Stimulus-specific hypothalamic encoding of a persistent defensive state.

Persistent neural activity in cortical, hippocampal, and motor networks has been described as mediating working memory for transiently encountered stimuli1,2. Internal emotional states, such as fear, also persist following exposure to an inciting stimulus3, but it is unclear whether slow neural dynamics are involved in this process. Neurons in the dorsomedial and central subdivisions of the ventromedial hypothalamus (VMHdm/c) that express the nuclear receptor protein NR5A1 (also known as SF1) are necessary for defensive responses to predators in mice4-7. Optogenetic activation of these neurons, referred to here as VMHdmSF1 neurons, elicits defensive behaviours that outlast stimulation5,8, which suggests the induction of a persistent internal state of fear or anxiety. Here we show that in response to naturalistic threatening stimuli, VMHdmSF1 neurons in mice exhibit activity that lasts for many tens of seconds. This persistent activity was correlated with, and required for, persistent defensive behaviour in an open-field assay, and depended on neurotransmitter release from VMHdmSF1 neurons. Stimulation and calcium imaging in acute slices showed that there is local excitatory connectivity between VMHdmSF1 neurons. Microendoscopic calcium imaging of VMHdmSF1 neurons revealed that persistent activity at the population level reflects heterogeneous dynamics among individual cells. Unexpectedly, distinct but overlapping VMHdmSF1 subpopulations were persistently activated by different modalities of threatening stimulus. Computational modelling suggests that neither recurrent excitation nor slow-acting neuromodulators alone can account for persistent activity that maintains stimulus identity. Our results show that stimulus-specific slow neural dynamics in the hypothalamus, on a time scale orders of magnitude longer than that of working memory in the cortex9,10, contribute to a persistent emotional state.

Genetic dissection of an amygdala microcircuit that gates conditioned fear.

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ(+) neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ(-) neurons in CEl. Electrical silencing of PKC-δ(+) neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called CEl(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.

Transcriptional regulation of Drosophila gonad formation.

The formation of the Drosophila embryonic gonad, involving the fusion of clusters of somatic gonadal precursor cells (SGPs) and their ensheathment of germ cells, provides a simple and genetically tractable model for the interplay between cells during organ formation. In a screen for mutants affecting gonad formation we identified a SGP cell autonomous role for Midline (Mid) and Longitudinals lacking (Lola). These transcriptional factors are required for multiple aspects of SGP behaviour including SGP cluster fusion, germ cell ensheathment and gonad compaction. The lola locus encodes more than 25 differentially spliced isoforms and we have identified an isoform specific requirement for lola in the gonad which is distinct from that in nervous system development. Mid and Lola work in parallel in gonad formation and surprisingly Mid overexpression in a lola background leads to additional SGPs at the expense of fat body cells. Our findings support the idea that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification or behaviour, they can also interact to impinge on both processes.

Lipid phosphate phosphatase activity regulates dispersal and bilateral sorting of embryonic germ cells in Drosophila.

In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. We find that wunens mediate germ cell-germ cell repulsion and that this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. We propose that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, we find that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. We suggest that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells.

Hedgehog does not guide migrating Drosophila germ cells.

In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies.

A noncoding RNA is required for the repression of RNApolII-dependent transcription in primordial germ cells.

RNApolII-dependent transcription is repressed in primordial germ cells of many animals during early development and is thought to be important for maintenance of germline fate by preventing somatic differentiation. Germ cell transcriptional repression occurs concurrently with inhibition of phosphorylation in the carboxy-terminal domain (CTD) of RNApolII, as well as with chromatin remodeling. The precise mechanisms involved are unknown. Here, we present evidence that a noncoding RNA transcribed by the gene polar granule component (pgc) regulates transcriptional repression in Drosophila germ cells. Germ cells lacking pgc RNA express genes important for differentiation of nearby somatic cells and show premature phosphorylation of RNApolII. We further show that germ cells lacking pgc show increased levels of K4, but not K9 histone H3 methylation, and that the chromatin remodeling Swi/Snf complex is required for a second stage in germ cell transcriptional repression. We propose that a noncoding RNA controls transcription in early germ cells by blocking the transition from preinitiation to transcriptional elongation. We further show that repression of somatic differentiation signals mediated by the Torso receptor-tyrosine kinase is important for germline development.

Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

Ventromedial hypothalamic neurons control a defensive emotion state.

Defensive behaviors reflect underlying emotion states, such as fear. The hypothalamus plays a role in such behaviors, but prevailing textbook views depict it as an effector of upstream emotion centers, such as the amygdala, rather than as an emotion center itself. We used optogenetic manipulations to probe the function of a specific hypothalamic cell type that mediates innate defensive responses. These neurons are sufficient to drive multiple defensive actions, and required for defensive behaviors in diverse contexts. The behavioral consequences of activating these neurons, moreover, exhibit properties characteristic of emotion states in general, including scalability, (negative) valence, generalization and persistence. Importantly, these neurons can also condition learned defensive behavior, further refuting long-standing claims that the hypothalamus is unable to support emotional learning and therefore is not an emotion center. These data indicate that the hypothalamus plays an integral role to instantiate emotion states, and is not simply a passive effector of upstream emotion centers.

The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.

Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction.

Tre1 GPCR initiates germ cell transepithelial migration by regulating Drosophila melanogaster E-cadherin.

Despite significant progress in identifying the guidance pathways that control cell migration, how a cell starts to move within an intact organism, acquires motility, and loses contact with its neighbors is poorly understood. We show that activation of the G protein-coupled receptor (GPCR) trapped in endoderm 1 (Tre1) directs the redistribution of the G protein Gbeta as well as adherens junction proteins and Rho guanosine triphosphatase from the cell periphery to the lagging tail of germ cells at the onset of Drosophila melanogaster germ cell migration. Subsequently, Tre1 activity triggers germ cell dispersal and orients them toward the midgut for directed transepithelial migration. A transition toward invasive migration is also a prerequisite for metastasis formation, which often correlates with down-regulation of adhesion proteins. We show that uniform down-regulation of E-cadherin causes germ cell dispersal but is not sufficient for transepithelial migration in the absence of Tre1. Our findings therefore suggest a new mechanism for GPCR function that links cell polarity, modulation of cell adhesion, and invasion.

In vivo migration: a germ cell perspective.

The basic concepts of the molecular machinery that mediates cell migration have been gleaned from cell culture systems. However, the three-dimensional environment within an organism presents migrating cells with a much greater challenge. They must move between and among other cells while interpreting multiple attractive and repulsive cues to choose their proper path. They must coordinate their cell adhesion with their surroundings and know when to start and stop moving. New insights into the control of these remaining mysteries have emerged from genetic dissection and live imaging of germ cell migration in Drosophila, zebrafish, and mouse embryos. In this review, we first describe germ cell migration in cellular and mechanistic detail in these different model systems. We then compare these systems to highlight the emerging principles. Finally, we contrast the migration of germ cells with that of immune and cancer cells to outline the conserved and different mechanisms.

Mixer/Bon and FoxH1/Sur have overlapping and divergent roles in Nodal signaling and mesendoderm induction.

Transcription factors belonging to the FoxH1 and Mixer families are required for facets of Nodal signaling during vertebrate mesendoderm induction. Here, we analyze whether zebrafish proteins related to FoxH1 [Schmalspur (Sur)] and Mixer [Bonnie and clyde (Bon)] act within or downstream of the Nodal signaling pathway, test whether these two factors have additive or overlapping activities, and determine whether FoxH1/Sur and Mixer/Bon can account for all Nodal signaling during embryogenesis. We find that sur expression is independent of Nodal signaling and that bon is expressed in the absence of Nodal signaling but requires Nodal signaling and Sur for enhanced, maintained expression. These results and the association of FoxH1 and Mixer/Bon with phosphorylated Smad2 support a role for these factors as components of the Nodal signaling pathway. In contrast to the relatively mild defects observed in single mutants, loss of both bon and sur results in a severe phenotype characterized by absence of prechordal plate, cardiac mesoderm, endoderm and ventral neuroectoderm. Analysis of Nodal-regulated proteins reveals that Bon and Sur have both distinct and overlapping regulatory roles. Some genes are regulated by both Bon and Sur, and others by either Bon or Sur. Complete loss of Nodal signaling results in a more severe phenotype than loss of both Bon and Sur, indicating that additional Smad-associated transcription factors remain to be identified that act as components of the Nodal signaling pathway.

The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival.

In mouse embryos, germ cells arise during gastrulation and migrate to the early gonad. First, they emerge from the primitive streak into the region of the endoderm that forms the hindgut. Later in development, a second phase of migration takes place in which they migrate out of the gut to the genital ridges. There, they co-assemble with somatic cells to form the gonad. In vitro studies in the mouse, and genetic studies in other organisms, suggest that at least part of this process is in response to secreted signals from other tissues. Recent genetic evidence in zebrafish has shown that the interaction between stromal cell-derived factor 1 (SDF1) and its G-protein-coupled receptor CXCR4, already known to control many types of normal and pathological cell migrations, is also required for the normal migration of primordial germ cells. We show that in the mouse, germ cell migration and survival requires the SDF1/CXCR4 interaction. First, migrating germ cells express CXCR4, whilst the body wall mesenchyme and genital ridges express the ligand SDF1. Second, the addition of exogenous SDF1 to living embryo cultures causes aberrant germ cell migration from the gut. Third, germ cells in embryos carrying targeted mutations in CXCR4 do not colonize the gonad normally. However, at earlier stages in the hindgut, germ cells are unaffected in CXCR4(-/-) embryos. Germ cell counts at different stages suggest that SDF1/CXCR4 interaction also mediates germ cell survival. These results show that the SDF1/CXCR4 interaction is specifically required for the colonization of the gonads by primordial germ cells, but not for earlier stages in germ cell migration. This demonstrates a high degree of evolutionary conservation of part of the mechanism, but also an area of evolutionary divergence.