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Mutations in GATA6 are associated with congenital heart disease, most notably conotruncal structural defects. However, how GATA6 regulates cardiac morphology during embryogenesis is undefined. We used knockout and conditional mutant zebrafish alleles to investigate the spatiotemporal role of gata6 during cardiogenesis. Loss of gata6 specifically impacts atrioventricular valve formation and recruitment of epicardium, with a prominent loss of arterial pole cardiac cells, including those of the ventricle and outflow tract. However, there are no obvious defects in cardiac progenitor cell specification, proliferation or death. Conditional loss of gata6 starting at 24 h is sufficient to disrupt the addition of late differentiating cardiomyocytes at the arterial pole, with decreased expression levels of anterior secondary heart field (SHF) markers spry4 and mef2cb. Conditional loss of gata6 in the endoderm is sufficient to phenocopy the straight knockout, resulting in a significant loss of ventricular and outflow tract tissue. Exposure to a Dusp6 inhibitor largely rescues the loss of ventricular cells in gata6-/- larvae. Thus, gata6 functions in endoderm are mediated by FGF signaling to regulate the addition of anterior SHF progenitor derivatives during heart formation., Competing Interests: Competing interests T.E. is a co-founder of OncoBeat, LLC., (© 2024. Published by The Company of Biologists Ltd.)

Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Rising in popularity as a model organism in the classroom, zebrafish have numerous characteristics that make them ideal for teaching. In this study, we describe an experiment that helps students better understand the concept of tissue regeneration and the genes that control it. This experiment utilizes a dominant negative transgene for fgfr1 and allows students to observe the consequences of its activation. The first part of the laboratory is hands-on, and includes details of the amputation of caudal fins, heat shocking, general fish care, and visual observations. Over the course of a week, students observed the differences between the activated and unactivated transgene in the zebrafish. The second part was literature based, in which students tried to determine which gene is responsible for inhibiting regeneration. This encouraged students to sharpen their skills of deductive reasoning and critical thinking as they conduct research based on the information they receive about dominant negative receptors and transgenes. Having both a hands-on and critical thinking component in the laboratory helped synthesize the learning goals and allowed students to actively participate.

Background: Kdm6b, a specific histone 3 lysine 27 (H3K27) demethylase, has been reported to be implicated in a variety of developmental processes including cell differentiation and cell fate determination and multiple organogenesis. Here, we regulated the transcript level of kdm6bb to study the potential role in controlling the hearing organ development of zebrafish., Methods: A morpholino antisense oligonucleotide (MO) strategy was used to induce Kdm6b deficiency; immunohistochemical staining and in situ hybridization analysis were conducted to figure out the morphologic alterations and embryonic mechanisms., Results: Kdm6bb is expressed in the primordium and neuromasts at the early stage of zebrafish embryogenesis, suggesting a potential function of Kdm6b in the development of mechanosensory organs. Knockdown of kdm6bb severely influences the cell migration and proliferation in posterior lateral line primordium, abates the number of neuromasts along the trunk, and mRNA-mediated rescue test can partially renew the neuromasts. Loss of kdm6bb might be related to aberrant expressions of chemokine genes encompassing cxcl12a and cxcr4b/cxcr7b in the migrating primordium. Moreover, inhibition of kdm6bb reduces the expression of genes in Fgf signaling pathway, while it increases the axin2 and lef1 expression level of Wnt/β-catenin signaling during the migrating stage., Conclusions: Collectively, our results revealed that Kdm6b plays an essential role in guiding the migration of primordium and in regulating the deposition of zebrafish neuromasts by mediating the gene expression of chemokines and Wnt and Fgf signaling pathway. Since histone methylation and demethylation are reversible, targeting Kdm6b may present as a novel therapeutic regimen for hearing disorders., (© 2021. The Author(s).)

Background and Objective: A key pathway controlling skeletal development is fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling. Major regulatory functions of FGF signaling are chondrogenesis, endochondral and intramembranous bone development. In this study we focus on fgfr2 , as mutations in this gene are found in patients with craniofacial malformations. The high degree of conservation between FGF signaling of human and zebrafish ( Danio rerio ) tempted us to investigate effects of the mutated fgfr2 allele in zebrafish on cartilage and bone formation.sa10729 allele in zebrafish on cartilage and bone formation., Methods: We stained cartilage and bone in 5 days post fertilization (dpf) zebrafish larvae and compared mutants with wildtypes. We also determined the expression of genes related to these processes. We further investigated whether pharmacological blocking of all FGFRs with the inhibitor BGJ398, during 0-12 and 24-36 h post fertilization (hpf), affected craniofacial structure development at 5 dpf., Results: We found only subtle differences in craniofacial morphology between wildtypes and mutants, likely because of receptor redundancy. After exposure to BGJ398, we found dose-dependent cartilage and bone malformations, with more severe defects in fish exposed during 0-12 hpf. These results suggest impairment of cranial neural crest cell survival and/or differentiation by FGFR inhibition. Compensatory reactions by upregulation of fgfr1a , fgfr1b, fgfr4 , sp7 and dlx2a were found in the 0-12 hpf group, while in the 24-36 hpf group only upregulation of fgf3 was found together with downregulation of fgfr1a and fgfr2 ., Conclusions: Pharmacological targeting of FGFR1-4 kinase signaling causes severe craniofacial malformations, whereas abrogation of FGFR2 kinase signaling alone does not induce craniofacial skeletal abnormalities. These findings enhance our understanding of the role of FGFRs in the etiology of craniofacial malformations., Competing Interests: The authors declare there are no competing interests., (©2022 Gebuijs et al.)

Ribosomal RNA (rRNA) transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies; however, the cellular and molecular reasons for these defects are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. ncl mutant (ncl-/-) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observed that ncl-/- mutants exhibited decreased rRNA synthesis and p53-dependent apoptosis, consistent with a role in ribosome biogenesis. However, we found that Nucleolin also performs functions not associated with ribosome biogenesis. We discovered that the half-life of fgf8a mRNA was reduced in ncl-/- mutants, which perturbed Fgf signaling, resulting in misregulated Sox9a-mediated chondrogenesis and Runx2-mediated osteogenesis. Consistent with this model, exogenous FGF8 treatment significantly rescued the cranioskeletal phenotype in ncl-/- zebrafish, suggesting that Nucleolin regulates osteochondroprogenitor differentiation. Our work has therefore uncovered tissue-specific functions for Nucleolin in rRNA transcription and post-transcriptional regulation of growth factor signaling during embryonic craniofacial development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Extracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development.

Somitic cells give rise to a variety of cell types in response to Hh, BMP, and FGF signaling. Cell position within the developing zebrafish somite is highly dynamic: how, when, and where these signals specify cell fate is largely unknown. Combining four-dimensional imaging with pathway perturbations, we characterize the spatiotemporal specification and localization of somitic cells. Muscle formation is guided by highly orchestrated waves of cell specification. We find that FGF directly and indirectly controls the differentiation of fast and slow-twitch muscle lineages, respectively. FGF signaling imposes tight temporal control on Shh induction of slow muscles by regulating the time at which fast-twitch progenitors displace slow-twitch progenitors from contacting the Shh-secreting notochord. Further, we find a reciprocal regulation of fast and slow muscle differentiation, morphogenesis, and migration. In conclusion, robust cell fate determination in the developing somite requires precise spatiotemporal coordination between distinct cell lineages and signaling pathways., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Growth factors induce and pattern sensory organs, but how their distribution is regulated by the extracellular matrix (ECM) is largely unclear. To address this question, we analyzed the diffusion behavior of Fgf10 molecules during sensory organ formation in the zebrafish posterior lateral line primordium. In this tissue, secreted Fgf10 induces organ formation at a distance from its source. We find that most Fgf10 molecules are highly diffusive and move rapidly through the ECM. We identify Anosmin1, which when mutated in humans causes Kallmann Syndrome, as an ECM protein that binds to Fgf10 and facilitates its diffusivity by increasing the pool of fast-moving Fgf10 molecules. In the absence of Anosmin1, Fgf10 levels are reduced and organ formation is impaired. Global overexpression of Anosmin1 slows the fast-moving Fgf10 molecules and results in Fgf10 dispersal. These results suggest that Anosmin1 liberates ECM-bound Fgf10 and shuttles it to increase its signaling range., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Zebrafish is the preferred vertebrate model for high throughput chemical screens to discover modulators of complex biological pathways. We adapted a transgenic zebrafish line, Tg(dusp6:EGFP) , which reports on fibroblast growth factor (Fgf)/Ras/Mapk activity, into a quantitative, high-content chemical screen to identify novel Fgf hyperactivators as chemical probes for zebrafish heart development and regeneration. We screened 10,000 compounds from the TimTec ActiProbe library, and identified several structurally distinct classes of molecules that enhanced Fgf/Ras/Mapk signaling. We chose three agents-ST020101, ST011282, and ST006994-for confirmatory and functional studies based on potency, repeatability with repurchased material, favorable whole organism toxicity, and evidence of structure⁻activity relationships. Functional follow-up assays confirmed that all three compounds induced the expression of Fgf target genes during zebrafish embryonic development. Moreover, these compounds increased cardiac progenitor populations by effecting a fate change from endothelial to cardiac progenitors that translated into increased numbers of cardiomyocytes. Interestingly, ST006994 augmented Fgf/Ras/Mapk signaling without increasing Erk phosphorylation, suggesting a molecular mechanism of action downstream of Erk. We posit that the ST006994 pharmacophore could become a unique chemical probe to uncover novel mechanisms of Fgf signaling during heart development and regeneration downstream of the Mapk signaling node.

Fibroblast growth factor binding proteins (FGFBPs) are a class of secreted proteoglycans that function as an extracellular chaperone for locally stored FGFs and enhance FGF signaling. To date, all three human FGFBP genes have been identified and one orthologue fgfbp1a has been studied in zebrafish embryos. Here, we described the cloning and expression patterns of four novel FGFBP orthologues in zebrafish, fgfbp1b, fgfbp2a, fgfbp2b, and fgfbp3. Quantitative PCR and whole-mount in situ hybridization results showed that all transcripts except fgfbp2a are initially expressed in a maternal manner. fgfbp1b, fgfbp2b and fgfbp2a transcripts are expressed broadly in the head at 24 h post-fertilization (hpf), and then become restricted to the pharyngeal tissue, pectoral fins, and liver, respectively. fgfbp3 is abundantly expressed in the central nervous system (CNS) throughout embryonic and larval development. In adults, fgfbp family manifests the tissue specific patterns of expression with fgfbp3 robustly expressed in muscle and heart. Our work offers a starting point to uncover roles of FGFBP family genes and the possible mechanisms of FGF-dependent and -independent actions of FGFBP in vertebrates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The ability of the Spemann organizer to induce dorsal axis formation is dependent on downstream factors of the maternal Wnt/β-catenin signaling pathway. The fibroblast growth factor (FGF) signaling pathway has been identified as one of the downstream components of the maternal Wnt/β-catenin signaling pathway. The ability of the FGF signaling pathway to induce the formation of a dorsal axis with a complete head structure requires chordin (chd) expression; however, the molecular mechanisms involved in this developmental process, due to activation of FGF signaling, remain unclear. In this study, we showed that activation of the FGF signaling pathway induced the formation of complete head structures through the expression of chd and dickkopf-1b (dkk1b). Using the organizer-deficient maternal mutant, ichabod, we identified dkk1b as a novel downstream factor in the FGF signaling pathway. We also demonstrate that dkk1b expression is necessary, after activation of the FGF signaling pathway, to induce neuroectoderm patterning along the anteroposterior (AP) axis and for formation of complete head structures. Co-injection of chd and dkk1b mRNA resulted in the formation of a dorsal axis with a complete head structure in ichabod embryos, confirming the role of these factors in this developmental process. Unexpectedly, we found that chd induced dkk1b expression in ichabod embryos at the shield stage. However, chd failed to maintain dkk1b expression levels in cells of the shield and, subsequently, in the cells of the prechordal plate after mid-gastrula stage. In contrast, activation of the FGF signaling pathway maintained the dkk1b expression from the beginning of gastrulation to early somitogenesis. In conclusion, activation of the FGF signaling pathway induces the formation of a dorsal axis with a complete head structure through the expression of chd and subsequent maintenance of dkk1b expression levels., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Histone deacetylases (HDACs) are involved in multiple developmental processes, but their functions in the development of mechanosensory organs are largely unknown. In the present study, we report the presence of HDAC3 in the zebrafish posterior lateral line primordium and newly deposited neuromasts. We used morpholinos to show that HDAC3 knockdown severely disrupts the development of the posterior lateral line and reduces the numbers of neuromasts and sensory hair cells within these organs. In HDAC3 morphants, we also observed decreased cell proliferation and increased apoptosis, which might lead to these defects. Finally, we show that HDAC3 deficiency results in attenuated Fgf signaling in the migrating primordium. In situ hybridizations indicate aberrant expression patterns of Notch signaling pathway genes in HDAC3 morphants. In addition, inhibition of HDAC3 function diminishes cxcr7b and alters cxcl12a expression in the migrating primordium. Our results indicate that HDAC3 plays a crucial role in regulating posterior lateral line (PLL) formation and provide evidence for epigenetic regulation in auditory organ development.

The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face., (© 2016. Published by The Company of Biologists Ltd.)

Background: During the course of development, the vertebrate nephric duct (ND) extends and migrates from the place of its initial formation, adjacent to the anterior somites, until it inserts into the bladder or cloaca in the posterior region of the embryo. The molecular mechanisms that guide ND migration are poorly understood., Results: A novel Gata3-enhancer-Gfp-based chick embryo live imaging system was developed that permits documentation of ND migration at the individual cell level for the first time. FGF Receptors and FGF response genes are expressed in the ND, and FGF ligands are expressed in surrounding tissues. FGF receptor inhibition blocked nephric duct migration. Individual inhibitors of the Erk, p38, or Jnk pathways did not affect duct migration, but inhibition of all three pathways together did inhibit migration of the duct. A localized source of FGF8 placed adjacent to the nephric duct did not affect the duct migration path., Conclusions: FGF signaling acts as a "motor" that is required for duct migration, but other signals are needed to determine the directionality of the duct migration pathway. Developmental Dynamics 244:157-167, 2015. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

How signaling gradients supply positional information in a field of moving cells is an unsolved question in patterning and morphogenesis. Here, we ask how a Wnt signaling gradient regulates the dynamics of a wavefront of cellular change in a flow of cells during somitogenesis. Using time-controlled perturbations of Wnt signaling in the zebrafish embryo, we changed segment length without altering the rate of somite formation or embryonic elongation. This result implies specific Wnt regulation of the wavefront velocity. The observed Wnt signaling gradient dynamics and timing of downstream events support a model for wavefront regulation in which cell flow plays a dominant role in transporting positional information.

Extracellular signal-regulated kinase (Erk) signaling dynamics elicit distinct cellular responses in a variety of contexts. The early zebrafish embryo is an ideal model to explore the role of Erk signaling dynamics in vivo , as a gradient of activated diphosphorylated Erk (P-Erk) is induced by fibroblast growth factor (Fgf) signaling at the blastula margin. Here, we describe an improved Erk-specific biosensor, which we term modified Erk kinase translocation reporter (modErk-KTR). We demonstrate the utility of this biosensor in vitro and in developing zebrafish and Drosophila embryos. Moreover, we show that Fgf/Erk signaling is dynamic and coupled to tissue growth during both early zebrafish and Drosophila development. Erk activity is rapidly extinguished just prior to mitosis, which we refer to as mitotic erasure, inducing periods of inactivity, thus providing a source of heterogeneity in an asynchronously dividing tissue. Our modified reporter and transgenic lines represent an important resource for interrogating the role of Erk signaling dynamics in vivo. [Display omitted] • Erk-KTR displays Cdk1-dependent activity that impairs Erk-specific readout • modErk-KTR represents an improved Erk-specific biosensor in zebrafish and Drosophila • Mitosis introduces periods of Erk inactivity in zebrafish and Drosophila embryos • Mitotic erasure introduces heterogeneity to Fgf signal interpretation Wilcockson et al. describe the development of an improved Erk-specific kinase translocation reporter (modErk-KTR). Live imaging of Erk dynamics in zebrafish and Drosophila embryos shows that Erk signaling is shut down prior to mitosis (mitotic erasure), which contributes to heterogeneity in Fgf signal interpretation in zebrafish embryos. [ABSTRACT FROM AUTHOR]

The identification of genetic mechanisms underlying evolutionary change is critical to our understanding of natural diversity, but is presently limited by the lack of genetic and genomic resources for most species. Here, we present a new comparative genomic approach that can be applied to a broad taxonomic sampling of nonmodel species to investigate the genetic basis of evolutionary change. Using our analysis pipeline, we show that duplication and divergence of fgfr1a is correlated with the reduction of scales within fishes of the genus Phoxinellus. As a parallel genetic mechanism is observed in scale-reduction within independent lineages of cypriniforms, our finding exposes significant developmental constraint guiding morphological evolution. In addition, we identified fixed variation in fgf20a within Phoxinellus and demonstrated that combinatorial loss-of-function of fgfr1a and fgf20a within zebrafish phenocopies the evolved scalation pattern. Together, these findings reveal epistatic interactions between fgfr1a and fgf20a as a developmental mechanism regulating skeletal variation among fishes. [ABSTRACT FROM AUTHOR]

Polycomb group (PcG) proteins are transcriptional repressors that mediate epigenetic gene silencing by chromatin modification. PcG-mediated gene repression is implicated in development, cell differentiation, stem-cell fate maintenance and cancer. However, analysis of the roles of PcG proteins in orchestrating vertebrate developmental programs in vivo has been hampered by the early embryonic lethality of several PcG gene knockouts in mice. Here, we demonstrate that zebrafish Ring1b, the E3 ligase in Polycomb Repressive Complex 1 (PRC1), is essential for pectoral fin development. We show that differentiation of lateral plate mesoderm (LPM) cells into presumptive pectoral fin precursors is initiated normally in ring1b mutants, but fin bud outgrowth is impaired. Fgf signaling, which is essential for migration, proliferation and cell-fate maintenance during fin development, is not sufficiently activated in ring1b mutants. Exogenous application of FGF4, as well as enhanced stimulation of Fgf signaling by overactivated Wnt signaling in apc mutants, partially restores the fin developmental program. These results reveal that, in the absence of functional Ring1b, fin bud cells fail to execute the pectoral fin developmental program. Together, our results demonstrate that PcG-mediated gene regulation is essential for sustained Fgf signaling in vertebrate limb development. [ABSTRACT FROM AUTHOR]

Abstract: Cardiomelic or heart–hand syndromes include congenital defects affecting both the forelimb and heart, suggesting a hypothesis where similar signals may coordinate their development. In support of this hypothesis, we have recently defined a mechanism by which retinoic acid (RA) signaling acts on the forelimb progenitors to indirectly restrict cardiac cell number. However, we still do not have a complete understanding of the mechanisms downstream of RA signaling that allow for the coordinated development of these structures. Here, we test the hypothesis that appropriate Fgf signaling in the cardiac progenitor field downstream of RA signaling is required for the coordinated development of the heart and forelimb. Consistent with this hypothesis, we find that increasing Fgf signaling can autonomously increase cardiac cell number and non-autonomously inhibit forelimb formation over the same time period that embryos are sensitive to loss of RA signaling. Furthermore, we find that Fgf8a, which is expressed in the cardiac progenitors, is expanded into the posterior in RA signaling-deficient zebrafish embryos. Reducing Fgf8a function in RA signaling-deficient embryos is able to rescue both heart and forelimb development. Together, these results are the first to directly support the hypothesis that RA signaling is required shortly after gastrulation in the forelimb field to temper Fgf8a signaling in the cardiac field, thus coordinating the development of the heart and forelimb. [Copyright &y& Elsevier]

Abstract: UDP-xylose synthase (Uxs1) is strongly conserved from bacteria to humans, but because no mutation has been studied in any animal, we do not understand its roles in development. Furthermore, no crystal structure has been published. Uxs1 synthesizes UDP-xylose, which initiates glycosaminoglycan attachment to a protein core during proteoglycan formation. Crystal structure and biochemical analyses revealed that an R233H substitution mutation in zebrafish uxs1 alters an arginine buried in the dimer interface, thereby destabilizing and, as enzyme assays show, inactivating the enzyme. Homozygous uxs1 mutants lack Alcian blue-positive, proteoglycan-rich extracellular matrix in cartilages of the neurocranium, pharyngeal arches, and pectoral girdle. Transcripts for uxs1 localize to skeletal domains at hatching. GFP-labeled neural crest cells revealed defective organization and morphogenesis of chondrocytes, perichondrium, and bone in uxs1 mutants. Proteoglycans were dramatically reduced and defectively localized in uxs1 mutants. Although col2a1a transcripts over-accumulated in uxs1 mutants, diminished quantities of Col2a1 protein suggested a role for proteoglycans in collagen secretion or localization. Expression of col10a1, indian hedgehog, and patched was disrupted in mutants, reflecting improper chondrocyte/perichondrium signaling. Up-regulation of sox9a, sox9b, and runx2b in mutants suggested a molecular mechanism consistent with a role for proteoglycans in regulating skeletal cell fate. Together, our data reveal time-dependent changes to gene expression in uxs1 mutants that support a signaling role for proteoglycans during at least two distinct phases of skeletal development. These investigations are the first to examine the effect of mutation on the structure and function of Uxs1 protein in any vertebrate embryos, and reveal that Uxs1 activity is essential for the production and organization of skeletal extracellular matrix, with consequent effects on cartilage, perichondral, and bone morphogenesis. [Copyright &y& Elsevier]

Abstract: The epibranchial placodes are cranial, ectodermal thickenings that give rise to sensory neurons of the peripheral nervous system. Despite their importance in the developing animal, the signals responsible for their induction remain unknown. Using the placodal marker, sox3, we have shown that the same Fgf signaling required for otic vesicle development is required for the development of the epibranchial placodes. Loss of both Fgf3 and Fgf8 is sufficient to block placode development. We further show that epibranchial sox3 expression is unaffected in mutants in which no otic placode forms, where dlx3b and dlx4b are knocked down, or deleted along with sox9a. However, the forkhead factor, Foxi1, is required for both otic and epibranchial placode development. Thus, both the otic and epibranchial placodes form in a common region of ectoderm under the influence of Fgfs, but these two structures subsequently develop independently. Although previous studies have investigated the signals that trigger neurogenesis from the epibranchial placodes, this represents the first demonstration of the signaling events that underlie the formation of the placodes themselves, and therefore, the process that determines which ectodermal cells will adopt a neural fate. [Copyright &y& Elsevier]

Notch and fibroblast growth factor (FGF) signaling pathways have been implicated in the establishment of proper periodicity of vertebrate somites. Here, we show evidence that a Hes6-related hairy/Enhancer of split-related gene, her13.2, links FGF signaling to the Notch-regulated oscillation machinery in zebrafish. Expression of her13.2 is induced by FGF-soaked beads and decreased by an FGF signaling inhibitor, her13.2 is required for periodic repression of the Notch-regulated genes her1 and her7 and for proper somite segmentation. Furthermore, Her13.2 augments autorepression of her1 in association with Her1 protein. Therefore, FGF signaling appears to maintain the oscillation machinery by supplying a binding partner, Her13.2, for Her1. [ABSTRACT FROM AUTHOR]

Abstract: We analyzed Polycomb group gene ph2α functionally in zebrafish embryos by a gene knock-down procedure using morpholino antisense oligos. Inhibition of ph2α message translation resulted in abnormal epibolic movements as well as a thick tailbud or incomplete covering of the yolk plug. At the 24hpf stage, morphants had short trunks and tails, phenotypes similar to those with disturbances in FGF signaling. Accordingly, we looked at the effects of ph2α expression upstream and downstream of the FGF pathway. Treatment with SU5402, an inhibitor of Fgfrs, or injection of dominant-negative Fgfr1 DNA markedly reduced ph2α expression in the tailbud. In addition, cells expressing mRNAs for no tail, spadetail, myoD, and papc, which are involved in FGF-related development of posterior mesoderm, were distributed abnormally. Collectively, the data argue that ph2α is required for epiboly and tailbud formation, acting downstream of the FGF signaling pathway. [Copyright &y& Elsevier]

The neural stem cell (NSC) reservoir can be harnessed for stem cell-based regenerative therapies. Zebrafish remarkably regenerate their brain by inducing NSC plasticity in a Amyloid-β-42 (Aβ42)-induced experimental Alzheimer's disease (AD) model. Interleukin-4 (IL-4) is also critical for AD-induced NSC proliferation. However, the mechanisms of this response have remained unknown. Using single-cell transcriptomics in the adult zebrafish brain, we identify distinct subtypes of NSCs and neurons and differentially regulated pathways and their gene ontologies and investigate how cell-cell communication is altered through ligand-receptor pairs in AD conditions. Our results propose the existence of heterogeneous and spatially organized stem cell populations that react distinctly to amyloid toxicity. This resource article provides an extensive database for the molecular basis of NSC plasticity in the AD model of the adult zebrafish brain. Further analyses of stem cell heterogeneity and neuro-regenerative ability at single-cell resolution could yield drug targets for mobilizing NSCs for endogenous neuro-regeneration in humans. • Single-cell transcriptomics reveals neural stem cell/glia heterogeneity in zebrafish • Different stem cell populations can be defined spatially and molecularly • Amyloid-β-42 and interleukin-4 induce plasticity of certain progenitor populations • Interaction mapping predicts the pathways that regulate neural stem cell plasticity The zebrafish brain combats Alzheimer's disease by producing more neurons, which are derived from neural stem cells. By using single-cell sequencing technology, Cosacak et al. identify distinct stem cell populations that react differently to Alzheimer's disease-like conditions in the adult zebrafish brain and develop tools to investigate their molecular programs. [ABSTRACT FROM AUTHOR]

An abstract of an article on the relationship between atoh1a expression and the effective morphogenesis of the posterior lateral line primordium in zebrafish is presented.

Phosphatidylinositol 4-kinases (PI4Ks) catalyze the first committed step in the synthesis of phosphoinositides, important lipid regulators of signaling and trafficking pathways. Here we cloned Pik4a, one of the zebrafish PI4K enzymes, and studied its role(s) in vertebrate development using morpholino oligonucleotide-based gene silencing in zebrafish. Downregulation of Pik4a led to multiple developmental abnormalities, affecting the brain, heart, trunk and most prominently causing loss of pectoral fins. Strikingly similar defects were caused by treatment of the developing embryos with the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. To investigate the cause of the pectoral fin developmental defect, we focused on fibroblast growth factor (FGF) signaling pathways because vertebrate limb development requires the concerted action of a series of FGF ligands. Using in situ hybridization, the pectoral fin defect was traced to disruption of the early FGF signaling loops that are crucial for the establishment of the sharp signaling center formed by the apical ectodermal ridge and the underlying mesenchyme. This, in turn caused a prominent loss of the induction of one of the mitogen-activated protein kinase (MAPK) phosphatases, Mkp3, an essential intermediate in vertebrate limb development. These changes were associated with impaired proliferation in the developing fin bud due to a loss of balance between the MAPK and PI3K branch of FGF-initiated signals. Our results identify Pik4a as an upstream partner of PI3Ks in the signaling cascade orchestrated by FGF receptors with a prominent role in forelimb development. [ABSTRACT FROM AUTHOR]

We have used the maternal effect mutant ichabod, which is deficient in maternal β-catenin signaling, to test for the epistatic relationship between β-catenin activation, FGF signaling and bozozok, squint and chordin expression. Injection of β-catenin RNA into ichabod embryos can completely rescue normal development. By contrast, when FGF signaling is inhibited, β-catenin did not induce goosecoid and chordin, repress bmp4 expression or induce a dorsal axis. These results demonstrate that FGF signaling is necessary for β-catenin induction of the zebrafish organizer. We show that FGFs function downstream of squint and bozozok to turn on chordin expression. Full rescue of ichabod by Squint is dependent on FGF signaling, and partial rescue by FGFs is completely dependent on chordin. By contrast, Bozozok can rescue the complete anteroposterior axis, but not notochord, in embryos blocked in FGF signaling. Surprisingly, accumulation of bozozok transcript in β-catenin RNA-injected ichabod embryos is also dependent on FGF signaling, indicating a role of FGFs in maintenance of bozozok RNA. These experiments show that FGF-dependent organizer function operates through both bozozok RNA accumulation and a pathway consisting of β-catenin→ Squint→ FGF→ Chordin, in which each component is sufficient for expression of the downstream factors of the pathway, and in which Nodal signaling is required for FGF gene expression and FGF signaling is required for Squint induction of chordin. [ABSTRACT FROM AUTHOR]