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

Gallardo, Viviana; Behra, Martine

doi:10.1016/j.ymeth.2013.06.005

Cited by 23 publications

(15 citation statements)

References 23 publications

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“…1a . For cell separation from ppv3b:GFP z ebrafish, embryos were dissociated as follows (previously reported 56 ): Five dpf larvae were dissociated by incubation in trypsin (Trypsin-EDTA 0.25%, Life Technologies) for 30 min with intermittent mechanical disruption by trituration. The reaction was stopped by adding HBSS supplemented with 10% FBS and 100 μg ml −1 DNaseI.…”

Section: Methodsmentioning

confidence: 99%

RFX transcription factors are essential for hearing in mice

et al. 2015

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Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.

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Section: Methodsmentioning

confidence: 99%

RFX transcription factors are essential for hearing in mice

et al. 2015

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“…Although previous transcriptomic screens have sought genes expressed in the lateral line, none has focused on mantle cells (18)(19)(20). The results of such studies reflect gene expression in several cell types, a complication that might mask gene expression in progenitors.…”

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confidence: 99%

Dynamic gene expression by putative hair-cell progenitors during regeneration in the zebrafish lateral line

Steiner

Kim

Cabot

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

107

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Hearing loss is most commonly caused by the destruction of mechanosensory hair cells in the ear. This condition is usually permanent: Despite the presence of putative hair-cell progenitors in the cochlea, hair cells are not naturally replenished in adult mammals. Unlike those of the mammalian ear, the progenitor cells of nonmammalian vertebrates can regenerate hair cells throughout life. The basis of this difference remains largely unexplored but may lie in molecular dissimilarities that affect how progenitors respond to hair-cell death. To approach this issue, we analyzed gene expression in hair-cell progenitors of the lateral-line system. We developed a transgenic line of zebrafish that expresses a red fluorescent protein in the presumptive hair-cell progenitors known as mantle cells. Fluorescence-activated cell sorting from the skins of transgenic larvae, followed by microarray-based expression analysis, revealed a constellation of transcripts that are specifically enriched in these cells. Gene expression analysis after hair-cell ablation uncovered a cohort of genes that are differentially regulated early in regeneration, suggesting possible roles in the response of progenitors to hair-cell death. These results provide a resource for studying hair-cell regeneration and the biology of sensory progenitor cells.alkaline phosphatase | auditory | neuromast | supporting cell

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“…As a first step, we tested whether miR-29-sponge mRNA was directly targeted by miR-29 family members by co-injecting its mRNA along with RFP control mRNA and miR-29 mimics into zebrafish embryos. At 24 hours post-fertilization (hpf), injected embryos were dissociated and the relative eGFP/RFP fluorescent ratio in dissociated cells was measured by fluorocytometry (according to [ 48 ]) (Fig. 2a ).…”

Section: Resultsmentioning

confidence: 99%

MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging

et al. 2017

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BackgroundA widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes.ResultsHere, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress.ConclusionsOur results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0354-x) contains supplementary material, which is available to authorized users.

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

Cited by 23 publications

References 23 publications

RFX transcription factors are essential for hearing in mice

RFX transcription factors are essential for hearing in mice

Dynamic gene expression by putative hair-cell progenitors during regeneration in the zebrafish lateral line

MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging

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