Regeneration is triggered by cells dying due to various types of injuries. However, it remains unclear whether different types of cell death elicit distinct regeneration responses. Here, we systematically profile dynamic transcriptional responses of macrophages and lateral line cells to different cell death modalities during zebrafish sensory hair cell regeneration. We show that chemogenetically induced programmed hair cell death triggers a diminished inflammatory response compared to pharmacologically induced cell lysis, characterized by minimal neutrophil recruitment, distinct transcriptional profiles in phagocytosing macrophages and reduced expression of injury-responsive genes in lateral line cells. Nevertheless, regeneration ultimately converges on a shared set of regeneration-specific genes. Importantly, preventing immune cell recruitment enhances injury-induced support cell proliferation in response to programmed, but not unprogrammed cell death, highlighting cell death-dependent regenerative outcomes following immune cell inhibition. Our findings demonstrate that different forms of cell death trigger distinct molecular events, with implications for tailoring regenerative therapies to specific injury contexts.

Vertebrate inner ear mechanosensory hair cells detect sound and gravitational forces. Additionally, fishes have homologous lateral line hair cells in the skin that detect water vibrations for orientation and predator avoidance. Hair cells in the lateral line and ear of fishes and other non-mammalian vertebrates regenerate readily after damage, but mammalians lack this ability, causing deafness and vestibular defects. As yet, experimental attempts at hair cell regeneration in mice result in incompletely differentiated and immature hair cells. Despite differences in regeneration capabilities, the gene regulatory networks (GRNs) driving hair cell maturation during development are highly similar across vertebrates. Here, we show that the transcription factor prdm1a plays a key role in the hair cell fate GRN in the zebrafish lateral line. Mutating prdm1a respecifies lateral line hair cells into ear hair cells, altering morphology and transcriptome. Understanding how transcription factors control diverse hair cell fates in zebrafish is crucial for understanding the yet unsolved regeneration of diverse hair cells in mammalian ears to restore hearing and balance.

Regeneration and homeostatic turnover of solid tissues depend on the proliferation of symmetrically dividing adult stem cells, which either remain stem cells or differentiate based on their niche position. Here we demonstrate that in zebrafish lateral line sensory organs, stem and progenitor cell proliferation are independently regulated by two cyclinD genes. Loss of ccnd2a impairs stem cell proliferation during development, while loss of ccndx disrupts hair cell progenitor proliferation but allows normal differentiation. Notably, ccnd2a can functionally replace ccndx, indicating that the respective effects of these Cyclins on proliferation are due to cell type-specific expression. However, even though hair cell progenitors differentiate normally in ccndx mutants, they are mispolarized due to hes2 and Emx2 downregulation. Thus, regulated proliferation ensures that equal numbers of hair cells are polarized in opposite directions. Our study reveals cell type-specific roles for cyclinD genes in regulating the different populations of symmetrically dividing cells governing organ development and regeneration, with implications for regenerative medicine and disease.

SUMMARY 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 Ca 2+ 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.

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.

SummaryLoss of sensory hair cells in the mammalian inner ear leads to permanent hearing and vestibular defects, whereas loss of hair cells in zebrafish results in their regeneration. We used scRNA-Seq to characterize the transcriptional dynamics of hair cell regeneration in zebrafish at unprecedented spatio-temporal resolution. We uncovered three, sequentially activated modules. First, an injury/inflammatory response and downregulation of progenitor/stem cell maintenance genes within minutes after hair cell loss. Second, the transient activation of regeneration-specific genes. And third, a robust reactivation of developmental gene programs, including hair cell specification, cell cycle activation, ribosome biogenesis, and a metabolic switch to oxidative phosphorylation. The results are not only relevant for our understanding of hair cell regeneration and how we might be able to trigger it in mammals but also for regenerative processes in general. The data is searchable and publicly accessible via a web-based interface.

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.

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.

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/β-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/β-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.

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. Developmental Dynamics 243:1187–1202, 2014 . © 2014 Wiley Periodicals, Inc.

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Proper orchestration of quiescence and activation of progenitor cells is crucial during embryonic development and adult homeostasis. We took advantage of the zebrafish sensory lateral line to define niche-progenitor interactions to understand how integration of diverse signaling pathways spatially and temporally regulates the coordination of these processes. Our previous studies demonstrated that Schwann cells play a crucial role in negatively regulating lateral line progenitor proliferation. Here we demonstrate that ErbB/Neuregulin signaling is not only required for Schwann cell migration but that it plays a continued role in postmigratory Schwann cells. ErbB expressing Schwann cells inhibit lateral line progenitor proliferation and differentiation through non-cell-autonomous inhibition of Wnt/β-catenin signaling. Subsequent activation of Fgf signaling controls sensory organ differentiation, but not progenitor proliferation. In addition to the lateral line, these findings have important implications for understanding how niche-progenitor cells segregate interactions during development, and how they may go wrong in disease states. https://doi.org/10.7554/eLife.01832.001 eLife digest All the different types of cells that make up the body of an animal are descended from a single fertilized egg. As this egg develops into an embryo, the cells divide and specialize to become a specific type of cell, such as: a liver cell, a muscle cell or a nerve cell. The cells in the embryo that are destined to become specific cell types are called progenitor cells. However, these cells are also found within adult tissues, where they wait until they are needed to replace old or damaged cells. Zebrafish are commonly used in scientific research and, like other fish, they have a ‘lateral line’ that runs along both sides of the body and contains cells that detect movements in the surrounding water. During its development, the lateral line contains many progenitors that are primed to form more of these sense organs. The lateral line is also connected to nerve cells that relay information about water movements to the central nervous system, while other cells called Schwann cells support the nerve cells. The local environment or ‘niche’ created by the Schwann cells is known to prevent the progenitor cells within the lateral line from becoming their specific cell type too early. However, the molecules that cause progenitor cells to stop dividing, and later restart dividing and change in to their predestined cell type is not well understood. Now Lush and Piotrowski have discovered that signaling through a protein called ErbB causes the Schwann cells to multiply, but has the opposite effect on nearby progenitor cells in the lateral line. ErbB signaling in the Schwann cells inhibited various signaling pathways in the progenitor cells; and whilst some of these pathways normally encourage the progenitors to multiply, others cause them to change into their specific cell type. The findings of Lush and Piotrowski have important implications for understanding how the interactions between progenitor cells and the cells around them affect their development. These findings may be useful for understanding diseases caused when the control of cell multiplication or cell-type changes goes awry—such as developmental abnormalities or cancer. https://doi.org/10.7554/eLife.01832.002 Introduction The cell signaling events that govern progenitor quiescence, activation and differentiation are incompletely understood, but emerging data in many tissues indicate that dynamic interactions between progenitors and specialized niche environments play key roles in regulating the properties of progenitor pools. Hence, understanding niche-progenitor interactions at the cellular level is crucial for building a general understanding of this process. The development of the zebrafish lateral line is an excellent model system to study progenitor cell regulation, as it consists of relatively few cells that are easily accessible and amenable to experimental manipulations. The sensory organs of the lateral line are called neuromasts. Neuromasts contain support and mechanosensory hair cells that detect water motion. The first set of neuromasts is laid down by a migrating primordium (primI) that develops from a placode just posterior to the otic vesicle. As the primordium migrates posteriorly along the trunk of the embryo it deposits five to six primary neuromasts and a chain of interneuromast cells that connects each neuromast (Ghysen and Dambly-Chaudiere, 2007). Before the placode becomes migratory, its anterior portion splits off and forms the posterior lateral line ganglion (Northcutt and Brandle, 1995). Lateral line axons closely follow the migrating primordium and eventually innervate deposited neuromasts (Gilmour et al., 2004). In turn, neural crest-derived Schwann cells migrate along the axons which they eventually myelinate (Gilmour et al., 2002; Lyons et al., 2005). Thus, interneuromast cells, axons and Schwann cells are in close contact during the early stages of lateral line development (see diagram in Figure 1A; Whitfield, 2005). Figure 1 with 4 supplements see all Download asset Open asset Illustration of cell types in the migrating lateral line. (A) As the primordium migrates it deposits neuromasts and a chain of interneuromast cells (green cells). Pioneer axons (yellow line) of the posterior lateral line ganglion grow out with the primordium. Schwann cells (red cells) migrate and proliferate along axons. nrg1-3z26 mutants and pharmacological inhibition of ErbB signaling mimics the erbb phenotype. (B–E) Double in situ hybridization was performed to label Schwann cells with myelin basic protein (mbp) and neuromasts with klf4 at 5 dpf. (B) Control siblings with Schwann cells (arrows) along the lateral line nerve and normal neuromast number. nrg1-3z26 mutants mimic erbb2 and erbb3b mutants in that they lack Schwann cells along the lateral line and have increased neuromast number (C). The brown cells along the midline in both sibling and nrg1-3z26 are pigment cells. (D and E) Double in situ hybridization for mbp and klf4 in DMSO or AG1478 treated larvae from 50 hpf. Compared to DMSO treatment (D), increased neuromasts are seen in AG1478 treated larvae (E). mbp expression along the midline shows that Schwann cells (arrows) are still present at 5 dpf when AG1478 was given at 50 hpf (E), compare to DMSO treated (D). https://doi.org/10.7554/eLife.01832.003 The adult posterior lateral line contains many more neuromasts than the 7–8 neuromasts initially laid down by primI. These additional ‘secondary’ neuromasts originate from several sources. (A) A second primordium, primII, develops at 40 hr post fertilization (hpf) and deposits neuromasts in between the previously deposited sensory organs (Sapede et al., 2002; Nunez et al., 2009). (B) Intercalary neuromasts arise during the first 2 weeks of development by proliferation and differentiation of primI deposited interneuromast cells (Sapede et al., 2002; Grant et al., 2005; Lopez-Schier and Hudspeth, 2005; Nunez et al., 2009). and (C) During juvenile stages neuromast stitches arise through budding from primary neuromasts (Ledent, 2002; Wada et al., 2013a). We and others have previously shown that Schwann cells play a crucial role in negatively regulating the timing of differentiation of interneuromast cells into intercalary neuromasts (Grant et al., 2005; Lopez-Schier and Hudspeth, 2005). In zebrafish that lack Schwann cells along the lateral line, such as in mutants for sox10 and the ErbB pathway members erbb2, erbb3b and nrg1-3, intercalary neuromasts form precociously (Grant et al., 2005; Rojas-Munoz et al., 2009; Perlin et al., 2011). As Schwann cells require axons for migration along the lateral line, neurogenin mutants that lack a posterior lateral line ganglion, also show extra neuromasts (Lopez-Schier and Hudspeth, 2005). Likewise, extra neuromasts form after posterior lateral line ganglion extirpation or Schwann cell ablation (Grant et al., 2005; Lopez-Schier and Hudspeth, 2005). These experiments suggest that Schwann cells contribute to an inhibitory niche that keeps lateral line progenitor cells from undergoing precocious proliferation and differentiation. The signaling pathways that orchestrate intercalary neuromast formation are currently unknown. In contrast, the early development of the migrating lateral line has been extensively studied. Complex cell signaling interactions between Wnt/β-catenin, Fgf, Notch and chemokine pathways regulate proliferation, neuromast formation and migration (Aman and Piotrowski, 2009; Ma and Raible, 2009; Chitnis et al., 2012). Wnt/β-catenin signaling in the leading region of the primordium initiates and restricts Fgf signaling to the trailing region. In turn, Fgf signaling upregulates dkk1b, a secreted Wnt/β-catenin inhibitor, that restricts Wnt/β-catenin signaling to the leading region (Aman and Piotrowski, 2008). Fgf signaling induces apical constriction in clusters of cells resulting in the morphogenesis of rosette shaped protoneuromasts (Lecaudey et al., 2008; Nechiporuk and Raible, 2008). Fgf signaling is also required for hair cell differentiation (Millimaki et al., 2007; Nechiporuk and Raible, 2008), and both Wnt/β-catenin and Fgf signaling are required for proliferation within the migrating primordium (Aman et al., 2011). This study focuses on the development of intercalary neuromasts to elucidate the molecules that regulate progenitor cell proliferation and development. We characterize the signaling pathways required for precocious intercalary neuromast formation downstream of ErbB signaling. In the absence of Schwann cells, or ErbB/Neuregulin signaling, Wnt/β-catenin and Fgf signaling are increased. Wnt/β-catenin signaling is required for interneuromast proliferation while Fgf signaling is required for subsequent rosette formation and cellular differentiation. Schwann cells maintain interneuromast cells as quiescent progenitors by expressing a, as yet unidentified Wnt/β-catenin inhibitor. These findings illustrate the intricate manner in which diverse signaling pathways coordinate distinct aspects of the niche-progenitor interaction needed to maintain the proper balance and timing of this dynamic cell population. Results Mutations in ErbB/Neuregulin pathway members cause precocious differentiation of intercalary neuromasts Intercalary neuromasts arise during a 2-week period from interneuromast cells, which are initially deposited by primI as a chain of single cells in between primary neuromasts (Grant et al., 2005). The cellular relationships within the migrating lateral line are outlined in Figure 1A. Deposited interneuromast cells are initially in close contact with Schwann cells (Figure 1A, green and red cells respectively). A variety of lines of genetic evidence, including our own, demonstrates that ErbB signaling plays a fundamental role in the migration of Schwann cells that control the proliferation of interneuromast cells. Mutations in ErbB receptors (row/erbb2, hps/erbb3b) cause a loss of Schwann cells along the lateral line nerve leading to precocious interneuromast proliferation and intercalary neuromast differentiation (Figure 1—figure supplement 1A–E; Grant et al., 2005; Lyons et al., 2005; Rojas-Munoz et al., 2009). We recently identified a mutation in the ErbB ligand neuregulin 1-3 (nrg1-3z26) that also lacks Schwann cell migration along lateral line axons (Perlin et al., 2011), and forms supernumerary neuromasts (Figure 1B–C). nrg1-3z26 mutants survive to adulthood but exhibit an adult pigment pattern and neuromast degeneration phenotype (Figure 1—figure supplement 2,3), similarly to erbb3b adult mutant fish (Budi et al., 2008; Honjo et al., 2011). Below we identified in which cell types different members of the ErbB/Neuregulin pathway are functioning to control Schwann cell migration and lateral line progenitor proliferation and differentiation. Pharmacological inhibition of ErbB signaling mimics the erbb2/3b mutant phenotype During development, signaling pathways are repeatedly employed. We therefore wanted to test if the extra neuromast phenotype is due solely to loss of Schwann cells along the lateral line, or if ErbB signaling plays an additional role in inhibiting proliferation of interneuromast cells. Therefore, ErbB signaling was inhibited with the ErbB tyrosine kinase inhibitor AG1478 (Osherov and Levitzki, 1994), before (24 hpf) and after (48 hpf) completion of Schwann cell migration, and neuromast number was assessed at 5 days post fertilization (dpf). As expected, inhibition of ErbB signaling at 24 hpf, when Schwann cells migrate, leads to a loss of Schwann cells and the formation of extra neuromasts (Figure 1—figure supplement 1F; Rojas-Munoz et al., 2009). Interestingly, ErbB inhibition is able to increase neuromast numbers even in the presence of Schwann cells, if supplied between 50–59 hpf (Figure 1D–E, Figure 1—figure supplement 1F). The presence of Schwann cells is based on detection of myelin basic protein (mbp) expression (Figure 1D–E, arrows). These data suggest that ErbB signaling not only regulates Schwann cell migration but also plays a continued role in post-migratory Schwann cells in inhibiting interneuromast cell proliferation. A potential caveat for that interpretation is that ErbB signaling is also required for Schwann cell proliferation (Lyons et al., 2005; Raphael et al., 2011), and pharmacologically lowering the number of Schwann cells could secondarily affect interneuromast proliferation. To test when Schwann cell numbers are reduced upon ErbB inhibition at 48 hpf we used the Tg(foxd3:gfp) zebrafish line that expresses EGFP in neural crest derived tissues including Schwann cells (Gilmour et al., 2002). Using BrdU labeling in control and AG1478 treated Tg(foxd3:gfp) fish, we counted BrdU positive Tg(foxd3:gfp) Schwann cells at 6, 14 or 24 hr post treatment. ErbB inhibition induces a decrease in BrdU incorporation in Schwann cells at 6 hr post treatment, however the total Schwann cell number remains unchanged (Figure 1—figure supplement 4A–B). A reduction in Schwann cell proliferation continues at 14 and 24 hr post treatment, at which point it is accompanied by a decrease in total Schwann cell numbers (Figure 1—figure supplement 4C–F). The finding that Schwann cell numbers are not affected at 6 hr post treatment is important, as the first molecular changes in interneuromast cells are already observed at this stage (see below, 'Wntβ-catenin signaling activation occurs prior to Notch and Fgf activation within interneuromast cells after ErbB inhibition'). This suggests that ErbB signaling affects lateral line proliferation directly, rather than indirectly via the regulation of Schwann cell number. Thus, ErbB signaling has independent functions in Schwann cell migration and lateral line progenitor proliferation and differentiation. To elucidate if ErbB signaling controls progenitor proliferation cell-autonomously or non-cell-autonomously, we performed transplantation experiments between mutant and wild type embryos. ErbB2 is required in Schwann cells and Nrg1-3 is required in lateral line axons to inhibit precocious formation of intercalary neuromasts Transplantation experiments revealed that erbb3b and sox10 act cell-autonomously in Schwann cells to regulate their migration and inhibit precocious interneuromast proliferation (Grant et al., 2005). As erbb2 is ubiquitously expressed and AG1478 blocks ErbB signaling globally, we wanted to clarify in which cell type ErbB2 signaling is required. We transplanted dextran-Alexa568 labeled cells from erbb2 mutant blastomere stage donor embryos into wild type Tg(foxd3:gfp) host embryos and analyzed clones that gave rise to interneuromast cells. erbb2 mutant interneuromast cell clones failed to produce extra neuromasts by 4 dpf, suggesting that ErbB2 signaling is not required in interneuromast cells to prevent intercalary neuromast formation (Figure 2A–A′, n = 0/4). On the other hand, when transplanted Tg(foxd3:gfp) wild type cells gave rise to Schwann cell clones in an erbb2 mutant host embryo, the extra neuromasts phenotype at 4 dpf was rescued (Figure 2B–B“’, n = 9/9). Rescue was only achieved when Schwann cell clones extended all the way along the trunk by 48 hpf. Transplanted cells that only gave rise to interneuromast cells, or a few Schwann cells that did not reach the tail tip, failed to rescue the erbb2 mutant phenotype (n = 0/12). These transplant experiments illustrate that, similar to ErbB3b, ErbB2 is required in Schwann cells to inhibit intercalary neuromast formation. Figure 2 Download asset Open asset Transplantation and transgenic analysis demonstrates that ErbB2 is required within Schwann cells and Nrg1-3 within lateral line neurons to inhibit extra neuromast formation. (A and A’) Alexa-568 dextran (red) labeled erbb2 mutant cells were transplanted into Tg(foxd3:gfp) (green) wild type fish. (A) High magnification view shows erbb2 interneuromast and mantle cells along the lateral line and around neuromasts (arrows). These erbb2 interneuromast cells fail to induce extra neuromasts by 4 dpf (A’). (B–B’’’) Alexa-568 dextran (red)/Tg(foxd3:gfp) (green) wild type cells were transplanted into erbb2 mutant host. At 4 dpf neuromast were labeled by DASPEI staining (green). (B and B’) On the untransplanted side there are no Schwann cells and eighteen neuromasts. (B’’ and B’’’) On the transplanted side you can see complete migration of wild type Schwann cells in an otherwise erbb2 mutant fish and rescue of neuromast number (arrows). (C and D) Dominant negative ErbB receptor expression in neural crest derived cells mimics erbb mutant phenotype. (C) Control Tg(SqET20:gfp) siblings at 4 dpf. (D) Tg(SqET20:gfp)/Tg(sox10:DNerbb4) showing extra neuromasts. (E–E’’’) Alexa-568 dextran (red)/Tg(clndB:lyngfp)(green) wild type cells were transplanted into nrg1-3z26 mutant host. (E–E’) At 4 dpf the untransplanted side has nineteen neuromasts. (E’’ and E’’’) On the transplanted side there are GFP labeled axons (arrowhead) along the entire length of the lateral line with rescue of neuromast number. All axons cannot be seen because some are obscured underneath pigment cells. https://doi.org/10.7554/eLife.01832.008 We confirmed these results genetically by generating a transgenic zebrafish line that drives human dominant-negative ErbB4 (DNErbB4) in neural crest-derived tissues, including Schwann cells, using the sox10 promoter. DNErbB4 blocks Neuregulin-induced signaling in cell culture and in vivo (Rio et al., 1997; Chen et al., 2006). We isolated a stable transgenic line for analysis designated Tg(sox10:DNhsaerbb4-rfp), from now on called Tg(sox10:DNerbb4). As expected, in transgenic embryos Schwann cells fail to migrate along the lateral line and they develop extra neuromasts (data not shown and Figure 2C–D). Transgenic fish survive to adulthood, are fertile and exhibit no other obvious phenotypes. Along with the transplantation experiments these results demonstrate that ErbB2/3b signaling is required in Schwann cells in order to non-cell-autonomously regulate neuromast number. nrg1-3 is expressed in lateral line ganglia suggesting that it is required in lateral line axons (Perlin et al., 2011). To functionally test in which cell type Nrg1-3 is required we performed transplantation experiments between wild type and nrg1-3z26 embryos. As donors we used Tg(cldnb:lyngfp) embryos that express EGFP in all lateral line cells including the ganglion (Haas and Gilmour, 2006). We rescued the extra neuromast phenotype in nrg1-3z26 mutants with transplanted wild type cells that gave rise to large posterior lateral line ganglion clones (Figure 2E–E’’’, n = 13/13). Transplanted cells that only contributed to interneuromast cells or to few lateral line ganglion neurons failed to rescue nrg1-3z26 (n = 0/19). This is consistent with prior findings that wild type posterior lateral line ganglion clones rescue Schwann cell migration in nrg1-3z26 mutant embryos (Perlin et al., 2011). Thus, the ligand Nrg1-3 is required in axons to induce migration and proliferation of ErbB expressing Schwann cells and inhibit precocious formation of intercalary neuromasts. Combined, these experiments revealed that the quiescent niche consists of axonal, membrane bound, Nrg1-3 that signals to ErbB receptors within Schwann cells. In response, Schwann cells send a signal to interneuromast cells that inhibits their precocious differentiation into neuromasts. The following experiments were designed to identify signaling pathways that are regulated in interneuromast cells in response to Schwann cell-derived signals. Proliferation is the first cellular response in interneuromast cells after abrogation of the ErbB pathway The identification of interneuromast cell behaviors that are inhibited by ErbB signaling provides clues to which signaling pathways might be regulated by ErbB signaling. To identify the earliest changes in lateral line cell behavior in response to the loss of ErbB signaling we performed time-lapse analyses. We imaged interneuromast cells in Schwann cell-depleted larvae derived from crosses between Tg(sox10:DNerbb4) and Tg(SqET20:gfp). Tg(SqET20:gfp) larvae express EGFP in neuromast mantle cells and interneuromast cells (Parinov et al., 2004). In a 40-hr time-lapse four intercalary neuromasts are formed from interneuromasts cells (Video 1). The time-lapse analyses revealed that interneuromast cell proliferation precedes clustering of interneuromast cells. In addition, interneuromast cells are highly motile and migrate into and out of the forming neuromasts. The clusters of interneuromast cells continue to proliferate and differentiate into neuromasts as evident by the mature pattern of a ring of Tg(SqET20:gfp) positive mantle cells that surrounds GFP-negative sensory hair cells. In contrast, control Tg(SqET20:gfp) larvae show no proliferation and little migration of interneuromast cells during the same time period (Video 2). In conclusion, the absence of Schwann cells, leads first to interneuromast cell proliferation, followed by an increase in migration and clustering of interneuromast cells that eventually differentiate into sensory hair and support cells. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse recording of Tg(sox10:DNerbb4)/Tg(SqET20:gfp)/Tg(clndB:H2B-mcherry) during intercalary neuromast formation. The time-lapse runs from 32 to 72 hpf. One frame was taken every 7 min. Four intercalary neuromasts form during this time. https://doi.org/10.7554/eLife.01832.012 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse recording of control Tg(SqET20:gfp)/(Tg(clndB:H2A-mcherry) from approximately 48–72 hpf. One frame was taken every 7 min. No interneuromast cell proliferation is seen during this time. https://doi.org/10.7554/eLife.01832.013 To be able to correlate cell behavior with gene expression changes (see below), we sought to determine how many hours after ErbB inhibition interneuromast proliferation begins. We added BrdU plus DMSO or AG1478 to Tg(SqET20:gfp) larvae at 48 hpf, after Schwann cell migration is completed. After 14 hr of ErbB signaling inhibition there is no significant increase in BrdU incorporation in GFP-positive interneuromast cells (Figure 3A–B,E). After 24 hr of treatment we detected a significant increase in BrdU labeling in interneuromast cells (Figure 3C–E). Concurrent with the increase in BrdU incorporation, an increase in interneuromast cells is observed after 24 hr of AG1478 treatment (Figure 3F). The increase in proliferation begins sometime between 14 and 24 hr post ErbB inhibition. Figure 3 Download asset Open asset ErbB inhibition, after Schwann cell lateral line migration is completed, induces proliferation of interneuromast cells. BrdU plus DMSO or AG1478 was given to Tg(SqET20:gfp) fish at 48 hpf then fixed at 14 or 24 hr post treatment. Immunohistochemistry for BrdU (red) and GFP (green) reveals no difference in BrdU incorporation within interneuromast cells between DMSO (A) or AG1478 (B) 14 hr post treatment. At 24 hr post treatment DMSO (C) treated fish show little BrdU incorporation while AG1478 (D) treated fish show increased BrdU incorporation and interneuromast cell number. Quantification of both BrdU index (E, Student’s t-test, p=0.18 for 14 hr and p=4.5E−18 for 24 hr time point) and interneuromast cell number (F, Student’s t-test, p=0.69 for 14 hr and p=0.003 for 24 hr time point) shows a significant increase with AG1478 only after 24 hr. https://doi.org/10.7554/eLife.01832.009 As ErbB signaling acts cell-autonomously in Schwann cells but proliferation occurs in interneuromast cells, we aimed to identify the signaling pathways that are activated in interneuromast cells when ErbB signaling is inhibited. Wnt/β-catenin, Fgf and Notch signaling pathways are upregulated in ErbB/Neuregulin pathway mutants The Wnt/β-catenin, Fgf and Notch signaling pathways are excellent candidates for being involved in intercalary neuromast formation as they regulate progenitor cell proliferation in several other organs, such as the CNS (Logan and Nusse, 2004; Guillemot and Zimmer, 2011; Koch et al., 2013). In addition, these three pathways play multiple, crucial roles in the primordium of the lateral line (reviewed in Aman and Piotrowski, 2009; Ma and Raible, 2009; Chitnis et al., 2012). Briefly, Wnt/β-catenin and Fgf signaling regulate cell proliferation, while Fgf is also required for neuromast rosette formation and hair cell differentiation (Aman and Piotrowski, 2008; Lecaudey et al., 2008; Nechiporuk and Raible, 2008; Aman et al., 2011). Notch signaling regulates sensory hair cell production and primordium cohesion (Itoh and Chitnis, 2001; Matsuda and Chitnis, 2010). To test if the Wnt/β-catenin, Fgf and Notch pathways are also involved in the extra neuromast phenotype we performed an in situ expression screen on 48 hpf nrg1-3z26 mutant larvae (Figure 4). The Wnt/β-catenin pathway members wnt10a, lef1, myca, and β-catenin-2 (ctnnb2) are upregulated in interneuromast cells in nrg1-3z26 larvae (Figure 4A–H). lef1 and ctnnb2 are also expressed at lower levels in interneuromast cells in control animals while wnt10a and myca show no expression. The expression of wnt10a correlates with the differentiation status of intercalary neuromasts. wnt10a is expressed in proliferating interneuromast cells but is down regulated in differentiating neuromasts (Figure 4—figure supplement 1A–D). The Notch receptor notch3 and the Notch target gene her4.1 are expressed in primary neuromasts in control animals but not in interneuromast cells (Figure 4I,K, arrows). notch3 is broadly induced in nrg1-3z26 interneuromast cells (Figure 4J). her4.1 is also induced in nrg1-3z26 but in a more discrete cluster of cells (Figure 4L, arrowhead). her4.1 is not expressed in proliferating interneuromast cells but is induced as intercalary neuromasts mature (Figure 4—figure supplement 1E–H). This suggests that Notch signaling is only active in differentiating neuromasts. Fgf signaling pathway components are also upregulated in interneuromast cells in nrg1-3z26 mutant larvae (Figure 4N,P,R,T). In control embryos, fgfr1a, fgf3, fgf10 and the Fgf target pea3 only show strong expression in primary neuromasts but not interneuromast cells (Figure 4M,O,Q,S, arrows), suggesting that, similar to Notch signaling, Fgf signaling might be involved in neuromast differentiation. Figure 4 with 3 supplements see all Download asset Open asset Increase in Wnt/β-catenin, Notch and Fgf signaling pathway gene expression in nrg1-3z26 mutant interneuromast cells. Control siblings and nrg1-3z26 mutants were processed for in situ hybridization at 48 hpf. A Wnt ligand, wnt10a, is not expressed in control interneuromast cells (A) but is increased in nrg1-3z26 (B). The Wnt/β-catenin target gene lef1 is expressed in interneuromast cells in control siblings (C) but is greatly increased in nrg1-3z26 (D). An additional Wnt/β-catenin target myca shows no expression in interneuromast cells (E) but strong expression in clumps of interneuromast cells in nrg1-3z26 (F). ctnnb2 shows weak expression in control interneuromast cells (G) which is upregulated in nrg1-3z26 (H). (I) In controls, notch3 is expressed in primary neuromasts (arrow) but not interneuromast cells. (J) notch3 is upregulated in mutant interneuromast cells. (K) In controls, the Notch target her4.1 is expressed in primary neuromasts (arrow) but not interneuromast cells. (L) In mutants, her4.1 is expressed in primary neuromasts (arrow) but is also increased in discrete clusters of cells (arrowhead). In controls the Fgf pathway genes including the receptor fgfr1a (M), the two ligands fgf3 (O) and fgf10 (Q) and the Fgf target gene pea3 (S) are all expressed in primary neuromasts (arrow) but not in

Proper orchestration of quiescence and activation of progenitor cells is crucial during embryonic development and adult homeostasis. We took advantage of the zebrafish sensory lateral line to define niche-progenitor interactions to understand how integration of diverse signaling pathways spatially and temporally regulates the coordination of these processes. Our previous studies demonstrated that Schwann cells play a crucial role in negatively regulating lateral line progenitor proliferation. Here we demonstrate that ErbB/Neuregulin signaling is not only required for Schwann cell migration but that it plays a continued role in postmigratory Schwann cells. ErbB expressing Schwann cells inhibit lateral line progenitor proliferation and differentiation through non-cell-autonomous inhibition of Wnt/β-catenin signaling. Subsequent activation of Fgf signaling controls sensory organ differentiation, but not progenitor proliferation. In addition to the lateral line, these findings have important implications for understanding how niche-progenitor cells segregate interactions during development, and how they may go wrong in disease states.

Olfactory sensory neurons expressing particular olfactory receptors project to specific reproducible locations within the bulb. The axonal guidance cues that organize this precise projection pattern are only beginning to be identified. To aid in their identification and characterization, we generated a transgenic zebrafish line, OR111-7:IRES:Gal4, in which a small subset of olfactory sensory neurons is labeled. Most sensory neurons expressing the OR111-7 transgene project to a specific location within the bulb, the central zone protoglomerulus, while a smaller number project to the lateral glomerulus 1 protoglomerulus. Inhibiting Netrin/DCC (deleted in colorectal cancer) signaling perturbs the ability of OR111-7-expressing axons to enter the olfactory bulb and alters their patterns of termination within the bulb. The Netrin receptor DCC is expressed in olfactory sensory neurons around the time that they elaborate their axons, netrin1a is expressed near the medial-most margin of the olfactory bulb, and netrin1b is expressed within the ventral region of the bulb. Loss of Netrin/DCC signaling components causes some OR111-7-expressing sensory axons to wander posteriorly after exiting the olfactory pit, away from netrin-expressing areas in the bulb. OR111-7-expressing axons that enter the bulb target the central zone less precisely than normal, spreading away from netrin-expressing regions. These pathfinding errors can be corrected by the reexpression of DCC within OR111-7 transgene-expressing neurons in DCC morphant embryos. These findings implicate Netrins as the only known attractants for olfactory sensory neurons, first drawing OR111-7-expressing axons into the bulb and then into the ventromedially positioned central zone protoglomerulus.

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.

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.

Zebrafish and the majority of other aquatic vertebrates possess hair cells not only in the vestibular sensory epithelia of the ear but also in their skin. These hair cells are part of a sensory system called the lateral line, so‐named because the sensory organs are arranged in lines on the trunk and around the eyes. The function of the sensory lateral line is to detect water motion and initiate the appropriate behavioral response for capturing prey, avoiding predators and schooling. The sensory organs are composed of support cells surrounding hair cells that are very similar to the hair cells of the inner ear of vertebrates. Importantly, despite the unusual location of the hair cells on the trunk, lateral line and ear hair cells develop and differentiate by similar stereotyped developmental mechanisms. For example, mutations in genes causing deafness in humans also disrupt hair cell function in the zebrafish lateral line and vestibular system (reviewed in Nicolson T., 2005). However, in contrast to human inner ear hair cells, the lateral line hair cells of zebrafish regenerate after damage and are accessible to experimental manipulation and visualization. We are therefore employing zebrafish as a valuable new model system to study signaling pathways that underly the development, as well as regeneration of hair cells.

The rodent barrel cortex is a useful system to study the role of genes and neuronal activity in the patterning of the nervous system. Several genes encoding either intracellular signaling molecules or neurotransmitter receptors are required for barrel formation. Neurofibromin is a tumor suppressor protein that has Ras GTPase activity, thus attenuating the MAPK (mitogen-activated protein kinase) and and PI-3 kinase (phosphatidylinositol 3-kinase) pathways, and is mutated in humans with the condition neurofibromatosis type 1 (NF1). Neurofibromin is widely expressed in the developing and adult nervous system, and a common feature of NF1 is deficits in intellectual development. In addition, NF1 is an uncommonly high disorder among individuals with autism. Thus, NF1 may have important roles in normal CNS development and function. To explore roles for neurofibromin in the development of the CNS, we took advantage of a mouse conditional allele. We show that mice that lack neurofibromin in the majority of cortical neurons and astrocytes fail to form cortical barrels in the somatosensory cortex, whereas segregation of thalamic axons within the somatosensory cortex appears unaffected.

Ras-mediated signaling pathways participate in multiple aspects of neural development and function. For example, Ras signaling lies downstream of neurotrophic factors and Trk family receptor tyrosine kinases to regulate neuronal survival and morphological differentiation, including axon extension and target innervation. Neurofibromin, the protein encoded by the tumor suppressor gene Nf1, is a negative regulator of Ras [ Ras- GAP ( GTPase- activating protein)], and we previously demonstrated that Nf1 null embryonic sensory and sympathetic neurons can survive and differentiate independent of neurotrophin support. In this report, we demonstrate that Nf1 loss in adult sensory neurons enhances their intrinsic capacity for neurite outgrowth and collateral branching in vitro and in vivo after dorsal root injury. In contrast to the permanent sensory deficits observed in control mice after dorsal rhizotomy, neuron- specific Nf1 mutant mice spontaneously recover proprioceptive function. This phenomenon appears to be mediated both by a cell- autonomous capacity of spared Nf1(-/-) DRG neurons for increased axonal sprouting, and by non- cell- autonomous contribution from Nf1(-/-) neurons in the denervated spinal cord.

TrkA, the high affinity receptor for nerve growth factor (NGF), is essential for the development of nociceptive sensory and sympathetic neurons. The zinc finger transcription factor Klf7 interacts with an important cis element of the TrkA minimal enhancer and is coexpressed with TrkA in these neurons. We show that Klf7 binds to the endogenous TrkA minimal enhancer and can activate transcription from the TrkA minimal enhancer in a sequence-dependent manner. In Klf7(-/-) newborn mice, we find a significant reduction in sensory neurons due to increased apoptosis. The neuronal loss is restricted to nociceptive neurons that normally depend on TrkA for neurotrophic support, while other populations of somatosensory neurons appear normal. The reduction of TrkA expression in sensory neurons is a direct effect of Klf7 gene ablation, rather than a secondary effect of cell death. As a result, Klf7(-/-) mice have deficient response to noxious stimuli. Finally, removal of one TrkA allele exacerbates the loss of TrkA(+) neurons in Klf7(-/-) mice. Thus, Klf7 specifically regulates TrkA gene expression and is required for the development of a subset of nociceptive sensory neurons.