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Dive into the research topics where Viviana Gallardo is active.

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Featured researches published by Viviana Gallardo.


BMC Biology | 2010

A high-throughput chemically induced inflammation assay in zebrafish

Claudia A d'Alençon; Oscar A Peña; Christine Wittmann; Viviana Gallardo; Rebecca Jones; Felix Loosli; Urban Liebel; Clemens Grabher; Miguel L. Allende

BackgroundStudies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening.ResultsHere we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction.ConclusionsThis approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay.See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.


PLOS Genetics | 2009

Phoenix Is Required for Mechanosensory Hair Cell Regeneration in the Zebrafish Lateral Line

Martine Behra; John Bradsher; Rachid Sougrat; Viviana Gallardo; Miguel L. Allende; Shawn M. Burgess

In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ears neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.


BMC Developmental Biology | 2010

Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish.

Viviana Gallardo; Jin Liang; Martine Behra; Abdel G. Elkahloun; Eduardo J. Villablanca; Vincenzo Russo; Miguel L. Allende; Shawn M. Burgess

BackgroundDevelopment of the posterior lateral line (PLL) system in zebrafish involves cell migration, proliferation and differentiation of mechanosensory cells. The PLL forms when cranial placodal cells delaminate and become a coherent, migratory primordium that traverses the length of the fish to form this sensory system. As it migrates, the primordium deposits groups of cells called neuromasts, the specialized organs that contain the mechanosensory hair cells. Therefore the primordium provides both a model for studying collective directional cell migration and the differentiation of sensory cells from multipotent progenitor cells.ResultsThrough the combined use of transgenic fish, Fluorescence Activated Cell Sorting and microarray analysis we identified a repertoire of key genes expressed in the migrating primordium and in differentiated neuromasts. We validated the specific expression in the primordium of a subset of the identified sequences by quantitative RT-PCR, and by in situ hybridization. We also show that interfering with the function of two genes, f11r and cd9b, defects in primordium migration are induced. Finally, pathway construction revealed functional relationships among the genes enriched in the migrating cell population.ConclusionsOur results demonstrate that this is a robust approach to globally analyze tissue-specific expression and we predict that many of the genes identified in this study will show critical functions in developmental events involving collective cell migration and possibly in pathological situations such as tumor metastasis.


Disease Models & Mechanisms | 2015

Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion

Viviana Gallardo; Gaurav K. Varshney; Minnkyong Lee; Sujata Bupp; Lisha Xu; Paul Shinn; Nigel P.S. Crawford; James Inglese; Shawn M. Burgess

ABSTRACT In the last decade, high-throughput chemical screening has become the dominant approach for discovering novel compounds with therapeutic properties. Automated screening using in vitro or cultured cell assays have yielded thousands of candidate drugs for a variety of biological targets, but these approaches have not resulted in an increase in drug discovery despite major increases in expenditures. In contrast, phenotype-driven screens have shown a much stronger success rate, which is why we developed an in vivo assay using transgenic zebrafish with a GFP-marked migrating posterior lateral line primordium (PLLp) to identify compounds that influence collective cell migration. We then conducted a high-throughput screen using a compound library of 2160 annotated bioactive synthetic compounds and 800 natural products to identify molecules that block normal PLLp migration. We identified 165 compounds that interfere with primordium migration without overt toxicity in vivo. Selected compounds were confirmed in their migration-blocking activity by using additional assays for cell migration. We then proved the screen to be successful in identifying anti-metastatic compounds active in vivo by performing orthotopic tumor implantation assays in mice. We demonstrated that the Src inhibitor SU6656, identified in our screen, can be used to suppress the metastatic capacity of a highly aggressive mammary tumor cell line. Finally, we used CRISPR/Cas9-targeted mutagenesis in zebrafish to genetically validate predicted targets of compounds. This approach demonstrates that the migrating PLLp in zebrafish can be used for large-scale, high-throughput screening for compounds that inhibit collective cell migration and, potentially, anti-metastatic compounds. Summary: We have developed a phenotype-driven screen for identifying new inhibitors of collective cell migration and demonstrated the screen can successfully identify compounds active in vivo and potentially new pathways for targeting cancer metastasis.


Biological Research | 2011

Sublethal concentrations of waterborne copper induce cellular stress and cell death in zebrafish embryos and larvae

Pedro P. Hernández; Cristian Undurraga; Viviana Gallardo; Natalia Mackenzie; Miguel L. Allende; Ariel E. Reyes

Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.


Methods | 2013

Fluorescent activated cell sorting (FACS) combined with gene expression microarrays for transcription enrichment profiling of zebrafish lateral line cells

Viviana Gallardo; Martine Behra

Transgenic lines carrying fluorescent reporter genes like GFP have been of great value in the elucidation of developmental features and physiological processes in various animal models, including zebrafish. The lateral line (LL), which is a fish specific superficial sensory organ, is an emerging organ model for studying complex cellular processes in the context of the whole living animal. Cell migration, mechanosensory cell development/differentiation and regeneration are some examples. This sensory system is made of superficial and sparse small sensory patches called neuromasts, with less than 50 cells in any given patch. The paucity of cells is a real problem in any effort to characterize those cells at the transcriptional level. We describe here a method which we applied to efficiently separate subpopulation of cells of the LL, using two distinct stable transgenic zebrafish lines, Tg(cldnb:gfp) and Tg(tnks1bp1:EGFP). In both cases, the GFP positive (GFP+) cells were separated from the remainder of the animal by using a Fluorescent Activated Cell Sorter (FACS). The transcripts of the GFP+ cells were subsequently analyzed on gene expression microarrays. The combination of FACS and microarrays is an efficient method to establish a transcriptional signature for discrete cell populations which would otherwise be masked in whole animal preparation.


bioRxiv | 2018

tbx5 and tbx15 mediate zebrafish posterior lateral line migration and development via a negative feedback loop

Heather R. Whitehurst; Viviana Gallardo; Shawn M. Burgess

The zebrafish posterior lateral line (pLL) is a mechanosensory organ enabling detection of movement in the aqueous environment. Evidence suggests that two T-box transcription factors, tbx5 and tbx15, participate in pLL migration and development. Lateral line migration and deposition defects are observed following tbx5 or tbx15 morpholino knockdown. Additional studies demonstrate that the tbx5 phenotype is partially rescued when tbx15 is also knocked down. pLL defects similar to those of tbx5 are noted following knockdown of camKII-ß2, a known downstream target of Tbx5, suggesting a potential mechanistic pathway. Ectopic expression of human CamKII-∂C partially rescues the tbx5 phenotype but exacerbates the tbx15 phenotype. These results, combined with in situ hybridization and qRT-PCR profiling, indicate a negative feedback loop controlling multiple primordium patterning markers, including cxcr4b, cxcr7b, fgf10a and notch3, which ultimately affects pLL neuromast migration and deposition.


Genome Research | 2015

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

Gaurav K. Varshney; Wuhong Pei; Matthew C. LaFave; Jennifer Idol; Lisha Xu; Viviana Gallardo; Blake Carrington; Kevin Bishop; MaryPat Jones; Mingyu Li; Ursula Harper; Sunny C. Huang; Anupam Prakash; Wenbiao Chen; Raman Sood; Johan Ledin; Shawn M. Burgess


Free Radical Biology and Medicine | 2004

Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells

Marco T. Núñez; Viviana Gallardo; Patricia Muñoz; Victoria Tapia; Andrés Esparza; Julio Salazar; Hernán Speisky


BMC Developmental Biology | 2012

Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

Martine Behra; Viviana Gallardo; John Bradsher; Aranza I. Torrado; Abdel G. Elkahloun; Jennifer Idol; Jessica Sheehy; Seth Zonies; Lisha Xu; Kenna M. Shaw; Chie Satou; Shin-ichi Higashijima; Brant M. Weinstein; Shawn M. Burgess

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Shawn M. Burgess

National Institutes of Health

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Martine Behra

University of Puerto Rico

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Lisha Xu

National Institutes of Health

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Jennifer Idol

National Institutes of Health

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John Bradsher

University of Puerto Rico

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Abdel G. Elkahloun

National Institutes of Health

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Blake Carrington

National Institutes of Health

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Gaurav K. Varshney

National Institutes of Health

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