stage NSCs to those of early-stage cells. Conversely, inactivation of Myc or forced p19ARF expression attenuates self-renewal and induces precocious gliogenesis through modulation of the responsiveness to gliogenic signals. These actions of p19ARF in NSCs are mainly mediated by p53. We propose that opposing actions of Myc and the p19ARF–p53 pathway have important functions in coordinated developmental control of self-renewal and cell fate choices in NSCs.

Xenbase: expansion and updates of the Xenopus model organism database

Xenbase (http://www.xenbase.org) is a model organism database that provides genomic, molecular, cellular and developmental biology content to biomedical researchers working with the frog, Xenopus and Xenopus data to workers using other model organisms. As an amphibian Xenopus serves as a useful evolutionary bridge between invertebrates and more complex vertebrates such as birds and mammals. Xenbase content is collated from a variety of external sources using automated and semi-automated pipelines then processed via a combination of automated and manual annotation. A link-matching system allows for the wide variety of synonyms used to describe biological data on unique features, such as a gene or an anatomical entity, to be used by the database in an equivalent manner. Recent updates to the database include the Xenopus laevis genome, a new Xenopus tropicalis genome build, epigenomic data, collections of RNA and protein sequences associated with genes, more powerful gene expression searches, a community and curated wiki, an extensive set of manually annotated gene expression patterns and a new database module that contains data on over 700 antibodies that are useful for exploring Xenopus cell and developmental biology.

Developmental and post‐injury cortical gliogenesis: A Genetic fate‐mapping study with Nestin‐CreER mice

The primary sources of cortical gliogenesis, either during development or after adult brain injury, remain uncertain. We previously generated Nestin‐CreER mice to fate‐map the progeny of radial glial cells (RG), a source of astrocytes and oligodendrocytes in the nervous system. Here, we show that Nestin‐CreER mice label another population of glial progenitors, namely the perinatal subventricular zone (SVZ) glioblasts, if they are crossed with stop‐floxed EGFP mice and receive tamoxifen in late embryogenesis (E16–E18). Quantification showed E18 tamoxifen‐induction labeled more perinatal SVZ glioblasts than RG and transitional RG combined in the newborn brain (54% vs. 22%). Time‐lapse microscopy showed SVZ‐glioblasts underwent complex metamorphosis and often‐reciprocal transformation into transitional RG. Surprisingly, the E10‐dosed RG progenitors produced astrocytes, but no oligodendrocytes, whereas E18‐induction fate‐mapped both astrocytes and NG2+ oligodendrocyte precursors in the postnatal brain. These results suggest that cortical oligodendrocytes mostly derive from perinatal SVZ glioblast progenitors. Further, by combining genetic fate‐mapping and BrdU‐labeling, we showed that cortical astrocytes cease proliferation soon after birth (

Xenbase: Core features, data acquisition, and data processing

Xenbase, the Xenopus model organism database (www.xenbase.org), is a cloud‐based, web‐accessible resource that integrates the diverse genomic and biological data from Xenopus research. Xenopus frogs are one of the major vertebrate animal models used for biomedical research, and Xenbase is the central repository for the enormous amount of data generated using this model tetrapod. The goal of Xenbase is to accelerate discovery by enabling investigators to make novel connections between molecular pathways in Xenopus and human disease. Our relational database and user‐friendly interface make these data easy to query and allows investigators to quickly interrogate and link different data types in ways that would otherwise be difficult, time consuming, or impossible. Xenbase also enhances the value of these data through high‐quality gene expression curation and data integration, by providing bioinformatics tools optimized for Xenopus experiments, and by linking Xenopus data to other model organisms and to human data. Xenbase draws in data via pipelines that download data, parse the content, and save them into appropriate files and database tables. Furthermore, Xenbase makes these data accessible to the broader biomedical community by continually providing annotated data updates to organizations such as NCBI, UniProtKB, and Ensembl. Here, we describe our bioinformatics, genome‐browsing tools, data acquisition and sharing, our community submitted and literature curation pipelines, text‐mining support, gene page features, and the curation of gene nomenclature and gene models. genesis 53:486–497, 2015.

Deleterious Effects of Plasminogen Activators in Neonatal Cerebral Hypoxia-Ischemia

The immature brains of newborns often respond differently from the brains of adults when exposed to similar insults. Previous studies have indicated that although hypoxia-ischemia (HI) induces persistent thrombosis in adult brains, it only modestly impairs blood perfusion in newborn brains. Here, we used the Vannucci model of HI encephalopathy to study age-related responses to cerebral HI in rat pups. We found that HI triggered fibrin deposition and impaired blood perfusion in both neonatal and adult brains. However, these effects were only transient in neonatal brains (<4 hours) and were accompanied by acute induction of both tissue-type and urinary-type plasminogen activators (tPA and uPA), which was not observed in adult brains subjected to the same insult. Interestingly, activation of the plasminogen system persisted up to 24 hours in neonatal brains, long after the clearance of fibrin-rich thrombi. Furthermore, astrocytes and macrophages outside blood vessels expressed tPA after HI, suggesting the possibility of tPA/plasmin-mediated cytotoxicity. Consistent with this hypothesis, injection of alpha2-antiplasmin into cerebral ventricles markedly ameliorated HI-induced damage to neurofilaments and white matter oligodendrocytes, providing a dose-response reduction of brain injury after 7 days of recovery. Conversely, ventricular injection of tPA increased HI-induced brain damage. Together, these results suggest that tPA/plasmin induction, which may contribute to acute fibrinolysis, is a critical component of extravascular proteolytic damage in immature brains, representing a new therapeutic target for the treatment of HI encephalopathy.

Enhanced XAO: the ontology of Xenopus anatomy and development underpins more accurate annotation of gene expression and queries on Xenbase

BackgroundThe African clawed frogs Xenopus laevis and Xenopus tropicalis are prominent animal model organisms. Xenopus research contributes to the understanding of genetic, developmental and molecular mechanisms underlying human disease. The Xenopus Anatomy Ontology (XAO) reflects the anatomy and embryological development of Xenopus. The XAO provides consistent terminology that can be applied to anatomical feature descriptions along with a set of relationships that indicate how each anatomical entity is related to others in the embryo, tadpole, or adult frog. The XAO is integral to the functionality of Xenbase (http://www.xenbase.org), the Xenopus model organism database.ResultsWe significantly expanded the XAO in the last five years by adding 612 anatomical terms, 2934 relationships between them, 640 synonyms, and 547 ontology cross-references. Each term now has a definition, so database users and curators can be certain they are selecting the correct term when specifying an anatomical entity. With developmental timing information now asserted for every anatomical term, the ontology provides internal checks that ensure high-quality gene expression and phenotype data annotation. The XAO, now with 1313 defined anatomical and developmental stage terms, has been integrated with Xenbase expression and anatomy term searches and it enables links between various data types including images, clones, and publications. Improvements to the XAO structure and anatomical definitions have also enhanced cross-references to anatomy ontologies of other model organisms and humans, providing a bridge between Xenopus data and other vertebrates. The ontology is free and open to all users.ConclusionsThe expanded and improved XAO allows enhanced capture of Xenopus research data and aids mechanisms for performing complex retrieval and analysis of gene expression, phenotypes, and antibodies through text-matching and manual curation. Its comprehensive references to ontologies across taxa help integrate these data for human disease modeling.