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.

Retinoic acid is required and Fgf, Wnt, and Bmp signaling inhibit posterior lateral line placode induction in zebrafish.

The lateral line system is a mechanosensory systems present in aquatic animals. The anterior and posterior lateral lines develop from anterior and posterior lateral line placodes (aLLp and pLLp), respectively. Although signaling molecules required for the induction of other cranial placodes have been well studied, the molecular mechanisms underlying formation of the lateral line placodes are unknown. In this study we tested the requirement of multiple signaling pathways, such as Wnt, Bmp Fgf, and Retinoic Acid for aLLp and pLLp induction. We determined that aLLp specification requires Fgf signaling, whilst pLLp specification requires retinoic acid which inhibits Fgf signaling. pLLp induction is also independent of Wnt and Bmp activities, even though these pathways limit the boundaries of the pLLp. This is the first report that the aLLp and pLLp depend on different inductive mechanisms and that pLLp induction requires the inhibition of Fgf, Wnt and Bmp signaling.

In toto imaging of the migrating Zebrafish lateral line primordium at single cell resolution.

The zebrafish Posterior Lateral Line primordium (PLLp) has emerged as an important model system for studying many aspects of development, including cell migration, cell type specification and tissue morphogenesis. Despite this, basic aspects of PLLp biology remain incompletely understood. The PLLp is a group of approximately 140 cells which pioneers the formation of the Posterior Lateral Line (LL) system by migrating along the length of the embryo, periodically depositing clusters of epithelial cells, which will go on to form the mature sense organs of the lateral line, called neuromasts. The neuromasts are formed within the migrating PLLp as protoneuromasts: the first protoneuromast is formed close to the trailing end and additional protoneuromasts are formed sequentially, progressively closer to the leading edge of the migrating collective. We imaged the migration of PLL primordia and tracked every cell in the lateral line system over the course of migration. From this data set we unambiguously determined the lineage and fate of every cell deposited by the migrating PLLp. We show that, on average, proliferation across the entire PLLp is weakly patterned, with leading cells tending to divide more slowly than trailing cells. Neuromasts are formed sequentially by local expansion of existing cells along the length of the PLLp, not by self-renewing stem cell-like divisions of a restricted leading population that is highly proliferative. The fate of deposited cells, either within neuromasts or as interneuromast cells (in between deposited neuromasts) is not determined by any obvious stereotyped lineages. Instead, it is determined somewhat stochasitcailly, as a function of a cells distance from the center of a maturing protoneuromast. Together, our data provide a rigorous baseline for the behavior of the PLLp, which can be used to inform further study of this important model system.

Glypican4 modulates lateral line collective cell migration non cell-autonomously.

Collective cell migration is an essential process during embryonic development and diseases such as cancer, and still much remains to be learned about how cell intrinsic and environmental cues are coordinated to guide cells to their targets. The migration-dependent development of the zebrafish sensory lateral line proves to be an excellent model to study how proteoglycans control collective cell migration in a vertebrate. Proteoglycans are extracellular matrix glycoproteins essential for the control of several signaling pathways including Wnt/ß-catenin, Fgf, BMP and Hh. In the lateral line primordium the modified sugar chains on proteoglycans are important regulators of cell polarity, ligand distribution and Fgf signaling. At least five proteoglycans show distinct expression patterns in the primordium; however, their individual functions have not been studied. Here, we describe the function of glypican4 during zebrafish lateral line development. glypican4 is expressed in neuromasts, interneuromast cells and muscle cells underlying the lateral line. knypekfr6/glypican4 mutants show severe primordium migration defects and the primordium often U-turns and migrates back toward the head. Our analysis shows that Glypican4 regulates the feedback loop between Wnt/ß-catenin/Fgf signaling in the primordium redundantly with other Heparan Sulfate Proteoglycans. In addition, the primordium migration defect is caused non-cell autonomously by the loss of cxcl12a-expressing muscle precursors along the myoseptum via downregulation of Hh. Our results show that glypican4 has distinct functions in primordium cells and cells in the environment and that both of these functions are essential for collective cell migration.

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line.

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/ß-catenin signaling following hair cell death. We propose that Wnt/ß-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals.

The development of lateral line placodes: taking a broader view.

The lateral line system of anamniote vertebrates enables the detection of local water movement and weak bioelectric fields. Ancestrally, it comprises neuromasts - small sense organs containing mechanosensory hair cells - distributed in characteristic lines over the head and trunk, flanked on the head by fields of electroreceptive ampullary organs, innervated by afferent neurons projecting respectively to the medial and dorsal octavolateral nuclei in the hindbrain. Given the independent loss of the electrosensory system in multiple lineages, the development and evolution of the mechanosensory and electrosensory components of the lateral line must be dissociable. Nevertheless, the entire system arises from a series of cranial lateral line placodes, which exhibit two modes of sensory organ formation: elongation to form sensory ridges that fragment (with neuromasts differentiating in the center of the ridge, and ampullary organs on the flanks), or migration as collectives of cells, depositing sense organs in their wake. Intensive study of the migrating posterior lateral line placode in zebrafish has yielded a wealth of information concerning the molecular control of migration and neuromast formation in this migrating placode, in this cypriniform teleost species. However, our mechanistic understanding of neuromast and ampullary organ formation by elongating lateral line placodes, and even of other zebrafish lateral line placodes, is sparse or non-existent. Here, we attempt to highlight the diversity of lateral line development and the limits of the current research focus on the zebrafish posterior lateral line placode. We hope this will stimulate a broader approach to this fascinating sensory system.

Hypoxia disruption of vertebrate CNS pathfinding through ephrinB2 Is rescued by magnesium.

The mechanisms of hypoxic injury to the developing human brain are poorly understood, despite being a major cause of chronic neurodevelopmental impairments. Recent work in the invertebrate Caenorhabditis elegans has shown that hypoxia causes discrete axon pathfinding errors in certain interneurons and motorneurons. However, it is unknown whether developmental hypoxia would have similar effects in a vertebrate nervous system. We have found that developmental hypoxic injury disrupts pathfinding of forebrain neurons in zebrafish (Danio rerio), leading to errors in which commissural axons fail to cross the midline. The pathfinding defects result from activation of the hypoxia-inducible transcription factor (hif1) pathway and are mimicked by chemical inducers of the hif1 pathway or by expression of constitutively active hif1α. Further, we found that blocking transcriptional activation by hif1α helped prevent the guidance defects. We identified ephrinB2a as a target of hif1 pathway activation, showed that knock-down of ephrinB2a rescued the guidance errors, and showed that the receptor ephA4a is expressed in a pattern complementary to the misrouting axons. By targeting a constitutively active form of ephrinB2a to specific neurons, we found that ephrinB2a mediates the pathfinding errors via a reverse-signaling mechanism. Finally, magnesium sulfate, used to improve neurodevelopmental outcomes in preterm births, protects against pathfinding errors by preventing upregulation of ephrinB2a. These results demonstrate that evolutionarily conserved genetic pathways regulate connectivity changes in the CNS in response to hypoxia, and they support a potential neuroprotective role for magnesium.

Sensory hair cell regeneration in the zebrafish lateral line.

BACKGROUND: Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. RESULTS: Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. CONCLUSIONS: Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish.

Neuronal Neuregulin 1 type III directs Schwann cell migration.

During peripheral nerve development, each segment of a myelinated axon is matched with a single Schwann cell. Tight regulation of Schwann cell movement, proliferation and differentiation is essential to ensure that these glial cells properly associate with axons. ErbB receptors are required for Schwann cell migration, but the operative ligand and its mechanism of action have remained unknown. We demonstrate that zebrafish Neuregulin 1 (Nrg1) type III, which signals through ErbB receptors, controls Schwann cell migration in addition to its previously known roles in proliferation and myelination. Chimera analyses indicate that ErbB receptors are required in all migrating Schwann cells, and that Nrg1 type III is required in neurons for migration. Surprisingly, expression of the ligand in a few axons is sufficient to induce migration along a chimeric nerve constituted largely of nrg1 type III mutant axons. These studies also reveal a mechanism that allows Schwann cells to fasciculate axons regardless of nrg1 type III expression. Time-lapse imaging of transgenic embryos demonstrated that misexpression of human NRG1 type III results in ectopic Schwann cell migration, allowing them to aberrantly enter the central nervous system. These results demonstrate that Nrg1 type III is an essential signal that controls Schwann cell migration to ensure that these glia are present in the correct numbers and positions in developing nerves.

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

Organisms adjust their physiology to cope with environmental fluctuations and maintain fitness. These adaptations occur via genetic changes over multiple generations or through acclimation, a set of reversible phenotypic changes that confer resilience to the individual. Aquatic organisms are subject to dramatic seasonal fluctuations in water salinity, which can affect the function of lateral line mechanosensory hair cells. To maintain hair cell function when salinity decreases, ion-regulating cells, Neuromast-associated ionocytes (Nm ionocytes), increase in number and invade lateral line neuromasts. How environmental changes trigger this adaptive differentiation of Nm ionocytes and how these cells are specified is still unknown. Here, we identify Nm ionocyte progenitors as foxi3a/foxi3b-expressing skin cells and show that their differentiation is associated with sequential activation of different Notch pathway components, which control ionocyte survival. We demonstrate that new Nm ionocytes are rapidly specified by absolute salinity levels, independently of stress response pathways. We further show that Nm ionocyte differentiation is selectively triggered by depletion of specific ions, such as Ca2+ and Na+/Cl-, but not by low K+ levels, and is independent of media osmolarity. Finally, we demonstrate that hair cell activity plays a role in Nm ionocyte recruitment and that systemic factors are not necessary for Nm ionocyte induction. In summary, we have identified how environmental changes activate a signaling cascade that triggers basal skin cell progenitors to differentiate into Nm ionocytes and invade lateral line organs. This adaptive behavior is an example of physiological plasticity that may prove essential for survival in changing climates.

Wnt/ß-catenin dependent cell proliferation underlies segmented lateral line morphogenesis.

Morphogenesis is a fascinating but complex and incompletely understood developmental process. The sensory lateral line system consists of only a few hundred cells and is experimentally accessible making it an excellent model system to interrogate the cellular and molecular mechanisms underlying segmental morphogenesis. The posterior lateral line primordium periodically deposits prosensory organs as it migrates to the tail tip. We demonstrate that periodic proneuromast deposition is governed by a fundamentally different developmental mechanism than the classical models of developmental periodicity represented by vertebrate somitogenesis and early Drosophila development. Our analysis demonstrates that proneuromast deposition is driven by periodic lengthening of the primordium and a stable Wnt/ß-catenin activation domain in the leading region of the primordium. The periodic lengthening of the primordium is controlled by Wnt/ß-catenin/Fgf-dependent proliferation. Once proneuromasts are displaced into the trailing Wnt/ß-catenin-free zone they are deposited. We have previously shown that Wnt/ß-catenin signaling induces Fgf signaling and that interactions between these two pathways regulate primordium migration and prosensory organ formation. Therefore, by coordinating migration, prosensory organ formation and proliferation, localized activation of Wnt/ß-catenin signaling in the leading zone of the primordium plays a crucial role in orchestrating lateral line morphogenesis.

An anti-inflammatory activation sequence governs macrophage transcriptional dynamics during tissue injury in zebrafish.

Macrophages are essential for tissue repair and regeneration. Yet, the molecular programs, as well as the timing of their activation during and after tissue injury are poorly defined. Using a high spatio-temporal resolution single cell analysis of macrophages coupled with live imaging after sensory hair cell death in zebrafish, we find that the same population of macrophages transitions through a sequence of three major anti-inflammatory activation states. Macrophages first show a signature of glucocorticoid activation, then IL-10 signaling and finally the induction of oxidative phosphorylation by IL-4/Polyamine signaling. Importantly, loss-of-function of glucocorticoid and IL-10 signaling shows that each step of the sequence is independently activated. Lastly, we show that IL-10 and IL-4 signaling act synergistically to promote synaptogenesis between hair cells and efferent neurons during regeneration. Our results show that macrophages, in addition to a switch from M1 to M2, sequentially and independently transition though three anti-inflammatory pathways in vivo during tissue injury in a regenerating organ.

Single-cell transcriptome analysis reveals three sequential phases of gene expression during zebrafish sensory hair cell regeneration.

Loss of sensory hair cells (HCs) in the mammalian inner ear leads to permanent hearing and vestibular defects, whereas loss of HCs in zebrafish results in their regeneration. We used single-cell RNA sequencing (scRNA-seq) to characterize the transcriptional dynamics of HC regeneration in zebrafish at unprecedented spatiotemporal resolution. We uncovered three sequentially activated modules: first, an injury/inflammatory response and downregulation of progenitor cell maintenance genes within minutes after HC loss; second, the transient activation of regeneration-specific genes; and third, a robust re-activation of developmental gene programs, including HC specification, cell-cycle activation, ribosome biogenesis, and a metabolic switch to oxidative phosphorylation. The results are relevant not only for our understanding of HC regeneration and how we might be able to trigger it in mammals but also for regenerative processes in general. The data are searchable and publicly accessible via a web-based interface.

Cell migration during morphogenesis.

During development, functional structures must form with the correct three-dimensional geometry composed of the correct cell types. In many cases cell types are specified at locations distant to where they will ultimately reside for normal biological function. Although cell migration is crucial for normal development and morphogenesis of animal body plans and organ systems, abnormal cell migration during adult life underlies pathological states such as invasion and metastasis of cancer. In both contexts cells migrate either individually, as loosely associated sheets or as clusters of cells. In this review, we summarize, compare and integrate knowledge gained from several in vivo model systems that have yielded insights into the regulation of morphogenic cell migration, such as the zebrafish lateral line primordium and primordial germ cells, Drosophila border cell clusters, vertebrate neural crest migration and angiogenic sprouts in the post-natal mouse retina. Because of its broad multicontextual and multiphylletic distribution, understanding cell migration in its various manifestations in vivo is likely to provide new insights into both the function and malfunction of key embryonic and postembryonic events. In this review, we will provide a succinct phenotypic description of the many model systems utilized to study cell migration in vivo. More importantly, we will highlight, compare and integrate recent advances in our understanding of how cell migration is regulated in these varied model systems with special emphasis on individual and collective cell movements.

Loss of adenomatous polyposis coli (apc) results in an expanded ciliary marginal zone in the zebrafish eye.

The distal region of neural retina (ciliary marginal zone [CMZ]) contains stem cells that produce non-neural and neuronal progenitors. We provide a detailed gene expression analysis of the eyes of apc mutant zebrafish where the Wnt/beta-catenin pathway is constitutively active. Wnt/beta-catenin signaling leads to an expansion of the CMZ accompanied by a central shift of the retinal identity gene sox2 and the proneural gene atoh7. This suggests an important role for peripheral Wnt/beta-catenin signaling in regulating the expression and localization of neurogenic genes in the central retina. Retinal identity genes rx1 and vsx2, as well as meis1 and pax6a act upstream of Wnt/beta-catenin pathway activation. Peripheral cells that likely contain stem cells can be identified by the expression of follistatin, otx1, and axin2 and the lack of expression of myca and cyclinD1. Our results introduce the zebrafish apc mutation as a new model to study signaling pathways regulating the CMZ.

Adaptive cell invasion maintains lateral line organ homeostasis in response to environmental changes.

Mammalian inner ear and fish lateral line sensory hair cells (HCs) detect fluid motion to transduce environmental signals. Actively maintained ionic homeostasis of the mammalian inner ear endolymph is essential for HC function. In contrast, fish lateral line HCs are exposed to the fluctuating ionic composition of the aqueous environment. Using lineage labeling, in vivo time-lapse imaging and scRNA-seq, we discovered highly motile skin-derived cells that invade mature mechanosensory organs of the zebrafish lateral line and differentiate into Neuromast-associated (Nm) ionocytes. This invasion is adaptive as it is triggered by environmental fluctuations. Our discovery of Nm ionocytes challenges the notion of an entirely placodally derived lateral line and identifies Nm ionocytes as likely regulators of HC function possibly by modulating the ionic microenvironment. Nm ionocytes provide an experimentally accessible in vivo system to study cell invasion and migration, as well as the physiological adaptation of vertebrate organs to changing environmental conditions.

Regulation of latent sensory hair cell precursors by glia in the zebrafish lateral line.

The lateral line is a placodally derived mechanosensory organ in anamniotes that detects the movement of water. In zebrafish embryos, a migrating primordium deposits seven to nine clusters of sensory hair cells, or neuromasts, at intervals along the trunk. Postembryonically, neuromasts continue to be added. We show that some secondary neuromasts arise from a pool of latent precursors that are deposited by the primordium between primary neuromasts. Interneuromast cells lie adjacent to the lateral line nerve and associated glia. These cells remain quiescent while they are juxtaposed with the glia; however, when they move away from the nerve they increase proliferation and form neuromasts. If glia are manually removed or genetically ablated by mutations in cls/sox10, hypersensitive (hps), or rowgain (rog), neuromasts precociously differentiate. Transplantation of wt glia into mutants rescues the appropriate temporal differentiation of interneuromast cells. Our studies reveal a role for glia in regulating sensory hair cell precursors.

Evolutionary Origin and Nomenclature of Vertebrate Wnt11-Family Genes.

To adequately connect zebrafish medical models to human biology, it is essential that gene nomenclature reflects gene orthology. Analysis of gene phylogenies and conserved syntenies shows that the zebrafish gene currently called wnt11 (ENSDARG00000004256, ZFIN ID: ZDB-GENE-990603-12) is not the ortholog of the human gene called WNT11 (ENSG00000085741); instead, the gene currently called wnt11r (ENSDARG00000014796, ZFIN ID: ZDB-GENE-980526-249) is the zebrafish ortholog of human WNT11. Genomic analysis of Wnt11-family genes suggests a model for the birth of Wnt11-family gene ohnologs in genome duplication events, provides a mechanism for the death of a Wnt11-family ohnolog in mammals after they diverged from birds, and suggests revised nomenclature to better connect teleost disease models to human biology.

PCP and Wnt pathway components act in parallel during zebrafish mechanosensory hair cell orientation.

Planar cell polarity (PCP) plays crucial roles in developmental processes such as gastrulation, neural tube closure and hearing. Wnt pathway mutants are often classified as PCP mutants due to similarities between their phenotypes. Here, we show that in the zebrafish lateral line, disruptions of the PCP and Wnt pathways have differential effects on hair cell orientations. While mutations in the PCP genes vangl2 and scrib cause random orientations of hair cells, mutations in wnt11f1, gpc4 and fzd7a/b induce hair cells to adopt a concentric pattern. This concentric pattern is not caused by defects in PCP but is due to misaligned support cells. The molecular basis of the support cell defect is unknown but we demonstrate that the PCP and Wnt pathways work in parallel to establish proper hair cell orientation. Consequently, hair cell orientation defects are not solely explained by defects in PCP signaling, and some hair cell phenotypes warrant re-evaluation.

scRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling.

Loss of sensory hair cells leads to deafness and balance deficiencies. In contrast to mammalian hair cells, zebrafish ear and lateral line hair cells regenerate from poorly characterized support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to differentiated hair cells. scRNA-Seq of lateral line organs uncovered five different support cell types, including quiescent and activated stem cells. Ordering of support cells along a developmental trajectory identified self-renewing cells and genes required for hair cell differentiation. scRNA-Seq analyses of fgf3 mutants, in which hair cell regeneration is increased, demonstrates that Fgf and Notch signaling inhibit proliferation of support cells in parallel by inhibiting Wnt signaling. Our scRNA-Seq analyses set the foundation for mechanistic studies of sensory organ regeneration and is crucial for identifying factors to trigger hair cell production in mammals. The data is searchable and publicly accessible via a web-based interface.