dnmt1function is required to maintain retinal stem cells within the ciliary marginal zone of the zebrafish eye

Abstract

The ciliary marginal zone (CMZ) of the zebrafish retina contains a population of actively proliferating resident stem cells, which generate retinal neurons throughout life. The maintenance methyltransferase, dnmt1, is expressed within the CMZ. Loss of dnmt1 function results in gene misregulation and cell death in a variety of developmental contexts, however, its role in retinal stem cell (RSC) maintenance is currently unknown. Here, we demonstrate that zebrafish dnmt1 s872 mutants possess severe defects in RSC maintenance within the CMZ. Using a combination of immunohistochemistry, in situ hybridization, and a transgenic reporter assay, our results demonstrate a requirement for dnmt1 activity in the regulation of RSC proliferation, gene expression and in the repression of endogenous retroelements (REs). Ultimately, cell death is elevated in the dnmt1 -/-CMZ, but in a p53-independent manner. Using a transgenic reporter for RE transposition activity, we demonstrate increased transposition in the dnmt1 -/-CMZ. Taken together our data identify a critical role for dnmt1 function in RSC maintenance in the vertebrate eye. Introduction:The distal region of the vertebrate retina, termed the ciliary marginal zone (CMZ), contains a population of resident retinal stem cells (RSCs). The CMZ remains proliferative throughout the life of fish, but it proliferates to a more limited extent during the lifetime of amphibians and birds 1-6 . Whether an analogous structure exists in mammals is debated, but there are distinct, progenitor-like cells in the periphery of the retina that are active during embryogenesis 7-9 . Mammalian RSCs can also be isolated from the adult ciliary margin, cultured in vitro, and stimulated to produce retinal neurons 10-13 .However, this activity has not been demonstrated in the mature mammalian retinae in vivo.Studies of the CMZ have primarily focused on zebrafish and Xenopus models to determine genetic pathways required for RSC identity 2,14-16 and to characterize the epigenetic networks which regulate RSC function 17,18 . By comparison, the mechanisms mediating RSC maintenance in vivo remain unknown. In studies of RSCs, the zebrafish has been advantageous given that it possesses a highly active RSC population and is tractable for genetic and pharmacological manipulations, transgenesis and in vivo imaging 19,20 .DNA methylation, a frequently studied epigenetic modification, is the process through which a methyl group is added to the fifth carbon of cytosine nucleotides and is commonly found at CpG dinucleotide sequences 21 . Members of the family of DNA methyltransferase (Dnmt) enzymes 22,23 catalyze this epigenetic modification. Dnmt1 serves as a maintenance methyltransferase, copying the methylation pattern from parent to daughter strand during DNA replication and its function is required for cell cycle progression [24][25][26] . Loss of Dnmt1 function results in genomic hypomethylation 27-29 and in developmental contexts and specific organ systems, this often compromises progenitor cell maintenance 24...

Dnmt1 is required for the development of auditory organs via cell cycle arrest and Fgf signalling

“…Further ISH experiment confirmed this result, that cdkn1a and tp53 were highly expressed in the OV of Dnmt1‐depleted animals. This finding broadly supports the previous work that the proliferation of retinal stem cell (RSC) is severely repressed and cell death is elevated after dnmt1 depletion 41 . However, this study demonstrated that knockdown of Dnmt1 increased the expression level of tp53 , while the study of Angileri et al 41 found that cell cycle inhibition was not in a tp53 ‐dependent manner.…”

Potential Endogenous Cell Sources for Retinal Regeneration in Vertebrates and Humans: Progenitor Traits and Specialization

“…Recently, information has been published on the epigenetic status of cells in the zebrafish eye CMZ [ 70 ]. The study considers the DNA methylation in CMZ cells using Dnmt1, one of the DNA methyltransferase (Dnmt) enzymes that catalyzes the DNA methylation process [ 70 ].…”

“…Recently, information has been published on the epigenetic status of cells in the zebrafish eye CMZ [ 70 ]. The study considers the DNA methylation in CMZ cells using Dnmt1, one of the DNA methyltransferase (Dnmt) enzymes that catalyzes the DNA methylation process [ 70 ]. It was previously known that the loss of this enzyme results in genomic hypomethylation, and that Dnmt1 is capable of maintaining the function of progenitor cells in self-renewing somatic tissues [ 71 ].…”

“…It was previously known that the loss of this enzyme results in genomic hypomethylation, and that Dnmt1 is capable of maintaining the function of progenitor cells in self-renewing somatic tissues [ 71 ]. Angileri and Gross [ 70 ], using a dnmt1 mutant zebrafish with abnormalities similar to those documented in Dnmt1 -/- conditional knockout mice, have established that the function of this gene is required within the CMZ cells to exhibit progenitor properties and maintain the retinal stem cell (RSC) population. Within the dnmt1 -deficient CMZ, a decrease in the number of RSCs, a reduced level of their proliferation, and aberrant gene expression, were detected.…”

Evaluation of the Potential Effects of Retinol and Alginate/Gelatin-Based Scaffolds on Differentiation Capacity of Mouse Mesenchymal Stem Cells (MSCs) into Retinal Cells

“…Retinal stem cells (RSCs) and progenitor cells (RPCs) are the adult multipotent cells which are capable of self-renewal and differentiation into almost all retinal cells (1). They were firstly discovered in lower vertebrates like urodele amphibians (2) and teleost fishes (3), which are located at a region called the ciliary marginal zone (CMZ) between neural retina and ciliary epithelium and present a high potential to proliferate and to generate retinal cells throughout life (4). Interestingly, it has been recognized that the stem cells resided in CMZ of frogs and fishes could be able to completely restore the retina after severe damages or retinal detachments (5).…”