The giant Mauthner (M) cell is the largest neuron known in the vertebrate brain. It has enabled major breakthroughs in neuroscience but its ultimate function remains surprisingly unclear: An actual survival value of M cell-mediated escapes has never been supported experimentally and ablating the cell repeatedly failed to eliminate all rapid escapes, suggesting that escapes can equally well be driven by smaller neurons. Here we applied techniques to simultaneously measure escape performance and the state of the giant M axon over an extended period following ablation of its soma. We discovered that the axon survives remarkably long and remains still fully capable of driving rapid escape behavior. By unilaterally removing one of the two M axons and comparing escapes in the same individual that could or could not recruit an M axon, we show that the giant M axon is essential for rapid escapes and that its loss means that rapid escapes are also lost forever. This allowed us to directly test the survival value of the M cell-mediated escapes and to show that the absence of this giant neuron directly affects survival in encounters with a natural predator. These findings not only offer a surprising solution to an old puzzle but demonstrate that even complex brains can trust vital functions to individual neurons. Our findings suggest that mechanisms must have evolved in parallel with the unique significance of these neurons to keep their axons alive and connected.

Proteolysis of mitotic regulators like securins and cyclins requires Fizzy(FZY)/Cdc20 and Fizzy-related(FZR)/Hct1/Cdh1 proteins. Budding yeast Cdh1 acts not only during G1, but is also required for B-type cyclin degradation during exit from mitosis when Cdh1 is a target of the mitotic exit network controlling progression through late mitosis and cytokinesis. In contrast, observations in frog and Drosophila embryos have suggested that the orthologous FZR is not involved during exit from mitosis. However, the potential involvement of minor amounts of maternally derived FZR was not excluded in these studies. Similarly, the reported absence of severe mitotic defects in chicken Cdh1(-/-) cells might be explained by the recent identification of multiple Cdh1 genes [10]. Here, we have carefully analyzed the FZR requirement during exit from mitosis in Drosophila, which, apart from fzr, has only one additional homolog. We find that this fzr2 gene, although expressed in the male germline, is not expressed during mitotic divisions. Moreover, by characterizing fzr alleles, we demonstrate that completion of mitosis including Cyclin B degradation does not require FZR. However, fzr is an essential gene corresponding to the rap locus, and FZR, which accumulates predominantly in the cytoplasm, is clearly required during G1.

The recently evolved field of synthetic biology has revolutionized the way we think of biology as an “engineerable” discipline. The newly sprouted branch is constantly in need of simple, cost-effective and automatable DNA-assembly methods. We have developed a reliable DNA-assembly system, ZeBRα (Zero-Background Redα), for cloning multiple DNA-fragments seamlessly with very high efficiency. The hallmarks of ZeBRα are the greatly reduced hands-on time and costs and yet excellent efficiency and flexibility. ZeBRα combines a “zero-background vector” with a highly efficient in vitro recombination method. The suicide-gene in the vector acts as placeholder, and is replaced by the fragments-of-interest, ensuring the exclusive survival of the successful recombinants. Thereby the background from uncut or re-ligated vector is absent and screening for recombinant colonies is unnecessary. Multiple fragments-of-interest can be assembled into the empty vector by a recombinogenic E. coli -lysate (SLiCE) with a total time requirement of less than 48 h. We have significantly simplified the preparation of the high recombination-competent E. coli -lysate compared to the original protocol. ZeBRα is the least labor intensive among comparable state-of-the-art assembly/cloning methods without a trade-off in efficiency.

Ammonia excretion in fish excretory epithelia is a complex interplay of multiple membrane transport proteins and mechanisms. Using the model system of zebrafish (Danio rerio) larvae, here we identified three paralogues of a novel ammonia transporter, hippocampus abundant transcript 1 (DrHIAT1), found also in most vertebrates. When functionally expressed in Xenopus laevis oocytes, DrHiat1a and DrHiat1b promoted methylamine uptake in a competitive manner with ammonia. In situ hybridization experiments showed that both transporters were expressed as early as the 4-cell stage in zebrafish embryos and could be identified in most tissues four days post fertilization. Larvae experiencing morpholino-mediated knockdown of DrHiat1b exhibited significantly lower whole body ammonia excretion rates compared to control larvae. Markedly decreased site-specific total ammonia excretion of up to 85% was observed in both the pharyngeal region (site of developing gills) and the yolk sac (region shown to have the highest NH4+ flux). This study is the first to identify especially DrHiat1b as an important contributor to ammonia excretion in larval zebrafish. Being evolutionarily conserved, these proteins are likely involved in multiple other general ammonia-handling mechanisms, making them worthy candidates for future studies on nitrogen regulation in fishes and across the animal kingdom.