In a genetic screen, we isolated a mutation that perturbed motor axon outgrowth, neurogenesis, and somitogenesis. Complementation tests revealed that this mutation is an allele of deadly seven (des). By creating genetic mosaics, we demonstrate that the motor axon defect is non-cell autonomous. In addition, we show that the pattern of migration for some neural crest cell populations is aberrant and crest-derived dorsal root ganglion neurons are misplaced. Furthermore, our analysis reveals that des mutant embryos exhibit a neurogenic phenotype. We find an increase in the number of primary motoneurons and in the number of three hindbrain reticulospinal neurons: Mauthner cells, RoL2 cells, and MiD3cm cells. We also find that the number of Rohon-Beard sensory neurons is decreased whereas neural crest-derived dorsal root ganglion neurons are increased in number supporting a previous hypothesis that Rohon-Beard neurons and neural crest form an equivalence group during development. Mutations in genes involved in Notch-Delta signaling result in defects in somitogenesis and neurogenesis. We found that overexpressing an activated form of Notch decreased the number of Mauthner cells in des mutants indicating that des functions via the Notch-Delta signaling pathway to control the production of specific cell types within the central and peripheral nervous systems.

Secondary sphere interactions are known to significantly impact catalytic rates within biological systems as well as synthetic molecular catalysts. The [NiFe] hydrogenase enzymes oxidize and produce molecular hydrogen at high turnover rates within a complex coordination environment. Nickel-substituted rubredoxin (NiRd) has been developed as a functional, protein-based mimic of the [NiFe] hydrogenase, providing an opportunity to understand the influence of the secondary coordination environment on proton reduction activity. In this work, a rationally designed series of mutants was generated to study the effects of outer-sphere interactions on catalysis. This library was characterized using quantitative protein film electrochemistry, optical spectroscopy, X-ray crystallography, and molecular dynamics simulations. Changing the secondary sphere residues modulates the redox activity of the nickel- and iron-bound rubredoxin proteins, alters the hydrogen-bonding network, and perturbs solvent accessibility of the active site, which correlates with catalytic turnover frequency. The effects on reactivity are dependent on the site of mutation and, when coupled to crystallographic and computational analyses, implicate one of the nickel-coordinating cysteine residues as the mechanistically relevant site of protonation. Introduction of a carboxylate residue, mimicking that found in the [NiFe] hydrogenase, significantly increases the overall catalytic rate, likely through installation of a proton transfer pathway into the active site. Apparent turnover frequencies within the mutant constructs range from 15 to 500 s–1 without imparting significant variation in overpotential, and many mutants break the typical scaling relationship between catalytic rates and overpotential that is often seen in small-molecule systems. These results demonstrate the substantial impact of the coordination environment on the hydrogen-producing activity of the artificial metalloenzyme, NiRd, and highlight the importance of such interactions within molecular catalysts.

The relatively simple neural circuit driving the escape response in zebrafish offers an excellent opportunity to study properties of neural circuit formation. The hindbrain Mauthner cell is an essential component of this circuit. Mutations in the zebrafish deadly seven/notch1a (des) gene result in supernumerary Mauthner cells. We addressed whether and how these extra cells are incorporated into the escape-response circuit. Calcium imaging revealed that all Mauthner cells in desb420 mutants were active during an elicited escape response. However, the kinematic performance of the escape response in mutant larvae was very similar to wild-type fish. Analysis of the relationship between Mauthner axon collaterals and spinal neurons revealed that there was a decrease in the number of axon collaterals per Mauthner axon in mutant larvae compared with wild-type larvae, indicative of a decrease in the number of synapses formed with target spinal neurons. Moreover, we show that Mauthner axons projecting on the same side of the nervous system have primarily nonoverlapping collaterals. These data support the hypothesis that excess Mauthner cells are incorporated into the escape-response circuit, but they divide their target territory to maintain a normal response, thus demonstrating plasticity in the formation of the escape-response circuit. Such plasticity may be key to the evolution of the startle responses in mammals, which use larger populations of neurons in circuits similar to those in the fish escape response.