Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.

Islet β ‐cell membrane excitability is a well‐established regulator of mammalian insulin secretion, and defects in β ‐cell excitability are linked to multiple forms of diabetes. Evolutionary conservation of islet excitability in lower organisms is largely unexplored. Here we show that adult zebrafish islet calcium levels rise in response to elevated extracellular [glucose], with similar concentration–response relationship to mammalian β ‐cells. However, zebrafish islet calcium transients are nor well coupled, with a shallower glucose‐dependence of cytoplasmic calcium concentration. We have also generated transgenic zebrafish that conditionally express gain‐of‐function mutations in ATP‐sensitive K + channels (K ATP ‐GOF) in β ‐cells. Following induction, these fish become profoundly diabetic, paralleling features of mammalian diabetes resulting from equivalent mutations. K ATP ‐GOF fish become severely hyperglycemic, with slowed growth, and their islets lose glucose‐induced calcium responses. These results indicate that, although lacking tight cell‐cell coupling of intracellular Ca 2+ , adult zebrafish islets recapitulate similar excitability‐driven β ‐cell glucose responsiveness to those in mammals, and exhibit profound susceptibility to diabetes as a result of inexcitability. While illustrating evolutionary conservation of islet excitability in lower vertebrates, these results also provide important validation of zebrafish as a suitable animal model in which to identify modulators of islet excitability and diabetes.

The development of the vertebrate jaw relies on a network of transcription factors that patterns the dorsal-ventral axis of the pharyngeal arches. Recent findings in both mouse and zebrafish illustrate that the basic-helix-loop-helix transcription factor, Hand2, is crucial in this patterning process. While Hand2 has functionally similar roles in these two species, little is known about the regulatory sequences controlling hand2 expression in zebrafish. Using bioinformatics and Tol2-mediated transgenesis, we have generated zebrafish transgenic reporter lines in which either the mouse or zebrafish arch-specific hand2 enhancer direct expression of a fluorescent reporter. We find that both the mouse and zebrafish enhancers drive early reporter expression in a hand2-specific pattern in the ventral pharyngeal arches of zebrafish embryos. These lines provide useful tools to follow ventral arch cells during vertebrate jaw development while also allowing dissection of hand2 transcriptional regulation during this process.

ATP‐sensitive potassium channels (KATP channels) are hetero‐octameric nucleotide‐gated ion channels that couple cellular metabolism to excitability in various tissues. In the heart, KATP channels are activated during ischaemia and potentially during adrenergic stimulation. In the vasculature, they are normally active at a low level, reducing vascular tone, but the ubiquitous nature of these channels leads to complex and poorly understood channelopathies as a result of gain‐ or loss‐of‐function mutations. Zebrafish (ZF) models of these channelopathies may provide insights to the link between molecular dysfunction and complex pathophysiology, but this requires understanding the tissue dependence of channel activity and subunit specificity. Thus far, direct analysis of ZF KATP expression and functional properties has only been performed in pancreatic β‐cells. Using a comprehensive combination of genetically modified fish, electrophysiology and gene expression analysis, we demonstrate that ZF cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. However, in contrast to mammalian cardiovascular KATP channels, ZF channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. The results provide a first characterization of the molecular properties of fish KATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9‐related Intellectual Disability and Myopathy syndrome. Key points Zebrafish cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. In contrast to mammalian cardiovascular KATP channels, zebrafish channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. We provide a first characterization of the molecular properties of fish KATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9‐related Intellectual Disability and Myopathy syndrome.

The myocyte enhancer factor-2 (MEF2) family of transcription factors plays key roles in the activation of muscle structural genes. In Drosophila, MEF2 accumulates at high levels in the embryonic muscles, where it activates target genes throughout the mesoderm. Here, we identify the Transglutaminase gene (Tg; CG7356) as a direct transcriptional target of MEF2 in the cardiac musculature. Tg is expressed in cells forming the inflow tracts of the dorsal vessel, and we identify the enhancer responsible for this expression. The enhancer contains three binding sites for MEF2, and can be activated by MEF2 in tissue culture and in vivo. Moreover, loss of MEF2 function, or removal of the MEF2 binding sites from the enhancer, results in loss of Tg expression. These studies identify a new MEF2 target in the cardiac musculature. These studies provide a possible mechanism for the activation of transglutaminase genes.