Gerald B. Downes

The G-Protein βγ Complex

Abstract The vast majority of signalling pathways in mammalian cells are mediated by heterotrimeric (αβγ) G proteins. Reviewed here is regulation of signal transduction by the βγ complex at different protein interfaces: subunit–subunit, receptor–G protein and G protein–effector. The role of diverse β and γ subunit types in achieving specificity in signalling and potentially unidentified functions for these subunits also are discussed.

Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis

BackgroundThe Deepwater Horizon disaster was the largest marine oil spill in history, and total vertical exposure of oil to the water column suggests it could impact an enormous diversity of ecosystems. The most vulnerable organisms are those encountering these pollutants during their early life stages. Water-soluble components of crude oil and specific polycyclic aromatic hydrocarbons have been shown to cause defects in cardiovascular and craniofacial development in a variety of teleost species, but the developmental origins of these defects have yet to be determined. We have adopted zebrafish, Danio rerio, as a model to test whether water accumulated fractions (WAF) of the Deepwater Horizon oil could impact specific embryonic developmental processes. While not a native species to the Gulf waters, the developmental biology of zebrafish has been well characterized and makes it a powerful model system to reveal the cellular and molecular mechanisms behind Macondo crude toxicity.ResultsWAF of Macondo crude oil sampled during the oil spill was used to treat zebrafish throughout embryonic and larval development. Our results indicate that the Macondo crude oil causes a variety of significant defects in zebrafish embryogenesis, but these defects have specific developmental origins. WAF treatments caused defects in craniofacial development and circulatory function similar to previous reports, but we extend these results to show they are likely derived from an earlier defect in neural crest cell development. Moreover, we demonstrate that exposure to WAFs causes a variety of novel deformations in specific developmental processes, including programmed cell death, locomotor behavior, sensory and motor axon pathfinding, somitogenesis and muscle patterning. Interestingly, the severity of cell death and muscle phenotypes decreased over several months of repeated analysis, which was correlated with a rapid drop-off in the aromatic and alkane hydrocarbon components of the oil.ConclusionsWhether these teratogenic effects are unique to the oil from the Deepwater Horizon oil spill or generalizable for most crude oil types remains to be determined. This work establishes a model for further investigation into the molecular mechanisms behind crude oil mediated deformations. In addition, due to the high conservation of genetic and cellular processes between zebrafish and other vertebrates, our work also provides a platform for more focused assessment of the impact that the Deepwater Horizon oil spill has had on the early life stages of native fish species in the Gulf of Mexico and the Atlantic Ocean.

Modular laboratory exercises to analyze the development of zebrafish motor behavior.

The embryonic zebrafish is an excellent research model to examine the neural networks that coordinate locomotive behavior. It demonstrates robust locomotive behavior early in development, its nervous system is relatively simple and accessible compared to mammalian systems, and there are mutants available with specific molecular and motor deficits. We have developed a series of four exercises that provide students with a basic understanding of locomotive behavior development, nervous system organization, development of neurotransmitter responsiveness, and genetics. The first two exercises can be performed in one 3-h laboratory period, and the third and fourth exercises, which build on the first two, can be completed in one or two subsequent periods. In the first exercise, students observe and quantify two distinct behaviors that characterize different developmental stages, spontaneous movement, and touch-evoked tail coiling. In the second, the students use a pharmacological approach to determine if the neurotransmitter glycine is required for the embryo to perform each behavior. In the third, they use simple lesions to assess whether the brain is required for each type of behavior. In the fourth, the students examine bandoneon, a zebrafish motility mutant that has a glycine receptor defect, by observing its behavior during spontaneous movement and touch-evoked tail coiling, performing lesions, and applying pharmacological drugs. These exercises are readily adaptable, such that portions can be omitted or expanded to examine other neurotransmitter systems or later stages of locomotive behavior development.

Disruption of Eaat2b, a glutamate transporter, results in abnormal motor behaviors in developing zebrafish.

Analysis of zebrafish mutants that have defects in motor behavior can allow entrée into the hindbrain and spinal cord networks that control locomotion. Here, we report that zebrafish techno trousers (tnt) locomotor mutants harbor a mutation in slc1a2b, which encodes Eaat2b, a plasma membrane glutamate transporter. We used tnt mutants to explore the effects of impaired glutamate transporter activity on locomotor network function. Wild-type larvae perform robust swimming behavior in response to touch stimuli at two and four days after fertilization. In contrast, tnt mutant larvae demonstrate aberrant, exaggerated body bends beginning two days after fertilization and they are almost paralyzed four days after fertilization. We show that slc1a2b is expressed in glial cells in a dynamic fashion across development, which may explain the abnormal sequence of motor behaviors demonstrated by tnt mutants. We also show that tnt larvae demonstrate enhanced excitation of neurons, consistent with the predicted effects of excessive glutamate. These findings illustrate the dynamic regulation and importance of glutamate transporters during development. Since glutamate toxicity caused by EAAT2 dysfunction is thought to promote several different neurological disorders in humans, including epilepsy and neurodegenerative diseases, tnt mutants hold promise as a new tool to better understand these pathologies.

Analysis of a zebrafish behavioral mutant reveals a dominant mutation in atp2a1/SERCA1.

Zebrafish embryos demonstrate robust swimming behavior, which consists of smooth, alternating body bends. In contrast, several motility mutants have been identified that perform sustained, bilateral trunk muscle contractions which result in abnormal body shortening. Unlike most of these mutants, accordion (acc)dta5 demonstrates a semidominant effect: Heterozygotes exhibit a distinct but less severe phenotype than homozygotes. Using molecular‐genetic mapping and candidate gene analysis, we determined that accdta5 mutants harbor a novel mutation in atp2a1, which encodes SERCA1, a calcium pump important for muscle relaxation. Previous studies have shown that eight other acc alleles compromise SERCA1 function, but these alleles were all reported to be recessive. Quantitative behavioral assays, complementation testing, and analysis of molecular models all indicate that the accdta5 mutation diminishes SERCA1 function to a greater degree than other acc alleles through either haploinsufficient or dominant‐negative molecular mechanisms. Since mutation of human ATP2A1 results in Brody disease, an exercise‐induced impairment of muscle relaxation, accdta5 mutants may provide a particularly sensitive model of this disorder. genesis, 48:354–361, 2010.

Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease.

SUMMARY Analysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que) mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt), a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD.

Gfap-positive radial glial cells are an essential progenitor population for later-born neurons and glia in the zebrafish spinal cord.

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial‐derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later‐born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter‐driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap‐negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood–brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170–1189

Vincristine and bortezomib cause axon outgrowth and behavioral defects in larval zebrafish.

Peripheral neuropathy is a common side effect of a number of pharmaceutical compounds, including several chemotherapy drugs. Among these are vincristine sulfate, a mitotic inhibitor used to treat a variety of leukemias, lymphomas, and other cancers, and bortezomib, a 26S proteasome inhibitor used primarily to treat relapsed multiple myeloma and mantle cell lymphoma. To gain insight into the mechanisms by which these compounds act, we tested their effects in zebrafish. Vincristine or bortezomib given during late embryonic development caused significant defects at both behavioral and cellular levels. Intriguingly, the effects of the two drugs appear to be distinct. Vincristine causes uncoordinated swimming behavior, which is coupled with a reduction in the density of sensory innervation and overall size of motor axon arbors. Bortezomib, in contrast, increases the duration and amplitude of muscle contractions associated with escape swimming, which is coupled with a preferential reduction in fine processes and branches of sensory and motor axons. These results demonstrate that zebrafish is a convenient in vivo assay system for screening potential pharmaceutical compounds for neurotoxic side effects, and they provide an important step toward understanding how vincristine and bortezomib cause peripheral neuropathy.

prdm12b specifies the p1 progenitor domain and reveals a role for V1 interneurons in swim movements

Proper functioning of the vertebrate central nervous system requires the precise positioning of many neuronal cell types. This positioning is established during early embryogenesis when gene regulatory networks pattern the neural tube along its anteroposterior and dorsoventral axes. Dorsoventral patterning of the embryonic neural tube gives rise to multiple progenitor cell domains that go on to differentiate unique classes of neurons and glia. While the genetic program is reasonably well understood for some lineages, such as ventrally derived motor neurons and glia, other lineages are much less characterized. Here we show that prdm12b, a member of the PR domain containing-family of transcriptional regulators, is expressed in the p1 progenitor domain of the zebrafish neural tube in response to Sonic Hedgehog signaling. We find that disruption of prdm12b function leads to dorsal expansion of nkx6.1 expression and loss of p1-derived eng1b-expressing V1 interneurons, while the adjacent p0 and p2 domains are unaffected. We also demonstrate that prdm12b-deficient fish exhibit an abnormal touch-evoked escape response with excessive body contractions and a prolonged response time, as well as an inability to coordinate swimming movements, thereby revealing a functional role for V1 interneurons in locomotor circuits. We conclude that prdm12b is required for V1 interneuron specification and that these neurons control swimming movements in zebrafish.

Expressing acetylcholine receptors after innervation suppresses spontaneous vesicle release and causes muscle fatigue

The formation and function of synapses are tightly orchestrated by the precise timing of expression of specific molecules during development. In this study, we determined how manipulating the timing of expression of postsynaptic acetylcholine receptors (AChRs) impacts presynaptic release by establishing a genetically engineered zebrafish line in which we can freely control the timing of AChR expression in an AChR-less fish background. With the delayed induction of AChR expression after an extensive period of AChR-less development, paralyzed fish displayed a remarkable level of recovery, exhibiting a robust escape response following developmental delay. Despite their apparent behavioral rescue, synapse formation in these fish was significantly altered as a result of delayed AChR expression. Motor neuron innervation determined the sites for AChR clustering, a complete reversal of normal neuromuscular junction (NMJ) development where AChR clustering precedes innervation. Most importantly, among the three modes of presynaptic vesicle release, only the spontaneous release machinery was strongly suppressed in these fish, while evoked vesicle release remained relatively unaffected. Such a specific presynaptic change, which may constitute a part of the compensatory mechanism in response to the absence of postsynaptic AChRs, may underlie symptoms of neuromuscular diseases characterized by reduced AChRs, such as myasthenia gravis.