When faced with starvation, the bacterium transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σ is exclusively activated in the smaller daughter cell. Compartment-specific activation of σ requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σ in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Persistent internal states are important for maintaining survival-promoting behaviors, such as aggression. In female Drosophila melanogaster, we have previously shown that individually activating either aIPg or pC1d cell types can induce aggression. Here we investigate further the individual roles of these cholinergic, sexually dimorphic cell types, and the reciprocal connections between them, in generating a persistent aggressive internal state. We find that a brief 30-second optogenetic stimulation of aIPg neurons was sufficient to promote an aggressive internal state lasting at least 10 minutes, whereas similar stimulation of pC1d neurons did not. While we previously showed that stimulation of pC1e alone does not evoke aggression, persistent behavior could be promoted through simultaneous stimulation of pC1d and pC1e, suggesting an unexpected synergy of these cell types in establishing a persistent aggressive state. Neither aIPg nor pC1d show persistent neuronal activity themselves, implying that the persistent internal state is maintained by other mechanisms. Moreover, inactivation of pC1d did not significantly reduce aIPg-evoked persistent aggression arguing that the aggressive state did not depend on pC1d-aIPg recurrent connectivity. Our results suggest the need for alternative models to explain persistent female aggression.

Many tools are available to analyse genomes but are often challenging to use in a cell type-specific context. We have developed a method similar to the isolation of nuclei tagged in a specific cell type (INTACT) technique [Deal,R.B. and Henikoff,S. (2010) A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell, 18, 1030-1040; Steiner,F.A., Talbert,P.B., Kasinathan,S., Deal,R.B. and Henikoff,S. (2012) Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res., doi:10.1101/gr.131748.111], first developed in plants, for use in Drosophila neurons. We profile gene expression and histone modifications in Kenyon cells and octopaminergic neurons in the adult brain. In addition to recovering known gene expression differences, we also observe significant cell type-specific chromatin modifications. In particular, a small subset of differentially expressed genes exhibits a striking anti-correlation between repressive and activating histone modifications. These genes are enriched for transcription factors, recovering those known to regulate mushroom body identity and predicting analogous regulators of octopaminergic neurons. Our results suggest that applying INTACT to specific neuronal populations can illuminate the transcriptional regulatory networks that underlie neuronal cell identity.

N-Methyl-D-aspartate receptors (NMDA-Rs) are ion channels that are important for synaptic plasticity, which is involved in learning and drug addiction. We show enzymatic targeting of an NMDA-R antagonist, MK801, to a molecularly defined neuronal population with the cell-type-selectivity of genetic methods and the temporal control of pharmacology. We find that NMDA-Rs on dopamine neurons are necessary for cocaine-induced synaptic potentiation, demonstrating that cell type-specific pharmacology can be used to dissect signaling pathways within complex brain circuits.

Molecular and cellular processes in neurons are critical for sensing and responding to energy deficit states, such as during weight-loss. AGRP neurons are a key hypothalamic population that is activated during energy deficit and increases appetite and weight-gain. Cell type-specific transcriptomics can be used to identify pathways that counteract weight-loss, and here we report high-quality gene expression profiles of AGRP neurons from well-fed and food-deprived young adult mice. For comparison, we also analyzed POMC neurons, an intermingled population that suppresses appetite and body weight. We find that AGRP neurons are considerably more sensitive to energy deficit than POMC neurons. Furthermore, we identify cell type-specific pathways involving endoplasmic reticulum-stress, circadian signaling, ion channels, neuropeptides, and receptors. Combined with methods to validate and manipulate these pathways, this resource greatly expands molecular insight into neuronal regulation of body weight, and may be useful for devising therapeutic strategies for obesity and eating disorders.

The striatum shows general topographic organization and regional differences in behavioral functions. How corticostriatal topography differs across cortical areas and cell types to support these distinct functions is unclear. This study contrasted corticostriatal projections from two layer 5 cell types, intratelencephalic (IT-type) and pyramidal tract (PT-type) neurons, using viral vectors expressing fluorescent reporters in Cre-driver mice. Long-range corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing somatotopic specificity as injections move from sensory to motor and frontal areas. Somatotopic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher somatotopic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. Thus, as predicted by a long-standing basal ganglia model, corticostriatal projections of interconnected cortical areas form parallel circuits in basal ganglia-thalamus-cortex loops.

Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism's internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. Here, we identify a population of sexually dimorphic aIPg neurons in the adult central brain whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated aIPg and pC1d neurons as core nodes regulating female aggression. Connectomic analysis demonstrated that aIPg neurons and pC1d are interconnected and suggest that aIPg neurons may exert part of their effect by gating the flow of visual information to descending neurons. Our work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation.

Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.