Dnmt1 is required for the development of auditory organs via cell cycle arrest and Fgf signalling

ObjectivesTo explore the role of DNA methyltransferase 1 (DNMT1) in the development of auditory system using zebrafish as experimental model.MethodsMorpholino oligonucleotide was used to induce Dnmt1 deficiency. RNA sequencing, in situ hybridization (ISH), whole genomic bisulfide sequencing (WGBS) and immunostaining were used to investigate the morphologic alterations and mechanisms.ResultsWe found that downregulation of Dnmt1 induced decreased number of neuromasts and repressed cell proliferation of primordium in the developing posterior lateral line system of zebrafish. The ISH data uncovered that Fgf signalling pathway was inhibited and the expression of chemokine members cxcr4b, cxcr7b and cxcl12a were interfered, while lef1 expression was increased after inhibiting Dnmt1. Additionally, Dnmt1 downregulation led to malformed otoliths and deformed semicircular canals, and hair cell differentiation in utricle and saccule was inhibited severely. The in situ staining of otic placode markers pax2/5 and fgf 3/8/10 was decreased when Dnmt1 downregulated. The WGBS analysis demonstrated that the global methylation status was markedly downregulated, and cell cycle genes were among those most differently expressed between Dnmt1 morphants and the controls. Further ISH analysis confirmed the findings by RNA‐seq and WGBS assay that cdkn1a and tp53 were both upregulated after knockdown of Dnmt1.ConclusionOur results revealed that Dnmt1 is essential for the development of zebrafish auditory organ through regulating cell cycle genes together with Wnt and Fgf signalling pathways.

     

Control of cell migration in the development of the posterior lateral line: antagonistic interactions between the chemokine receptors CXCR4 and CXCR7/RDC1

Background  

The formation of the posterior lateral line of teleosts depends on the migration of a primordium that originates near the otic vesicle and moves to the tip of the tail. Groups of cells at the trailing edge of the primordium slow down at regular intervals and eventually settle to differentiate as sense organs. The migration of the primordium is driven by the chemokine SDF1 and by its receptor CXCR4, encoded respectively by the genes sdf1a and cxcr4b. cxcr4b is expressed in the migrating cells and is down-regulated in the trailing cells of the primordium. sdf1a is expressed along the path of migration. There is no evidence for a gradient of sdf1a expression, however, and the origin of the directionality of migration is not known.     

Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line

The shape of most complex organ systems arises from the directed migration of cohesive groups of cells. Here, we dissect the role of the chemokine guidance receptor Cxcr4b in regulating the collective migration of one such cohesive tissue, the zebrafish lateral line primordium. Using in vivo imaging, we show that the shape and organization of the primordium is surprisingly labile, and that internal cell movements are uncoordinated in embryos with reduced Cxcr4b signaling. Genetic mosaic experiments reveal that single cxcr4b mutant cells can migrate in a directional manner when placed in wild-type primordia, but that they are specifically excluded from the leading edge. Moreover, a remarkably small number of SDF1a-responsive cells are able to organize an entire cxcr4b mutant primordium to restore migration and organogenesis in the lateral line. These results reveal a role for chemokine signaling in mediating the self-organizing migration of tissues during morphogenesis.     

The Nogo-C2/Nogo Receptor Complex Regulates the Morphogenesis of Zebrafish Lateral Line Primordium through Modulating the Expression of dkk1b,a Wnt Signal Inhibitor

The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium.     

Chemokine signaling regulates sensory cell migration in zebrafish

Chemokines play an important role in the migration of a variety of cells during development. Recent investigations have begun to elucidate the importance of chemokine signaling within the developing nervous system. To better appreciate the neural function of chemokines in vivo, the role of signaling by SDF-1 through its CXCR4 receptor was analyzed in zebrafish. The SDF-1-CXCR4 expression pattern suggested that SDF-1-CXCR4 signaling was important for guiding migration by sensory cells known as the migrating primordium of the posterior lateral line. Ubiquitous induction of the ligand in transgenic embryos, antisense knockdown of the ligand or receptor, and a genetic receptor mutation all disrupted migration by the primordium. Furthermore, in embryos in which endogenous SDF-1 was knocked down, the primordium migrated towards exogenous sources of SDF-1. These data demonstrate that SDF-1 signaling mediated via CXCR4 functions as a chemoattractant for the migrating primordium and that chemokine signaling is both necessary and sufficient for directing primordium migration.     

Zebrafish Prion Protein PrP2 Controls Collective Migration Process during Lateral Line Sensory System Development

Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, PrP1 and PrP2, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in PrP2 morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in PrP2 morphants as well as in PrP2^−/− mutants. Rosette formation defects were observed in PrP2 morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion.     

Cell-cell signaling interactions coordinate multiple cell behaviors that drive morphogenesis of the lateral line

The zebrafish sensory lateral line system has emerged as a powerful model for the mechanistic study of collective cell migration and morphogenesis. Recent work has uncovered the details of a signaling network involving the Wnt/β-catenin, Fgf and Delta-Notch pathways that patterns the migrating lateral line primordium into distinct regions. Cells within these regions exhibit different fundamental behaviors that together orchestrate normal lateral line morphogenesis. In this review, we summarize the signaling network that patterns the migrating lateral line primordium and describe how this patterning coordinates crucial morphogenic cell behaviors.Key words: lateral line, Wnt signaling, Fgf signaling, collective migration, morphogenesis     

BDNF-TrkB Axis Regulates Migration of the Lateral Line Primordium and Modulates the Maintenance of Mechanoreceptor Progenitors

BDNF and its specialized receptor TrkB are expressed in the developing lateral line system of zebrafish, but their role in this organ is unknown. To tackle this problem in vivo, we used transgenic animals expressing fluorescent markers in different cell types of the lateral line and combined a BDNF gain-of-function approach by BDNF mRNA overexpression and by soaking embryos in a solution of BDNF, with a loss-of-function approach by injecting the antisence ntrk2b-morpholino and treating embryos with the specific Trk inhibitor K252a. Subsequent analysis demonstrated that the BDNF-TrkB axis regulates migration of the lateral line primordium. In particular, BDNF-TrkB influences the expression level of components of chemokine signaling including Cxcr4b, and the generation of progenitors of mechanoreceptors, at the level of expression of Atoh1a-Atp2b1a.     

Wnt/β-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/β-catenin activation domain in the leading region of the primordium. The periodic lengthening of the primordium is controlled by Wnt/β-catenin/Fgf-dependent proliferation. Once proneuromasts are displaced into the trailing Wnt/β-catenin-free zone they are deposited. We have previously shown that Wnt/β-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/β-catenin signaling in the leading zone of the primordium plays a crucial role in orchestrating lateral line morphogenesis.     

Lef1 is required for progenitor cell identity in the zebrafish lateral line primordium

The zebrafish posterior lateral line (pLL) is a sensory system that comprises clusters of mechanosensory organs called neuromasts (NMs) that are stereotypically positioned along the surface of the trunk. The NMs are deposited by a migrating pLL primordium, which is organized into polarized rosettes (proto-NMs). During migration, mature proto-NMs are deposited from the trailing part of the primordium, while progenitor cells in the leading part give rise to new proto-NMs. Wnt signaling is active in the leading zone of the primordium and global Wnt inactivation leads to dramatic disorganization of the primordium and a loss of proto-NM formation. However, the exact cellular events that are regulated by the Wnt pathway are not known. We identified a mutant strain, lef1(nl2), that contains a lesion in the Wnt effector gene lef1. lef1(nl2) mutants lack posterior NMs and live imaging reveals that rosette renewal fails during later stages of migration. Surprisingly, the overall primordium patterning, as assayed by the expression of various markers, appears unaltered in lef1(nl2) mutants. Lineage tracing and mosaic analyses revealed that the leading cells (presumptive progenitors) move out of the primordium and are incorporated into NMs; this results in a decrease in the number of proliferating progenitor cells and eventual primordium disorganization. We concluded that Lef1 function is not required for initial primordium organization or migration, but is necessary for proto-NM renewal during later stages of pLL formation. These findings revealed a novel role for the Wnt signaling pathway during mechanosensory organ formation in zebrafish.     

Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.     

Cell-cell signaling interactions coordinate multiple cell behaviors that drive morphogenesis of the lateral line

The zebrafish sensory lateral line system has emerged as a powerful model for the mechanistic study of collective cell migration and morphogenesis. Recent work has uncovered the details of a signaling network involving the Wnt/β-catenin, Fgf and Delta-Notch pathways that patterns the migrating lateral line primordium into distinct regions. Cells within these regions exhibit different fundamental behaviors that together orchestrate normal lateral line morphogenesis. In this review, we summarize the signaling network that patterns the migrating lateral line primordium and describe how this patterning coordinates crucial morphogenic cell behaviors.     

Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.     

Duplicate sfrp1 genes in zebrafish: sfrp1a is dynamically expressed in the developing central nervous system,gut and lateral line

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.     

A Systematic Survey of Expression and Function of Zebrafish frizzled Genes

Wnt signaling is crucial for the regulation of numerous processes in development. Consistent with this, the gene families for both the ligands (Wnts) and receptors (Frizzleds) are very large. Surprisingly, while we have a reasonable understanding of the Wnt ligands likely to mediate specific Wnt-dependent processes, the corresponding receptors usually remain to be elucidated. Taking advantage of the zebrafish model''s excellent genomic and genetic properties, we undertook a comprehensive analysis of the expression patterns of frizzled (fzd) genes in zebrafish. To explore their functions, we focused on testing their requirement in several developmental events known to be regulated by Wnt signaling, convergent extension movements of gastrulation, neural crest induction, and melanocyte specification. We found fourteen distinct fzd genes in the zebrafish genome. Systematic analysis of their expression patterns between 1-somite and 30 hours post-fertilization revealed complex, dynamic and overlapping expression patterns. This analysis demonstrated that only fzd3a, fzd9b, and fzd10 are expressed in the dorsal neural tube at stages corresponding to the timing of melanocyte specification. Surprisingly, however, morpholino knockdown of these, alone or in combination, gave no indication of reduction of melanocytes, suggesting the important involvement of untested fzds or another type of Wnt receptor in this process. Likewise, we found only fzd7b and fzd10 expressed at the border of the neural plate at stages appropriate for neural crest induction. However, neural crest markers were not reduced by knockdown of these receptors. Instead, these morpholino knockdown studies showed that fzd7a and fzd7b work co-operatively to regulate convergent extension movement during gastrulation. Furthermore, we show that the two fzd7 genes function together with fzd10 to regulate epiboly movements and mesoderm differentiation.     

Role of zebrafish lbx2 in embryonic lateral line development

Background

The zebrafish ladybird homeobox homologous gene 2 (lbx2) has been suggested to play a key role in the regulation of hypaxial myogenic precursor cell migration. Unlike their lbx counterparts in mammals, the function of teleost lbx genes beyond myogenesis during embryonic development remains unexplored.

Principal Findings

Abrogation of lbx2 function using a specific independent morpholino oligonucleotide (MO) or truncated lbx2 mRNA with an engrailed domain deletion (lbx2^eh-) resulted in defective formation of the zebrafish posterior lateral line (PLL). Migration of the PLL primordium was altered and accompanied by increased cell death in the primordium of lbx2-MO-injected embryos. A decreased number of muscle pioneer cells and impaired expression pattern of sdf1a in the horizontal myoseptum was observed in lbx2 morphants.

Significance

Injection of lbx2 MO or lbx2^eh- mRNA resulted in defective PPL formation and altered sdf1a expression, confirming an important function for lbx2 in sdf1a-dependent migration. In addition, the disassociation of PPL nerve extension with PLL primordial migration in some lbx2 morphants suggests that pathfinding of the PLL primordium and the lateral line nerve may be regulated independently.     

Building the posterior lateral line system in zebrafish

The posterior lateral line (pLL) in zebrafish has emerged as an excellent system to study how a sensory organ system develops. Here we review recent studies that illustrate how interactions between multiple signaling pathways coordinate cell fate,morphogenesis, and collective migration of cells in the posterior lateral line primordium. These studies also illustrate how the pLL system is contributing much more broadly to our understanding of mechanisms operating during the growth, regeneration, and self-organization of other organ systems during development and disease.