The recent description of virus-induced fish IFNs has raised questions about the evolution of this complex antiviral system. Identification of the receptor of the zebrafish virus-induced IFN (zIFN) was sought to help resolve these questions. We set up an experimental system to study the zIFN system in the course of a viral infection of zebrafish embryos. In this setting, zIFN was induced by viral infection, and we identified zIFN-dependent induced transcripts. Embryos quickly died from the infection, but zIFN overexpression increased their survival. We took advantage of this experimental system to perform in vivo loss and gain of function analysis of candidate receptors of the class II helical receptor family and identified zCRFB1 and zCRFB5 as the two subunits of the zebrafish IFN receptor. Based on the organization of the zIFN gene and the protein structure of the identified receptor components, the virus-induced fish IFNs appear as orthologs of mammalian IFN-λ, specifying type III IFN as the ancestral antiviral system of vertebrates.

The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology.

Background Although the overall brain organization is shared in vertebrates, there are significant differences within subregions among different groups, notably between Sarcopterygii (lobe-finned fish) and Actinopterygii (ray-finned fish). Recent comparative studies focusing on the ventricular morphology have revealed a large diversity of the hypothalamus. Here, we study the development of the inferior lobe (IL), a prominent structure forming a bump on the ventral surface of the teleost brain. Based on its position, IL has been thought to be part of the hypothalamus (therefore forebrain). Results Taking advantage of genetic lineage-tracing techniques in zebrafish, we reveal that cells originating from her5 -expressing progenitors in the midbrain-hindbrain boundary (MHB) participate in the formation of a large part of the IL. 3D visualization demonstrated how IL develops in relation to the ventricular system. We found that IL is constituted by two developmental components: the periventricular zone of hypothalamic origin and the external zone of mesencephalic origin. The mesencephalic external zone grows progressively until adulthood by adding new cells throughout development. Conclusion Our results disprove a homology between the IL and the mammalian lateral hypothalamus. We suggest that the IL is likely to be involved in multimodal sensory integration rather than feeding motivation. The teleost brain is not a simpler version of the mammalian brain, and our study highlights the evolutionary plasticity of the brain which gives rise to novel structures. Electronic supplementary material The online version of this article (10.1186/s12915-019-0631-y) contains supplementary material, which is available to authorized users.

The inhibitory glycine receptor is a pentameric membrane protein composed of alpha and beta subunits. In the postsynaptic membrane, the glycine receptor and the copurifying peripheral membrane protein gephyrin are clustered underneath glycine-releasing nerve terminals. Here, we describe the expression of gephyrin and the neonatal and adult glycine receptor alpha subunit isoforms alpha1 and alpha2 during in vitro differentiation of rat spinal neurons. Analysis by immunoassays and the reverse transcriptase-polymerase chain reaction showed that gephyrin and alpha subunit mRNA and protein levels exhibited a marked increase from 1 to 5 days in vitro, i.e. prior to the formation of functional synaptic contacts. Using confocal and standard immunofluorescence, we determined the number of immunoreactive cells and the cellular localization of the alpha subunits and gephyrin. At 3 days in vitro, glycine receptor immunoreactivity revealed by the monoclonal antibody mAb4a was found in < 10% of cells and was mainly localized intracellularly; in contrast, gephyrin was detected in in vitro, gephyrin was essentially localized at the neuronal surface. At this stage, the number of glycine receptor-positive cells approached that of gephyrin-containing neurons (50%), and glycine receptor antigen was found both intracellularly and at the periphery of the cells. The antibody mAb2b, which binds exclusively to the alpha1 subunit, revealed aggregates at the surface of a few neurons. At 10 days in vitro, glycine receptor and gephyrin staining was localized in clusters at the periphery of the soma and the neurites. This quantitative analysis corroborates temporal differences in the cellular distribution of gephyrin and glycine receptor alpha subunits, the former being accumulated first at the neuronal surface.

Dopaminergic (DA) neurons located in the preoptico-hypothalamic region of the brain exert a major neuroendocrine control on reproduction, growth, and homeostasis by regulating the secretion of anterior pituitary (or adenohypophysis) hormones. Here, using a retrograde tract tracing experiment, we identified the neurons playing this role in the zebrafish. The DA cells projecting directly to the anterior pituitary are localized in the most anteroventral part of the preoptic area, and we named them preoptico-hypophyseal DA (POHDA) neurons. During development, these neurons do not appear before 72 hours postfertilization (hpf) and are the last dopaminergic cell group to differentiate. We found that the number of neurons in this cell population continues to increase throughout life proportionally to the growth of the fish. 5-Bromo-2'-deoxyuridine incorporation analysis suggested that this increase is due to continuous neurogenesis and not due to a phenotypic change in already-existing neurons. Finally, expression profiles of several genes (foxg1a, dlx2a, and nr4a2a/b) were different in the POHDA compared with the adjacent suprachiasmatic DA neurons, suggesting that POHDA neurons develop as a distinct DA cell population in the preoptic area. This study offers some insights into the regional identity of the preoptic area and provides the first bases for future functional genetic studies on the development of DA neurons controlling anterior pituitary functions.

Ascending visual projections similar to the mammalian thalamocortical pathway are found in a wide range of vertebrate species, but their homology is debated. To get better insights into their evolutionary origin, we examined the developmental origin of a thalamic-like sensory structure of teleosts, the preglomerular complex (PG), focusing on the visual projection neurons. Similarly to the tectofugal thalamic nuclei in amniotes, the lateral nucleus of PG receives tectal information and projects to the pallium. However, our cell lineage study in zebrafish reveals that the majority of PG cells are derived from the midbrain, unlike the amniote thalamus. We also demonstrate that the PG projection neurons develop gradually until late juvenile stages. Our data suggest that teleost PG, as a whole, is not homologous to the amniote thalamus. Thus, the thalamocortical-like projections evolved from a non-forebrain cell population, which indicates a surprising degree of variation in the vertebrate sensory systems.

The sequence of events leading to the chemical matching of presynaptic neurotransmitters and postsynaptic transmitter receptors is investigated here in vivo for the spinal glycine receptor (GlyR) by using immunocytochemical methods. In the ventral horn of adult rat spinal cord, GlyRs are only present at glycinergic postsynaptic differentiations where they are stabilized by the associated protein gephyrin. With quantitative confocal microscopy, we found that gephyrin is detected before GlyRs at embryonic day (E)13-E14 and at E15, respectively, inside the cytoplasm and at plasmalemmal loci. Around the time of birth, the number of cell surface gephyrin-immunoreactive (-IR) spots exceeds that of GlyR. They first match 10 days after birth. The densities of postsynaptic gephyrin- and GlyR-IR were quantified between birth and the adult stage with post-embedding immunogold staining. Immunostaining for gephyrin and GlyR was not detected in the extrasynaptic membrane. The density of staining in postsynaptic membrane increased progressively with development. The inhibitory amino-acid content of the presynaptic terminal boutons opposed to gephyrin-IR sites was also analyzed. In the newborn, postnatal day 10, and adult, more than 90% of these boutons were immunostained for glycine. As seen with serial sections, 38% and 51.2% of the terminals also contained gamma-aminobutyric acid (GABA) in neonate and adult, respectively. These data indicate that around the time of birth, most glycine-containing boutons, some also containing GABA, are opposed to gephyrin-IR postsynaptic densities, whereas GlyRs are not present. Our results suggest that gephyrin determines subsynaptic loci on the plasma membrane where GlyR will subsequently accumulate.

and their biomolecular interactions with high sensitivity on the cellular and subcellular level in vitro and in vivo. [1] Autofluorescence of endogenous biological components in cells can cause significant fluorescence background, [2] which is most prominent in tissues and living organisms. [3] Several physical, chemical, and biological approaches have been developed to avoid autofluorescence, [4-6] including nanoparticle-based probes that absorb and/or emit in the infrared spectral region. [7,8] Because autofluorescence is short-lived (nano-to microseconds), pulsed excitation and time-gated (TG) detection with a delay that exceeds the autofluorescence background is another possibility for its efficient removal. [9] Nanotechnology has played an important role in such approaches by providing semiconductor, silicon, or lanthanide nanoparticles with long excited-state lifetimes for TG imaging. [10-12] Autofluorescence background is by far not the only problem of fluorescence imaging in the complex environment of living organisms. Fluorescent probes should be bright, resistant to photobleaching, and nontoxic, have minimal interference The zebrafish is an important vertebrate model for disease, drug discovery, toxicity, embryogenesis, and neuroscience. In vivo fluorescence microscopy can reveal cellular and subcellular details down to the molecular level with fluorescent proteins (FPs) currently the main tool for zebrafish imaging. However, long maturation times, low brightness, photobleaching, broad emission spectra, and sample autofluorescence are disadvantages that cannot be easily overcome by FPs. Here, a bright and photostable terbiumto-quantum dot (QD) Förster resonance energy transfer (FRET) nanoprobe with narrow and tunable emission bands for intracellular in vivo imaging is presented. The long photoluminescence (PL) lifetime enables time-gated (TG) detection without autofluorescence background. Intracellular four-color multiplexing with a single excitation wavelength and in situ assembly and FRET to mCherry demonstrate the versatility of the TG-FRET nanoprobes and the possibility of in vivo bioconjugation to FPs and combined nanoprobe-FP FRET sensing. Upon injection at the one-cell stage, FRET nanoprobes can be imaged in developing zebrafish embryos over seven days with toxicity similar to injected RNA and strongly improved signal-to-background ratios compared to non-TG imaging. This work provides a strategy for advancing in vivo fluorescence imaging applications beyond the capabilities of FPs.