During embryogenesis, blood vessels are remodelled in response to blood flow. Nicoli et al. describe a genetic pathway that explains how this mechanosensory stimulus is integrated with early developmental signals to remodel aortic arch vessels in zebrafish. The flow-induced transcription factor klf2a is required for the induction of an endothelial cell-specific microRNA, miR-126, which promotes VEGF signalling and angiogenesis through repressing Spred1, an inhibitor of VEGF signalling. This demonstrates how blood flow modulates endothelial cell-signalling pathways and implicates a microRNA as a central integration point during this process. During embryonic development, blood vessels remodel in response to blood flow. Here, a genetic pathway is described through which this mechanosensory stimulus is integrated with early developmental signals to remodel vessels of the aortic arch in zebrafish. It is found that the flow-induced transcription factor klf2a is required to induce the expression of an endothelial-specific microRNA, activating signalling through the growth factor Vegf. Within the circulatory system, blood flow regulates vascular remodelling1, stimulates blood stem cell formation2, and has a role in the pathology of vascular disease3. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation4. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system4. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow1. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a5,6,7 induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.

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Clonal selection and transcriptional reprogramming (e.g., epithelial-mesenchymal transition or phenotype switching) are the predominant theories thought to underlie tumor progression. However, a "division of labor" leading to cooperation among tumor-cell subpopulations could be an additional catalyst of progression. Using a zebrafish-melanoma xenograft model, we found that in a heterogeneous setting, inherently invasive cells, which possess protease activity and deposit extracellular matrix (ECM), co-invade with subpopulations of poorly invasive cells, a phenomenon we term "cooperative invasion". Whereas the poorly invasive cells benefit from heterogeneity, the invasive cells switch from protease-independent to an MT1-MMP-dependent mode of invasion. We did not observe changes in expression of the melanoma phenotype determinant MITF during cooperative invasion, thus ruling out the necessity for phenotype switching for invasion. Altogether, our data suggest that cooperation can drive melanoma progression without the need for clonal selection or phenotype switching and can account for the preservation of heterogeneity seen throughout tumor progression.

It has not previously been possible to live image the earliest interactions between the host environment and oncogene-transformed cells as they initiate formation of cancers within an organism. Here we take advantage of the translucency of zebrafish larvae to observe the host innate immune cell response as oncogene-transformed melanoblasts and goblet cells multiply within the larval skin. Our studies indicate activation of leukocytes at very early stages in larvae carrying a transformed cell burden. Locally, we see recruitment of neutrophils and macrophages by 48 h post-fertilization, when transformed cells are still only singletons or doublets, and soon after this we see intimate associations between immune and transformed cells and frequent examples of cytoplasmic tethers linking the two cell types, as well as engulfment of transformed cells by both neutrophils and macrophages. We show that a major component of the signal drawing inflammatory cells to oncogenic HRAS^G12V-transformed cells is H₂O₂, which is also a key damage cue responsible for recruiting neutrophils to a wound. Our short-term blocking experiments show that preventing recruitment of immune cells at these early stages results in reduced growth of transformed cell clones and suggests that immune cells may provide a source of trophic support to the transformed cells just as they do at a site of tissue repair. These parallels between the inflammatory responses to transformed cells and to wounds reinforce the suggestion by others that cancers resemble non-healing wounds. The translucency of zebrafish larvae allows us to live image the earliest dynamic interactions between host innate immune cells and oncogene-transformed cell clones as they first establish themselves as the precursors of full-blown cancer. These early associations manifest via cytoplasmic tethers between an immune cell and a transformed cell, and occasional phagocytic engulfment. Immune cells are first attracted to transformed cells at surprisingly early stages, before transformed cells have had a chance to form clones and are thus still singletons or doublets. We show that the key attractant is hydrogen peroxide (H₂O₂), which was also recently shown to be the essential early damage signal responsible for drawing neutrophils to wounds. Tissue transplantation experiments allow us to test which cells are responsible for generating the H₂O₂ attractant, and we show that both transformed cells and their otherwise healthy neighbors contribute. Blocking H₂O₂ synthesis, either pharmacologically or by morpholino-mediated knockdown of DUOX, the enzyme responsible for H₂O₂ synthesis in larval skin, very significantly reduces the numbers of neutrophils and macrophages drawn to transformed cell clones, and this results in reduced numbers of transformed cells, suggesting that innate immune cells play a trophic and/or support role in early transformed cell growth.