Comparing Sensory Organs to Define the Path for Hair Cell Regeneration.

Deafness or hearing deficits are debilitating conditions. They are often caused by loss of sensory hair cells or defects in their function. In contrast to mammals, nonmammalian vertebrates robustly regenerate hair cells after injury. Studying the molecular and cellular basis of nonmammalian vertebrate hair cell regeneration provides valuable insights into developing cures for human deafness. In this review, we discuss the current literature on hair cell regeneration in the context of other models for sensory cell regeneration, such as the retina and the olfactory epithelium. This comparison reveals commonalities with, as well as differences between, the different regenerating systems, which begin to define a cellular and molecular blueprint of regeneration. In addition, we propose how new technical advances can address outstanding questions in the field.

Spherical harmonics analysis reveals cell shape-fate relationships in zebrafish lateral line neuromasts.

Cell shape is a powerful readout of cell state, fate and function. We describe a custom workflow to perform semi-automated, 3D cell and nucleus segmentation, and spherical harmonics and principal components analysis to distill cell and nuclear shape variation into discrete biologically meaningful parameters. We apply these methods to analyze shape in the neuromast cells of the zebrafish lateral line system, finding that shapes vary with cell location and identity. The distinction between hair cells and support cells accounted for much of the variation, which allowed us to train classifiers to predict cell identity from shape features. Using transgenic markers for support cell subpopulations, we found that subtypes had different shapes from each other. To investigate how loss of a neuromast cell type altered cell shape distributions, we examined atoh1a mutants that lack hair cells. We found that mutant neuromasts lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare and classify cells in a living developing organism.

Environmental and molecular control of tissue-specific ionocyte differentiation in zebrafish.

Organisms cope with environmental fluctuations and maintain fitness in part via reversible phenotypic changes (acclimation). Aquatic animals are subject to dramatic seasonal fluctuations in water salinity, which affect osmolarity of their cells and consequently cellular function. Mechanosensory lateral line hair cells detect water motion for swimming behavior and are especially susceptible to salinity changes due to their direct contact with the environment. To maintain hair cell function when salinity decreases, neuromast (Nm)-associated ionocytes differentiate and invade lateral line neuromasts. The signals that trigger the adaptive differentiation of Nm ionocytes are unknown. We demonstrate that new Nm ionocytes are rapidly specified and selectively triggered to proliferate by low Ca2+ and Na+/Cl- levels. We further show that Nm ionocyte recruitment and induction is affected by hair cell activity. Once specified, Nm ionocyte differentiation and survival are associated with sequential activation of different Notch pathway components, a process different from other tissue-specific ionocytes. In summary, we show how environmental changes activate a signaling cascade that leads to physiological adaptation. This may prove essential for survival not only in seasonal changing environments but also changing climates.

RhoA GEF Mcf2lb regulates rosette integrity during collective cell migration.

Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of ∼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.

Tadpoles rely on mechanosensory stimuli for communication when visual capabilities are poor.

The ways in which animals sense the world changes throughout development. For example, young of many species have limited visual capabilities, but still make social decisions, likely based on information gathered through other sensory modalities. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems relative to adults. Alternatively, other sensory modalities, such as the lateral line system, are functional at hatching in frogs and may guide social decisions while other sensory systems mature. Here, we examined development of the mechanosensory lateral line and visual systems in tadpoles of the mimic poison frog (Ranitomeya imitator) that use vibrational begging displays to stimulate egg feeding from their mothers. We found that tadpoles hatch with a fully developed lateral line system. While begging behavior increases with development, ablating the lateral line system inhibited begging in pre-metamorphic tadpoles, but not in metamorphic tadpoles. We also found that the increase in begging and decrease in reliance on the lateral line co-occurs with increased retinal neural activity and gene expression associated with eye development. Using the neural tracer neurobiotin, we found that axonal innervations from the eye to the brain proliferate during metamorphosis, with few retinotectal connections in recently-hatched tadpoles. We then tested visual function in a phototaxis assay and found tadpoles prefer darker environments. The strength of this preference increased with developmental stage, but eyes were not required for this behavior, possibly indicating a role for the pineal gland. Together, these data suggest that tadpoles rely on different sensory modalities for social interactions across development and that the development of sensory systems in socially complex poison frog tadpoles is similar to that of other frog species.

Pharmacological reprogramming of zebrafish lateral line supporting cells to a migratory progenitor state.

In the zebrafish lateral line, non-sensory supporting cells readily re-enter the cell cycle to generate new hair cells and supporting cells during homeostatic maintenance and following damage to hair cells. This contrasts with supporting cells from mammalian vestibular and auditory sensory epithelia which rarely re-enter the cell cycle, and hence loss of hair cells results in permanent sensory deficit. Lateral line supporting cells are derived from multipotent progenitor cells that migrate down the trunk midline as a primordium and are deposited to differentiate into a neuromast. We have found that we can revert zebrafish support cells back to a migratory progenitor state by pharmacologically altering the signaling environment to mimic that of the migratory primordium, with active Wnt signaling and repressed FGF signaling. The reverted supporting cells migrate anteriorly and posteriorly along the horizontal myoseptum and will re-epithelialize to form an increased number of neuromasts along the midline when the pharmacological agents are removed. These data demonstrate that supporting cells can be readily reprogrammed to a migratory multipotent progenitor state that can form new sensory neuromasts, which has important implications for our understanding of how the lateral line system matures and expands in fish and also suggest avenues for returning mammalian supporting cells back to a proliferative state.

Macrophages enhance regeneration of lateral line neuromast derived from interneuromast cells through TGF-ß in zebrafish.

Macrophages play a pivotal role in the response to injury, contributing significantly to the repair and regrowth of damaged tissues. The external lateral line system in aquatic organisms offers a practical model for studying regeneration, featuring interneuromast cells connecting sensory neuromasts. Under normal conditions, these cells remain dormant, but their transformation into neuromasts occurs when overcoming inhibitory signals from Schwann cells and posterior lateral line nerves. The mechanism enabling interneuromast cells to evade inhibition by Schwann cells remains unclear. Previous observations suggest that macrophages physically interact with neuromasts, nerves, and Schwann cells during regeneration. This interaction leads to the regeneration of neuromasts in a subset of zebrafish with ablated neuromasts. To explore whether macrophages achieve this effect through secreted cytokines, we conducted experiments involving tail amputation in zebrafish larvae and tested the impact of cytokine inhibitors on neuromast regeneration. Most injured larvae remarkably regenerated a neuromast within 4 days post-amputation. Intriguingly, removal of macrophages and inhibition of the anti-inflammatory cytokine transforming growth factor-beta (TGF-ß) significantly delayed neuromast regeneration. Conversely, inhibition of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) had minor effects on the regeneration process. This study provides insights into how macrophages activate interneuromast cells, elucidating the pathways underlying neuromast regeneration.

Representation of bulk water flow in the goldfish (Carassius auratus) midbrain.

With the mechanosensory lateral line system, fish and semi-aquatic amphibians detect water movements and pressure gradients. Hydrodynamic information picked up by the lateral line receptors is relayed via peripheral nerves to the lateral line brainstem and from there to the midbrain torus semicircularis. Most prior electrophysiological studies of the lateral line were done under still-water conditions, even though natural environments encountered by fish include bulk-flow. Flow velocity and direction sensing are likely important to fish as they navigate variable, turbulent environments, but to date, only few studies have gathered information on the processing of bulk water flow by midbrain units. Here, we recorded from lateral line units in the torus semicircularis while presenting various bulk flow velocities in anterior-to-posterior and posterior-to-anterior flow directions. We studied (1) the temporal spike patterns of mechanosensory midbrain units, (2) the processing of bulk water flow velocity by these units, and (3) the processing of bulk water flow direction. We found that midbrain mechanosensory units alter their discharge rate during bulk water flow - some units responded to flow by increasing their discharge rate but did not vary this rate significantly with flow velocity, while others exhibited increasing discharge rates with increasing flow velocity. Units directly coding for flow direction were not found.

The incomparable fascination of comparative physiology: 40 years with animals in the field and laboratory.

This paper is not meant to be a review article. Instead, it gives an overview of the major research projects that the author, together with his students, colleagues and collaborators, has worked on. Although the main focus of the author's work has always been the fish lateral line, this paper is mainly about all the other research projects he did or that were done in his laboratory. These include studies on fishing spiders, weakly electric fish, seals, water rats, bottom dwelling sharks, freshwater rays, venomous snakes, birds of prey, fire loving beetles and backswimmers. The reasons for this diversity of research projects? Simple. The authors's lifelong enthusiasm for animals, and nature's ingenuity in inventing new biological solutions. Indeed, this most certainly was a principal reason why Karl von Frisch and Alfred Kühn founded the Zeitschrift für vergleichende Physiologie (now Journal of Comparative Physiology A) 100 years ago.

The role of vision and lateral line sensing for schooling in giant danios (Devario aequipinnatus).

Schooling is a collective behavior that relies on a fish's ability to sense and respond to the other fish around it. Previous work has identified 'rules' of schooling - attraction to neighbors that are far away, repulsion from neighbors that are too close and alignment with neighbors at the correct distance - but we do not understand well how these rules emerge from the sensory physiology and behavior of individual fish. In particular, fish use both vision and their lateral lines to sense each other, but it is unclear how much they rely on information from these sensory modalities to coordinate schooling behavior. To address this question, we studied how the schooling of giant danios (Devario aequipinnatus) changes when they are unable to see or use their lateral lines. We found that giant danios were able to school without their lateral lines but did not school in darkness. Surprisingly, giant danios in darkness had the same attraction properties as fish in light when they were in close proximity, indicating that they could sense nearby fish with their lateral lines. However, they were not attracted to more distant fish, suggesting that long-distance attraction through vision is important for maintaining a cohesive school. These results help us expand our understanding of the roles that vision and the lateral line play in the schooling of some fish species.

Fish communicate with water flow to enhance a school's social network.

Schooling fish rely on a social network created through signaling between its members to interact with their environment. Previous studies have established that vision is necessary for schooling and that flow sensing by the lateral line system may aid in a school's cohesion. However, it remains unclear to what extent flow provides a channel of communication between schooling fish. Based on kinematic measurements of the speed and heading of schooling tetras (Petitella rhodostoma), we found that compromising the lateral line by chemical treatment reduced the mutual information between individuals by ∼13%. This relatively small reduction in pairwise communication propagated through schools of varying size to reduce the degree and connectivity of the social network by more than half. Treated schools additionally showed more than twice the spatial heterogeneity of fish with unaltered flow sensing. These effects were much more substantial than the changes that we measured in the nearest-neighbor distance, speed and intermittency of individual fish by compromising flow sensing. Therefore, flow serves as a valuable supplement to visual communication in a manner that is revealed through a school's network properties.

Local tissue interactions govern pLL patterning in medaka.

Vertebrate organs are arranged in a stereotypic, species-specific position along the animal body plan. Substantial morphological variation exists between related species, especially so in the vastly diversified teleost clade. It is still unclear how tissues, organs and systems can accommodate such diverse scaffolds. Here, we use the distinctive arrangement of neuromasts in the posterior lateral line (pLL) system of medaka fish to address the tissue-interactions defining a pattern. We show that patterning in this peripheral nervous system is established by autonomous organ precursors independent of neuronal wiring. In addition, we target the keratin 15 gene to generate stuck-in-the-midline (siml) mutants, which display epithelial lesions and a disrupted pLL patterning. By using siml/wt chimeras, we determine that the aberrant siml pLL pattern depends on the mutant epithelium, since a wild type epithelium can rescue the siml phenotype. Inducing epithelial lesions by 2-photon laser ablation during pLL morphogenesis phenocopies siml genetic mutants and reveals that epithelial integrity defines the final position of the embryonic pLL neuromasts. Our results using the medaka pLL disentangle intrinsic from extrinsic properties during the establishment of a sensory system. We speculate that intrinsic programs guarantee proper organ morphogenesis, while instructive interactions from surrounding tissues facilitates the accommodation of sensory organs to the diverse body plans found among teleosts.

Peripheral nerve development in zebrafish requires muscle patterning by tcf15/paraxis.

The vertebrate peripheral nervous system (PNS) is an intricate network that conveys sensory and motor information throughout the body. During development, extracellular cues direct the migration of axons and glia through peripheral tissues. Currently, the suite of molecules that govern PNS axon-glial patterning is incompletely understood. To elucidate factors that are critical for peripheral nerve development, we characterized the novel zebrafish mutant, stl159, that exhibits abnormalities in PNS patterning. In these mutants, motor and sensory nerves that develop adjacent to axial muscle fail to extend normally, and neuromasts in the posterior lateral line system, as well as neural crest-derived melanocytes, are incorrectly positioned. The stl159 genetic lesion lies in the basic helix-loop-helix (bHLH) transcription factor tcf15, which has been previously implicated in proper development of axial muscles. We find that targeted loss of tcf15 via CRISPR-Cas9 genome editing results in the PNS patterning abnormalities observed in stl159 mutants. Because tcf15 is expressed in developing muscle prior to nerve extension, rather than in neurons or glia, we predict that tcf15 non-cell-autonomously promotes peripheral nerve patterning in zebrafish through regulation of extracellular patterning cues. Our work underscores the importance of muscle-derived factors in PNS development.

Underwater Biomimetic Lateral Line Sensor Based on Triboelectric Nanogenerator for Dynamic Pressure Monitoring and Trajectory Perception.

Developing desirable sensors is crucial for underwater perceptions and operations. The perceiving organs of marine creatures have greatly evolved to react accurately and promptly underwater. Inspired by the fish lateral line, this study proposes a triboelectric dynamic pressure sensor for underwater perception. The biomimetic lateral line sensor (BLLS) has high sensitivity to the disturbance amplitude/frequency, good adaptability to underwater environments and (relative) low cost. The sensors are deployed at the bottom of the test basin to perceive various moving objects, such as a robotic fish, robotic seal, etc. By analyzing the electrical signal of the sensor, the motion parameters of the objects passed over can be obtained. By monitoring signal variations across multiple sensors, the ability to sense different disturbance movement trajectories, including linear and angular trajectories, is achievable. The study will prove significant in forming an unconventional underwater perceiving method, which can back-up the sonic/optical sensors when are impaired in complex underwater environments.

Molecular mechanisms of Eda-mediated adaptation to freshwater in threespine stickleback.

A main goal of evolutionary biology is to understand the genetic basis of adaptive evolution. Although the genes that underlie some adaptive phenotypes are now known, the molecular pathways and regulatory mechanisms mediating the phenotypic effects of those genes often remain a black box. Unveiling this black box is necessary to fully understand the genetic basis of adaptive phenotypes, and to understand why particular genes might be used during phenotypic evolution. Here, we investigated which genes and regulatory mechanisms are mediating the phenotypic effects of the Eda haplotype, a locus responsible for the loss of lateral plates and changes in the sensory lateral line of freshwater threespine stickleback (Gasterosteus aculeatus) populations. Using a combination of RNAseq and a cross design that isolated the Eda haplotype on a fixed genomic background, we found that the Eda haplotype affects both gene expression and alternative splicing of genes related to bone development, neuronal development and immunity. These include genes in conserved pathways, like the BMP, netrin and bradykinin signalling pathways, known to play a role in these biological processes. Furthermore, we found that differentially expressed and differentially spliced genes had different levels of connectivity and expression, suggesting that these factors might influence which regulatory mechanisms are used during phenotypic evolution. Taken together, these results provide a better understanding of the mechanisms mediating the effects of an important adaptive locus in stickleback and suggest that alternative splicing could be an important regulatory mechanism mediating adaptive phenotypes.

The oral sensory organs in Bathochordaeus stygius (Tunicata Appendicularia) are unique in structure and homologous to the coronal organ.

BACKGROUND: Appendicularia consists of approximately 70 purely marine species that belong to Tunicata the probable sister taxon to Craniota. Therefore, Appendicularia plays a pivotal role for our understanding of chordate evolution. In addition, appendicularians are an important part of the epipelagic marine plankton. Nevertheless, little is known about appendicularian species, especially from deeper water. RESULTS: Using µCT, scanning electron microscopy, and digital 3D-reconstruction techniques we describe three pairs of complex oral sensory organs in the mesopelagic appendicularian Bathochordaeus stygius. The oral sensory organs are situated at the anterior and lateral margin of the mouth and inside the mouth cavity. A single organ consists of 22-90 secondary receptor cells that project apical cilia through a narrow hole in the epidermis. The receptor cells are innervated by branches of the second brain nerve. CONCLUSIONS: Based on position, morphology, and innervation we suggest that the oral sensory organs are homologues of the coronal organs in other tunicates. We discuss the hypothesized homology of coronal organs and the lateral line system of primary aquatic vertebrates. The complex oral sensory organs of B. stygius are unique in tunicates and could be adaptations to the more muffled environment of the mesopelagic.

Sensorimotor control of swimming Polypterus senegalus is preserved during sensory deprivation conditions across altered environments.

Control of locomotion involves the interplay of sensory signals and motor commands. Sensory information is essential for adjusting locomotion in response to environmental changes. A previous study using mathematical modelling of lamprey swimming has shown that, in the absence of sensory feedback, increasing fluid viscosity constrains swimming kinematics, limiting tail amplitude and body wavelength, resulting in decreased swimming speed. In contrast, previous experiments with Polypterus senegalus reported increased magnitude swimming kinematics (increased body curvature, body wave speed and frequency, and pectoral fin frequency) in high viscosity water suggesting that sensory information is used to adjust swimming form. It is not known what sensory systems are providing the necessary information to respond to these environmental changes. We tested the hypothesis that lateral line and visual input are responsible for the sensory-driven increase in swimming kinematics in response to experimentally increased fluid viscosity. The kinematics of five P. senegalus were recorded in two different viscosities of water while removing lateral line and visual sensory feedback. Unlike the mathematical model devoid of sensory feedback, P. senegalus with lateral line and/or visual senses removed did not reduce the magnitude of swimming kinematic variables, suggesting that additional sensory feedback mechanisms are present in these fish to help overcome increased fluid viscosity. Increases in swimming speed when both lateral line and visual sensory feedback were removed suggest that lateral line and visual information may be used to regulate swimming speed in P. senegalus, possibly using an internal model of predictions to adjust swimming form.

A sensation for inflation: initial swim bladder inflation in larval zebrafish is mediated by the mechanosensory lateral line.

Larval zebrafish achieve neutral buoyancy by swimming up to the surface and taking in air through their mouths to inflate their swim bladders. We define this behavior as 'surfacing'. Little is known about the sensory basis for this underappreciated behavior of larval fish. A strong candidate is the mechanosensory lateral line, a hair cell-based sensory system that detects hydrodynamic information from sources such as water currents, predators, prey and surface waves. However, a role for the lateral line in mediating initial inflation of the swim bladder has not been reported. To explore the connection between the lateral line and surfacing, we used a genetic mutant (lhfpl5b-/-) that renders the zebrafish lateral line insensitive to mechanical stimuli. We observed that approximately half of these lateral line mutants over-inflate their swim bladders during initial inflation and become positively buoyant. Thus, we hypothesized that larval zebrafish use their lateral line to moderate interactions with the air-water interface during surfacing to regulate swim bladder inflation. To test the hypothesis that lateral line defects are responsible for swim bladder over-inflation, we showed that exogenous air is required for the hyperinflation phenotype and transgenic rescue of hair cell function restores normal inflation. We also found that chemical ablation of anterior lateral line hair cells in wild-type larvae causes hyperinflation. Furthermore, we show that manipulation of lateral line sensory information results in abnormal inflation. Finally, we report spatial and temporal differences in the surfacing behavior between wild-type and lateral line mutant larvae. In summary, we propose a novel sensory basis for achieving neutral buoyancy where larval zebrafish use their lateral line to sense the air-water interface and regulate initial swim bladder inflation.

Evolution of left-right asymmetry in the sensory system and foraging behavior during adaptation to food-sparse cave environments.

BACKGROUND: Laterality in relation to behavior and sensory systems is found commonly in a variety of animal taxa. Despite the advantages conferred by laterality (e.g., the startle response and complex motor activities), little is known about the evolution of laterality and its plasticity in response to ecological demands. In the present study, a comparative study model, the Mexican tetra (Astyanax mexicanus), composed of two morphotypes, i.e., riverine surface fish and cave-dwelling cavefish, was used to address the relationship between environment and laterality. RESULTS: The use of a machine learning-based fish posture detection system and sensory ablation revealed that the left cranial lateral line significantly supports one type of foraging behavior, i.e., vibration attraction behavior, in one cave population. Additionally, left-right asymmetric approaches toward a vibrating rod became symmetrical after fasting in one cave population but not in the other populations. CONCLUSION: Based on these findings, we propose a model explaining how the observed sensory laterality and behavioral shift could help adaptation in terms of the tradeoff in energy gain and loss during foraging according to differences in food availability among caves.

Cellular projections from sensory hair cells form polarity-specific scaffolds during synaptogenesis.

The assembly of a nervous system requires the extension of axons and dendrites to specific regions where they are matched with appropriate synaptic targets. Although the cues that guide long-range outgrowth have been characterized extensively, additional mechanisms are required to explain short-range guidance in neural development. Using a complementary combination of time-lapse imaging by fluorescence confocal microscopy and serial block-face electron microscopy, we identified a novel type of presynaptic projection that participates in the assembly of the vertebrate nervous system. Synapse formation by each hair cell of the zebrafish's lateral line occurs during a particular interval after the cell's birth. During the same period, projections emerge from the cellular soma, extending toward a specific subpopulation of mature hair cells and interacting with polarity-specific afferent nerve terminals. The terminals then extend along the projections to reach appropriately matched presynaptic sites, after which the projections recede. Our results suggest that presynaptic projections act as transient scaffolds for short-range partner matching, a mechanism that may occur elsewhere in the nervous system.