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Regeneration and tissue homeostasis require accurate production of missing cell lineages. Cell production is driven by changes to gene expression, which is shaped by multiple layers of regulation. Here, we find that the ubiquitous mRNA base-modification, m6A, is required for proper cell fate choice and cellular maturation in planarian stem cells (neoblasts). We mapped m6A-enriched regions in 7,600 planarian genes and found that perturbation of the m6A pathway resulted in progressive deterioration of tissues and death. Using single-cell RNA sequencing of20,000 cells following perturbation of the m6A pathway, we identified an increase in expression of noncanonical histone variants, and that inhibition of the pathway resulted in accumulation of undifferentiated cells throughout the animal in an abnormal transcriptional state. Analysis of1,000 planarian gene expression datasets revealed that the inhibition of the chromatin modifying complex NuRD had almost indistinguishable consequences, unraveling an unappreciated link between m6A and chromatin modifications. Our findings reveal that m6A is critical for planarian stem cell homeostasis and gene regulation in tissue maintenance and regeneration.

Optic cups are a structural feature of diverse eyes, from simple pit eyes to camera eyes of vertebrates and cephalopods. We used the planarian prototypic eye as a model to study the genetic control of optic cup formation and regeneration. We identified two genes encoding transcription factors, sp6-9 and dlx, that were expressed in the eye specifically in the optic cup and not the photoreceptor neurons. RNAi of these genes prevented formation of visible optic cups during regeneration. Planarian regeneration requires an adult proliferative cell population with stem cell-like properties called the neoblasts. We found that optic cup formation occurred only after migration of progressively differentiating progenitor cells from the neoblast population. The eye regeneration defect caused by dlx and sp6-9 RNAi can be explained by a failure to generate these early optic cup progenitors. Dlx and Sp6-9 genes function as a module during the development of diverse animal appendages, including vertebrate and insect limbs. Our work reveals a novel function for this gene pair in the development of a fundamental eye component, and it utilizes these genes to demonstrate a mechanism for total organ regeneration in which extensive cell movement separates new cell specification from organ morphogenesis., United States. National Institutes of Health (NIH R01GM080639), W. M. Keck Foundation, Howard Hughes Medical Institute