Eugene Bell Center for Regenerative Biology and Tissue Engineering
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The ability of many animals to spontaneously regenerate their body parts has intrigued scientific observers for centuries. Although humans share the same basic genes and pathways, we have somehow lost these regenerative capacities, which leads to significant health costs. An understanding of tissue and organ regeneration in lower animals holds great promise for translating to medical treatments for serious human conditions, including spinal cord injury, diabetes, organ failure, and degenerative neural diseases such as Alzheimer’s.
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PreprintDevelopment of xenopus resource centers : the national xenopus resource and the european xenopus resource center( 2011-07) Pearl, Esther J. ; Grainger, Robert M. ; Guille, Matthew ; Horb, Marko E.Xenopus is an essential vertebrate model system for biomedical research that has contributed to important discoveries in many disciplines, including cell biology, molecular biology, physiology, developmental biology and neurobiology. However, unlike other model systems no central repository/stock center for Xenopus had been established until recently. Similar to mouse, zebrafish and fly communities, which have established stock centers, Xenopus researchers need to maintain and distribute rapidly growing numbers of inbred, mutant and transgenic frog strains, along with DNA and protein resources, and individual laboratories struggle to accomplish this efficiently. In the last five years two resource centers were founded to address this need: the European Xenopus Resource Center (EXRC) at the University of Portsmouth in England, and the National Xenopus Resource (NXR) at the Marine Biological Laboratory (MBL) in Woods Hole, MA, USA. These two centers work together to provide resources and support to the Xenopus research community. The EXRC and NXR serve as stock centers and acquire, produce, maintain and distribute mutant, inbred and transgenic X. laevis and X. tropicalis lines. Independently, the EXRC is a repository for Xenopus cDNAs, fosmids and antibodies; it also provides oocytes and wild type frogs within the UK. The NXR will complement these services by providing research training and promoting intellectual interchange through hosting minicourses and workshops and offering space for researchers to perform short-term projects at the MBL. Together the EXRC and NXR will enable researchers to improve productivity by providing resources and expertise to all levels, from graduate students to experienced PIs. These two centers will also enable investigators that use other animal systems to take advantage of Xenopus’ unique experimental features to complement their studies.
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ArticleExtracellular electrical fields direct wound healing and regeneration(Marine Biological Laboratory, 2011-08) Messerli, Mark A. ; Graham, David M.Endogenous DC electric fields (EFs) are important, fundamental components of development, regeneration, and wound healing. The fields are the result of polarized ion transport and current flow through electrically conductive pathways. Nullification of endogenous EFs with pharmacological agents or applied EFs of opposite polarity disturbs the aforementioned processes, while enhancement increases the rate of wound closure and the extent of regeneration. EFs are applied to humans in the clinic, to provide an overwhelming signal for the enhancement of healing of chronic wounds. Although clinical trials, spanning a course of decades, have shown that applied EFs enhance healing of chronic wounds, the mechanisms by which cells sense and respond to these weak cues remains unknown. EFs are thought to influence many different processes in vivo. However, under more rigorously controlled conditions in vitro, applied EFs induce cellular polarity and direct migration and outgrowth. Here we review the generation of endogenous EFs, the results of their alteration, and the mechanisms by which cells may sense these weak fields. Understanding the mechanisms by which native and applied EFs direct development and repair will enable current and future therapeutic applications to be optimized.
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ArticleRegeneration in the era of functional genomics and gene network analysis(Marine Biological Laboratory, 2011-08) Smith, Joel ; Morgan, Jennifer R. ; Zottoli, Steven J. ; Smith, Peter J. S. ; Buxbaum, Joseph D. ; Bloom, Ona E.What gives an organism the ability to regrow tissues and to recover function where another organism fails is the central problem of regenerative biology. The challenge is to describe the mechanisms of regeneration at the molecular level, delivering detailed insights into the many components that are cross-regulated. In other words, a broad, yet deep dissection of the system-wide network of molecular interactions is needed. Functional genomics has been used to elucidate gene regulatory networks (GRNs) in developing tissues, which, like regeneration, are complex systems. Therefore, we reason that the GRN approach, aided by next generation technologies, can also be applied to study the molecular mechanisms underlying the complex functions of regeneration. We ask what characteristics a model system must have to support a GRN analysis. Our discussion focuses on regeneration in the central nervous system, where loss of function has particularly devastating consequences for an organism. We examine a cohort of cells conserved across all vertebrates, the reticulospinal (RS) neurons, which lend themselves well to experimental manipulations. In the lamprey, a jawless vertebrate, there are giant RS neurons whose large size and ability to regenerate make them particularly suited for a GRN analysis. Adding to their value, a distinct subset of lamprey RS neurons reproducibly fail to regenerate, presenting an opportunity for side-by-side comparison of gene networks that promote or inhibit regeneration. Thus, determining the GRN for regeneration in RS neurons will provide a mechanistic understanding of the fundamental cues that lead to success or failure to regenerate.
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ArticleModels and mechanisms of regenerative biology across phylogeny : introduction to a virtual symposium in The Biological Bulletin(Marine Biological Laboratory, 2011-08-01) Smith, Joel ; Olds, James L.This virtual symposium issue of The Biological Bulletin celebrates a major milestone for our publisher, The Marine Biological Laboratory, as it opens the new Eugene Bell Center for Regenerative Biology and Tissue Engineering on its Woods Hole campus. As with recent virtual symposia published by the journal, the current issue brings together a set of invited reviews, original research reports, and a position paper that offers a coherent and current window into some of the major contemporary trends in animal regeneration research.
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PreprintXenopus staufen2 is required for anterior endodermal organ formation( 2011-11) Bilogan, Cassandra K. ; Horb, Marko E.Defining the regulatory molecular networks involved in patterning the developing anterior endoderm is essential to understanding how the pancreas, liver, stomach and duodenum are discretely specified from each other. In this study, we analyzed the expression and function of the double-stranded RNA-binding protein Staufen2 in Xenopus laevis endoderm. We found that staufen2 was broadly expressed within the developing endoderm beginning at gastrulation becoming localized to the anterior endoderm at later stages. Through morpholino-mediated knockdown, we demonstrate that Staufen2 function is required for proper formation of the stomach, liver and pancreas. We define that its function is required during gastrulation for proper patterning of the dorsal-ventral axis and that it acts to regulate expression of BMP signaling components.
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PreprintTransient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells( 2011-11) Oropeza, Daniel ; Horb, Marko E.Promoting ectopic development of pancreatic beta cells from other cell types is one of the strategies being pursued for the treatment of diabetes. To achieve this, a detailed outline of the molecular lineage that operates in pancreatic progenitor cells to generate beta cells over other endocrine cell types is necessary. Here, we demonstrate that early transient expression of the endocrine progenitor bHLH protein Neurogenin 3 (Ngn3) favors the promotion of pancreatic beta and delta cell fates over an alpha cell fate, while later transient expression promotes ectopic development of all three endocrine cell fates. We found that short-term activation of Ngn3 in Xenopus laevis endoderm just after gastrulation was sufficient to promote both early and ectopic development of beta and delta cells. By examining gene expression changes four hours after Ngn3 activation we identified several new downstream targets of Ngn3. We show that several of these are required for the promotion of ectopic beta cells by Ngn3 as well as for normal beta cell development. These results provide new detail regarding the Ngn3 transcriptional network operating in endocrine progenitor cells to specify a beta cell phenotype and should help define new approaches to promote ectopic development of beta cells for diabetes therapy.
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ArticleSpatial manipulation of cells and organelles using single electrode dielectrophoresis(Informa Healthcare USA, 2012-01) Graham, David M. ; Messerli, Mark A. ; Pethig, RonaldThe selection, isolation, and accurate positioning of single cells in three dimensions are increasingly desirable in many areas of cell biology and tissue engineering. We describe the application of a simple and low cost dielectrophoretic device for picking out and relocating single target cells. The device consists of a single metal electrode and an AC signal generator. It does not require microfabrication technologies or sophisticated electronics. The dielectrophoretic manipulator also discriminates between live and dead cells and is capable of redistributing intracellular organelles.
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PreprintMicroarray analysis of Xenopus endoderm expressing Ptf1a( 2012-07) Bilogan, Cassandra K. ; Horb, Marko E.Pancreas specific transcription factor 1a (Ptf1a), a bHLH transcription factor, has two temporally distinct functions during pancreas development; initially it is required for early specification of the entire pancreas, while later it is required for proper differentiation and maintenance of only acinar cells. The importance of Ptf1a function was revealed by the fact that loss of Ptf1a leads to pancreas agenesis in humans. While Ptf1a is one of the most important pancreatic transcription factors, little is known about the differences between the regulatory networks it controls during initial specification of the pancreas as opposed to acinar cell development, and to date no comprehensive analysis of its downstream targets has been published. In this paper, we use Xenopus embryos to identify putative downstream targets of Ptf1a. We isolated anterior endoderm tissue overexpressing Ptf1a at two early stages, NF32 and NF36, and compared their gene expression profiles using microarrays. Our results revealed that Ptf1a regulates genes with a wide variety of functions, providing insight into the complexity of the regulatory network required for pancreas specification.
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DatasetNematostella Embryonic Transcriptome( 2012-12-12) Tulin, Sarah ; Aguiar, Derek ; Istrail, Sorin ; Smith, JoelRNA-Seq was performed on Nematostella Embryos at 5 timepoints during early development: 0hrs, 6hrs, 12hrs, 18hrs, 24hrs after fertilization. Embryos were harvested, lysed and mRNAs were selected using Dynabeads. Directional sequencing libraries were constructed using the ScriptSeq kit from Epicentre. A control RNA set from the National Institute of Standards and Technology (NIST) were spiked-into the library preparation. Libraries were sequenced using the Illumina HiSeq 1000, version 3 chemistry, 100bp paired-end reads and produced over 200million reads. The reads were quality controlled filtered and assembled using the Trinity Assembler. This database contains the raw read files and the assembled Transcriptome.
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ArticleCharacterization of differential transcript abundance through time during Nematostella vectensis development(BioMed Central, 2013-04-19) Helm, Rebecca R. ; Siebert, Stefan ; Tulin, Sarah ; Smith, Joel ; Dunn, Casey W.Nematostella vectensis, a burrowing sea anemone, has become a popular species for the study of cnidarian development. In previous studies, the expression of a variety of genes has been characterized during N. vectensis development with in situ mRNA hybridization. This has provided detailed spatial resolution and a qualitative perspective on changes in expression. However, little is known about broad transcriptome-level patterns of gene expression through time. Here we examine the expression of N. vectensis genes through the course of development with quantitative RNA-seq. We provide an overview of changes in the transcriptome through development, and examine the maternal to zygotic transition, which has been difficult to investigate with other tools. We measured transcript abundance in N. vectensis with RNA-seq at six time points in development: zygote (2 hours post fertilization (HPF)), early blastula (7 HPF), mid-blastula (12 HPF), gastrula (24 HPF), planula (5 days post fertilization (DPF)) and young polyp (10 DPF). The major wave of zygotic expression appears between 7–12 HPF, though some changes occur between 2–7 HPF. The most dynamic changes in transcript abundance occur between the late blastula and early gastrula stages. More transcripts are upregulated between the gastrula and planula than downregulated, and a comparatively lower number of transcripts significantly change between planula and polyp. Within the maternal to zygotic transition, we identified a subset of maternal factors that decrease early in development, and likely play a role in suppressing zygotic gene expression. Among the first genes to be expressed zygotically are genes whose proteins may be involved in the degradation of maternal RNA. The approach presented here is highly complementary to prior studies on spatial patterns of gene expression, as it provides a quantitative perspective on a broad set of genes through time but lacks spatial resolution. In addition to addressing the problems identified above, our work provides an annotated matrix that other investigators can use to examine genes and developmental events that we do not examine in detail here.
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ArticleA role for an Hsp70 nucleotide exchange factor in the regulation of synaptic vesicle endocytosis(Society for Neuroscience, 2013-05-01) Morgan, Jennifer R. ; Jiang, Jianwen ; Oliphint, Paul A. ; Jin, Suping ; Gimenez, Luis E. ; Busch, David J. ; Foldes, Andrea E. ; Zhuo, Yue ; Sousa, Rui ; Lafer, Eileen M.Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. After the internalization of a clathrin-coated vesicle, the vesicle must uncoat to replenish the pool of SVs. Clathrin-coated vesicle uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo and whether they play such roles in SV endocytosis in particular is unknown. To address this question, we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:co-chaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so that it could no longer bind and inhibit Hsp110 (a NEF that we find to be highly abundant in brain cytosol), its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis.
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ArticleA quantitative reference transcriptome for Nematostella vectensis early embryonic development : a pipeline for de novo assembly in emerging model systems(BioMed Central, 2013-06-03) Tulin, Sarah ; Aguiar, Derek ; Istrail, Sorin ; Smith, JoelThe de novo assembly of transcriptomes from short shotgun sequences raises challenges due to random and non-random sequencing biases and inherent transcript complexity. We sought to define a pipeline for de novo transcriptome assembly to aid researchers working with emerging model systems where well annotated genome assemblies are not available as a reference. To detail this experimental and computational method, we used early embryos of the sea anemone, Nematostella vectensis, an emerging model system for studies of animal body plan evolution. We performed RNA-seq on embryos up to 24 h of development using Illumina HiSeq technology and evaluated independent de novo assembly methods. The resulting reads were assembled using either the Trinity assembler on all quality controlled reads or both the Velvet and Oases assemblers on reads passing a stringent digital normalization filter. A control set of mRNA standards from the National Institute of Standards and Technology (NIST) was included in our experimental pipeline to invest our transcriptome with quantitative information on absolute transcript levels and to provide additional quality control. We generated >200 million paired-end reads from directional cDNA libraries representing well over 20 Gb of sequence. The Trinity assembler pipeline, including preliminary quality control steps, resulted in more than 86% of reads aligning with the reference transcriptome thus generated. Nevertheless, digital normalization combined with assembly by Velvet and Oases required far less computing power and decreased processing time while still mapping 82% of reads. We have made the raw sequencing reads and assembled transcriptome publically available. Nematostella vectensis was chosen for its strategic position in the tree of life for studies into the origins of the animal body plan, however, the challenge of reference-free transcriptome assembly is relevant to all systems for which well annotated gene models and independently verified genome assembly may not be available. To navigate this new territory, we have constructed a pipeline for library preparation and computational analysis for de novo transcriptome assembly. The gene models defined by this reference transcriptome define the set of genes transcribed in early Nematostella development and will provide a valuable dataset for further gene regulatory network investigations.
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DatasetNematostella High-density RNAseq time-course( 2013-06-14) Fischer, Antje H. L. ; Smith, JoelRNA-Seq was performed on Nematostella embryos at 20 timepoints: unfertilized eggs, 1hour post fertilization (hpf), 2hpf, 3hpf, 4hpf, 5hpf, 6hpf, 7hpf, 8hpf, 9hpf, 10hpf, 11hpf, 12hpf, 13hpf, 14hpf, 15hpf, 16hpf, 17hpf, 18hpf, 19hpf. Two samples (A and B) were collected for each time point. All embryos were fertilized at the same time and 300 embryos were collected for each sample. After harvesting the embroys, they were lysed and the mRNAs was extracted using Dynabeads (Invitrogen). Directional sequencing libraries were constructed using the ScriptSeq V2 kit (Epicentre). The libraries were size selected with PippinPrep to 300bp insert size. Two different control RNA sets from the National Institute of Standards and Technology (NIST) (ERCC Spike-in, Ambion) were added into the m-RNA prior to the library preparation. Libraries were sequenced using the Illumina HiSeq 1000, version 3 chemistry, 100bp paired-end reads and produced over 1.4 billion reads. The reads were quality controlled filtered and mapped against the Nematostella embryonic transcriptome (Tulin et al.). This database contains the raw read files for each library.
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PreprintEmploying BAC-reporter constructs in the sea anemone Nematostella vectensis( 2013-07-05) Fischer, Antje H. L. ; Tulin, Sarah ; Fredman, David ; Smith, JoelChanges in the expression and function of genes drive evolutionary change. Comparing how genes are regulated in different species is therefore becoming an important part of evo-devo studies. A key tool for investigating the regulation of genes is represented by Bacterial Artificial Chromosomes-reporter constructs (BAC). BACs are large insert libraries, often >>100 kb, which thus capture the genomic sequences surrounding a gene of interest, including all, or nearly all, of the elements underpinning regulation. Recombinant BACs, containing a reporter gene in place of the endogenous coding sequence of genes, can be utilized to drive the expression of reporter genes under the regulatory control of the gene of interest while still embedded within its genomic context. Systematic deletions within the BAC reporter construct can be used to identify the minimal reporter in an unbiased way, avoiding the risk of overlooking regulatory elements that may be many kilobases away from the transcription start-site. Nematostella vectensis (Edwardsiidae, Anthozoa, Cnidaria) has become an important model in regenerative biology, ecology, and especially in studies of evo-devo and gene-regulatory networks due to its interesting phylogenetic position and amenability to molecular techniques. The increasing interest in this rising model system also led to a demand for methods that can be used to study the regulation of genes in Nematostella. Here we present our progress in employing BAC reporter constructs to visualize gene-expression in Nematostella. Using a new Nematostella-specific recombination cassette, we made nine different BAC reporter constructs. Although five BAC recombinants gave variable effects, three constructs, namely Nv-bra:eGFP::L10 BAC, Nv-dpp:eGFP::L10 BAC, and Nv-grm:eGFP::L10 BAC, delivered promising results. We show that these three constructs express the reporter gene eGFP in 10.4% – 17.2% of all analyzed larvae, out of which 26.2 – 41.9% express GFP in a mosaic fashion within the expected domain. In addition to the expression within the known domains, we also observed cases of misexpression of eGFP and examples that could represent actual expression outside the described domain. Furthermore, we deep-sequenced and assembled five different BACs containing Nv-chordin, Nv-foxa, Nv-dpp, Nv-wnta, and Nvwnt1, to improve assembly around these genes. The use of BAC reporter constructs will foster cis-regulatory analyses in Nematostella and thus help to improve our understanding of the regulatory network in this cnidarian system. Ultimately, this will advance the comparison of gene-regulation across species and lead to a much better understanding of evolutionary changes and novelties.
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DatasetNematostella vectensis BAC sequences( 2013-07-11) Fischer, Antje H. L. ; Tulin, Sarah ; Fredman, David ; Smith, JoelA key tool for investigating the regulation of genes is represented by Bacterial Artificial Chromosomes-reporter constructs (BAC). BACs are large insert libraries, often >>100 kb, which thus capture the genomic sequences surrounding a gene of interest, including all, or nearly all, of the elements underpinning regulation. Recombinant BACs, containing a reporter gene in place of the endogenous coding sequence of genes, can be utilized to drive the expression of reporter genes under the regulatory control of the gene of interest while still embedded within its genomic context. Systematic deletions within the BAC reporter construct can be used to identify the minimal reporter in an unbiased way, avoiding the risk of overlooking regulatory elements that may be many kilobases away from the transcription start-site. Nematostella vectensis (Edwardsiidae, Anthozoa, Cnidaria) has become an important model in regenerative biology, ecology, and especially in studies of evo-devo and gene-regulatory networks due to its interesting phylogenetic position and amenability to molecular techniques. This dataset represents the sequencing of 5 Nematostella vectensis BACs containing the genes of interest: NvDpp, NvChordin, NvFoxa, NvWnta, and NvWnt1.
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PreprintClosing the wounds : one hundred and twenty five years of regenerative biology in the ascidian Ciona intestinalis( 2014-06) Jeffery, William R.This year marks the 125th anniversary of the beginning of regeneration research in the ascidian Ciona intestinalis. A brief note was published in 1891 reporting the regeneration of the Ciona neural complex and siphons. This launched an active period of Ciona regeneration research culminating in the demonstration of partial body regeneration: the ability of proximal body parts to regenerate distal ones, but not vice versa. In a process resembling regeneration, wounds in the siphon tube were discovered to result in the formation of an ectopic siphon. Ciona regeneration research then lapsed into a period of relative inactivity following the purported demonstration of the inheritance of acquired characters using siphon regeneration as a model. Around the turn of the present century, Ciona regeneration research experienced a new blossoming. The current studies established the morphological and physiological integrity of the regeneration process and its resemblance to ontogeny. They also determined some of the cell types responsible for tissue and organ replacement and their sources in the body. Finally, they showed that regenerative capacity is reduced with age. Many other aspects of regeneration now can be studied at the mechanistic level because of the extensive molecular tools available in Ciona.
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PreprintKinesin family member 6 (kif6) is necessary for spine development in zebrafish( 2014-06) Buchan, Jillian G. ; Gray, Ryan S. ; Gansner, John M. ; Alvarado, David M. ; Burgert, Lydia ; Gitlin, Jonathan D. ; Gurnett, Christina A. ; Goldsmith, Matthew I.Idiopathic scoliosis is a form of spinal deformity that affects 2–3% of children and results in curvature of the spine without structural defects of the vertebral units. The pathogenesis of idiopathic scoliosis remains poorly understood, in part due to the lack of a relevant animal model. We performed a forward mutagenesis screen in zebrafish to identify new models for idiopathic scoliosis. We isolated a recessive zebrafish mutant, called skolios, which develops isolated spinal curvature that arises independent of vertebral malformations. Using meiotic mapping and whole genome sequencing, we identified a nonsense mutation in kinesin family member 6 (kif6gw326) unique to skolios mutants. Three additional kif6 frameshift alleles (gw327, gw328, gw329) were generated with transcription activator-like effector nucleases (TALENs). Zebrafish homozygous or compound heterozygous for kif6 frameshift mutations developed a scoliosis phenotype indistinguishable from skolios mutants, confirming that skolios is caused by the loss of kif6. Although kif6 may play a role in cilia, no evidence for cilia dysfunction was seen in kif6gw326 mutants. Overall, these findings demonstrate a novel role for kif6 in spinal development and identify a new candidate gene for human idiopathic scoliosis.
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ArticleThe tunicate Ciona : a model system for understanding the relationship between regeneration and aging(Taylor & Francis, 2014-06-10) Jeffery, William R.The use of the tunicate Ciona intestinalis as a model system to study the relationship between regeneration and aging is reviewed. Ciona has powerful regeneration capacities, which fade with age. Some additional benefits are a relatively short life span, the ability to study regeneration in vitro, the close phylogenetic relationship between tunicates and vertebrates, and the host of molecular tools already established in this system. The neural complex (NC), the oral siphon (OS), and the oral siphon pigment organs (OPO) have high capacities for regeneration. However, these organs show an inverse relationship between rate of regeneration and age. The ability to regenerate a complete OS disappears in the oldest animals of a natural population, probably due to the inability to form a blastema at the wound site. Effects on blastema formation could also be involved in the reduction of NC regeneration capacity. The fidelity of OPO restoration is also compromised by excess differentiation of precursor cells in local siphon niches in the oldest animals. The Ciona model provides a pathway to understand the molecular basis of these phenomena.
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ArticleFormation of the statolith in the ctenophore Mnemiopsis leidyi(Marine Biological Laboratory, 2014-08-01) Tamm, Sidney L.The aboral sensory organ (apical organ) of ctenophores contains a statocyst with a single large statolith. The statolith comprises living cells (lithocytes), each containing a large membrane-bound concretion. The statolith is supported on the distal ends of four compound motile mechanoresponsive cilia (balancers) which control the beat frequencies of the eight locomotory comb rows, and thereby the orientation of animals to gravity. In Mnemiopsis leidyi and Pleurobrachia pileus, lithocytes arise in the thickened epithelial floor of the apical organ on opposite sides along the tentacular plane. Lithocytes progressively differentiate and migrate toward the apical surface where they bud off next to the bases of the balancers. New lithocytes are transported up the balancers by ciliary surface motility to form the statolith (Noda, 2013). The statolith has a superellipsoidal shape due to the rectangular arrangement of the four balancers and the addition of new lithocytes to its ends via the balancers. The size of the statolith increases with animal size, starting at the highest rate of growth in younger stages and gradually decreasing in larger animals. The total number of developing lithocytes in the epithelial floor increases rapidly in smaller animals and reaches a plateau range in larger animals. Lithocytes are therefore produced continually throughout life for enlargement of the statolith and possibly for turnover and replacement of existing lithocytes. The dome cilia enclosing the statocyst were observed to propagate slow, low-ampitude waves distally. The dome cilia may act as an undulating screen to prevent foreign objects in the seawater from being transported non-specifically up the balancers to make a defective statolith.
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ArticleAcute increase of α-synuclein inhibits synaptic vesicle recycling evoked during intense stimulation(American Society for Cell Biology, 2014-10-01) Busch, David J. ; Oliphint, Paul A. ; Walsh, Rylie B. ; Banks, Susan M. L. ; Woods, Wendy S. ; George, Julia M. ; Morgan, Jennifer R.Parkinson's disease is associated with multiplication of the α-synuclein gene and abnormal accumulation of the protein. In animal models, α-synuclein overexpression broadly impairs synaptic vesicle trafficking. However, the exact steps of the vesicle trafficking pathway affected by excess α-synuclein and the underlying molecular mechanisms remain unknown. Therefore we acutely increased synuclein levels at a vertebrate synapse and performed a detailed ultrastructural analysis of the effects on presynaptic membranes. At stimulated synapses (20 Hz), excess synuclein caused a loss of synaptic vesicles and an expansion of the plasma membrane, indicating an impairment of vesicle recycling. The N-terminal domain (NTD) of synuclein, which folds into an α-helix, was sufficient to reproduce these effects. In contrast, α-synuclein mutants with a disrupted N-terminal α-helix (T6K and A30P) had little effect under identical conditions. Further supporting this model, another α-synuclein mutant (A53T) with a properly folded NTD phenocopied the synaptic vesicle recycling defects observed with wild type. Interestingly, the vesicle recycling defects were not observed when the stimulation frequency was reduced (5 Hz). Thus excess α-synuclein impairs synaptic vesicle recycling evoked during intense stimulation via a mechanism that requires a properly folded N-terminal α-helix.