Developmental Effects of Oxytocin Neurons on Social Affiliation and Processing of Social Information.

Hormones regulate behavior either through activational effects that facilitate the acute expression of specific behaviors or through organizational effects that shape the development of the nervous system thereby altering adult behavior. Much research has implicated the neuropeptide oxytocin (OXT) in acute modulation of various aspects of social behaviors across vertebrate species, and OXT signaling is associated with the developmental social deficits observed in autism spectrum disorders (ASDs); however, little is known about the role of OXT in the neurodevelopment of the social brain. We show that perturbation of OXT neurons during early zebrafish development led to a loss of dopaminergic neurons, associated with visual processing and reward, and blunted the neuronal response to social stimuli in the adult brain. Ultimately, adult fish whose OXT neurons were ablated in early life, displayed altered functional connectivity within social decision-making brain nuclei both in naive state and in response to social stimulus and became less social. We propose that OXT neurons have an organizational role, namely, to shape forebrain neuroarchitecture during development and to acquire an affiliative response toward conspecifics.SIGNIFICANCE STATEMENT Social behavior is developed over the lifetime of an organism and the neuropeptide oxytocin (OXT) modulates social behaviors across vertebrate species, and is associated with neuro-developmental social deficits such as autism. However, whether OXT plays a role in the developmental maturation of neural systems that are necessary for social behavior remains poorly explored. We show that proper behavioral and neural response to social stimuli depends on a developmental process orchestrated by OXT neurons. Animals whose OXT system is ablated in early life show blunted neuronal and behavioral responses to social stimuli as well as wide ranging disruptions in the functional connectivity of the social brain. We provide a window into the mechanisms underlying OXT-dependent developmental processes that implement adult sociality.

PINK1 deficiency impairs adult neurogenesis of dopaminergic neurons.

Recent evidence suggests neurogenesis is on-going throughout life but the relevance of these findings for neurodegenerative disorders such as Parkinson's disease (PD) is poorly understood. Biallelic PINK1 mutations cause early onset, Mendelian inherited PD. We studied the effect of PINK1 deficiency on adult neurogenesis of dopaminergic (DA) neurons in two complementary model systems. Zebrafish are a widely-used model to study neurogenesis in development and through adulthood. Using EdU analyses and lineage-tracing studies, we first demonstrate that a subset of ascending DA neurons and adjacent local-projecting DA neurons are each generated into adulthood in wild type zebrafish at a rate that decreases with age. Pink1-deficiency impedes DA neurogenesis in these populations, most significantly in early adult life. Pink1 already exerts an early effect on Th1+ progenitor cells rather than on differentiated DA neurons only. In addition, we investigate the effect of PINK1 deficiency in a human isogenic organoid model. Global neuronal differentiation in PINK1-deficient organoids and isogenic controls is similar, but PINK1-deficient organoids display impeded DA neurogenesis. The observation of impaired adult dopaminergic neurogenesis in Pink1 deficiency in two complementing model systems may have significant consequences for future therapeutic approaches in human PD patients with biallelic PINK1 mutations.

Genetic variation in the social environment affects behavioral phenotypes of oxytocin receptor mutants in zebrafish.

Oxytocin-like peptides have been implicated in the regulation of a wide range of social behaviors across taxa. On the other hand, the social environment, which is composed of conspecifics that may vary in their genotypes, also influences social behavior, creating the possibility for indirect genetic effects. Here, we used a zebrafish oxytocin receptor knockout line to investigate how the genotypic composition of the social environment (Gs) interacts with the oxytocin genotype of the focal individual (Gi) in the regulation of its social behavior. For this purpose, we have raised wild-type or knock-out zebrafish in either wild-type or knock-out shoals and tested different components of social behavior in adults. GixGs effects were detected in some behaviors, highlighting the need to control for GixGs effects when interpreting results of experiments using genetically modified animals, since the genotypic composition of the social environment can either rescue or promote phenotypes associated with specific genes.

Perceptual mechanisms of social affiliation in zebrafish.

Social living animals need to recognize the presence of conspecifics in the environment in order to engage in adaptive social interactions. Social cues can be detected through different sensory modalities, including vision. Two main visual features can convey information about the presence of conspecifics: body form and biological motion (BM). Given the role that oxytocin plays in social behavior regulation across vertebrates, particularly in the salience and reward values of social stimuli, we hypothesized that it may also be involved in the modulation of perceptual mechanisms for conspecific detection. Here, using videoplaybacks, we assessed the role of conspecific form and BM in zebrafish social affiliation, and how oxytocin regulates the perception of these cues. We demonstrated that while each visual cue is important for social attraction, BM promotes a higher fish engagement than the static conspecific form alone. Moreover, using a mutant line for one of the two oxytocin receptors, we show that oxytocin signaling is involved in the regulation of BM detection but not conspecific form recognition. In summary, our results indicate that, apart from oxytocin role in the regulation of social behaviors through its effect on higher-order cognitive mechanisms, it may regulate social behavior by modulating very basic perceptual mechanisms underlying the detection of socially-relevant cues.

Single-Cell Molecular and Cellular Architecture of the Mouse Neurohypophysis.

The neurohypophysis (NH), located at the posterior lobe of the pituitary, is a major neuroendocrine tissue, which mediates osmotic balance, blood pressure, reproduction, and lactation by means of releasing the neurohormones oxytocin (OXT) and arginine-vasopressin (AVP) from the brain into the peripheral blood circulation. The major cellular components of the NH are hypothalamic axonal termini, fenestrated endothelia and pituicytes, the resident astroglia. However, despite the physiological importance of the NH, the exact molecular signature defining neurohypophyseal cell types and in particular the pituicytes, remains unclear. Using single-cell RNA sequencing (scRNA-Seq), we captured seven distinct cell types in the NH and intermediate lobe (IL) of adult male mouse. We revealed novel pituicyte markers showing higher specificity than previously reported. Bioinformatics analysis demonstrated that pituicyte is an astrocytic cell type whose transcriptome resembles that of tanycyte. Single molecule in situ hybridization revealed spatial organization of the major cell types implying intercellular communications. We present a comprehensive molecular and cellular characterization of neurohypophyseal cell types serving as a valuable resource for further functional research.

The fenestrae-associated protein Plvap regulates the rate of blood-borne protein passage into the hypophysis.

To maintain body homeostasis, endocrine systems must detect and integrate blood-borne peripheral signals. This is mediated by fenestrae, specialized permeable pores in the endothelial membrane. Plasmalemma vesicle-associated protein (Plvap) is located in the fenestral diaphragm and is thought to play a role in the passage of proteins through the fenestrae. However, this suggested function has yet to be demonstrated directly. We studied the development of fenestrated capillaries in the hypophysis, a major neuroendocrine interface between the blood and brain. Using a transgenic biosensor to visualize the vascular excretion of the genetically tagged plasma protein DBP-EGFP, we show that the developmental acquisition of vascular permeability coincides with differential expression of zebrafish plvap orthologs in the hypophysis versus brain. Ultrastructural analysis revealed that plvapb mutants display deficiencies in fenestral diaphragms and increased density of hypophyseal fenestrae. Measurements of DBP-EGFP extravasation in plvapb mutants provided direct proof that Plvap limits the rate of blood-borne protein passage through fenestrated endothelia. We present the regulatory role of Plvap in the development of blood-borne protein detection machinery at a neuroendocrine interface through which hormones are released to the general circulation.

Robo2 regulates synaptic oxytocin content by affecting actin dynamics.

The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT) in the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were enriched with polymerized actin. Perturbation of actin filaments by either cytochalasin-D or conditional Cofilin expression resulted in decreased synaptic OXT levels. Genetic loss of robo2 or slit3 displayed decreased synaptic OXT content and robo2 mutants displayed reduced mobility of the actin probe Lifeact-EGFP in OXT synapses. Using a novel transgenic reporter allowing real-time monitoring of OXT-loaded vesicles, we show that robo2 mutants display slower rate of vesicles accumulation. OXT-specific expression of dominant-negative Cdc42, which is a key regulator of actin dynamics and a downstream effector of Robo2, led to a dose-dependent increase in OXT content in WT, and a dampened effect in robo2 mutants. Our results link Slit3-Robo2-Cdc42, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels.

Pituicyte Cues Regulate the Development of Permeable Neuro-Vascular Interfaces.

The hypothalamo-neurohypophyseal system (HNS) regulates homeostasis through the passage of neurohormones and blood-borne proteins via permeable blood capillaries that lack the blood-brain barrier (BBB). Why neurohypophyseal capillaries become permeable while the neighboring vasculature of the brain forms BBB remains unclear. We show that pituicytes, the resident astroglial cells of the neurohypophysis, express genes that are associated with BBB breakdown during neuroinflammation. Pituicyte-enriched factors provide a local microenvironment that instructs a permeable neurovascular conduit. Thus, genetic and pharmacological perturbations of Vegfa and Tgfß3 affected HNS vascular morphogenesis and permeability and impaired the expression of the fenestral marker plvap. The anti-inflammatory agent dexamethasone decreased HNS permeability and downregulated the pituicyte-specific cyp26b gene, encoding a retinoic acid catabolic enzyme. Inhibition of Cyp26b activity led to upregulation of tight junction protein Claudin-5 and decreased permeability. We conclude that pituicyte-derived factors regulate the "decision" of endothelial cells to adopt a permeable endothelial fate instead of forming a BBB.

Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin.

Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.

The not-so-long history of zebrafish research in Israel.

The zebrafish has become a model of choice in fundamental and applied life sciences and is widely used in various fields of biomedical research as a human disease model for cancer, metabolic and neurodegenerative diseases, and regenerative medicine. The transparency of the zebrafish embryo allows real-time visualization of the development and morphogenesis of practically all of its tissues and organs. Zebrafish are amenable to genetic manipulation, for which innovative genetic and molecular techniques are constantly being introduced. These include the study of gene function and regulation using gene knockdown, knockout and knock-in, as well as transgenesis and tissue-specific genetic perturbations. Complementing this genetic toolbox, the zebrafish exhibits measurable behavioral and hormonal responses already at the larval stages, providing a viable vertebrate animal model for high-throughput drug screening and chemical genetics. With the available tools of the genomic era and the abundance of disease-associated human genes yet to be explored, the zebrafish model is becoming the preferred choice in many studies. Its advantages and potential are being increasingly recognized within the Israeli scientific community, and its use as a model system for basic and applied science has expanded in Israel in recent years. Since the first zebrafish-focused laboratory was introduced at Tel Aviv University 16 years ago, seven more zebrafish-centric research groups have been established, along with more than two dozen academic research groups and three bio-medical companies that are now utilizing this model.

Homeodomain protein Otp affects developmental neuropeptide switching in oxytocin neurons associated with a long-term effect on social behavior.

Proper response to stress and social stimuli depends on orchestrated development of hypothalamic neuronal circuits. Here we address the effects of the developmental transcription factor orthopedia (Otp) on hypothalamic development and function. We show that developmental mutations in the zebrafish paralogous gene otpa but not otpb affect both stress response and social preference. These behavioral phenotypes were associated with developmental alterations in oxytocinergic (OXT) neurons. Thus, otpa and otpb differentially regulate neuropeptide switching in a newly identified subset of OXT neurons that co-express the corticotropin-releasing hormone (CRH). Single-cell analysis revealed that these neurons project mostly to the hindbrain and spinal cord. Ablation of this neuronal subset specifically reduced adult social preference without affecting stress behavior, thereby uncoupling the contribution of a specific OXT cluster to social behavior from the general otpa-/- deficits. Our findings reveal a new role for Otp in controlling developmental neuropeptide balance in a discrete OXT circuit whose disrupted development affects social behavior.

Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity.

Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

Homeodomain protein otp and activity-dependent splicing modulate neuronal adaptation to stress.

Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal's adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing.

The hypothalamic neuropeptide oxytocin is required for formation of the neurovascular interface of the pituitary.

The hypothalamo-neurohypophyseal system (HNS) is the neurovascular structure through which the hypothalamic neuropeptides oxytocin and arginine-vasopressin exit the brain into the bloodstream, where they go on to affect peripheral physiology. Here, we investigate the molecular cues that regulate the neurovascular contact between hypothalamic axons and neurohypophyseal capillaries of the zebrafish. We developed a transgenic system in which both hypothalamic axons and neurohypophyseal vasculature can be analyzed in vivo. We identified the cellular organization of the zebrafish HNS as well as the dynamic processes that contribute to formation of the HNS neurovascular interface. We show that formation of this interface is regulated during development by local release of oxytocin, which affects endothelial morphogenesis. This cell communication process is essential for the establishment of a tight axovasal interface between the neurons and blood vessels of the HNS. We present a unique example of axons affecting endothelial morphogenesis through secretion of a neuropeptide.

The metabolic regulator PGC-1α directly controls the expression of the hypothalamic neuropeptide oxytocin.

The transcriptional coactivator PGC-1α is a key regulator of cellular energy expenditure in peripheral tissues. Recent studies report that PGC-1α-null mice develop late-onset obesity and that the neuronal inactivation of PGC-1α causes increased food intake. However, the exact role of PGC-1α in the CNS remains unclear. Here we show that PGC-1α directly regulates the expression of the hypothalamic neuropeptide oxytocin, a known central regulator of appetite. We developed a unique genetic approach in the zebrafish, allowing us to monitor and manipulate PGC-1α activity in oxytocinergic neurons. We found that PGC-1α is coexpressed with oxytocin in the zebrafish hypothalamus. Targeted knockdown of the zebrafish PGC-1α gene activity caused a marked decrease in oxytocin mRNA levels and inhibited the expression of a transgenic GFP reporter driven by the oxytocin promoter. The effect of PGC-1α loss of function on oxytocin gene activity was rescued by tissue-specific re-expression of either PGC-1α or oxytocin precursor in zebrafish oxytocinergic neurons. PGC-1α activated the oxytocin promoter in a heterologous cell culture system, and overexpression of PGC-1α induced ectopic expression of oxytocin in muscles and neurons. Finally, PGC-1α forms an in vivo complex with the oxytocin promoter in fed but not fasted animals. These findings demonstrate that PGC-1α is both necessary and sufficient for the production of oxytocin, implicating hypothalamic PGC-1α in the direct activation of a hypothalamic hormone known to control energy intake.

Dopaminergic neuronal cluster size is determined during early forebrain patterning.

We have explored the effects of robust neural plate patterning signals, such as canonical Wnt, on the differentiation and configuration of neuronal subtypes in the zebrafish diencephalon at single-cell resolution. Surprisingly, perturbation of Wnt signaling did not have an overall effect on the specification of diencephalic fates, but selectively affected the number of dopaminergic (DA) neurons. We identified the DA progenitor zone in the diencephalic anlage of the neural plate using a two-photon-based uncaging method and showed that the number of non-DA neurons derived from this progenitor zone is not altered by Wnt attenuation. Using birthdating analysis, we determined the timing of the last cell division of DA progenitors and revealed that the change in DA cell number following Wnt inhibition is not due to changes in cell cycle exit kinetics. Conditional inhibition of Wnt and of cell proliferation demonstrated that Wnt restricts the number of DA progenitors during a window of plasticity, which occurs at primary neurogenesis. Finally, we demonstrated that Wnt8b is a modulator of DA cell number that acts through the Fz8a (Fzd8a) receptor and its downstream effector Lef1, and which requires the activity of the Fezl (Fezf2) transcription factor for this process. Our data show that the differential response of distinct neuronal populations to the Wnt signal is not a simple interpretation of their relative anteroposterior position. This study also shows, for the first time, that diencephalic DA population size is modulated inside the neural plate much earlier than expected, concomitant with Wnt-mediated regional patterning events.

Specification of hypothalamic neurons by dual regulation of the homeodomain protein Orthopedia.

In the developing hypothalamus, a variety of neurons are generated adjacent to each other in a highly coordinated, but poorly understood process. A critical question that remains unanswered is how coordinated development of multiple neuronal types is achieved in this relatively narrow anatomical region. We focus on dopaminergic (DA) and oxytocinergic (OT) neurons as a paradigm for development of two prominent hypothalamic cell types. We report that the development of DA and OT-like neurons in the zebrafish is orchestrated by two novel pathways that regulate the expression of the homeodomain-containing protein Orthopedia (Otp), a key determinant of hypothalamic neural differentiation. Genetic analysis showed that the G-protein-coupled receptor PAC1 and the zinc finger-containing transcription factor Fezl act upstream to Otp. In vivo and in vitro experiments demonstrated that Fezl and PAC1 regulate Otp at the transcriptional and the post-transcriptional levels, respectively. Our data reveal a new genetic network controlling the specification of hypothalamic neurons in vertebrates, and places Otp as a critical determinant underlying Fezl- and PAC1-mediated differentiation.

Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition.

Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.

Autoregulation of E-cadherin expression by cadherin-cadherin interactions: the roles of beta-catenin signaling, Slug, and MAPK.

Transcriptional repression of E-cadherin, characteristic of epithelial to mesenchymal transition, is often found also during tumor cell invasion. At metastases, migratory fibroblasts sometimes revert to an epithelial phenotype, by a process involving regulation of the E-cadherin-beta-catenin complex. We investigated the molecular basis of this regulation, using human colon cancer cells with aberrantly activated beta-catenin signaling. Sparse cultures mimicked invasive tumor cells, displaying low levels of E-cadherin due to transcriptional repression of E-cadherin by Slug. Slug was induced by beta-catenin signaling and, independently, by ERK. Dense cultures resembled a differentiated epithelium with high levels of E-cadherin and beta-catenin in adherens junctions. In such cells, beta-catenin signaling, ErbB-1/2 levels, and ERK activation were reduced and Slug was undetectable. Disruption of E-cadherin-mediated contacts resulted in nuclear localization and signaling by beta-catenin, induction of Slug and inhibition of E-cadherin transcription, without changes in ErbB-1/2 and ERK activation. This autoregulation of E-cadherin by cell-cell adhesion involving Slug, beta-catenin and ERK could be important in tumorigenesis.