Skin cells undergo asynthetic fission to expand body surfaces in zebrafish.

As an animal's surface area expands during development, skin cell populations must quickly respond to maintain sufficient epithelial coverage. Despite much progress in understanding of skin cell behaviours in vivo1,2, it remains unclear how cells collectively act to satisfy coverage demands at an organismic level. Here we created a multicolour cell membrane tagging system, palmskin, to monitor the entire population of superficial epithelial cells (SECs) in developing zebrafish larvae. Using time-lapse imaging, we found that many SECs readily divide on the animal body surface; during a specific developmental window, a single SEC can produce a maximum of four progeny cells over its lifetime on the surface of the animal. Remarkably, EdU assays, DNA staining and hydroxyurea treatment showed that these terminally differentiated skin cells continue splitting despite an absence of DNA replication, causing up to 50% of SECs to exhibit reduced genome size. On the basis of a simple mathematical model and quantitative analyses of cell volumes and apical surface areas, we propose that 'asynthetic fission' is used as an efficient mechanism for expanding epithelial coverage during rapid growth. Furthermore, global or local manipulation of body surface growth affects the extent and mode of SEC division, presumably through tension-mediated activation of stretch-activated ion channels. We speculate that this frugal yet flexible mode of cell proliferation might also occur in contexts other than zebrafish skin expansion.

Whole-body clonal mapping identifies giant dominant clones in zebrafish skin epidermis.

Skin expansion during development is predominantly driven by growth of basal epithelial cell (BEC)-derived clonal populations, which often display varied sizes and shapes. However, little is known about the causes of clonal heterogeneity and the maximum size to which a single clone can grow. Here, we created a zebrafish model, basebow, for capturing clonal growth behavior in the BEC population on a whole-body, centimeter scale. By tracking 222 BECs over the course of a 28-fold expansion of body surface area, we determined that most BECs survive and grow clonal populations with an average size of 0.013 mm2. An extensive survey of 742 sparsely labeled BECs further revealed that giant dominant clones occasionally arise on specific body regions, covering up to 0.6% of the surface area. Additionally, a growth-induced extracellular matrix component, Lamb1a, mediates clonal growth in a cell-autonomous manner. Altogether, our findings demonstrate how clonal heterogeneity and clonal dominance may emerge to enable post-embryonic growth of a vertebrate organ, highlighting key cellular mechanisms that may only become evident when visualizing single cell behavior at the whole-animal level.

The RNA helicase Ddx52 functions as a growth switch in juvenile zebrafish.

Vertebrate animals usually display robust growth trajectories during juvenile stages, and reversible suspension of this growth momentum by a single genetic determinant has not been reported. Here, we report a single genetic factor that is essential for juvenile growth in zebrafish. Using a forward genetic screen, we recovered a temperature-sensitive allele, pan (after Peter Pan), that suspends whole-organism growth at juvenile stages. Remarkably, even after growth is halted for a full 8-week period, pan mutants are able to resume a robust growth trajectory after release from the restrictive temperature, eventually growing into fertile adults without apparent adverse phenotypes. Positional cloning and complementation assays revealed that pan encodes a probable ATP-dependent RNA helicase (DEAD-Box Helicase 52; ddx52) that maintains the level of 47S precursor ribosomal RNA. Furthermore, genetic silencing of ddx52 and pharmacological inhibition of bulk RNA transcription similarly suspend the growth of flies, zebrafish and mice. Our findings reveal evidence that safe, reversible pauses of juvenile growth can be mediated by targeting the activity of a single gene, and that its pausing mechanism has high evolutionary conservation.

Caspase dependent apoptosis is required for anterior regeneration in Aeolosoma viride and its related gene expressions are regulated by the Wnt signaling pathway.

Although apoptosis has been widely observed during the regenerative process, the mechanisms by which it is regulated and its roles in regeneration remained unclear. In this study, we introduced Aeolosoma viride, a fresh water annelid with an extraordinary regenerative ability as our model organism to study the functions and regulations of apoptotic caspases. Here we showed that major events of apoptosis were detected near the wounded area and showed spatial correlation with the expression patterns of caspase gene namely Avi-caspase X and two apoptosis regulators namely Avi-Bax and Avi-Bcl-xL. Next, we investigated how Avi-caspase X gene expression and apoptosis influence regeneration following head amputation. RNA interference of Avi-caspase X reduced the amounts of apoptotic cells, as well as the percentage of successful regeneration, suggesting a critical role for apoptosis in anterior regeneration of A. viride. In addition, we also discovered that the expression of apoptotic caspases was regulated by the canonical Wnt signaling pathway. Together, our study showed that caspase dependent apoptosis was critical to the anterior regeneration of A. viride, and could be regulated by the canonical Wnt signaling pathway.

Genetic Reprogramming of Positional Memory in a Regenerating Appendage.

Certain vertebrates such as salamanders and zebrafish are able to regenerate complex tissues (e.g., limbs and fins) with remarkable fidelity. However, how positional information of the missing structure is recalled by appendage stump cells has puzzled researchers for centuries. Here, we report that sizing information for adult zebrafish tailfins is encoded within proliferating blastema cells during a critical period of regeneration. Using a chemical mutagenesis screen, we identified a temperature-sensitive allele of the gene encoding DNA polymerase alpha subunit 2 (pola2) that disrupts fin regeneration in zebrafish. Temperature shift assays revealed a 48-h window of regeneration, during which positional identities could be disrupted in pola2 mutants, leading to regeneration of miniaturized appendages. These fins retained memory of the new size in subsequent rounds of amputation and regeneration. Similar effects were observed upon transient genetic or pharmacological disruption of progenitor cell proliferation after plucking of zebrafish scales or head or tail amputation in amphioxus and annelids. Our results provide evidence that positional information in regenerating tissues is not hardwired but malleable, based on regulatory mechanisms that appear to be evolutionarily conserved across distantly related phyla.