Temperature-sensing riboceptors.

Understanding how cells sense temperature is a fundamental question in biology and is pivotal for the evolution of life. In numerous organisms, temperature is not only sensed but also generated due to cellular processes. Consequently, the mechanisms governing temperature sensation in various organisms have been experimentally elucidated. Extending upon others' proposals and demonstration of protein- and nucleic acid-based thermosensors, and utilizing a colonial India 'punkah-wallahs' analogy, I present my rationale for the necessity of temperature sensing in every organelle in a cell. Finally, I propose temperature-sensing riboceptors (ribonucleic acid receptors) to integrate all the RNA molecules (mRNA, non-coding RNA, and so forth) capable of sensing temperature and triggering a signaling event, which I call as thermocrine signaling. This approach could enable the identification of riboceptors in every cell of almost every organism, not only for temperature but also for other classes of ligands, including gaseous solutes, and water.

Gas-sensing riboceptors.

Understanding how cells sense gases or gaseous solutes is a fundamental question in biology and is pivotal for the evolution of molecular and organismal life. In numerous organisms, gases can diffuse into cells, be transported, generated, and sensed. Controlling gases in the cellular environment is essential to prevent cellular and molecular damage due to interactions with gas-dependent free radicals. Consequently, the mechanisms governing acute gas sensing are evolutionarily conserved and have been experimentally elucidated in various organisms. However, the scientific literature on direct gas sensing is largely based on hemoprotein-based gasoreceptors (or sensors). As RNA-based G-quadruplex (G4) structures can also bind to heme, I propose that some ribozymes can act as gas-sensing riboceptors (ribonucleic acid receptors). Additionally, I present a few other ideas for non-heme metal ion- or metal cluster-based gas-sensing riboceptors. Studying riboceptors can help understand the evolutionary origins of cellular and gasocrine signaling.

Oxygen is an essential gasotransmitter directly sensed via protein gasoreceptors.

The current restrictive criteria for gasotransmitters exclude oxygen (O2) as a gasotransmitter in vertebrates. In this manuscript, I propose a revision of gasotransmitter criteria to include O2 per se as a signaling molecule and 'essential gasotransmitter' for vertebrates. This revision would enable us to search for protein-based O2-binding sensors (gasoreceptors) in all cells in the brain or other tissues rather than specialized tissues such as the carotid body or gills. If microorganisms have protein-based O2-binding sensors or gasoreceptors such as DosP or FixL or FNR with diverse signaling domains, then eukaryotic cells must also have O2-binding sensors or gasoreceptors. Just as there are protein-based receptor(s) for nitric oxide (GUCY1A, GUCY1B, CLOCK, NR1D2) in cells of diverse tissues, it is reasonable to consider that there are protein-based receptors for O2 in cells of diverse tissues as well. In mammals, O2 must be acting as a gasotransmitter or gaseous signaling molecule via protein-based gasoreceptors such as androglobin that very likely mediate acute sensing of O2. Accepting O2 as an essential gasotransmitter will enable us to search for gasoreceptors not only for O2 but also for other nonessential gasotransmitters such as hydrogen sulfide, ammonia, methane, and ethylene. It will also allow us to investigate the role of environment-derived metal ions in acute gas (or solute) sensing within and between organisms. Finally, accepting O2 per se as a signaling molecule acting via gasoreceptors will open up the field of gasocrinology.

Heme-based oxygen gasoreceptors.

To investigate gasocrine signaling, there is a critical need to identify gasoreceptors for the essential gasotransmitters like O2. Based on existing scientific literature, I propose that heme-based O2 sensors, featuring diverse signaling domains across genera, should be explicitly designated as O2 gasoreceptors. Acknowledging that O2 gasoreceptors are likely to belong to multiple protein classes with diverse signaling domains and pathways will facilitate a comprehensive search for O2 gasoreceptors in all organisms and across every cell type. This approach will broaden the investigation beyond specialized tissues or cells, encompassing a systemic exploration.

"Blind men and an elephant": The need for animals in research, drug safety studies, and understanding civilizational diseases.

Animal-based research and drug safety studies are essential to understanding the mysteries of nature and the long-term survival of humans. Due to the rapid increase in the global human population, conflict- and economically driven human migration, tourism-related activities, densely populated metropolitan areas, and local policies, humans will be affected by a multitude of novel disease-causing microorganisms and civilizational diseases. Despite disparities among countries, recent and planned changes in regulations concerning animal research and drug safety studies could have detrimental effects on both the animal research community and nations lacking sufficient social support systems. Based on existing scientific literature, I argue that we need animal research encompassing aspects such as animal development, behavior, drug safety studies, and for the understanding of future civilizational diseases. Depending on the nature of the research questions and local challenges, a suitable animal model organism should be made mandatory.

A ligand-receptor interactome atlas of the zebrafish.

Studies in zebrafish can unravel the functions of cellular communication and thus identify novel bench-to-bedside drugs targeting cellular communication signaling molecules. Due to the incomplete annotation of zebrafish proteome, the knowledge of zebrafish receptors, ligands, and tools to explore their interactome is limited. To address this gap, we de novo predicted the cellular localization of zebrafish reference proteome using deep learning algorithm. We combined the predicted and existing annotations on cellular localization of zebrafish proteins and created repositories of zebrafish ligands, membrane receptome, and interactome as well as associated diseases and targeting drugs. Unlike other tools, our interactome atlas is based on both the physical interaction data of zebrafish proteome and existing human ligand-receptor pair databases. The resources are available as R and Python scripts. DanioTalk provides a novel resource for researchers interested in targeting cellular communication in zebrafish, as we demonstrate in applications studying synapse and axo-glial interactome. DanioTalk methodology can be applied to build and explore the ligand-receptor atlas of other non-mammalian model organisms.

CRISPR-Cas9 F0 knockout approach using predesigned in vitro transcribed guide RNAs partially recapitulates Rx3 function in eye morphogenesis.

CRISPR-Cas9-based F0 knockout (KO) approach permits relatively simple and rapid generation of homozygous KOs and allows quick investigation of gene functions in zebrafish. However, F0 KO studies are largely performed using commercial synthetic guide RNAs (gRNAs) which are unaffordable by majority of the researchers. We tested (i) how effective is the CRISPR-Cas9-based F0 KO approach using in vitro transcribed gRNAs; (ii) how penetrant are the resulting phenotype at the later developmental stages and (iii) whether Coughlin's group pre-designed gRNAs are functional even without validating the gRNAs or testing for lack of SNP's in target loci. We targeted the rx3 gene that is required for the formation of the eye, a structure that exhibits robustness and can quickly recover from early phenotypes. Our results indicate that, in the majority of the samples, injection of Cas9 protein complex with four different in vitro transcribed gRNAs; targeting rx3 results in lack of eyes or disrupted eye development. Thus, the CRISPR-Cas9-based F0 KO approach using pre-designed, quadruple in vitro transcribed gRNAs can recapitulate the function of a gene at least until 5-dpf stage of larval zebrafish.

Stress resilience is established during development and is regulated by complement factors.

Individuals in a population respond differently to stressful situations. While resilient individuals recover efficiently, others are susceptible to the same stressors. However, it remains challenging to determine if resilience is established as a trait during development or acquired later in life. Using a behavioral paradigm in zebrafish larvae, we show that resilience is a stable and heritable trait, which is determined and exhibited early in life. Resilient larvae show unique stress-induced transcriptional response, and larvae with mutations in resilience-associated genes, such as neuropeptide Y and miR218, are less resilient. Transcriptome analysis shows that resilient larvae downregulate multiple factors of the innate immune complement cascade in response to stress. Perturbation of critical complement factors leads to an increase in resilience. We conclude that resilience is established as a stable trait early during development and that neuropeptides and the complement pathway play positive and negative roles in determining resilience, respectively.

Endocrine cross-talk between the gut microbiome and glial cells in development and disease.

Glial cells make up the major cellular component of the nervous system. Glial development is usually investigated through perturbations of host genetics, although non-host-derived signalling molecules can also regulate glial cells. Indeed, gut microbiome colonisation and the presence of microbiome-derived factors in the blood coincide with glial cell development. Emerging data suggest that the gut microbiome can regulate gliogenesis, myelination and glial epigenetics. Neurodegenerative diseases are characterised by changes in the gut microbiome and glial dysfunction. This perspective discusses the ways in which microbiome-derived molecules can engage in cross-talk with glial cells during development and in dysfunctional glial diseases.

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.

Oxytocin receptor signalling modulates novelty recognition but not social preference in zebrafish.

Sociality is a complex phenomenon that involves the individual´s motivation to approach their conspecifics, along with social cognitive functions that enable individuals to interact and survive. The nonapeptide oxytocin (OXT) is known to regulate sociality in many species. However, the role of OXT in specific aspects of sociality is still not well understood. In the present study, we investigated the contribution of the OXT receptor (OXTR) signalling in two different aspects of zebrafish social behaviour: social preference, by measuring their motivation to approach a shoal of conspecifics, and social recognition, by measuring their ability to discriminate between a novel and familiar fish, using a mutant zebrafish lacking a functional OXTR. Although oxtr mutant zebrafish displayed normal attraction to a shoal of conspecifics, they exhibited reduced social recognition. We further investigated whether this effect would be social-domain specific by replacing conspecific fish by objects. Although no differences were observed in object approach, oxtr mutant fish also exhibited impaired object recognition. Our findings suggest that OXTR signalling regulates a more general memory recognition of familiar vs novel entities, not only in social but also in a non-social domain, in zebrafish.

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.

Tbf1 and Vid22 promote resection and non-homologous end joining of DNA double-strand break ends.

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. The Saccharomyces cerevisiae protein Tbf1, which is characterized by a Myb domain and is related to mammalian TRF1 and TRF2, has been proposed to act as a transcriptional activator. Here, we show that Tbf1 and its interacting protein Vid22 are new players in the response to DSBs. Inactivation of either TBF1 or VID22 causes hypersensitivity to DSB-inducing agents and shows strong negative interactions with mutations affecting homologous recombination. Furthermore, Tbf1 and Vid22 are recruited to an HO-induced DSB, where they promote both resection of DNA ends and repair by non-homologous end joining. Finally, inactivation of either Tbf1 or Vid22 impairs nucleosome eviction around the DSB, suggesting that these proteins promote efficient repair of the break by influencing chromatin identity in its surroundings.

The role of shelterin in maintaining telomere integrity.

The ends of eukaryotic chromosomes need to be protected from detection as DNA double strand breaks by the DNA damage response pathways. Failure to do so would have devastating consequences for genome integrity. Packaging of chromosome ends into protective structures called telomeres prevents checkpoint activation and DNA repair/recombination activities. Several studies on a variety of organisms have revealed that protein complexes with specificity for telomeric DNA protect chromosome ends from being recognized as DNA double-strand breaks and regulate telomere maintenance by the telomerase. In this review, we will discuss the consequences of telomere dysfunction and our understanding of how telomere integrity is maintained.

Rif1 supports the function of the CST complex in yeast telomere capping.

Telomere integrity in budding yeast depends on the CST (Cdc13-Stn1-Ten1) and shelterin-like (Rap1-Rif1-Rif2) complexes, which are thought to act independently from each other. Here we show that a specific functional interaction indeed exists among components of the two complexes. In particular, unlike RIF2 deletion, the lack of Rif1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. This synthetic interaction between Rif1 and the CST complex occurs independently of rif1Δ-induced alterations in telomere length. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, both DNA damage checkpoint activation and lethality in cdc13 rif1Δ cells are partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. The functional interaction between Rif1 and the CST complex is specific, because RIF1 deletion does not enhance checkpoint activation in case of CST-independent telomere capping deficiencies, such as those caused by the absence of Yku or telomerase. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex.

Shelterin-like proteins and Yku inhibit nucleolytic processing of Saccharomyces cerevisiae telomeres.

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. Here we investigate the role of key telomeric proteins in protecting budding yeast telomeres from degradation. We show that the Saccharomyces cerevisiae shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres, where Yku plays the major role in preventing initiation, whereas Rif1, Rif2, and Rap1 act primarily by limiting extensive resection. In fact, exonucleolytic degradation of a de novo telomere is more efficient in yku70Delta than in rif2Delta G1 cells, but generation of ssDNA in Yku-lacking cells is limited to DNA regions close to the telomere tip. This limited processing is due to the inhibitory action of Rap1, Rif1, and Rif2, as their inactivation allows extensive telomere resection not only in wild-type but also in yku70Delta G1 cells. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases.