Contribution of the eye and of opn4xa function to circadian photoentrainment in the diurnal zebrafish.

The eye is instrumental for controlling circadian rhythms in mice and human. Here, we address the conservation of this function in the zebrafish, a diurnal vertebrate. Using lakritz (lak) mutant larvae, which lack retinal ganglion cells (RGCs), we show that while a functional eye contributes to masking, it is largely dispensable for the establishment of circadian rhythms of locomotor activity. Furthermore, the eye is dispensable for the induction of a phase delay following a pulse of white light at CT 16 but contributes to the induction of a phase advance upon a pulse of white light at CT21. Melanopsin photopigments are important mediators of photoentrainment, as shown in nocturnal mammals. One of the zebrafish melanopsin genes, opn4xa, is expressed in RGCs but also in photosensitive projection neurons in the pineal gland. Pineal opn4xa+ projection neurons function in a LIGHT ON manner in contrast to other projection neurons which function in a LIGHT OFF mode. We generated an opn4xa mutant in which the pineal LIGHT ON response is impaired. This mutation has no effect on masking and circadian rhythms of locomotor activity, or for the induction of phase shifts, but slightly modifies period length when larvae are subjected to constant light. Finally, analysis of opn4xa;lak double mutant larvae did not reveal redundancy between the function of the eye and opn4xa in the pineal for the control of phase shifts after light pulses. Our results support the idea that the eye is not the sole mediator of light influences on circadian rhythms of locomotor activity and highlight differences in the circadian system and photoentrainment of behaviour between different animal models.

Cartilage regeneration in zebrafish depends on Nrg1/ErbB signaling pathway.

Objective: Cartilage, as the majority of adult mammalian tissues, has limited regeneration capacity. Cartilage degradation consecutive to joint injury or aging then leads to irreversible joint damage and diseases. In contrast, several vertebrate species such as the zebrafish have the remarkable capacity to spontaneously regenerate skeletal structures after severe injuries. The objective of our study was to test the regenerative capacity of Meckel's cartilage (MC) upon mechanical injury in zebrafish and to identify the mechanisms underlying this process. Methods and Results: Cartilage regenerative capacity in zebrafish larvae was investigated after mechanical injuries of the lower jaw MC in TgBAC(col2a1a:mCherry), to visualize the loss and recovery of cartilage. Confocal analysis revealed the formation of new chondrocytes and complete regeneration of MC at 14 days post-injury (dpi) via chondrocyte cell cycle re-entry and proliferation of pre-existing MC chondrocytes near the wound. Through expression analyses, we showed an increase of nrg1 expression in the regenerating lower jaw, which also expresses Nrg1 receptors, ErbB3 and ErbB2. Pharmacological inhibition of the ErbB pathway and specific knockdown of Nrg1 affected MC regeneration indicating the pivotal role of this pathway for cartilage regeneration. Finally, addition of exogenous NRG1 in an in vitro model of osteoarthritic (OA)-like chondrocytes induced by IL1ß suggests that Nrg1/ErbB pathway is functional in mammalian chondrocytes and alleviates the increased expression of catabolic markers characteristic of OA-like chondrocytes. Conclusion: Our results show that the Nrg1/ErbB pathway is required for spontaneous cartilage regeneration in zebrafish and is of interest to design new therapeutic approaches to promote cartilage regeneration in mammals.

Animal Models of Osteoarthritis Part 1-Preclinical Small Animal Models: Challenges and Opportunities for Drug Development.

Osteoarthritis (OA) is the most common form of arthritis and a major source of pain and disability in the adult population. There is a significant unmet medical need for the development of effective pharmacological therapies for the treatment of OA. In addition to spontaneously occurring animal models of OA, many experimental animal models have been developed to provide insights into mechanisms of pathogenesis and progression. Many of these animal models are also being used in the drug development pipeline. Here, we provide an overview of commonly used and emerging preclinical small animal models of OA and highlight the strengths and limitations of small animal models in the context of translational drug development. There is limited information in the published literature regarding the technical reliability of these small animal models and their ability to accurately predict clinical drug development outcomes. The cost and complexity of the available models however is an important consideration for pharmaceutical companies, biotechnology startups, and contract research organizations wishing to incorporate preclinical models in target validation, discovery, and development pipelines. Further considerations relevant to industry include timelines, methods of induction, the key issue of reproducibility, and appropriate outcome measures needed to objectively assess outcomes of experimental therapeutics. Preclinical small animal models are indispensable tools that will shine some light on the pathogenesis of OA and its molecular endotypes in the context of drug development. This paper will focus on small animal models used in preclinical OA research. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Exploring Macrophage-Dependent Wound Regeneration During Mycobacterial Infection in Zebrafish.

The molecular and cellular mechanisms associated with tissue degradation or regeneration in an infectious context are poorly defined. Herein, we explored the role of macrophages in orchestrating either tissue regeneration or degradation in zebrafish embryos pre-infected with the fish pathogen Mycobacterium marinum. Zebrafish were inoculated with different infectious doses of M. marinum prior to fin resection. While mild infection accelerated fin regeneration, moderate or severe infection delayed this process by reducing blastemal cell proliferation and impeding tissue morphogenesis. This was correlated with impaired macrophage recruitment at the wound of the larvae receiving high infectious doses. Macrophage activation characterized, in part, by a high expression level of tnfa was exacerbated in severely infected fish during the early phase of the regeneration process, leading to macrophage necrosis and their complete absence in the later phase. Our results demonstrate how a mycobacterial infection influences the macrophage response and tissue regenerative processes.

Functional heterogeneity in the pineal projection neurons of zebrafish.

The zebrafish pineal organ is a photoreceptive structure containing two main neuronal populations (photoreceptors and projections neurons). Here we describe a subpopulation of projection neurons that expresses the melanopsin gene, opn4xa. This new pineal cell type, that displays characteristics of both projection neurons and photoreceptors, share a similar dependency for BMP and Notch signalling pathways with classical non-photosensitive projection neurons (PN). Functionally, however, whereas classical, opn4xa-negative PNs display an achromatic LIGHT OFF response, the novel cell type we describe exhibit a LIGHT ON character that is elicited by green and blue light. Taken together, our data suggest a previously unanticipated heterogeneity in the projection neuron population in the zebrafish pineal organ raising the question of the importance of these differences in pineal function.

The pineal gland from development to function.

The pineal gland is a small neuroendocrine organ whose main and most conserved function is the nighttime secretion of melatonin. In lower vertebrates, the pineal gland is directly photosensitive. In contrast, in higher vertebrates, the direct photosensitivity of the pineal gland had been lost. Rather, the action of this gland as a relay between environmental light conditions and body functions involves reception of light information by the retina. In parallel to this sensory regression, the pineal gland (and its accessory organs) appears to have lost several functions in relation to light and temperature, which are important in lower vertebrate species. In humans, the functions of the pineal gland overlap with the functions of melatonin. They are extremely widespread and include general effects both on cell protection and on more precise functions, such as sleep and immunity. Recently, the role of melatonin has received a considerable amount of attention due to increased cancer risk in shift workers and the discovery that patients suffering from neurodegenerative diseases, autism, or depression exhibit abnormal melatonin rhythms.

Cell lineage analysis reveals three different progenitor pools for neurosensory elements in the otic vesicle.

In the inner ear, sensory versus neuronal specification is achieved through few well-defined bHLH transcription factors. However, the molecular mechanisms regulating the generation of the appropriate cell type in the correct place and at the correct time are not completely understood yet. Various studies have shown that hair cell- and neuron-specifying genes partially overlap in the otic territory, suggesting that mutual interactions among these bHLH factors could direct the generation of the two cell types from a common neurosensory progenitor. Although there is little evidence for a clonal relationship between macular hair cells and sensory neurons, the existence of a single progenitor able to give both sensory and neuronal cell types remains an open question. Here, we identified a population of common neurosensory progenitors in the zebrafish inner ear and studied the proneural requirement for cell fate decision within this population. Expression analysis reveals that proneural genes for hair cells and neurons overlap within the posteromedial otic epithelium. Combined results from single-cell lineage and functional studies on neurog1 and neuroD1 further demonstrate the following: (1) in the anterior region of the ear, neuronal and sensory lineages have already segregated at the onset of proneural gene expression and are committed to a given fate very early; (2) in contrast, the posteromedial part of the ear harbors a population of common progenitors giving both neurons and hair cells until late stages; and finally (3) neuroD1 is required within this pool of bipotent progenitors to generate the hair cell fate.

Origin and early development of the posterior lateral line system of zebrafish.

The lateral line system of teleosts has recently become a model system to study patterning and morphogenesis. However, its embryonic origins are still not well understood. In zebrafish, the posterior lateral line (PLL) system is formed in two waves, one that generates the embryonic line of seven to eight neuromasts and 20 afferent neurons and a second one that generates three additional lines during larval development. The embryonic line originates from a postotic placode that produces both a migrating sensory primordium and afferent neurons. Nothing is known about the origin and innervation of the larval lines. Here we show that a "secondary" placode can be detected at 24 h postfertilization (hpf), shortly after the primary placode has given rise to the embryonic primordium and ganglion. The secondary placode generates two additional sensory primordia, primD and primII, as well as afferent neurons. The primary and secondary placodes require retinoic acid signaling at the same stage of late gastrulation, suggesting that they share a common origin. Neither primary nor secondary neurons show intrinsic specificity for neuromasts derived from their own placode, but the sequence of neuromast deposition ensures that neuromasts are primarily innervated by neurons derived from the cognate placode. The delayed formation of secondary afferent neurons accounts for the capability of the fish to form a new PLL ganglion after ablation of the embryonic ganglion at 24 hpf.

Hedgehog signaling governs the development of otic sensory epithelium and its associated innervation in zebrafish.

The inner ear is responsible for the perception of motion and sound in vertebrates. Its functional unit, the sensory patch, contains mechanosensory hair cells innervated by sensory neurons from the statoacoustic ganglion (SAG) that project to the corresponding nuclei in the brainstem. How hair cells develop at specific positions, and how otic neurons are sorted to specifically innervate each endorgan and to convey the extracted information to the hindbrain is not completely understood. In this work, we study the generation of macular sensory patches and investigate the role of Hedgehog (Hh) signaling in the production of their neurosensory elements. Using zebrafish transgenic lines to visualize the dynamics of hair cell and neuron production, we show that the development of the anterior and posterior maculae is asynchronic, suggesting they are independently regulated. Tracing experiments demonstrate the SAG is topologically organized in two different neuronal subpopulations, which are spatially segregated and innervate specifically each macula. Functional experiments identify the Hh pathway as crucial in coordinating the production of hair cells in the posterior macula, and the formation of its specific innervation. Finally, gene expression analyses suggest that Hh influences the balance between different SAG neuronal subpopulations. These results lead to a model in which Hh orients functionally the development of inner ear towards an auditory fate in all vertebrate species.

CXCR4 and CXCR7 cooperate during tangential migration of facial motoneurons.

Migration of facial motoneurons in the zebrafish hindbrain depends on SDF1/CXCL12 signaling. Recent studies demonstrated that SDF1 can bind two chemokine receptors, CXCR4 and CXCR7. Here we explore the expression and function of the cxcr7b gene in zebrafish hindbrain development. By the time cxcr4b-expressing motoneurons migrate from rhombomere (r) r4 to r6, expression of cxcr7b is rapidly restricted to the ventral part of r5. Inactivation of either cxcr7b or cxcr4b impairs motoneuron migration, with however different phenotypes. Facial motoneurons preferentially accumulate in r5 in cxcr7b morphant embryos, while they are distributed between r4, r5 and r6 in cxcr4b morphants. Simultaneous inactivation of both receptors leads to yet a third phenotype, with motoneurons mostly distributed between r4 and r5. The latter phenotype resembles that of sdf1a morphant embryos. Double inactivation of sdf1a and cxcr7b indeed did not lead to a complete arrest of migration but rather to a partial rescue of r5 arrest of motoneuron migration. This result is in accordance with the functional hypothesis that SDF1 might interact with CXCR7 and that they have an antagonistic effect within r5. The ectopic expression of a truncated CXCR7 receptor leads to a motoneuron migration defect. Altogether, we show that CXCR7 is required, for proper tangential migration of facial motoneurons, by determining a permissive migration pathway through r5.

Control of cell migration in the zebrafish lateral line: implication of the gene "tumour-associated calcium signal transducer," tacstd.

The sensory organs of the zebrafish lateral-line system (neuromasts) originate from migrating primordia that move along precise pathways. The posterior primordium, which deposits the neuromasts on the body and tail of the embryo, migrates along the horizontal myoseptum from the otic region to the tip of the tail. This migration is controlled by the chemokine SDF1, which is expressed along the prospective pathway, and by its receptor CXCR4, which is expressed by the migrating cells. In this report, we describe another zebrafish gene that is heterogeneously expressed in the migrating cells, tacstd. This gene codes for a membrane protein that is homologous to the TACSTD1/2 mammalian proteins. Inactivation of the zebrafish tacstd gene results in a decrease in proneuromast deposition, suggesting that tacstd is required for the deposition process.

Role of SDF1 chemokine in the development of lateral line efferent and facial motor neurons.

Most sensory systems are innervated by efferent neurons as well as by afferent neurons. The efferent innervation modulates the sensitivity of the receptor cells or of the sensory terminals. In the posterior lateral line system of the zebrafish, two efferent nuclei have been described in the hindbrain. Here we examine the development of the efferent neurons. We show that their axons are guided toward the target organ along the lateral line nerve while their cell bodies migrate posteriorward across rhombomeres to achieve their final position in rhombomeres 6/7. This migration depends on the SDF1 chemokine. We show that the migration of motor neurons of the facial nucleus from rhombomere 4 to 6 is also affected in sdf1a morphants (embryos injected with morpholine-conjugated antisense oligonucleotides). We propose that SDF1/CXCR4-mediated cell migration is preferentially associated with movement along the anteroposterior axis of the animal.

The lateral line of zebrafish: a model system for the analysis of morphogenesis and neural development in vertebrates.

The lateral line of the zebrafish has many of the advantages that made the sensory organs of Drosophila a very productive model system: 1) it comprises a set of discrete sense organs (neuromasts) arranged in a defined, species-specific pattern, such that each organ can be individually recognized; 2) the neuromasts are superficial and easy to visualize, and the innervating neurons are easy to label; 3) the sensory projection is simple yet reproducibly organized. Here we describe some of the tools that can be used to investigate the development of this system, and we illustrate their usefulness with specific examples. We conclude that the lateral line is uniquely suited among vertebrate sensory systems for a molecular, cellular and genetic analysis of pattern formation and of neural development.

Molecular basis of cell migration in the fish lateral line: role of the chemokine receptor CXCR4 and of its ligand, SDF1.

Cell migration plays an essential role in many morphogenetic processes, and its deregulation has many dramatic consequences. Yet how migration is controlled during normal development is still a largely unresolved question. We examined this process in the case of the posterior lateral line (PLL), a mechanosensory system present in fish and amphibians. In zebrafish, the embryonic PLL comprises seven to eight sense organs (neuromasts) aligned from head to tail along the flank of the animal and is formed by a primordium that originates from a cephalic placode. This primordium migrates along a stereotyped pathway toward the tip of the tail and deposits in its wake discrete groups of cells, each of which will become a neuromast. We show that a trail of SDF1-like chemokine is present along the pathway of the primordium and that a CXCR4-like chemokine receptor is expressed by the migrating cells. The inactivation of either the ligand or its receptor blocks migration, whereas in mutants in which the normal SDF1 trail is absent, the primordium path is redirected to the next, more ventral sdf1 expression domain. In all cases, the sensory axons remain associated to the primordium, indicating that the extension of the neurites to form the PLL nerve depends on the movement of the primordium. We conclude that both the formation and the innervation of this system depend on the SDF1-CXCR4 system, which has also been implicated in several migration events in humans, including metastasis formation and lymphocyte homing.

Cell migration in the postembryonic development of the fish lateral line.

We examine at the cellular level the postembryonic development of the posterior lateral line in the zebrafish. We show that the first wave of secondary neuromasts is laid down by a migrating primordium, primII. This primordium originates from a cephalic region much like the primordium that formed the primary line during embryogenesis. PrimII contributes to both the lateral and the dorsal branches of the posterior lateral line. Once they are deposited by the primordium, the differentiating neuromasts induce the specialisation of overlying epidermal cells into a pore-forming annulus, and the entire structure begins to migrate ventrally across the epithelium. Thus the final two-dimensional pattern depends on the combination of two orthogonal processes: anteroposterior waves of neuromast formation and dorsoventral migration of individual neuromasts. Finally, we examine how general these migratory processes can be by describing two fish species with very different adult patterns, Astyanax fasciatus (Mexican blind cavefish) and Oryzias latipes (medaka). We show that their primary patterns are nearly identical to that observed in zebrafish embryos, and that their postembryonic growth relies on the same combination of migratory processes that we documented in the case of the zebrafish.