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.Fundação para a Ciência e a Tecnologia - FCT, United States-Israel BinationalScience Foundation; Lisboa Regional Operational Programme; Israel Science Foundationinfo:eu-repo/semantics/publishedVersio

Autoregulation of E-cadherin expression by cadherin–cadherin interactions: the roles of β-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–β-catenin complex. We investigated the molecular basis of this regulation, using human colon cancer cells with aberrantly activated β-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 β-catenin signaling and, independently, by ERK. Dense cultures resembled a differentiated epithelium with high levels of E-cadherin and β-catenin in adherens junctions. In such cells, β-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 β-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, β-catenin and ERK could be important in tumorigenesis

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

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.Fundação para a Ciência e Tecnologia - FCTinfo:eu-repo/semantics/publishedVersio

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

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

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