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| Status |
Public on Nov 24, 2017 |
| Title |
Transcriptome and Functional Analyses Reveal Roles For Regulators of Epigenetic States, Micro RNA Processing, And Long Non-Coding RNA In Myocyte Dedifferentiation: Insights Into Reprogramming A “Post-Mitotic” Cell |
| Organism |
Danio rerio |
| Experiment type |
Expression profiling by high throughput sequencing
Non-coding RNA profiling by high throughput sequencing
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| Summary |
Purpose: The ability of adult zebrafish tissues to undergo dedifferentiation provides an opportunity to probe the molecular underpinnings of cell identity and reprogramming. Zebafish muscle regeneration utilizes dedifferentiation to reprogram mature multinucleated myocytes into dedifferentiated myoblast that re-enter the cell cycle. A unique advantage of this system is that the regenerating cell mass is large and fairly homogenous, facilitating genomics approaches to uncovering the underlying biology.
Methods: To better understand cellular reprogramming of mature myocytes, we temporally analyzed the changing transcriptome leading up to the proliferative switch. RNA was obtained after Laser Micro-dissection (LMD) of Control, 9 hour post-injury (HPI) or 18 HPI using Trizol and micro column purification. Illumina's TruSeq Stranded mRNA Library Prep Kit and 0.1 - 4 µg total mRNA from pooled purified RNA samples were used for performing ribosomal-depletion (Ribo-Zero Gold rRNA Removal Kit, Illumina) and library preparation. Sequencing was performed by the UM DNA Sequencing Core, using an Illumina Hi-Seq 2000 (50-cycle, single end read) platform.
Results: Clustering and functional annotation of differentially expressed genes highlighted the importance of catabolic and phagocytic processes upregulation at 9 and 18 hours post injury (hpi). Furthermore, genes encoding principle regulators of chromatin states were actively re-regulated during the reprogramming process. Utilizing the accessibility of these tissues in the zebrafish model, kKnockdown experiments enabled in vivo validation and phenotypic analysis of candidate genes and pathways for their roles in genomic and cellular reprogramming. Additionally, we found that despite of their low expression levels, lncRNAs were highly represented in gene clusters with dynamic, “switch-like” expression profiles, and that miRNA processing was also found important for reprogramming
Conclusions: We conclude that reprogramming of a “post-mitotic” myocyte into a dedifferentiated myoblast requires both heritable yet nuanced epigenetic alterations and molecular switches that involve transcription factors, miRNA and lncRNA, while maintaining the lineage restriction of the cell of origin.
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| Overall design |
Early time points post injury (9 & 18 hours) mRNA and lncRNA profiles of Zebrafish lateral eye muscle (EOM) were generated by deep sequencing, in quadruplicate, using Illumina Hi-seq.
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| Contributor(s) |
Saera-Vila A, Kish PE, Kahana A |
| Citation(s) |
29121865 |
| Submission date |
Dec 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Alon Kahana |
| E-mail(s) |
akahana@med.umich.edu
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| Phone |
(734) 936-8654
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| Organization name |
University of Michigan
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| Department |
Ophthalmology and Visual Sciences
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| Lab |
Kellogg Eye Center
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| Street address |
1000 Wall St.
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48105 |
| Country |
USA |
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| Platforms (1) |
| GPL14875 |
Illumina HiSeq 2000 (Danio rerio) |
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| Samples (14)
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| Relations |
| BioProject |
PRJNA357683 |
| SRA |
SRP095238 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE92489_CON_v_H18_DE.txt.gz |
1.9 Mb |
(ftp)(http) |
TXT |
| GSE92489_CON_v_H18_gene.foldchange.1.5_annotated.txt.gz |
2.0 Mb |
(ftp)(http) |
TXT |
| GSE92489_CON_v_H9_DE.txt.gz |
1.9 Mb |
(ftp)(http) |
TXT |
| GSE92489_CON_v_H9_gene.foldchange.1.5_annotated.txt.gz |
2.0 Mb |
(ftp)(http) |
TXT |
| GSE92489_CuffDiff_rawCountsMatrixGEO.txt.gz |
699.3 Kb |
(ftp)(http) |
TXT |
| GSE92489_H9_v_H18_DE.txt.gz |
1.8 Mb |
(ftp)(http) |
TXT |
| GSE92489_H9_v_H18_gene.foldchange.1.5_annotated.txt.gz |
1.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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