Unknown,Transcriptomics,Genomics,Proteomics

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Early depletion of primordial germ cells in zebrafish promotes testis formation

ABSTRACT: The requirement for primordial germ cells (PGCs) during sexual differentiation is variable among vertebrates. It has been shown that in zebrafish complete loss of PGCs in the embryos causes exclusive male development. Further, transplantation of a single PGC into a germline deficient zebrafish embryos generates male exclusively, suggesting PGC number might be important for the ovarian fate.To explore how PGC number might regulate sexual development in zebrafish, we experimentally manipulated its number by injecting dnd MO into embryos to generate fish containing a spectrum of PGC number. The experiment was designed to compare transcriptomes between the developing trunk regions of wild type and dnd morphants at different developmental stages. The dnd MO was microinjected into one cell stage embryos to generate zebrafish with a range of PGC numbers. Transcriptomes of developing trunk regions of wild type and dnd morphants at 14 dpf and 22 dpf were analyzed.

ORGANISM(S): Danio rerio

SUBMITTER: Keh-Weei Tzung

PROVIDER: E-GEOD-57046 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Early depletion of primordial germ cells in zebrafish promotes testis formation.

Tzung Keh-Weei KW Goto Rie R Saju Jolly M JM Sreenivasan Rajini R Saito Taiju T Arai Katsutoshi K Yamaha Etsuro E Hossain Mohammad Sorowar MS Calvert Meredith E K MEK Orbán László L

Stem cell reports 20141126 1

As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcri ...[more]

PMID: 25434820

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Project description:Vitamin D receptors (VDR) are abundantly expressed in developing zebrafish as early as 48 hours post-fertilization, and prior to the development of a mineralized skeleton, and mature intestine and kidney. We probed the role of VDR in zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D3, 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3). We observed significant changes in RNAs encoding proteins of fatty acid, amino acid, and xenobiotic metabolism pathways, and RNAs of transcription factors, leptin, peptide hormones, receptor-activator of NFkB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early small, and subsequent massive changes in >10% of expressed cellular RNAs were observed. At day 2 (24h 1a,25(OH)2D3-treatment), only 5 RNAs were differentially expressed (hormone vs. vehicle). On day 4 (72h-treatment), 78 RNAs; on day 6 (120h-treatment) 1040 RNAs; and on day 7 (144h-treatment), 1755 RNAs were differentially expressed in response to 1a,25(OH)2D3. Fewer RNAs (n = 482) were altered in day 7 embryos treated for 24h with 1a,25(OH)2D3 vs. those treated with hormone for 144h. At 7 days, in 1a,25(OH)2D3-treated embryos, pharyngeal cartilage was larger and mineralization was greater. Changes in expression of RNAs for transcription factors, peptide hormones, and RNAs encoding proteins integral to fatty acid, amino acid, leptin, calcitonin-like ligand receptor, RANKL and xenobiotic metabolism pathways, demonstrate heretofore unrecognized mechanisms by which 1a,25(OH)2D3 functions in vivo in developing eukaryotes. Zebrafish embryos were obtained from mating of Segrest wild-type (SWT) parents under controlled barrier conditions, in the Mayo Clinic Zebrafish Core Facility, in Instant Ocean media . Zebrafish embryos (25-30) were placed in 20 mL embryo medium (pH 7.2) containing 1-phenyl-2-thiourea (PTU) (0.003% (w/v) and were maintained at 28-30 oC. At 24 hpf (1 day post fertilization, dpf), 10 microliters of 1a,25(OH)2D3 in ethanol was added to embryos maintained in 20 mL fresh embryo medium with PTU. The final concentration of 1a,25(OH)2D3 was 300 pM. Control zebrafish were treated with 10 microliters ethanol alone (vehicle controls). The medium containing either 300 pM 1a,25(OH)2D3 or vehicle was changed every 24 h . In experiment 1, at 2, 4, 6 and 7 dpf embryos/larvae were removed and immediately frozen at -80 0C for later RNA preparations. 25-30 embryos per set were used for preparation on RNA. At the same times, 7-12 embryos were fixed in 4% paraformaldehyde in 0.75 X DulbeccoM-bM-^@M-^Ys phosphate buffered saline (DPBS). In experiment 2, 6 dpf larvae were treated with 1a,25(OH)2D3 (300 pM) or vehicle for 24 h. RNA was prepared from three sets of larvae.

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Early depletion of primordial germ cells in zebrafish promotes testis formation

Publications

Early depletion of primordial germ cells in zebrafish promotes testis formation.

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