SUMMARYUnlike the adult mammalian retina, Müller glia (MG) in the adult zebrafish retina are able to dedifferentiate into a ‘‘stem cell’’-like state and give rise to multipotent progenitor cells upon retinal damage. We show that miR-216a is downregulated in MG after constant intense light lesioning and that miR-216a suppression is necessary and sufficient for MG dedifferentiation and proliferation during retina regeneration. miR-216a targets the H3K79 methyltransferase Dot1l, which is upregulated in proliferating MG after retinal damage. Loss-of-function experiments show that Dot1l is necessary for MG reprogramming and mediates MG proliferation downstream of miR-216a. We further demonstrate that miR-216a and Dot1l regulate MG-mediated retina regeneration through canonical Wnt signaling. This article reports a regulatory mechanism upstream of Wnt signaling during retina regeneration and provides potential targets for enhancing regeneration in the adult mammalian retina.

BackgroundPh- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage. JAK2V617F, an activation mutation form of tyrosine kinase JAK2, is found in the majority of patients with MPNs. Studies have demonstrated that JAK2V617F can cause MPNs, and various methods have been developed to detect JAK2V617F for diagnostic purposes. However, a highly sensitive method is still needed for the earliest possible detection and for disease prevention and treatment.MethodsIn the present study, we developed a method dubbed restriction fragment nested allele-specific PCR (RFN-AS-PCR). The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the JAK2V617F mutation site, 2) digestion of the PCR products with restriction enzyme BsaXI which only cleaves the wild type allele, and 3) detection of JAK2V617F by allele-specific PCR with nested primers.ResultsWe tested the sensitivity of the method by using purified plasmid DNAs and blood cell DNAs containing known proportions of JAK2V617F. We were able to detect JAK2V617F with a sensitivity of 0.001%. We further analyzed blood cell DNA samples from 105 healthy donors with normal blood cell counts and found three JAK2V617F-positive cases, which would have remained undetected using a less sensitive method.ConclusionsWe have developed a highly sensitive method that will allow for detection of JAK2V617F at a very early stage. This method may have major implications in diagnosis and prevention of MPNs and related diseases.

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