Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing

Khuansuwan, Sataree; Gamse, Joshua T.

doi:10.1016/j.ydbio.2014.08.015

Cited by 12 publications

(12 citation statements)

References 57 publications

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“…One of the powerful features of the zebrafish model is the possibility to use transgenic reporter lines for the isolation of specific cell types through fluorescence-based cell sorting and subsequent tissue and cell specific transcriptome analysis on in vivo samples [ 13 – 15 , 33 , 34 ]. Of note, tissue dissociation and cell sorting may induce cellular stress responses that influence cell viability and quality of the isolated RNA [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

et al. 2019

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BackgroundTransgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis.ResultsWe evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results.ConclusionsThe presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-5608-2) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

et al. 2019

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“…Despite their crucial and well-described role in the establishment of left-right asymmetry, functional characterization of PP cells is sparse. However, recent transcriptome analyses suggest that PP cells represent a specialized form of PhR cells (Khuansuwan and Gamse, 2014).…”

Section: Introductionmentioning

confidence: 99%

Bsx controls pineal complex development

Schredelseker

Driever

2018

Development

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Neuroendocrine cells in the pineal gland release melatonin during the night and, in teleosts, are directly photoreceptive. During development of the pineal complex, a small number of cells migrate leftward away from the pineal anlage to form the parapineal cell cluster, a process that is crucial for asymmetrical development of the bilateral habenular nuclei. Here, we show that, throughout zebrafish embryonic development, the () gene is expressed in all cell types of the pineal complex. We identified Bmp and Noto/Flh as major regulators of expression in the pineal complex. Upon loss of Bsx through the generation of a targeted mutation, embryos fail to form a parapineal organ and develop right-isomerized habenulae. Crucial enzymes in the melatonin biosynthesis pathway are not expressed, suggesting the absence of melatonin from the pineal gland in mutants. Several genes involved in rod-like or cone-like phototransduction are also abnormally expressed, indicating that Bsx has a pivotal role in the differentiation of multiple cell types in the zebrafish pineal complex.

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“…Elucidation of Tbx2b-responsive genes will enable a better understanding of how these parapineal neurons are able to migrate precisely to their target destination. Transcriptome analysis between wild-type and tbx2b morphant pineal complexes at 24 hpf identified several differentially expressed genes ( Khuansuwan and Gamse, 2014 ). Further investigation of these differentially expressed genes should provide insight into how Tbx2b regulates parapineal migration.…”

Section: Discussionmentioning

confidence: 99%

A transcription factor network controls cell migration and fate decisions in the developing zebrafish pineal complex

et al. 2016

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The zebrafish pineal complex consists of four cell types (rod and cone photoreceptors, projection neurons and parapineal neurons) that are derived from a single pineal complex anlage. After specification, parapineal neurons migrate unilaterally away from the rest of the pineal complex whereas rods, cones and projection neurons are non-migratory. The transcription factor Tbx2b is important for both the correct number and migration of parapineal neurons. We find that two additional transcription factors, Flh and Nr2e3, negatively regulate parapineal formation. Flh induces non-migratory neuron fates and limits the extent of parapineal specification, in part by activation of Nr2e3 expression. Tbx2b is positively regulated by Flh, but opposes Flh action during specification of parapineal neurons. Loss of parapineal neuron specification in Tbx2b-deficient embryos can be partially rescued by loss of Nr2e3 or Flh function; however, parapineal migration absolutely requires Tbx2b activity. We conclude that cell specification and migration in the pineal complex are regulated by a network of at least three transcription factors.

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Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing

Cited by 12 publications

References 57 publications

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Bsx controls pineal complex development

A transcription factor network controls cell migration and fate decisions in the developing zebrafish pineal complex

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