A single genetic locus lengthens deer mouse burrows via motor pattern evolution.

The question of how evolution builds complex behaviors has long fascinated biologists. To address this question from a genetic perspective, we capitalize on variation in innate burrowing behavior between two sister species of Peromyscus mice: P. maniculatus that construct short, simple burrows and P. polionotus that uniquely construct long, elaborate burrows. We identify three regions of the genome associated with differences in burrow length and then narrow in on one large-effect 12-Mb locus on chromosome 4. By introgressing the P. polionotus allele into a P. maniculatus background, we demonstrate this locus, on its own, increases burrow length by 20%. Next, by recording mice digging in a transparent tube, we find this locus has specific effects on burrowing behavior. This locus does not affect time spent digging or latency to dig, but rather affects usage of only two of the primary digging behaviors that differ between the focal species: forelimb digging, which loosens substrate, and hindlimb kicking, which powerfully ejects substrate. This locus has an especially large effect on hindkicking, explaining 56% and 22% of interspecific differences in latency and proportion of hindkicks, respectively. Together, these data provide genetic support for the hierarchical organization of complex behaviors, offering evolution the opportunity to tinker with specific behavioral components.

cis-Regulatory changes in locomotor genes are associated with the evolution of burrowing behavior.

How evolution modifies complex, innate behaviors is largely unknown. Divergence in many morphological traits, and some behaviors, is linked to cis-regulatory changes in gene expression. Given this, we compare brain gene expression of two interfertile sister species of Peromyscus mice that show large and heritable differences in burrowing behavior. Species-level differential expression and allele-specific expression in F1 hybrids indicate a preponderance of cis-regulatory divergence, including many genes whose cis-regulation is affected by burrowing behavior. Genes related to locomotor coordination show the strongest signals of lineage-specific selection on burrowing-induced cis-regulatory changes. Furthermore, genetic markers closest to these candidate genes associate with variation in burrow shape in a genetic cross, suggesting an enrichment for loci affecting burrowing behavior near these candidate locomotor genes. Our results provide insight into how cis-regulated gene expression can depend on behavioral context and how this dynamic regulatory divergence between species may contribute to behavioral evolution.

Corrigendum: Commentary: Arginine Vasotocin Preprohormone Is Expressed in Surprising Regions of the Teleost Forebrain.

[This corrects the article on p. 63 in vol. 9, PMID: 29545774.].

Peromyscus burrowing: A model system for behavioral evolution.

A major challenge to understanding the genetic basis of complex behavioral evolution is the quantification of complex behaviors themselves. Deer mice of the genus Peromyscus vary in their burrowing behavior, which leaves behind a physical trace that is easily preserved and measured. Moreover, natural burrowing behaviors are recapitulated in the lab, and there is a strong heritable component. Here we discuss potential mechanisms driving variation in burrows with an emphasis on two sister species: P. maniculatus, which digs a simple, short burrow, and P. polionotus, which digs a long burrow with a complex architecture. A forward-genetic cross between these two species identified several genomic regions associated with burrow traits, suggesting this complex behavior has evolved in a modular fashion. Because burrow differences are most likely due to differences in behavioral circuits, Peromyscus burrowing offers an exciting opportunity to link genetic variation between natural populations to evolutionary changes in neural circuits.

Identification of prohormones and pituitary neuropeptides in the African cichlid, Astatotilapia burtoni.

BACKGROUND: Cichlid fishes have evolved remarkably diverse reproductive, social, and feeding behaviors. Cell-to-cell signaling molecules, notably neuropeptides and peptide hormones, are known to regulate these behaviors across vertebrates. This class of signaling molecules derives from prohormone genes that have undergone multiple duplications and losses in fishes. Whether and how subfunctionalization, neofunctionalization, or losses of neuropeptides and peptide hormones have contributed to fish behavioral diversity is largely unknown. Information on fish prohormones has been limited and is complicated by the whole genome duplication of the teleost ancestor. We combined bioinformatics, mass spectrometry-enabled peptidomics, and molecular techniques to identify the suite of neuropeptide prohormones and pituitary peptide products in Astatotilapia burtoni, a well-studied member of the diverse African cichlid clade. RESULTS: Utilizing the A. burtoni genome, we identified 148 prohormone genes, with 21 identified as a single copy and 39 with at least 2 duplicated copies. Retention of prohormone duplicates was therefore 41 %, which is markedly above previous reports for the genome-wide average in teleosts. Beyond the expected whole genome duplication, differences between cichlids and mammals can be attributed to gene loss in tetrapods and additional duplication after divergence. Mass spectrometric analysis of the pituitary identified 620 unique peptide sequences that were matched to 120 unique proteins. Finally, we used in situ hybridization to localize the expression of galanin, a prohormone with exceptional sequence divergence in cichlids, as well as the expression of a proopiomelanocortin, prohormone that has undergone an additional duplication in some bony fish lineages. CONCLUSION: We characterized the A. burtoni prohormone complement. Two thirds of prohormone families contain duplications either from the teleost whole genome duplication or a more recent duplication. Our bioinformatic and mass spectrometric findings provide information on a major vertebrate clade that will further our understanding of the functional ramifications of these prohormone losses, duplications, and sequence changes across vertebrate evolution. In the context of the cichlid radiation, these findings will also facilitate the exploration of neuropeptide and peptide hormone function in behavioral diversity both within A. burtoni and across cichlid and other fish species.

Electrical synapses connect a network of gonadotropin releasing hormone neurons in a cichlid fish.

Initiating and regulating vertebrate reproduction requires pulsatile release of gonadotropin-releasing hormone (GnRH1) from the hypothalamus. Coordinated GnRH1 release, not simply elevated absolute levels, effects the release of pituitary gonadotropins that drive steroid production in the gonads. However, the mechanisms underlying synchronization of GnRH1 neurons are unknown. Control of synchronicity by gap junctions between GnRH1 neurons has been proposed but not previously found. We recorded simultaneously from pairs of transgenically labeled GnRH1 neurons in adult male Astatotilapia burtoni cichlid fish. We report that GnRH1 neurons are strongly and uniformly interconnected by electrical synapses that can drive spiking in connected cells and can be reversibly blocked by meclofenamic acid. Our results suggest that electrical synapses could promote coordinated spike firing in a cellular assemblage of GnRH1 neurons to produce the pulsatile output necessary for activation of the pituitary and reproduction.

Ancient origins and evolutionary conservation of intracellular and neural signaling pathways engaged by the leptin receptor.

In mammals, leptin acts on leptin receptor (LepR) -expressing neurons in the brain to suppress food intake and stimulate whole-body metabolism. A similar action of leptin on food intake has been reported in the frog Xenopus laevis and in several bony fishes. However, the intracellular signaling and neural pathways by which leptin regulates energy balance have not been investigated outside of mammals. Using reporter assays and site-directed mutagenesis we show that the frog LepR signals via signal transducer and activator of transcription (STAT) 3 and STAT5 through evolutionarily conserved tyrosine residues in the LepR cytoplasmic domain. In situ hybridization histochemistry for LepR mRNA in brain and pituitary showed strong expression in the magno- and parvocellular divisions of the anterior preoptic area (homologous to the mammalian paraventricular nucleus), the suprachiasmatic nucleus, ventral hypothalamus, and pars intermedia and pars distalis of the anterior pituitary. Leptin injection increased phosphorylated STAT3 immunoreactivity in LepR mRNA-positive cells, and induced socs3 and pomc mRNAs. Microarray analysis of preoptic area/hypothalamus/pituitary 2 hours after leptin injection identified leptin-regulated genes that included c-fos, a known leptin-activated gene; pituitary follicle-stimulating hormone subunit ß, suggesting an important role for leptin in the reproductive axis of frogs; and B-cell translocation factor 2, which has important functions in neurogenesis. Our findings support that the intracellular signaling pathways and neural substrates that mediate leptin actions on energy balance were present in the common ancestor of modern amphibians and amniotes and have been conserved over 350 million years of evolutionary time.

Tol2-mediated generation of a transgenic haplochromine cichlid, Astatotilapia burtoni.

Cichlid fishes represent one of the most species-rich and rapid radiations of a vertebrate family. These ~2200 species, predominantly found in the East African Great Lakes, exhibit dramatic differences in anatomy, physiology, and behavior. However, the genetic bases for this radiation, and for the control of their divergent traits, are unknown. A flood of genomic and transcriptomic data promises to suggest mechanisms underlying the diversity, but transgenic technology will be needed to rigorously test the hypotheses generated. Here we demonstrate the successful use of the Tol2 transposon system to generate transgenic Astatotilapia burtoni, a haplochromine cichlid from Lake Tanganyika, carrying the GFP transgene under the control of the ubiquitous EF1α promoter. The transgene integrates into the genome, is successfully passed through the germline, and the widespread GFP expression pattern is stable across siblings and multiple generations. The stable inheritance and expression patterns indicate that the Tol2 system can be applied to generate A. burtoni transgenic lines. Transgenesis has proven to be a powerful technology for manipulating genes and cells in other model organisms and we anticipate that transgenic A. burtoni and other cichlids will be used to test the mechanisms underlying behavior and speciation.

Glucocorticoid signaling in myeloid cells worsens acute CNS injury and inflammation.

Glucocorticoid stress hormones (GCs) are well known for being anti-inflammatory, but some reports suggest that GCs can also augment aspects of inflammation during acute brain injury. Because the GC receptor (GR) is ubiquitously expressed throughout the brain, it is difficult to know which cell types might mediate these unusual "proinflammatory" GC actions. We examined this with cell type-specific deletion or overexpression of GR in mice experiencing seizure or ischemia. Counter to their classical anti-inflammatory actions, GR signaling in myeloid cells increased Iba-1 and CD68 staining as well as nuclear p65 levels in the injured tissue. GCs also reduced levels of occludin, claudin 5, and caveolin 1, proteins central to blood-brain-barrier integrity; these effects required GR in endothelial cells. Finally, GCs compromised neuron survival, an effect mediated by GR in myeloid and endothelial cells to a greater extent than by neuronal GR.

The role of hepatic invariant NKT cells in systemic/local inflammation and mortality during polymicrobial septic shock.

NKT cells have been described as innate regulatory cells because of their rapid response to conserved glycolipids presented on CD1d via their invariant TCR. However, little is known about the contribution of the hepatic NKT cell to the development of a local and/or systemic immune response to acute septic challenge (cecal ligation and puncture (CLP)). We found not only that mice deficient in invariant NKT cells (Jalpha18(-/-)) had a marked attenuation in CLP-induced mortality, but also exhibited an oblation of the systemic inflammatory response (with little effect on splenic/peritoneal immune responsiveness). Flow cytometric data indicated that following CLP, there was a marked decline in the percentage of CD3(+)alpha-galactosylceramide CD1d tetramer(+) cells in the mouse C57BL/6J and BALB/c liver nonparenchymal cell population. This was associated with the marked activation of these cells (increased expression of CD69 and CD25) as well as a rise in the frequency of NKT cells positive for both Th1 and Th2 intracellular cytokines. In this respect, when mice were pretreated in vivo with anti-CD1d-blocking Ab, we observed not only that this inhibited the systemic rise of IL-6 and IL-10 levels in septic mice and improved overall septic survival, but that the CLP-induced changes in liver macrophage IL-6 and IL-10 expressions were inversely effected by this treatment. Together, these findings suggest that the activation of hepatic invariant NKT cells plays a critical role in regulating the innate immune/systemic inflammatory response and survival in a model of acute septic shock.