Teleost Hox code defines regional identities competent for the formation of dorsal and anal fins.

The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position.

Target-selective vertebrate motor axon regeneration depends on interaction with glial cells at a peripheral nerve plexus.

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from 3 nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real-time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

Neural signatures of sleep in zebrafish.

Slow-wave sleep and rapid eye movement (or paradoxical) sleep have been found in mammals, birds and lizards, but it is unclear whether these neuronal signatures are found in non-amniotic vertebrates. Here we develop non-invasive fluorescence-based polysomnography for zebrafish, and show-using unbiased, brain-wide activity recording coupled with assessment of eye movement, muscle dynamics and heart rate-that there are at least two major sleep signatures in zebrafish. These signatures, which we term slow bursting sleep and propagating wave sleep, share commonalities with those of slow-wave sleep and paradoxical or rapid eye movement sleep, respectively. Further, we find that melanin-concentrating hormone signalling (which is involved in mammalian sleep) also regulates propagating wave sleep signatures and the overall amount of sleep in zebrafish, probably via activation of ependymal cells. These observations suggest that common neural signatures of sleep may have emerged in the vertebrate brain over 450 million years ago.

A novel gene-trap line reveals the dynamic patterns and essential roles of cysteine and glycine-rich protein 3 in zebrafish heart development and regeneration.

Mutations in cysteine and glycine-rich protein 3 (CSRP3)/muscle LIM protein (MLP), a key regulator of striated muscle function, have been linked to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, the roles of CSRP3 in heart development and regeneration are not completely understood. In this study, we characterized a novel zebrafish gene-trap line, gSAIzGFFM218A, which harbors an insertion in the csrp3 genomic locus, heterozygous fish served as a csrp3 expression reporter line and homozygous fish served as a csrp3 mutant line. We discovered that csrp3 is specifically expressed in larval ventricular cardiomyocytes (CMs) and that csrp3 deficiency leads to excessive trabeculation, a common feature of CSRP3-related HCM and DCM. We further revealed that csrp3 expression increased in response to different cardiac injuries and was regulated by several signaling pathways vital for heart regeneration. Csrp3 deficiency impeded zebrafish heart regeneration by impairing CM dedifferentiation, hindering sarcomere reassembly, and reducing CM proliferation while aggravating apoptosis. Csrp3 overexpression promoted CM proliferation after injury and ameliorated the impairment of ventricle regeneration caused by pharmacological inhibition of multiple signaling pathways. Our study highlights the critical role of Csrp3 in both zebrafish heart development and regeneration, and provides a valuable animal model for further functional exploration that will shed light on the molecular pathogenesis of CSRP3-related human cardiac diseases.

The dwarf neon rainbowfish Melanotaenia praecox, a small spiny-rayed fish with potential as a new Acanthomorpha model fish: II. Establishment of a microinjection procedure for genetic engineering.

BACKGROUND: Rainbowfish is a clade of colorful freshwater fish. Melanotaenia praecox is a small rainbowfish species with biological characteristics that make it potentially useful as an experimental model species. We anticipate that M. praecox could become a new model used in various fields, such as ecology, evolution, and developmental biology. However, few previous studies have described experimental set-ups needed to understand the molecular and genetic mechanisms within this species. RESULTS: We describe detailed procedures for genetic engineering in the rainbowfish M. praecox. By using these procedures, we successfully demonstrated CRISPR/Cas-mediated knockout and Tol2 transposon-mediated transgenesis in this species. Regarding the CRISPR/Cas system, we disrupted the tyrosinase gene and then showed that injected embryos lacked pigmentation over much of their body. We also demonstrated that a Tol2 construct, including a GFP gene driven by a ubiquitous promoter, was efficiently integrated into the genome of M. praecox embryos. CONCLUSIONS: The establishment of procedures for genetic engineering in M. praecox enables investigation of the genetic mechanisms behind a broad range of biological phenomena in this species. Thus, we suggest that M. praecox can be used as a new model species in various experimental biology fields.

The enteric nervous system in zebrafish larvae can regenerate via migration into the ablated area and proliferation of neural crest-derived cells.

The enteric nervous system (ENS), which is derived from neural crest, is essential for gut function, and its deficiency causes severe congenital diseases. Since the capacity for ENS regeneration in mammals is limited, additional complementary models would be useful. Here, we show that the ENS in zebrafish larvae at 10-15 days postfertilization is highly regenerative. After laser ablation, the number of enteric neurons recovered to ∼50% of the control by 10 days post-ablation (dpa). Using transgenic lines in which enteric neural crest-derived cells (ENCDCs) and enteric neurons are labeled with fluorescent proteins, we live imaged the regeneration process and found covering by neurites that extended from the unablated area and entry of ENCDCs into the ablated areas by 1-3 dpa. BrdU assays suggested that ∼80% of the enteric neurons and ∼90% of the Sox10-positive ENCDCs therein at 7 dpa were generated through proliferation. Thus, ENS regeneration involves proliferation, entrance and neurogenesis of ENCDCs. This is the first report regarding the regeneration process of the zebrafish ENS. Our findings provide a basis for further in vivo research at single-cell resolution in this vertebrate model.

Remodeling of the hyomandibular skeleton and facial nerve positioning during embryonic and postembryonic development of teleost fish.

The vertebrate skeleton changes its shape during development through the activities of chondrocytes, osteoblasts and osteoclasts. Although much is known about the mechanisms for differentiation in these cells, it is less understood how they behave in a region-specific manner to acquire unique bone shapes. To address this question, we investigated the development of the hyomandibular (Hm) system in zebrafish. The Hm originates as cartilage carrying a single foramen (the Hm foramen), through which the facial (VII) nerve passes. We reveal that Schwann cells, which myelinate the VII nerve, regulate rearrangement of the chondrocytes to enlarge the Hm foramen. The Hm cartilage then becomes ossified in the perichondrium, where the marrow chondrocytes are replaced by adipocytes. Then, the bone matrix along the VII nerve is resorbed by osteoclasts, generating a gateway to the bone marrow. Subsequent movement of the VII nerve into the marrow, followed by deposition of new bone matrix, isolates the nerve from the jaw muscle insertion. Genetic ablation of osteoblasts and osteoclasts reveals specific roles of these cells during remodeling processes. Interestingly, the VII nerve relocation does not occur in medaka; instead, bone deposition distinct from those in zebrafish separates the VII nerve from the muscle insertion. Our results define novel mechanisms for skeletal remodeling, by which the bone shapes in a region- and species-specific manner.

A novel gene trap line for visualization and manipulation of erbb3b+ neural crest and glial cells in zebrafish.

Glia are a diverse and essential cell type in the vertebrate nervous system. Transgenic tools and fluorescent reporter lines are critical resources to investigate how glial subtypes develop and function. However, despite the many lines available in zebrafish, the community still lacks the ability to label all unique stages of glial development and specific subpopulations of cells. To address this issue, we screened zebrafish gene and enhancer trap lines to find a novel reporter for peripheral glial subtypes. From these, we generated the gSAIzGFFD37A transgenic line that expresses GFP in neural crest cells and central and peripheral glia. We found that the gene trap construct is located within an intron of erbb3b, a gene essential for glial development. Additionally, we confirmed that GFP+ â€‹cells express erbb3b along with sox10, a known glial marker. From our screen, we have identified the gSAIzGFFD37A line as a novel and powerful tool for studying glia in the developing zebrafish, as well as a new resource to manipulate erbb3b+ â€‹cells.

Gsx2 is required for specification of neurons in the inferior olivary nuclei from Ptf1a-expressing neural progenitors in zebrafish.

Neurons in the inferior olivary nuclei (IO neurons) send climbing fibers to Purkinje cells to elicit functions of the cerebellum. IO neurons and Purkinje cells are derived from neural progenitors expressing the proneural gene ptf1a In this study, we found that the homeobox gene gsx2 was co-expressed with ptf1a in IO progenitors in zebrafish. Both gsx2 and ptf1a zebrafish mutants showed a strong reduction or loss of IO neurons. The expression of ptf1a was not affected in gsx2 mutants, and vice versa. In IO progenitors, the ptf1a mutation increased apoptosis whereas the gsx2 mutation did not, suggesting that ptf1a and gsx2 are regulated independently of each other and have distinct roles. The fibroblast growth factors (Fgf) 3 and 8a, and retinoic acid signals negatively and positively, respectively, regulated gsx2 expression and thereby the development of IO neurons. mafba and Hox genes are at least partly involved in the Fgf- and retinoic acid-dependent regulation of IO neuronal development. Our results indicate that gsx2 mediates the rostro-caudal positional signals to specify the identity of IO neurons from ptf1a-expressing neural progenitors.

Transient and lineage-restricted requirement of Ebf3 for sternum ossification.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.

A virtual reality system to analyze neural activity and behavior in adult zebrafish.

Virtual realities are powerful tools to analyze and manipulate interactions between animals and their environment and to enable measurements of neuronal activity during behavior. In many species, however, optical access to the brain and/or the behavioral repertoire are limited. We developed a high-resolution virtual reality for head-restrained adult zebrafish, which exhibit cognitive behaviors not shown by larvae. We noninvasively measured activity throughout the dorsal telencephalon by multiphoton calcium imaging. Fish in the virtual reality showed regular swimming patterns and were attracted to animations of conspecifics. Manipulations of visuo-motor feedback revealed neurons that responded selectively to the mismatch between the expected and the actual visual consequences of motor output. Such error signals were prominent in multiple telencephalic areas, consistent with models of predictive processing. A virtual reality system for adult zebrafish therefore provides opportunities to analyze neuronal processing mechanisms underlying higher brain functions including decision making, associative learning, and social interactions.

Dysregulated TDP-43 proteostasis perturbs excitability of spinal motor neurons during brainstem-mediated fictive locomotion in zebrafish.

Spinal motor neurons (SMNs) are the primary target of degeneration in amyotrophic lateral sclerosis (ALS). Degenerating motor neurons accumulate cytoplasmic TAR DNA-binding protein 43 (TDP-43) aggregates in most ALS cases. This SMN pathology can occur without mutation in the coding sequence of the TDP-43-encoding gene, TARDBP. Whether and how wild-type TDP-43 drives pathological changes in SMNs in vivo remains largely unexplored. In this study, we develop a two-photon calcium imaging setup in which tactile-evoked neural responses of motor neurons in the brainstem and spinal cord can be monitored using the calcium indicator GCaMP. We devise a piezo-assisted tactile stimulator that reproducibly evokes a brainstem descending neuron upon tactile stimulation of the head. A direct comparison between caudal primary motor neurons (CaPs) with or without TDP-43 overexpression in contiguous spinal segments demonstrates that CaPs overexpressing TDP-43 display attenuated Ca2+ transients during fictive escape locomotion evoked by the tactile stimulation. These results show that excessive amounts of TDP-43 protein reduce the neuronal excitability of SMNs and potentially contribute to asymptomatic pathological lesions of SMNs and movement disorders in patients with ALS.

Retinoic acid signaling regulates late stages of semicircular canal morphogenesis and otolith maintenance in the zebrafish inner ear.

BACKGROUND: The vitamin A derivative all-trans retinoic acid (RA) regulates early stages of inner ear development. As the early disruption of the RA pathway results in profound mispatterning of the developing inner ear, this confounds analyses of specific roles in later stages. Therefore, we used the temporal-specific exposure of all-trans RA or diethylaminobenzaldehyde to evaluate RA functions in late otic development. RESULTS: Perturbing late RA signaling causes behavioral defects analogous to those expected in larvae suffering from vestibular dysfunction. These larvae also demonstrate malformations of the semi-circular canals, as visualized through (a) use of the transgenic strain nkhspdmc12a, a fluorescent reporter expressed in otic epithelium; and (b) injection of the fluorescent lipophilic dye DiI. We also noted the altered expression of genes encoding ECM proteins or modifying enzymes. Other malformations of the inner ear observed in our work include the loss or reduced size of the utricular and saccular otoliths, suggesting a role for RA in otolith maintenance. CONCLUSION: Our work has identified a previously undescribed late phase of RA activity in otic development, demonstrating that vestibular defects observed in human patients in relation to perturbed RA signaling are not solely due to its early disruption in otic development.

Multi-phaseted problems of TDP-43 in selective neuronal vulnerability in ALS.

Transactive response DNA-binding protein 43 kDa (TDP-43) encoded by the TARDBP gene is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) that regulates multiple steps of RNA metabolism, and its cytoplasmic aggregation characterizes degenerating motor neurons in amyotrophic lateral sclerosis (ALS). In most ALS cases, cytoplasmic TDP-43 aggregation occurs in the absence of mutations in the coding sequence of TARDBP. Thus, a major challenge in ALS research is to understand the nature of pathological changes occurring in wild-type TDP-43 and to explore upstream events in intracellular and extracellular milieu that promote the pathological transition of TDP-43. Despite the inherent obstacles to analyzing TDP-43 dynamics in in vivo motor neurons due to their anatomical complexity and inaccessibility, recent studies using cellular and animal models have provided important mechanistic insights into potential links between TDP-43 and motor neuron vulnerability in ALS. This review is intended to provide an overview of the current literature on the function and regulation of TDP-43-containing RNP granules or membraneless organelles, as revealed by various models, and to discuss the potential mechanisms by which TDP-43 can cause selective vulnerability of motor neurons in ALS.

Six6 and Six7 coordinately regulate expression of middle-wavelength opsins in zebrafish.

Color discrimination in the vertebrate retina is mediated by a combination of spectrally distinct cone photoreceptors, each expressing one of multiple cone opsins. The opsin genes diverged early in vertebrate evolution into four classes maximally sensitive to varying wavelengths of light: UV (SWS1), blue (SWS2), green (RH2), and red (LWS) opsins. Although the tetrachromatic cone system is retained in most nonmammalian vertebrate lineages, the transcriptional mechanism underlying gene expression of the cone opsins remains elusive, particularly for SWS2 and RH2 opsins, both of which have been lost in the mammalian lineage. In zebrafish, which have all four cone subtypes, rh2 opsin gene expression depends on a homeobox transcription factor, sine oculis homeobox 7 (Six7). However, the six7 gene is found only in the ray-finned fish lineage, suggesting the existence of another evolutionarily conserved transcriptional factor(s) controlling rh2 opsin expression in vertebrates. Here, we found that the reduced rh2 expression caused by six7 deficiency was rescued by forced expression of six6b, which is a six7-related transcription factor conserved widely among vertebrates. The compensatory role of six6b was reinforced by ChIP-sequencing analysis, which revealed a similar pattern of Six6b- and Six7-binding sites within and near the cone opsin genes. TAL effector nuclease-induced genetic ablation of six6b and six7 revealed that they coordinately regulate SWS2 opsin gene expression. Mutant larvae deficient for these transcription factors showed severely impaired visually driven foraging behavior. These results demonstrate that in zebrafish, six6b and six7 govern expression of the SWS2 and RH2 opsins responsible for middle-wavelength sensitivity, which would be physiologically important for daylight vision.

Neuronal Circuits That Control Rhythmic Pectoral Fin Movements in Zebrafish.

The most basic form of locomotion in limbed vertebrates consists of alternating activities of the flexor and extensor muscles within each limb coupled with left/right limb alternation. Although larval zebrafish are not limbed, their pectoral fin movements exhibit the following fundamental aspects of this basic movement: abductor/adductor alternation (corresponding to flexor/extensor alternation) and left/right fin alternation. Because of the simplicity of their movements and the compact neural organization of their spinal cords, zebrafish can serve as a good model to identify the neuronal networks of the central pattern generator (CPG) that controls rhythmic appendage movements. Here, we set out to investigate neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish, using transgenic fish that specifically express GFP in abductor or adductor motor neurons (MNs) and candidate CPG neurons. First, we showed that spiking activities of abductor and adductor MNs were essentially alternating. Second, both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively, indicating that the MN spiking activities are controlled in a push-pull manner. Further, we obtained the following evidence that dmrt3a-expressing commissural inhibitory neurons are involved in regulating the activities of abductor MNs: (1) strong inhibitory synaptic connections were found from dmrt3a neurons to abductor MNs; and (2) ablation of dmrt3a neurons shifted the spike timing of abductor MNs. Thus, in this simple system of abductor/adductor alternation, the last-order inhibitory inputs originating from the contralaterally located neurons play an important role in controlling the firing timings of MNs.SIGNIFICANCE STATEMENT Pectoral fin movements in larval zebrafish exhibit fundamental aspects of basic rhythmic appendage movement: alternation of the abductor and adductor (corresponding to flexor-extensor alternation) coupled with left-right alternation. We set out to investigate the neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish. We showed that both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively. This indicates that MN activities are controlled in a push-pull manner. We further obtained evidence that dmrt3a-expressing commissural inhibitory neurons exert an inhibitory effect on abductor MNs. The current study marks the first step toward the identification of central pattern generator organization for rhythmic fin movements.

Zebrafish can regenerate endoskeleton in larval pectoral fin but the regenerative ability declines.

We show for the first time endoskeletal regeneration in the developing pectoral fin of zebrafish. The developing pectoral fin contains an aggregation plate of differentiated chondrocytes (endochondral disc; primordium for endoskeletal components, proximal radials). The endochondral disc can be regenerated after amputation in the middle of the disc. The regenerated disc sufficiently forms endoskeletal patterns. Early in the process of regenerating the endochondral disc, epithelium with apical ectodermal ridge (AER) marker expression rapidly covers the amputation plane, and mesenchymal cells start to actively proliferate. Taken together with re-expression of a blastema marker gene, msxb, and other developmental genes, it is likely that regeneration of the endochondral disc recaptures fin development as epimorphic limb regeneration does. The ability of endoskeletal regeneration declines during larval growth, and adult zebrafish eventually lose the ability to regenerate endoskeletal components such that amputated endoskeletons become enlarged. Endoskeletal regeneration in the zebrafish pectoral fin will serve as a new model system for successful appendage regeneration in mammals.

Electrophysiological and pharmacological characterization of spreading depolarization in the adult zebrafish tectum.

Spreading depolarization (SD) is a slowly propagating wave of neuronal and glial depolarization. A growing number of studies show that SD and SD-like phenomena play a role in neurological disorders such as migraine, stroke, and traumatic brain injury. Despite the clinical importance of SD, its underlying molecular and cellular mechanisms remain elusive, possibly because of insufficient animal model allowing genetic manipulation. Such a model would also allow high-throughput screening for SD-suppressing drug development. To address this, we developed a novel experimental system to study SD using zebrafish. Electrophysiological recordings in the immobilized adult zebrafish revealed that increasing extracellular potassium concentration elicited SD with a large and long-lasting negative shift of direct current (DC) potential in the optic tectum. It also reduced the oscillatory activity in the extracellular field potential and increased the expression of the immediate early gene c-fos. Pharmacological blocking of the N-methyl-d-aspartate (NMDA) glutamate receptor attenuated the propagation of SD, suggesting that glutamatergic neurotransmission mediated tectal SD in zebrafish. Our analyses revealed that the zebrafish tectum and rodent cortex had similar SD kinetics. The current study provides electrophysiological and pharmacological evidence that zebrafish SD and mammal SD are comparable. This zebrafish SD model is suitable for genetic manipulation and cost-effective high-throughput screening. It could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.NEW & NOTEWORTHY Previous studies have implicated spreading depolarization (SD) in stroke and migraine. Here, we demonstrate SD, for the first time, in the adult zebrafish tectum showing waveform kinetics, c-fos expression, and attenuation by N-methyl-d-aspartate glutamate receptor blocker as observed in the rodent cortex. Since the zebrafish is an animal model amenable to genetic manipulation and chemical screening, this result could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.

Haploinsufficiency of PRR12 causes a spectrum of neurodevelopmental, eye, and multisystem abnormalities.

PURPOSE: Proline Rich 12 (PRR12) is a gene of unknown function with suspected DNA-binding activity, expressed in developing mice and human brains. Predicted loss-of-function variants in this gene are extremely rare, indicating high intolerance of haploinsufficiency. METHODS: Three individuals with intellectual disability and iris anomalies and truncating de novo PRR12 variants were described previously. We add 21 individuals with similar PRR12 variants identified via matchmaking platforms, bringing the total number to 24. RESULTS: We observed 12 frameshift, 6 nonsense, 1 splice-site, and 2 missense variants and one patient with a gross deletion involving PRR12. Three individuals had additional genetic findings, possibly confounding the phenotype. All patients had developmental impairment. Variable structural eye defects were observed in 12/24 individuals (50%) including anophthalmia, microphthalmia, colobomas, optic nerve and iris abnormalities. Additional common features included hypotonia (61%), heart defects (52%), growth failure (54%), and kidney anomalies (35%). PrediXcan analysis showed that phecodes most strongly associated with reduced predicted PRR12 expression were enriched for eye- (7/30) and kidney- (4/30) phenotypes, such as wet macular degeneration and chronic kidney disease. CONCLUSION: These findings support PRR12 haploinsufficiency as a cause for a novel disorder with a wide clinical spectrum marked chiefly by neurodevelopmental and eye abnormalities.

Development of the anterior lateral line system through local tissue-tissue interactions in the zebrafish head.

BACKGROUND: The distribution of sensory organs is important for detecting environmental signals efficiently. The mechanosensory receptors of the lateral line system, neuromasts, are stereotypically distributed over the head and body surface of fish, although how neuromasts arise in these predetermined positions during development remains unclear. RESULTS: We investigated the development of the anterior lateral line (ALL) system in zebrafish head. The ALL neuromasts formed in the predetermined positions through proliferation and differentiation of (a) nonmigratory lateral line primordia, (b) migratory primordia, (c) interneuromast cells connecting preexisting neuromasts, and (d) budding primordia. We demonstrated that R-spondin2 (Rspo2), an activator of Wnt/ß-catenin signaling, is required for the development of a particular set of neuromasts associated with hyomandibular cartilage. Further genetic analyses suggested that Rspo2, which emanates from the hyoid mesenchyme, acts on the adjacent neuromast progenitor cells to stimulate their proliferation through activating Wnt/ß-catenin signaling. CONCLUSION: This study has revealed novel mechanisms for neuromast positioning through local tissue-tissue interactions, providing insights into the development and evolution of the vertebrate head.