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Viviparous rockfishes (Sebastes spp., family Scorpaenidae) mate and store sperm in the ovaries for several months prior to fertilization, as oocytes develop for the parturition season. Although multiple paternity has been documented in single-brooding rockfishes, paternity in consecutive broods of multiple-brooding species has not been studied. Analyses of multilocus microsatellite genotypes in both residual larvae left in the ovary from a previous parturition and upcoming fertilized broods in the same ovary demonstrated evidence of the same sires in consecutive broods in chilipepper (Sebastes goodei) and speckled (Sebastes ovalis) rockfishes. One S. goodei mother showed evidence of multiple paternity from the same two sires in both consecutive broods. The ability to retain sperm, even after a parturition event, for use in subsequent broods, confers an advantage to ensure fertilization and allows for extension of the parturition season. This life-history strategy provides a bet-hedging advantage in the California Current system, an environmentally dynamic ecosystem where larval survivorship and subsequent recruitment to adult populations can vary temporally by orders of magnitude.

Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.

Fluid intake is an essential innate behaviour that is mainly caused by two distinct types of thirst1-3. Increased blood osmolality induces osmotic thirst that drives animals to consume pure water. Conversely, the loss of body fluid induces hypovolaemic thirst, in which animals seek both water and minerals (salts) to recover blood volume. Circumventricular organs in the lamina terminalis are critical sites for sensing both types of thirst-inducing stimulus4-6. However, how different thirst modalities are encoded in the brain remains unknown. Here we employed stimulus-to-cell-type mapping using single-cell RNA sequencing to identify the cellular substrates that underlie distinct types of thirst. These studies revealed diverse types of excitatory and inhibitory neuron in each circumventricular organ structure. We show that unique combinations of these neuron types are activated under osmotic and hypovolaemic stresses. These results elucidate the cellular logic that underlies distinct thirst modalities. Furthermore, optogenetic gain of function in thirst-modality-specific cell types recapitulated water-specific and non-specific fluid appetite caused by the two distinct dipsogenic stimuli. Together, these results show that thirst is a multimodal physiological state, and that different thirst states are mediated by specific neuron types in the mammalian brain.

A warming climate poses a fundamental problem for embryos that develop within eggs because their demand for oxygen (O2) increases much more rapidly with temperature than their capacity for supply, which is constrained by diffusion across the egg surface. Thus, as temperatures rise, eggs may experience O2 limitation due to an imbalance between O2 supply and demand. Here, we formulate a mathematical model of O2 limitation and experimentally test whether this mechanism underlies the upper thermal tolerance in large aquatic eggs. Using Chinook salmon (Oncorhynchus tshawytscha) as a model system, we show that the thermal tolerance of eggs varies systematically with features of the organism and environment. Importantly, this variation can be precisely predicted by the degree to which these features shift the balance between O2 supply and demand. Equipped with this mechanistic understanding, we predict and experimentally confirm that the thermal tolerance of these embryos in their natural habitat is substantially lower than expected from laboratory experiments performed under normoxia. More broadly, our biophysical model of O2 limitation provides a mechanistic explanation for the elevated thermal sensitivity of fish embryos relative to other life stages, global patterns in egg size and the extreme fecundity of large teleosts.

The neocortex is functionally organized into layers. Layer four receives the densest bottom up sensory inputs, while layers 2/3 and 5 receive top down inputs that may convey predictive information. A subset of cortical somatostatin (SST) neurons, the Martinotti cells, gate top down input by inhibiting the apical dendrites of pyramidal cells in layers 2/3 and 5, but it is unknown whether an analogous inhibitory mechanism controls activity in layer 4. Using high precision circuit mapping, in vivo optogenetic perturbations, and single cell transcriptional profiling, we reveal complementary circuits in the mouse barrel cortex involving genetically distinct SST subtypes that specifically and reciprocally interconnect with excitatory cells in different layers: Martinotti cells connect with layers 2/3 and 5, whereas non-Martinotti cells connect with layer 4. By enforcing layer-specific inhibition, these parallel SST subnetworks could independently regulate the balance between bottom up and top down input.

To define a complete catalog of the genes that are activated during mouse sclerotome formation, we sequenced RNA from embryonic mouse tissue directed to form sclerotome in culture. In addition to well-known early markers of sclerotome, such as Pax1, Pax9, and the Bapx2/Nkx3-2 homolog Nkx3-1, the long-noncoding RNA PEAT (Pax1 enhancer antisense transcript) was induced in sclerotome-directed samples. Strikingly, PEAT is located just upstream of the Pax1 gene. Using CRISPR/Cas9, we generated a mouse line bearing a complete deletion of the PEAT-transcribed unit. RNA-seq on PEAT mutant embryos showed that loss of PEAT modestly increases bone morphogenetic protein target gene expression and also elevates the expression of a large subset of ribosomal protein mRNAs.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

When compared to single mutants for Follistatin or Noggin, we find that double mutants display a dramatic further reduction in trunk cartilage formation, particularly in the vertebral bodies and proximal ribs. Consistent with these observations, expression of the early sclerotome markers Pax1 and Uncx is diminished in Noggin;Follistatin compound mutants. In contrast, Sim1 expression expands medially in double mutants. As the onset of Follistatin expression coincides with sclerotome specification, we argue that the effect of Follistatin occurs after sclerotome induction. We hypothesize that Follistatin aids in maintaining proper somite size, and consequently sclerotome progenitor numbers, by preventing paraxial mesoderm from adopting an intermediate/lateral plate mesodermal fate in the Noggin-deficient state.

The effects of two anorectic drugs, dexfenfluramine and phentermine, on food intake under different food-access conditions were examined. Experiment 1 compared the effects of these drugs on food intake under a progressive-ratio (PR) schedule and free-access conditions. Dexfenfluramine decreased food intake under both conditions, but the doses required to decrease intake under free-access conditions were higher than those required to reduce intake under the PR condition. Intermediate doses of phentermine sometimes increased breaking points, and higher doses decreased them. Phentermine decreased food intake at the same doses under both access conditions. Thus the potency of dexfenfluramine, but not phentermine, to decrease food-maintained behavior depended upon the food-access condition. Experiment 2 used a novel mixed progressive-ratio schedule of food delivery to study the duration of drug effects. Sessions consisted of five components separated by 3-hr timeouts. The ratio requirement reset at the beginning of each component and a new breaking point was obtained. Both dexfenfluramine and phentermine dose-dependently decreased breaking points early in the session. In some rats, compensatory increases in breaking point were observed. That is, breaking points later in the session increased over control levels, resulting in no change in the total number of food pellets earned for the session compared to control. The present findings suggest that the effects of some anorectic drugs depend upon the access conditions for food; increasing the effort to obtain food may enhance their ability to decrease food-maintained behavior. (Contains 1 table and 7 figures.)

Reinforcement schedules are some of the most potent tools in experimental psychology as they both establish and maintain lawful behavioral patterns and also may determine to a large degree how other variables will influence behavior. A study involving pigeons was conducted to describe in detail the effects of step size and break-point criterion on breaking points reached by pigeons on an arithmetically increasing progressive ratio schedule of food delivery. The results are discussed.

The effects of two anorectic drugs, dexfenfluramine and phentermine, on food intake under different food-access are examined. The effects of some anorectic drugs depend upon the access conditions for food and increasing the effort to obtain food may enhance their ability to decrease food-maintain behavior.

To define a complete catalog of the genes that are activated during mouse sclerotome formation, we sequenced RNA from embryonic mouse tissue directed to form sclerotome in culture. In addition to well-known early markers of sclerotome, such as Pax1, Pax9, and the Bapx2/Nkx3-2 homolog Nkx3-1, the long-noncoding RNA PEAT (Pax1 enhancer antisense transcript) was induced in sclerotome-directed samples. Strikingly, PEAT is located just upstream of the Pax1 gene. Using CRISPR/Cas9, we generated a mouse line bearing a complete deletion of the PEAT-transcribed unit. RNA-seq on PEAT mutant embryos showed that loss of PEAT modestly increases bone morphogenetic protein target gene expression and also elevates the expression of a large subset of ribosomal protein mRNAs. [ABSTRACT FROM AUTHOR]