Publications
Recent publications by ARMI researchers
Discerning Different
In vivo Roles of MicroRNAs by Experimental Approaches in Zebrafish
Pase,L.,*
1 and
Lieschke, G.J*
1
*Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, 3800, Australia
1Walter and Eliza Hall Institute, Parkville, Victoria, 3050, Australia
Methods in Cell Biology 2011. Oxford: Academic Press; 2011, p. 353-378.
Abstract: MicroRNAs (miRNAs) are small endogenous RNAs of approximately 23 nucleotides that regulate the cellular transcriptome by binding to target mRNAs in a sequence-restricted manner, thereby modulating target transcript translation and turnover. Although the direct repressive trans-acting action of miRNAs is to cause a net reduction in the total amount of protein generated from their target mRNAs, developmental and physiological processes have combined this with the flexibility of spatial and temporal regulation of both the miRNAs and their targets to employ miRNAs in a range of regulatory roles. These different roles achieve diverse regulatory
outcomes. Five common in vivo regulatory roles of miRNAs are summarized, along with their key defining attributes that could be experimentally addressed to distinguish between them. Methods utilizing zebrafish that are suitable for determining the functional role of a particular miRNA of interest are outlined.
Berger. J, Berger. S, Jacoby, A.S., Wilton, S.D., and
Currie, P.D.
J Cell Mol Med 2011. E-pubCategory: Medicine, Research & Experimental Impact Factor: 4.608 Ranking: 18/106
Abstract: Duchenne Muscular Dystrophy is a severe muscle wasting disease caused by mutations in the dystrophin gene. By utilising antisense oligonucleotides, splicing of the dystrophin transcript can be altered so that exons harbouring a mutation are excluded from the mature mRNA. While this approach has been shown to be effective to restore partially functional dystrophin protein, the level of dystrophin protein that is necessary to rescue a severe muscle pathology has not been addressed. As zebrafish dystrophin mutants (dmd) resemble the severe muscle pathology of human patients, we have utilised this model to evaluate exon-skipping. Novel dmd mutations were identified to enable the design of phenotype rescue studies via morpholino administration. Correlation of induced exon-skipping efficiency and the level of phenotype rescue suggests that relatively robust levels of exon-skipping are required to achieve significant therapeutic ameliorations and that pre-screening analysis of exon-skipping drugs in zebrafish may help to more accurately predict clinical trials for therapies of Duchenne Muscular Dystrophy.
Mor, E., Cabilly,
Goldshmit. Y, Y., Zalts, H., Modai, S., Edry, L., Elroy-Stein, O., and Shomron, N.
Nucleic Acids Res 2011. 39(9): p. 3710-3723 Category: Biochemistry & Molecular Biology Impact Factor: 7.836 Ranking: 30/286
Abstract: MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in regulation of gene expression by binding to target genes. Many miRNAs were associated with the function of the central nervous system (CNS) in health and disease. Astrocytes are the CNS most abundant glia cells, providing support by maintaining homeostasis and by regulating neuronal signaling, survival and synaptic plasticity. Astrocytes play a key role in repair of brain insults, as part of local immune reactivity triggered by inflammatory or pathological conditions. Thus, astrocyte activation, or astrogliosis, is an important outcome of the innate immune response, which can be elicited by endotoxins such as lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-γ). The involvement of miRNAs in inflammation and stress led us to hypothesize that astrogliosis is mediated by miRNA function. In this study, we compared the miRNA regulatory layer expressed in primary cultured astrocyte derived from rodents (mice) and primates (marmosets) brains upon exposure to LPS and IFN-γ. We identified subsets of differentially expressed miRNAs some of which are shared with other immunological related systems while others, surprisingly, are mouse and rat specific. Of interest, these specific miRNAs regulate genes involved in the tumor necrosis factor-alpha (TNF-α) signaling pathway, indicating a miRNA-based species-specific regulation. Our data suggests that miRNA function is more significant in the mechanisms governing astrocyte activation in rodents compared to primates.
Pacary, E.,
Heng, J., Azzarelli, R., Riou, P., Castro, D., Lebel-Potter, M., Parras, C., Bell, D.M., Ridley, A.J., Parsons, M., and Guillemot, F.
Neuron 2011. 69(6): p. 1069-84 Category: Neurosciences Impact Factor: 14.027 Ranking: 6/237
Abstract: Little is known of the intracellular machinery that controls the
motility of newborn neurons. We have previously shown that the proneural
protein Neurog2 promotes the migration of nascent cortical neurons by
inducing the expression of the atypical Rho GTPase Rnd2. Here, we show
that another proneural factor, Ascl1, promotes neuronal migration in the
cortex through direct regulation of a second Rnd family member, Rnd3.
Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA
signaling, but they control distinct steps of the migratory process,
multipolar to bipolar transition in the intermediate zone and locomotion
in the cortical plate, respectively. Interestingly, these divergent
functions directly result from the distinct subcellular distributions of
the two Rnd proteins. Because Rnd proteins also regulate progenitor
divisions and neurite outgrowth, we propose that proneural factors,
through spatiotemporal regulation of Rnd proteins, integrate the process
of neuronal migration with other events in the neurogenic program.
Rios, A.C.,
Serralbo, O.,
Salgado, D., and
Marcelle, C.
Nature 2011. 473(7348): p. 532-535 Category: Multidisciplinary Sciences Impact Factor: 36.101 Ranking: 1/57
Abstract: How dynamic signalling and extensive tissue rearrangements interact to generate complex patterns and shapes during embryogenesis is poorly understood1, 2, 3. Here we characterize the signalling events taking place during early morphogenesis of chick skeletal muscles. We show that muscle progenitors present in somites require the transient activation of NOTCH signalling to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. Our results indicate that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool.
Smedley, D., Salimova, E., and
Rosenthal.N
Methods 2011. 53(4): p. 411-6 Category: Biochemical Research Methods Impact Factor: 4.527 Ranking:14/71
Abstract: Large scale international activities for systematic conditional mouse mutagenesis, exploiting advances in the sophisticated manipulation of the mouse genome, has established the mouse as the premier organism for developing models of human disease and drug action. Conditional mutagenesis is critical for the elucidation of the gene functions that exert pleiotropic effects in a variety of cell types and tissues throughout the life of the animal. The majority of new mouse mutants are therefore designed as conditional, activated only in a specific tissue (spatial control) and/or life stage (temporal control) through biogenic Cre/loxP technologies. The full power of conditional mutant mice can therefore only be exploited with the availability of well characterized mouse lines expressing Cre-recombinase in tissue, organ and cell type-specific patterns, to allow the creation of somatic mutations in defined genes. This chapter provides an update on the current state of Cre driver mouse lines worldwide, and reviews the available public databases and portals that capture critical details of Cre driver lines such as the efficiency of recombination, cell tissue specificity, or genetic background effects. The continuously changing landscape of these mouse resources reflects the rapid progression of research and development in conditional and inducible mouse mutagenesis.
Ellett, F.,
Luke Pase1, Hayman.J.,
Alex Andrianopoulos2 and Lieschke, G.J.
Blood First Edition Paper, prepublished
online November 17, 2010; DOI 10.1182/blood-2010-10-314120
1Cancer and Haematology Division, The Walter and Eliza Hall
Institute of Medical Research, Melbourne, Victoria, Australia;
2Department of Genetics, University of Melbourne,
Melbourne, Victoria, Australia
Abstract: Macrophages and neutrophils play important roles during the innate
immune response, phagocytosing invading microbes and delivering
antimicrobial compounds to the site of injury. Functional analyses
of the cellular innate immune response in zebrafish
infection/inflammation
models have been aided by transgenic lines with
fluorophore-marked neutrophils. However, it has not been
possible to study macrophage behaviors and neutrophil/macrophage
interactions
in vivo directly because there has been no macrophage-only reporter
line.
To remove this roadblock, a macrophage-specific marker
was identified (mpeg1) and its promoter used in mpeg1-driven
transgenes. mpeg1-driven transgenes are expressed in
macrophage-lineage cells that do not express
neutrophil-marking transgenes. Using these lines, the
different dynamic behaviors of neutrophils and macrophages
following wounding were compared side-by-side in compound
transgenics. Macrophage/neutrophil interactions, such as
phagocytosis of senescent neutrophils were readily observed in
real time. These zebrafish transgenes provide a new resource that
will contribute to the fields of inflammation, infection and
leukocyte biology.
Source:
http://bloodjournal.hematologylibrary.org/cgi/content/abstract/blood-2010-10-314120v1
Berger, J., Berger. S.,
Hall, T.E., Lieschke, G.J., Currie, P.D.
Neuromuscul Disord. 2010 Dec;20(12):826-32. Epub 2010 Sep 17.
Abstract: Duchenne muscular dystrophy is caused by mutations in the dystrophin gene. As in humans, zebrafish dystrophin is initially expressed at the peripheral ends of the myofibres adjacent to the myotendinous junction and gradually shifts to non-junctional sites. Dystrophin-deficient zebrafish larvae are characterised by abundant necrotic fibres being replaced by mono-nucleated infiltrates, extensive fibrosis accompanied by inflammation, and a broader variation in muscle fibre cross-sectional areas. Muscle progenitor proliferation cannot compensate for the extensive skeletal muscle loss. Live imaging of dystrophin-deficient zebrafish larvae documents detaching myofibres elicited by muscle contraction. Correspondingly, the progressive phenotype of dystrophin-deficient zebrafish resembles many aspects of the human disease, suggesting that specific advantages of the zebrafish model system, such as the ability to undertake in vivo drug screens and real time analysis of muscle fibre loss, could be used to make novel insights relevant to understanding and treating the pathological basis of dystrophin deficient muscular dystrophy.
Homman-Ludiye,J., Manger
PR,
Bourne.J.A.
J Comp Neurol. 2010 Nov
1;518(21):4439-62.
Abstract: Electrophysiological mapping of the adult ferret visual cortex has until
now determined the existence of 12 retinotopically distinct areas;
however, in the cat, another member of the Carnivora, 20 distinct visual
areas have been identified by using retinotopic mapping and
immunolabeling. In the present study, the immunohistochemical approach
to demarcate the areal boundaries of the adult ferret visual cortex was
applied in order to overcome the difficulties in accessing the sulcal
surfaces of a small, gyrencephalic brain. Nonphosphorylated
neurofilament (NNF) expression profiles were compared with another
classical immunostain of cortical nuclei, Cat-301 chondroitin sulfate
proteoglycan (CSPG). Together, these two markers reliably demarcated the
borders of the 12 previously defined areas and revealed further
arealization beyond those borders to a total of 19 areas: 21a and 21b;
the anterolateral, posterolateral, dorsal, and ventral lateral
suprasylvian areas (ALLS, PLLS, DLS, and VLS, respectively); and the
splenial and cingulate visual areas (SVA and CVA). NNF expression
profile and location of the newly defined areas correlate with
previously defined areas in the cat. Moreover, NNF and Cat-301 together
revealed discrete expression domains in the posteroparietal (PP) cortex,
demarcating four subdivisions in the caudal lateral and medial domains
(PPcL and PPcM) and rostral lateral and medial domains (PPrL and PPrM),
where only two retinotopic maps have been previously identified (PPc and
PPr). Taken together, these studies suggest that NNF and Cat-301 can
illustrate the homology between cortical areas in different species and
draw out the principles that have driven evolution of the visual cortex.
Goldshmit,Y., and
Bourne,J.A.
Journal of Neurotrauma 27:1321–1332 (July 2010)
Abstract: Glial scar formation occurs in response to brain injury in mammalian models and inhibits axonal growth. Identification of molecules that may mediate reactivity of astrocytes has become a leading therapeutic goal in the field of neurotrauma. In adult rodent brain and spinal cord, many of the Eph receptors and their ephrin ligands have been demonstrated to be upregulated on reactive astrocytes at the injury site; however, little is known about the expression of these molecules in nonhuman primate injury models.
This study examines the role of the tyrosine kinase EphA4 receptor, which predominantly binds most ephrin ligands, after injury in marmoset monkey brain. Following lesioning of the primary visual cortex (V1) in the adult marmoset, EphA4 is strongly upregulated on reactive astrocytes around the lesion site, which secrete extracellular matrix molecules such as chondroitin sulfate proteoglycans, which are known for their inhibitory effect on axonal growth and regeneration. This astrocyte reactivity was also associated with neuronal death in the area adjacent to the lesion site. EphA4 activation induced by clustered ephrin A5-Fc–mediated astrocyte proliferation and glial fibrillary acidic protein expression in vitro, as demonstrated by closure of scratched wound and MTT assays, occurs via two potential signaling pathways, the mitogen-activated protein kinase and Rho pathways. These results in a nonhuman primate model highlight the importance of developing pharmacotherapeutic approaches to block these molecules following brain injury.
Rios, A.C., Nicolas Denans,
Marcelle,C.
Developmental Dynamics, Volume 239, Issue 1, Pages 346 - 353, Published Online: 15 Dec 2009
Abstract: A critical mediator of cell-cell signaling events during embryogenesis is the highly conserved Wnt family of secreted proteins. Reporter constructs containing multimerized TCF DNA binding sites have been used to detect Wnt beta-catenin dependent activity during animal development. In this report, we have constructed and compared several TCF green fluorescent protein (GFP) reporter constructs. They contained 3, 8, or 12 TCF binding sites upstream of a minimal promoter driving native or destabilized enhanced GFP (EGFP). We have used the electroporation of somites in the chick embryo as a paradigm to test them in vivo. We have verified that they all respond to Wnt signaling in vivo. We have then assessed their efficiency at reflecting the activity of the Wnt pathway. Using destabilized EGFP reporter constructs, we show that somite cells dynamically regulate Wnt/beta-catenin-dependent signaling, a finding that was confirmed by performing time-lapse video confocal observation of electroporated embryos. Developmental Dynamics 239:346-353, 2010. © 2009 Wiley-Liss, Inc.
Davis, N., C. Yoffe, S. Raviv, R. Antes,
Berger, J., S. Holzmann, A. Stoykova, P.A. Overbeek, E.R. Tamm, and R. Ashery-Padan,
Dev Biol, 2009. 333(1): p. 132-142 Category: Developmental Biology Impact Factor: 4.416 Ranking: 7/37
Abstract: Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.
Goldshmit.Y., S. Galley, D. Foo, E. Sernagor, and
Bourne, J.A.
Neurosciences Impact Factor: 3.556 Ranking: 67/221 E-Pub: p. 1-13 Category:
Abstract: Mutations in the human cone-rod homeobox (Crx) gene are associated with retinal dystrophies such as Leber Congenital Amaurosis (LCA), characterized by complete or near complete absence of vision from birth. The photoreceptors of Crx−/− mice lack outer segments, and therefore cannot capture light signals through rods and cones, thus resulting in a lack of normal retinal ganglion cell activity from birth. Using specific antibodies to subsets of neurons and markers of activity, we examined the impact of this absence of sensory input on the development of the primary visual cortex (V1) in early postnatal Crx−/− mice, before wiring of the visual system is complete, and in adulthood. We revealed that Crx−/− mice did not exhibit gross anatomical differences in V1; however, they exhibited significantly fewer calcium-binding protein (parvalbumin and calbindin-D28k) expressing interneurons, as well as reduced nonphosphorylated neurofilament expression in V1. These results reveal that the Crx mutation and lack of light stimulation through the photoreceptor pathway regulate the development and phenotype of different neuronal populations in V1 but not its general morphology. We conclude, therefore, that photoreceptor-mediated visual input during development is crucial for the normal postnatal development and maturation of subsets of cortical neurons.
Jacoby, A.S., E. Busch-Nentwich, R.J. Bryson-Richardson, Hall, T.E.,
Berger,J.,
Berger,S.,
Sonntag, C., C. Sachs, R. Geisler, D.L. Stemple, and
Currie, P.D
Development. 136(19): p. 3367-76 Category: Developmental Biology Impact Factor: 6.812 Ranking: 5/38
Abstract: The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene, identifying laminin beta2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss.