Loss of sensory hair cells in the mammalian inner ear leads to permanent hearing and vestibular defects, whereas loss of hair cells in zebrafish results in their regeneration. We used scRNA-Seq to characterize the transcriptional dynamics of hair cell regeneration in zebrafish at unprecedented spatio-temporal resolution. We uncovered three, sequentially activated modules. First, an injury/inflammatory response and downregulation of progenitor/stem cell maintenance genes within minutes after hair cell loss. Second, the transient activation of regeneration-specific genes. And third, a robust reactivation of developmental gene programs, including hair cell specification, cell cycle activation, ribosome biogenesis, and a metabolic switch to oxidative phosphorylation. The results are not only relevant for our understanding of hair cell regeneration and how we might be able to trigger it in mammals but also for regenerative processes in general. The data is searchable and publicly accessible via a web-based interface.

Macrophages are essential for tissue repair and regeneration. Yet, the molecular programs, as well as the timing of their activation during and after tissue injury are poorly defined. Using a high spatio-temporal resolution single cell analysis of macrophages coupled with live imaging after sensory hair cell death in zebrafish, we find that the same population of macrophages transitions through a sequence of three major anti-inflammatory activation states. Macrophages first show a signature of glucocorticoid activation, then IL10 signaling and finally the induction of oxidative phosphorylation by IL4/Polyamine signaling. Importantly, loss-of-function of glucocorticoid and IL10 signaling shows that each step of the sequence is independently activated. Our results provide the first evidence that macrophages, in addition to a switch from M1 to M2, sequentially and independently transition though three anti-inflammatory pathways in vivo during tissue injury in a regenerating organ.