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Published on: October 24, 2013
Molecular and cellular characterization of a zebrafish optic pathway tumor line implicates glia-derived progenitors
Staci L Solin1, Ying Wang1, Joshua Mauldin1
1Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa, United States of America.
Abstract:
In this study we describe the molecular and cellular characterization of a zebrafish mutant that develops tumors in the optic pathway. Heterozygous Tg(flk1:RFP)is18 transgenic adults develop tumors of the retina, optic nerve and optic tract. Molecular and genetic mapping demonstrate the tumor phenotype is linked to a high copy number transgene array integrated in the lincRNA gene lincRNAis18/Zv9_00007276 on chromosome 3. TALENs were used to isolate a 147 kb deletion allele that removes exons 2-5 of the lincRNAis18 gene. Deletion allele homozygotes are viable and do not develop tumors, indicating loss of function of the lincRNAis18 locus is not the trigger for tumor onset. Optic pathway tumors in the Tg(flk1:RFP)is18 mutant occur with a penetrance of 80-100% by 1 year of age. The retinal tumors are highly vascularized and composed of rosettes of various sizes embedded in a fibrous matrix. Immunohistochemical analysis showed increased expression of the glial markers GFAP and BLBP throughout retinal tumors and in dysplastic optic nerve. We performed transcriptome analysis of pre-tumorous retina and retinal tumor tissue and found changes in gene expression signatures of radial glia and astrocytes (slc1a3), activated glia (atf3, blbp, apoeb), proliferating neural progenitors (foxd3, nestin, cdh2, her9/hes1), and glioma markers (S100β, vim). The transcriptome also revealed activation of cAMP, Stat3 and Wnt signal transduction pathways. qRT-PCR confirmed >10-fold overexpression of the Wnt pathway components hbegfa, ascl1a, and insm1a. Together the data indicate Müller glia and/or astrocyte-derived progenitors could contribute to the zebrafish Tg(flk1:RFP)is18 optic pathway tumors.
Insights
Zebrafish with a transgene array develop optic pathway tumors, linked to the lincRNA gene. Loss of this gene doesn't cause tumors, suggesting other mechanisms drive optic pathway glioma development.
Area of Science:
- Developmental biology
- Cancer research
- Zebrafish models
Background:
- Transgenic zebrafish (Tg(flk1:RFP)is18) exhibit spontaneous optic pathway tumors.
- Tumor phenotype is associated with a transgene array disrupting the lincRNA gene lincRNAis18 on chromosome 3.
Purpose of the Study:
- To characterize the molecular and cellular basis of optic pathway tumors in Tg(flk1:RFP)is18 zebrafish.
- To investigate the role of the lincRNAis18 gene in tumor development.
Main Methods:
- Molecular and genetic mapping to identify the causative genetic element.
- TALENs used to create a deletion allele of lincRNAis18.
- Immunohistochemistry to analyze glial marker expression.
- Transcriptome analysis (RNA-seq) of tumor and pre-tumorous tissues.
- Quantitative reverse transcription PCR (qRT-PCR) for pathway gene validation.
Main Results:
- A 147 kb deletion in lincRNAis18 did not prevent tumor formation, indicating the gene itself is not essential for tumor initiation.
- Retinal tumors are highly vascularized, showing increased glial markers (GFAP, BLBP) and expression of neural progenitor, activated glia, and glioma markers.
- Transcriptome analysis revealed activation of cAMP, Stat3, and Wnt signaling pathways, with significant overexpression of Wnt components (hbegfa, ascl1a, insm1a).
Conclusions:
- The Tg(flk1:RFP)is18 zebrafish optic pathway tumors are complex, highly vascularized neoplasms.
- Müller glia and/or astrocyte-derived progenitors are implicated as potential cell contributors.
- Aberrant Wnt pathway activation is a key feature of these optic pathway gliomas.
