ABSTRACT
In the central nervous system injury induces cellular reprogramming and progenitor proliferation, but the molecular mechanisms that limit regeneration and prevent tumorigenesis are not completely understood. We previously described a zebrafish optic pathway tumor model in which transgenic Tg(flk1:RFP)is18/+ adults develop nonmalignant retinal tumors. Key pathways driving injury-induced glial reprogramming and regeneration contributed to tumor formation. In this study, we examine a time course of proliferation and present new analyses of the Tg(flk1:RFP)is18/+ dysplastic retina and tumor transcriptomes. Retinal dysplasia was first detected in 3-month-old adults, but was not limited to a specific stem cell or progenitor niche. Pathway analyses suggested a decrease in cellular respiration and increased expression of components of Hif1-α, VEGF, mTOR, NFκß, and multiple interleukin pathways are associated with early retinal dysplasia. Hif-α targets VEGFA (vegfab) and Leptin (lepb) were both highly upregulated in dysplastic retina; however, each showed distinct expression patterns in neurons and glia, respectively. Phospho-S6 immunolabeling indicated that mTOR signaling is activated in multiple cell populations in wild-type retina and in the dysplastic retina and advanced tumor. Our results suggest that multiple pathways may contribute to the continuous proliferation of retinal progenitors and tumor growth in this optic pathway tumor model. Further investigation of these signaling pathways may yield insight into potential mechanisms to control the proliferative response during regeneration in the nervous system.
Subject(s)
Cell Proliferation , Eye Neoplasms/pathology , Leptin/metabolism , Retinal Dysplasia/pathology , Vascular Endothelial Growth Factor A/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Eye Neoplasms/genetics , Eye Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Leptin/genetics , Retinal Dysplasia/genetics , Retinal Dysplasia/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/genetics , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/geneticsABSTRACT
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 (S100ß, 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.
