The body-brain axis is emerging as a principal conductor of organismal physiology. It senses and controls organ function 1,2 , metabolism 3 and nutritional state 4-6 . Here we show that a peripheral immune insult strongly activates the body-brain axis to regulate immune responses. We demonstrate that pro-inflammatory and anti-inflammatory cytokines communicate with distinct populations of vagal neurons to inform the brain of an emerging inflammatory response. In turn, the brain tightly modulates the course of the peripheral immune response. Genetic silencing of this body-brain circuit produced unregulated and out-of-control inflammatory responses. By contrast, activating, rather than silencing, this circuit affords neural control of immune responses. We used single-cell RNA sequencing, combined with functional imaging, to identify the circuit components of this neuroimmune axis, and showed that its selective manipulation can effectively suppress the pro-inflammatory response while enhancing an anti-inflammatory state. The brain-evoked transformation of the course of an immune response offers new possibilities in the modulation of a wide range of immune disorders, from autoimmune diseases to cytokine storm and shock. The body-brain axis regulates body pro-inflammatory and anti-inflammatory immune responses following an immune insult.

Understanding the neural mechanisms underlying sleep state transitions is a fundamental goal of neurobiology and important for the development of new treatments for insomnia and other sleep disorders. Yet, brain circuits controlling this process remain poorly understood. Here we identify a population of sleep-active glutamatergic neurons in the ventrolateral medulla (VLM) that project to the preoptic area (POA), a prominent sleep-promoting region, in mice. Microendoscopic calcium imaging demonstrate that these VLM glutamatergic neurons display increased activity during the transitions from wakefulness to Non-Rapid Eye Movement (NREM) sleep. Chemogenetic silencing of POA-projecting VLM neurons suppresses NREM sleep, whereas chemogenetic activation of these neurons promotes NREM sleep. Moreover, we show that optogenetic activation of VLM glutamatergic neurons or their projections in the POA initiates NREM sleep in awake mice. Together, our findings uncover an excitatory brainstem-hypothalamic circuit that controls the wake-sleep transitions.

Hardwired circuits encoding innate responses have emerged as an essential feature of the mammalian brain. Sweet and bitter evoke opposing predetermined behaviors. Sweet drives appetitive responses and consumption of energy-rich food sources, whereas bitter prevents ingestion of toxic chemicals. Here we identified and characterized the neurons in the brainstem that transmit sweet and bitter signals from the tongue to the cortex. Next we examined how the brain modulates this hardwired circuit to control taste behaviors. We dissect the basis for bitter-evoked suppression of sweet taste and show that the taste cortex and amygdala exert strong positive and negative feedback onto incoming bitter and sweet signals in the brainstem. Finally we demonstrate that blocking the feedback markedly alters responses to ethologically relevant taste stimuli. These results illustrate how hardwired circuits can be finely regulated by top-down control and reveal the neural basis of an indispensable behavioral response for all animals.

SUMMARYSleep is a ubiquitous behavior in animal species. Yet, brain circuits controlling sleep remain poorly understood. Previous studies have identified several brain structures that promote sleep, but whether these structures are involved in sleep initiation or sleep maintenance remains largely unknown. Here we identified a population of glutamatergic neurons in the medulla that project to the preoptic area (POA), a prominent sleep-promoting region. Chemogenetic silencing of POA-projecting medulla neurons disrupts the transitions from wakefulness to Non-Rapid Eye Movement (NREM) sleep, whereas chemogenetic activation of these neurons promotes NREM sleep. Moreover, we show that optogenetic activation of medulla glutamatergic neurons or their projections in the POA reliably initiates long-lasting NREM sleep in awake mice. Together, our findings uncover a novel excitatory brainstem-hypothalamic circuit that controls the wake-sleep transitions.

Neutrophils play an essential role in the innate immune defense system in vertebrates. During hematopoiesis, the full function of neutrophils involves maturation of granules and related enzymes. Yet, transcription regulators that promote neutrophil maturation remain largely undefined. Here, two hematopoiesis-defective zebrafish mutants, runx1(w84x) and c-myb(hkz3), were used to investigate the in vivo roles of Runx1 in cooperation with c-Myb in regulating neutrophil maturation. Loss of runx1 impairs primitive neutrophil development. Additional regulation of c-myb(+/-) and c-myb(-/-) induces a more severe phenotypes suggesting a synergistic genetic interaction between c-myb and runx1 in neutrophil maturation. Further studies revealed that the two transcription factors act cooperatively to control neutrophil maturation processes via transactivating a series of neutrophil maturation-related genes. These data reveal the in vivo roles of Runx1 in regulating primitive neutrophil maturation while also indicating a novel genetic and molecular orchestration of Runx1 and c-Myb in myeloid cell development. The study will provide new evidence on the regulation of neutrophil maturation during hematopoiesis.

The taste of sugar is one of the most basic sensory percepts for humans and other animals. Animals can develop a strong preference for sugar even if they lack sweet taste receptors, indicating a mechanism independent of taste1–3. Here we examined the neural basis for sugar preference and demonstrate that a population of neurons in the vagal ganglia and brainstem are activated via the gut–brain axis to create preference for sugar. These neurons are stimulated in response to sugar but not artificial sweeteners, and are activated by direct delivery of sugar to the gut. Using functional imaging we monitored activity of the gut–brain axis, and identified the vagal neurons activated by intestinal delivery of glucose. Next, we engineered mice in which synaptic activity in this gut-to-brain circuit was genetically silenced, and prevented the development of behavioural preference for sugar. Moreover, we show that co-opting this circuit by chemogenetic activation can create preferences to otherwise less-preferred stimuli. Together, these findings reveal a gut-to-brain post-ingestive sugar-sensing pathway critical for the development of sugar preference. In addition, they explain the neural basis for differences in the behavioural effects of sweeteners versus sugar, and uncover an essential circuit underlying the highly appetitive effects of sugar. Experiments in mice show that a population of neurons in the vagal ganglia respond to the presence of glucose in the gut and connect to neurons in the brainstem, revealing the circuit that underlies the neural basis for the behavioural preference for sugar.

The ability to sense sour provides an important sensory signal to prevent the ingestion of unripe, spoiled, or fermented foods. Taste and somatosensory receptors in the oral cavity trigger aversive behaviors in response to acid stimuli. Here, we show that the ion channel Otopetrin-1, a proton-selective channel normally involved in the sensation of gravity in the vestibular system, is essential for sour sensing in the taste system. We demonstrate that knockout of Otop1 eliminates acid responses from sour-sensing taste receptor cells (TRCs). In addition, we show that mice engineered to express otopetrin-1 in sweet TRCs have sweet cells that also respond to sour stimuli. Next, we genetically identified the taste ganglion neurons mediating each of the five basic taste qualities and demonstrate that sour taste uses its own dedicated labeled line from TRCs in the tongue to finely tuned taste neurons in the brain to trigger aversive behaviors.

Neutrophils are the key effectors for generating innate immunity in response to pathogenic infection and tissue injury in vertebrates. Dysregulation of neutrophil development and function is known to associate with various human disorders. Yet, the genetic network that orchestrates lineage commitment, differentiation, and maturation of neutrophils remains incompletely defined. Here, we present an in vivo study to delineate the genetic program underlying neutrophil development during zebrafish embryonic myelopoiesis. We show that loss of c-Myb function has no effect on macrophages but severely impairs neutrophil terminal differentiation, resulting in the accumulation of neutrophils with unsegmented nuclei and scant granule. This neutrophilic defect, which resembles the neutrophil-specific granule deficiency (SGD) caused by the mutations in CCAAT/enhancer-binding protein ε (C/EBPε) in humans, is attributed, at least in part, to the downregulation of the granule protein transcription. Likewise, genetic inactivation of Cebp1, the zebrafish functional homolog of mammalian C/EBPε, also leads to a similar SGD-like phenotype in zebrafish. Genetic epistasis and biochemical analysis further reveals that c-Myb and Cebp1 act in parallel and cooperatively to control neutrophil differentiation by directly regulating granule protein gene transcription. Our study indicates that c-MYB is an intrinsic master regulator for neutrophil terminal differentiation and a potential target in SGD patients.

State Key Laboratory of Molecular Neuroscience, Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, People’s Republic of China; Key Laboratory of Zebrafish Modeling and Drug Screening for Human Diseases of Guangdong Higher Education Institutes, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, People’s Republic of China; and The Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, State Key Laboratory Breeding Base of Eco-Environments and Bio-Resources of the Three Gorges Area, School of Life Science, Southwest University, Chongqing, People’s Republic of China

Taste is responsible for evaluating the nutritious content of food, guiding essential appetitive behaviours, preventing the ingestion of toxic substances, and helping to ensure the maintenance of a healthy diet. Sweet and bitter are two of the most salient sensory percepts for humans and other animals; sweet taste allows the identification of energy-rich nutrients whereas bitter warns against the intake of potentially noxious chemicals. In mammals, information from taste receptor cells in the tongue is transmitted through multiple neural stations to the primary gustatory cortex in the brain. Recent imaging studies have shown that sweet and bitter are represented in the primary gustatory cortex by neurons organized in a spatial map, with each taste quality encoded by distinct cortical fields. Here we demonstrate that by manipulating the brain fields representing sweet and bitter taste we directly control an animal's internal representation, sensory perception, and behavioural actions. These results substantiate the segregation of taste qualities in the cortex, expose the innate nature of appetitive and aversive taste responses, and illustrate the ability of gustatory cortex to recapitulate complex behaviours in the absence of sensory input.

Proper cell fate choice in myelopoiesis is essential for generating correct numbers of distinct myeloid subsets manifesting a wide spectrum of subset-specific activities during development and adulthood. Studies have suggested that myeloid fate choice is primarily regulated by transcription factors; however, new intrinsic regulators and their underlying mechanisms remain to be elucidated. Zebrafish embryonic myelopoiesis gives rise to neutrophils and macrophages and represents a promising system to derive new regulatory mechanisms for myeloid fate decision in vertebrates. Here we present an in vivo study of cell fate specification during zebrafish embryonic myelopoiesis through characterization of the embryos with altered Pu.1, Runx1 activity alone, or their combinations. Genetic analysis shows that low and high Pu.1 activities determine embryonic neutrophilic granulocyte and macrophage fate, respectively. Inactivation and overexpression of Runx1 in zebrafish uncover Runx1 as a key embryonic myeloid fate determinant that favors neutrophil over macrophage fate. Runx1 is induced by high Pu.1 level and in turn transrepresses pu.1 expression, thus constituting a negative feedback loop that fashions a favorable Pu.1 level required for balanced fate commitment to neutrophils versus macrophages. Our findings define a Pu.1-Runx1 regulatory loop that governs the equilibrium between distinct myeloid fates by assuring an appropriate Pu.1 dosage.

The establishment of the HSC pool in vertebrates depends not only on the formation and the propagation of these stem cells but also on their proper trafficking among the defined hematopoietic organs. However, the physiologic mechanisms that regulate HSC mobilization remain elusive. Through analysis of the zebrafish cmyb mutant cmyb(hkz3), we show that the suppression of cMyb function abrogates larval and adult hematopoiesis, with concomitant accumulation of hematopoietic stem/progenitor cells (HSPCs) in their birthplace, the ventral wall of the dorsal aorta (VDA). Cell tracking and time-lapse recording reveal that the accumulation of HSPCs in cmyb(hkz3) mutants is caused by the impairment of HSPC egression from the VDA. Further analysis demonstrates that the HSPC migratory defects in cmyb(hkz3) mutants are at least partly because of adversely elevated levels of chemokine stromal cell-derived factor 1a (Sdf1a). Our study reveals that cMyb plays a hitherto unidentified role in dictating physiologic HSPC migration by modulating Sdf1a signaling.

In our previous study (Jin et al., 2009), we presented evidence indicating that, in zebrafish embryos, hematopoietic stem cells (HSCs) in the ventral wall of the dorsal aorta (VDA), where HSCs originate, and in the caudal hematopoietic tissue (CHT)/posterior blood island (PBI), where HSCs later home, possess distinct differentiation outputs. Using expression analysis and fate mapping, we showed that HSCs only give rise to myeloid cells and not to erythroid cells in the VDA, whereas HSCs differentiate into erythroid cells upon migrating to the CHT/PBI (Jin et al., 2009). By analyzing the differentiation of HSCs in embryos in which HSCs were trapped in the VDA, we further showed that the erythropoietic ability of developing HSCs is dependent on the CHT/PBI (Jin et al., 2009).In their Correspondence (Falenta and Rodaway, 2011), Falenta and Rodaway have provided additional evidence to support their earlier finding (Zhang and Rodaway, 2007) and questioned the non-erythropoietic nature of the VDA because they have detected αe1- and βe1-globin-expressing cells (using αe1- and βe1-globin probes) and cells positive for hemoglobin peroxidase activity [using diaminofluorene (DAF) staining] in the trunk of zebrafish embryos. As suggested by Falenta and Rodaway, we stained 4 days post-fertilization (dpf) embryos for a longer time period of 24 hours (see Appendix S1 in the supplementary material), rather than 2 hours as in the original paper, by whole-mount in situ hybridization (WISH) to detect αe1- and βe1-globin. Whereas staining for 2 hours in our original study failed to detect αe1- and βe1-globin expression in the trunk, staining for 24 hours allowed the detection of αe1- and βe1-globin expression in the trunk of some 4 dpf embryos. However, these αe1- or βe1-globin-positive cells in the trunk were rare in comparison with the numbers of αe1- and βe1-globin-positive cells that were present in the CHT/PBI and a significant portion of 4 dpf embryos that were stained for 24 hours with αe1- or βe1-globin probes lacked staining in the trunk (see Table S1 in the supplementary material). Furthermore, `per cell' staining intensity for αe1- and βe1-globin was much weaker in the trunk compared with that in CHT/PBI (see Fig. S1A,B in the supplementary material). By contrast, all the embryos stained with lyz or mpx, two myeloid markers, for a short period of 2 hours had detectable signals in the trunk (see Appendix S1 and Table S1 in the supplementary material) and `per cell' staining intensity was comparable in the trunk and CHT/PBI (see Fig. S1C,D in the supplementary material). Because of the finding that staining intensity for αe1- and βe1-globin in the trunk was weaker than that observed in the CHT/PBI, whereas staining intensity for lyz and mpx was comparable in these two sites, we think that cells in the trunk might express genuinely low levels of αe1- and βe1-globin. Additionally, in these embryos stained for 24 hours, primitive erythrocytes were also weakly stained by αe1-globin and βe1-globin probes (see Fig. S1A,B in the supplementary material), which raises the possibility that the erythroid cells reported by Falenta and Rodaway to be present in the trunk are primitive erythrocytes. It is also possible that these rare erythrocytes come from the CHT/PBI and stop randomly in the blood vessel during the fixation step of WISH. Therefore, the nature and origin of these cells weakly positive for αe1- and βe1-globin in the trunk are not clearly addressed. Specifically, it is unknown whether these cells are derived from the differentiation of HSCs in the VDA, from primitive erythrocytes that carry residual globin transcripts, or from other sources. A lineage-tracing study would help to resolve the origin of these trunk erythrocytes in the future. Irrespective of the origin of trunk erythrocytes, embryos at 4 dpf, on average, contained many fewer αe1- and βe1-globin+ clusters in the trunk than lyz+ or mpx+ clusters (see Table S1 in the supplementary material). Moreover, Falenta and Rodaway mentioned that there was no detectable hemoglobin expression between the DA and the posterior cardinal vein (PCV) at 2 dpf, which is consistent with our previous finding that myeloid cells but not erythroid cells are generated in situ at this time point in the VDA in our previous report (Jin et al., 2009). Altogether, the contention that HSCs in the VDA produce erythroid cells in situ is not well supported.Falenta and Rodaway also raised concerns over the requirement of CHT/PBI for definitive erythropoiesis. They suggested that definitive erythrocytes could arise in the absence of CHT/PBI because the proportion of rugby ball-shaped `definitive' erythrocytes was normal in the circulation of 7 dpf tail-transected embryos. Although the tail transection experiment is elegant, it suffers from two potential drawbacks. First, in embryos in which the CHT/PBI has been removed, other alternative definitive hematopoietic compartments might still function, such as the pronephros, which could compensate for the loss of the CHT/PBI. Second, the identification of rugby ball-shaped circulating cells as definitive erythrocytes is still questionable and is not generally accepted within the field, as these cells have not been shown to be lost from embryos that lack definitive hematopoiesis. Indeed, the high percentage (∼94%) of definitive-like cells among the circulating erythrocytes of 7 dpf wild-type embryos reported by Falenta and Rodaway conflicts with findings reported by Weinstein et al. that 75% of circulating cells are primitive erythrocytes at 7 dpf, and that at 10 dpf, 50% of circulating cells remain as primitive cells (Weinstein et al., 1996). Given such ambiguity, we revisited the identity of these rugby ball-shaped `definitive' cells in this study. If these cells are of definitive origin, we anticipate that they would be absent or greatly reduced in runx1 mutant embryos (runx1w84x), in which definitive hematopoiesis is reported to be blocked at an early stage (Jin et al., 2009; Sood et al., 2010). All runx1w84x mutants lack HSC- and lineage-specific markers in the AGM, CHT and thymus during early definitive hematopoiesis and, consequently, all runx1w84x mutants become bloodless after 11-12 dpf, although 20% of runx1w84x mutants regain circulating blood cells after 15 dpf (Jin et al., 2009; Sood et al., 2010). Thus, the runx1w84x mutant is an appropriate system to evaluate whether rugby ball-shaped cells at 7 dpf are definitive hematopoietic cells. Largely consistent with the finding reported by Falenta and Rodaway, we found that 82.9% of circulating blood cells isolated from 7 dpf wild-type embryos were of rugby ball shape (see Appendix S1 and Fig. S1E,F in the supplementary material). However, the percentage of rugby ball-shaped cells did not decrease, but was instead maintained, in 7 dpf runx1w84x mutants (in which 95.5% of circulating blood cells were of rugby ball shape), which would not be predicted if they are definitive in origin (see Appendix S1 and Fig. S1E,F in the supplementary material). Thus, it appears more likely that the rugby ball-shaped cells observed at 7 dpf reflect a more matured state of primitive erythrocytes rather than a definitive erythroid population. Our data also suggest that rugby ball shape is not a reliable criterion for scoring definitive erythrocytes in related experiments.As a result of these findings, we stand by our original conclusions.

Runx1 is required for the emergence of hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, its role in the generation and maintenance of HSCs during adult hematopoiesis remains uncertain. Here, we present analysis of a zebrafish mutant line carrying a truncation mutation, W84X, in runx1. The runx1(W84X/W84X) embryos showed blockage in the initiation of definitive hematopoiesis, but some embryos were able to recover from a larval "bloodless" phase and develop to fertile adults with multilineage hematopoiesis. Using cd41-green fluorescent protein transgenic zebrafish and lineage tracing, we demonstrated that the runx1(W84X/W84X) embryos developed cd41(+) HSCs in the aorta-gonad-mesonephros region, which later migrated to the kidney, the site of adult hematopoiesis. Overall, our data suggest that in zebrafish adult HSCs can be formed without an intact runx1.

Runx1 is essential for the generation of hematopoietic stem cells (HSCs) and is frequently mutated in human leukemias. However, the cis-regulatory mechanisms modulating the Runx1 gene expression remain to be elucidated. Herewith, we report the identification of an intronic Runx1 enhancer, Runx1 +24 mouse conserved noncoding element (mCNE), using a combinatorial in silico approach involving comparative genomics and retroviral integration sites mapping. The Runx1 +24 mCNE was found to possess hematopoietic-specific enhancer activity in both zebrafish and mouse models. Significantly, this enhancer is active specifically in hemogenic endothelial cells (ECs) at sites where the de novo generation of HSCs occurs. The activity of this enhancer is also strictly restricted to HSCs within the hematopoietic compartment of the adult bone marrow. We anticipate that Runx1 +24 mCNE HSC enhancer will serve as a molecular handle for tracing and/or manipulating hemogenic ECs/HSCs behavior in vivo, and consequently become an invaluable tool for research on stem cell and cancer biology.

In vertebrates, myeloid cells comprise polymorphonuclear and mononuclear lineages that arise from 2 successive waves of development: a transitory primitive wave giving rise to limited myeloid cells during embryonic stage and a definitive wave capable of producing myeloid cells throughout the fetal and adult life. One key unresolved question is what factors dictate polymorphonuclear versus mononuclear lineage fates during myelopoiesis. Here we show that during zebrafish embryogenesis interferon regulatory factor-8 (irf8) is expressed specifically in macrophages but not neutrophils. Suppression of Irf8 function in zebrafish causes a depletion of macrophages and an enhanced output of neutrophils but does not affect the overall number, proliferation, and survival of primitive myeloid cells. These data indicate that the skewed myeloid lineage development in Irf8 knockdown embryos results from a cell-fate switching. Such a conclusion is further supported by the observation showing that overexpression of Irf8 promotes macrophage formation at the expense of neutrophil development. Genetic epistasis analysis reveals that Irf8 acts downstream of Pu.1 but is insufficient to promote macrophage development in the absence of Pu.1. Our findings demonstrate that Irf8 is a critical determinant for neutrophil versus macrophage fate choice during zebrafish primitive myelopoiesis.

The cytoplasmic domains of UNC5 are responsible for its netrin-mediated signaling events in axonal migrations, blood vessel patterning, and apoptosis, although the molecular mechanisms governing these processes are unknown. To provide a foundation for the elucidation of the UNC5-mediated signaling mechanism, we determined the crystal structure of the cytoplasmic portion of UNC5b. We found that it contains three distinctly folded domains, namely ZU5, UPA, and death domain (DD). These three domains form a structural supramodule, with ZU5 binding to both UPA and DD, thereby locking the ZU5-UPA-DD supramodule in a closed conformation and suppressing its biological activities. Release of the closed conformation of the ZU5-UPA-DD supramodule leads to the activation of the receptor in the promotion of apoptosis and blood vessel patterning. Finally, we provide evidence showing that the supramodular nature of UNC5 ZU5-UPA-DD is likely to be shared by the ankyrin and PIDD families of scaffold proteins.

One unique feature of vertebrate definitive hematopoiesis is the ontogenic switching of hematopoietic stem cells from one anatomical compartment or niche to another. In mice, hematopoietic stem cells are believed to originate in the aorta-gonadmesonephros ( AGM), subsequently migrate to the fetal liver (FL) and finally colonize the bone marrow ( BM). Yet, the differentiation potential of hematopoietic stem cells within early niches such as the AGM and FL remains incompletely defined. Here, we present in vivo analysis to delineate the differentiation potential of definitive hematopoietic stem/progenitor cells (HSPCs) in the zebrafish AGM and FL analogies, namely the ventral wall of dorsal aorta (VDA) and the posterior blood island (PBI), respectively. Cell fate mapping and analysis of zebrafish runx1(w84x) and vlad tepes ( vlt(m651)) mutants revealed that HSPCs in the PBI gave rise to both erythroid and myeloid lineages. However, we surprisingly found that HSPCs in the VDA were not quiescent but were uniquely adapted to generate myeloid but not erythroid lineage cells. We further showed that such distinct differentiation output of HSPCs was, at least in part, ascribed to the different micro-environments present in these two niches. Our results highlight the importance of niche in shaping the differentiation output of developing HSPCs.

The development of vertebrate definitive hematopoiesis is featured by temporally and spatially dynamic distribution of hematopoietic stem/progenitor cells (HSPCs). It is proposed that the migration of definitive HSPCs, at least in part, accounts for this unique characteristic; however, compelling in vivo lineage evidence is still lacking. Here we present an in vivo analysis to delineate the migration route of definitive HSPCs in the early zebrafish embryo. Cell-marking analysis was able to first map definitive HSPCs to the ventral wall of dorsal aorta (DA). These cells were subsequently found to migrate to a previously unappreciated organ, posterior blood island (PBI), located between the caudal artery and caudal vein, and finally populate the kidney, the adult hematopoietic organ. These findings demonstrate that the PBI acts as an intermediate hematopoietic organ in a manner analogous to the mammalian fetal liver to sustain definitive hematopoiesis before adult kidney hematopoiesis occurs. Thus our study unambiguously documents the in vivo trafficking of definitive HSPCs among developmentally successive hematopoietic compartments and underscores the ontogenic conservation of definitive hematopoiesis between zebrafish and mammals.