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

AbstractProper 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

Developmental exposures to perfluorooctanesulfonic acid (PFOS) impact embryonic nutrition, pancreatic morphology, and adiposity in the zebrafish, \u3cem\u3eDanio rerio\u3c/em\u3e

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental contaminant previously found in consumer surfactants and industrial fire-fighting foams. PFOS has been widely implicated in metabolic dysfunction across the lifespan, including diabetes and obesity. However, the contributions of the embryonic environment to metabolic disease remain uncharacterized. This study seeks to identify perturbations in embryonic metabolism, pancreas development, and adiposity due to developmental and subchronic PFOS exposures and their persistence into later larval and juvenile periods. Zebrafish embryos were exposed to 16 or 32 μM PFOS developmentally (1–5 days post fertilization; dpf) or subchronically (1–15 dpf). Embryonic fatty acid and macronutrient concentrations and expression of peroxisome proliferator-activated receptor (PPAR) isoforms were quantified in embryos. Pancreatic islet morphometry was assessed at 15 and 30 dpf, and adiposity and fish behavior were assessed at 15 dpf. Concentrations of lauric (C12:0) and myristic (C14:0) saturated fatty acids were increased by PFOS at 4 dpf, and PPAR gene expression was reduced. Incidence of aberrant islet morphologies, principal islet areas, and adiposity were increased in 15 dpf larvae and 30 dpf juvenile fish. Together, these data suggest that the embryonic period is a susceptible window of metabolic programming in response to PFOS exposures, and that these early exposures alone can have persisting effects later in the lifecourse

Zebrafish prdm12b acts independently of nkx6.1 repression to promote eng1b expression in the neural tube p1 domain

Abstract Background Functioning of the adult nervous system depends on the establishment of neural circuits during embryogenesis. In vertebrates, neurons that make up motor circuits form in distinct domains along the dorsoventral axis of the neural tube. Each domain is characterized by a unique combination of transcription factors (TFs) that promote a specific fate, while repressing fates of adjacent domains. The prdm12 TF is required for the expression of eng1b and the generation of V1 interneurons in the p1 domain, but the details of its function remain unclear. Methods We used CRISPR/Cas9 to generate the first germline mutants for prdm12 and employed this resource, together with classical luciferase reporter assays and co-immunoprecipitation experiments, to study prdm12b function in zebrafish. We also generated germline mutants for bhlhe22 and nkx6.1 to examine how these TFs act with prdm12b to control p1 formation. Results We find that prdm12b mutants lack eng1b expression in the p1 domain and also possess an abnormal touch-evoked escape response. Using luciferase reporter assays, we demonstrate that Prdm12b acts as a transcriptional repressor. We also show that the Bhlhe22 TF binds via the Prdm12b zinc finger domain to form a complex. However, bhlhe22 mutants display normal eng1b expression in the p1 domain. While prdm12 has been proposed to promote p1 fates by repressing expression of the nkx6.1 TF, we do not observe an expansion of the nkx6.1 domain upon loss of prdm12b function, nor is eng1b expression restored upon simultaneous loss of prdm12b and nkx6.1. Conclusions We conclude that prdm12b germline mutations produce a phenotype that is indistinguishable from that of morpholino-mediated loss of prdm12 function. In terms of prdm12b function, our results indicate that Prdm12b acts as transcriptional repressor and interacts with both EHMT2/G9a and Bhlhe22. However, bhlhe22 function is not required for eng1b expression in vivo, perhaps indicating that other bhlh genes can compensate during embryogenesis. Lastly, we do not find evidence for nkx6.1 and prdm12b acting as a repressive pair in formation of the p1 domain – suggesting that prdm12b is not solely required to repress non-p1 fates, but is specifically needed to promote p1 fates

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

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

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

Expression of GABA_A receptor α subunits at 24, 48, and 96 hours post fertilization.

<p>Expression of GABA_A receptor α subunits at 24, 48, and 96 hours post fertilization.</p

Expression of the eight GABA_A receptor α subunits in the developing zebrafish central nervous system

<div><p>GABA is a robust regulator of both developing and mature neural networks. It exerts many of its effects through GABA_A receptors, which are heteropentamers assembled from a large array of subunits encoded by distinct genes. In mammals, there are 19 different GABA_A subunit types, which are divided into the α, β, γ, δ, ε, π, θ and ρ subfamilies. The immense diversity of GABA_A receptors is not fully understood. However, it is known that specific isoforms, with their distinct biophysical properties and expression profiles, tune responses to GABA. Although larval zebrafish are well-established as a model system for neural circuit analysis, little is known about GABA_A receptors diversity and expression in this system. Here, using database analysis, we show that the zebrafish genome contains at least 23 subunits. All but the mammalian θ and ε subunits have at least one zebrafish ortholog, while five mammalian GABA_A receptor subunits have two zebrafish orthologs. Zebrafish contain one subunit, β4, which does not have a clear mammalian ortholog. Similar to mammalian GABA_A receptors, the zebrafish α subfamily is the largest and most diverse of the subfamilies. In zebrafish there are eight α subunits, and RNA <i>in situ</i> hybridization across early zebrafish development revealed that they demonstrate distinct patterns of expression in the brain, spinal cord, and retina. Some subunits were very broadly distributed, whereas others were restricted to small populations of cells. Subunit-specific expression patterns in zebrafish resembled were those found in frogs and rodents, which suggests that the roles of different GABA_A receptor isoforms are largely conserved among vertebrates. This study provides a platform to examine isoform specific roles of GABA_A receptors within zebrafish neural circuits and it highlights the potential of this system to better understand the remarkable heterogeneity of GABA_A receptors.</p></div

Phylogenetic analysis shows that the zebrafish GABA_A subunit gene family is similar in size, diversity, and organization to the mouse GABA_A subunit gene family.

<p>Amino acid sequence alignments were used to generate a consensus tree using 100 bootstrap replicates. The genes that encode the GABA_A subunits are shown at the tip of each branch and bootstrap proportions are shown at the branch points. The zebrafish and mouse GABA_A subunit sequences showed high amino acid identity and grouped into the α, β, γ, δ, π, and ρ subfamilies. Most mouse GABA_A subunits have a single zebrafish ortholog, while six mouse GABA_A subunits have two zebrafish orthologs, likely due to gene duplication.</p