Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the fundamental role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel // regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by chromosomal rearrangement that generated new -regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying hindbrain boundary-specific-regulatory elements. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.

Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fi sh. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fi sh. Here, acute toxicity tests and LC50 curves have been generated for zebrafi sh larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentrationdependent manner, resulting in changes in gene expression. Using a transgenic fi sh line that expresses enhanced green fl uorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specifi c stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.

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53The recovery of injured or lost sensory neurons after trauma, disease or aging is a major 54 scientific challenge. Upon hearing loss or balance disorder, regeneration of mechanosensory 55 hair cells has been observed in fish, some amphibians and under special circumstances in 56 birds, but is absent in adult mammals. In aquatic vertebrates, hair cells are not only present in 57 the inner ear but also in neuromasts of the lateral line system. The zebrafish lateral line 58 neuromast has an almost unlimited capacity to regenerate hair cells. This remarkable ability 59is possible due to the presence of neural stem/progenitor cells within neuromasts. In order to 60 further characterize these stem cells, we use the expression of the neural progenitor markers 61Sox2 and Sox3, transgenic reporter lines, and morphological and topological analysis of the 62 different cell types within the neuromast. We reveal new sub-populations of supporting cells, 63 the sustentacular supporting cells and the neuromast stem cells. In addition, using loss-of-64 function and mutants of sox2 and sox3, we find that the combined activity of both genes is 65 essential for lateral line development and regeneration. The capability of sox2/sox3 expressing 66 stem cells to produce new hair cells, hair cell-precursors, and supporting cells after damage 67 was analyzed in detail by time-lapse microscopy and immunofluorescence. We are able to 68 provide evidence that sox2/3 expressing cells are the main contributors to the regenerated 69 neuromast, and that their daughter cells are able to differentiate into most cell types of the 70 neuromast.