Deformación de la aleta caudal en alevín de salmón del atlántico (Salmo salar), en etapa de saco vitelino / Deformation of the caudal fin in atlantic salmon fry (Salmo salar), in the yolk sac stage

RESUMEN: En este estudio se analiza una deformación que afectó la aleta caudal de los alevines de salmón del Atlántico (Salmo salar) y que les proporcionó un aspecto de "cola aguzada". Al momento de la eclosión se observaron completamente normales pero la deformación se detectó con posterioridad. El objetivo de este trabajo es describir los cambios anatómicos e histológicos de la aleta caudal deformada del alevín de salmón del Atlántico (Salmo salar). Para esto se utilizaron 60 alevines para realizar análisis de laboratorio y descartar la presencia de patógenos virales o bacterianos. Otros 60 alevines con un estado de desarrollo entre 600 y 700 Unidades térmicas acumuladas (UTAs) fueron anestesiados con Benzocaína 5 %, fijados en formalina al 10 % pesados y medidos. De estos un grupo de 30 alevines (15 normales y 15 deformes) fueron sometidos a la técnica de Hanken y Wassersug para evaluar Lepidotriquias. Los otros 30 alevines (15 normales y 15 deformes), fueron procesados mediante las técnicas histoquímicas: H&E/azul de Alcián para evaluar las características histológicas generales. Adicionalmente se utilizó técnicas inmunohistoquímicas para reconocer la ubicación y la presencia de los centros de señales Sonic hedgehog (Shh) para la formación de Lepidotriquias. A los valores obtenidos para las variables cuantitativas peso y longitud de cuerpo, largo y ancho de aleta caudal, se les realizó estadística descriptiva y fueron sometidos a prueba de normalidad de Shapiro-Wilk. Las diferencias observadas entre peces normales y deformes, fueron analizadas mediante prueba t de Student o U de Mann Whitney, utilizando el paquete estadístico IBM SPSS 20.0. La deformación se observó desde las 600 UTA. El peso de los alevines deformes fue similar al de los peces normales (p>0,05), lo mismo sucedió con la longitud de la aleta (p>0,05). Por el contrario, el ancho de la aleta de los deformes fue muy reducida (p<0,05). El fenotipo de aleta aguzada presentó un cambio en la integridad de los bordes, fracturas de Lepidotriquias. La epidermis de la aleta caudal de los alevines deformados presentó sus centros de señalización Shh activos, pero el blastema interrayos presentó vasodilatación, congestión y hemorragias. La presentación de este caso se relacionó con incrementos bruscos de temperatura peri eclosional.

SUMMARY: This study analyzes the deformity of Atlantic salmon fry (Salmo salar) caudal fin, which gives it a "pointed tail" appearance. Although at hatching specimens were normal, the deformities were detected later. The objective of this work is to describe the anatomical and histological changes of the deformed caudal fin of the Atlantic salmon fry (Salmo salar). In this analysis we used 60 specimens for laboratory analyses, to rule out the presence of viral or bacterial pathogens. Another 60 fry, developmental stage between 600 and 700 Accumulated Thermal Units (UTAs) were anesthetized with 5 % Benzocaine, fixed in 10 % formalin, weighed and measured. Of these, a group of 30 fry (15 normal and 15 deformed) underwent the Hanken and Wassersug technique to evaluate lepidotrychia or dermal rays. The remaining 30 fry (15 normal and 15 deformed) were processed using H & E / Alcián blue histochemical techniques to evaluate general histological characteristics. Additionally, immunohistochemical techniques were used to determine the location and presence of Sonic hedgehog (Shh) signal centers for lepidotrychia development. The values obtained for the quantitative variables body weight, length and width of the tail fin were described using the arithmetic mean and standard deviation. The deformity was observed from 600 UTA. Weight of deformed fry was less than normal fish, length of the fin was similar in normal and deformed fish. In contrast, width of the deformed fin was significantly reduced. The sharp fin phenotype presented a change in the conformity of the edges, lepidotrychia fractures. The epidermis presented active Shh signaling centers, but the interray blastema showed vasodilation, congestion and hemorrhages. The presentation of this case was related to sudden increases in perieclosional temperature.

Expression of nucleosome assembly protein 1 like genes in zebrafish embryos.

Nucleosome assembly protein 1-like (Nap1l) family plays numerous biological roles including nucleosome assembly, transcriptional regulation, and cell cycle progression. However, the tissue specific in vivo functions of the Nap1l family members remain largely unknown. In this study, we finished the complete expression patterns of nap1l1 and nap1l4a in zebrafish embryos by whole-mount in situ hybridization. We observed maternal existence of nap1l1 transcript and that its zygotic expression is abundant and not spatially restricted at 6 somite stage, while nap1l4a mRNA is not detectable until 6 somite stage when it is weakly transcribed throughout the embryo. At 24 h post-fertilization (hpf), nap1l1 is predominantly expressed in the central nervous system, neural tube, ventral mesoderm, branchial arches, and pectoral fins, while nap1l4a mRNA is throughout the embryo, enriched in the eyes, tectum, and myotomes. As the embryo develops, nap1l1 expression maintains throughout the head, with gradually enriched in the tectum, olfactory vesicle, lens, optic cups, heart, branchial arches, pectoral fins, axial vasculature, pronephros, and lateral line neuromasts, whereas nap1l4a expression is weak in the tectum, branchial arches, and pectoral fins. Overall, these expression analyses provide a valuable basis for the functional study of nap1l family in zebrafish development.

Deficiency of lrp4 in zebrafish and human LRP4 mutation induce aberrant activation of Jagged-Notch signaling in fin and limb development.

Low-density lipoprotein receptor-related protein 4 (LRP4) is a multi-functional protein implicated in bone, kidney and neurological diseases including Cenani-Lenz syndactyly (CLS), sclerosteosis, osteoporosis, congenital myasthenic syndrome and myasthenia gravis. Why different LRP4 mutation alleles cause distinct and even contrasting disease phenotypes remain unclear. Herein, we utilized the zebrafish model to search for pathways affected by a deficiency of LRP4. The lrp4 knockdown in zebrafish embryos exhibits cyst formations at fin structures and the caudal vein plexus, malformed pectoral fins, defective bone formation and compromised kidney morphogenesis; which partially phenocopied the human LRP4 mutations and were reminiscent of phenotypes resulting form a perturbed Notch signaling pathway. We discovered that the Lrp4-deficient zebrafish manifested increased Notch outputs in addition to enhanced Wnt signaling, with the expression of Notch ligand jagged1b being significantly elevated at the fin structures. To examine conservatism of signaling mechanisms, the effect of LRP4 missense mutations and siRNA knockdowns, including a novel missense mutation c.1117C > T (p.R373W) of LRP4, were tested in mammalian kidney and osteoblast cells. The results showed that LRP4 suppressed both Wnt/ß-Catenin and Notch signaling pathways, and these activities were perturbed either by LRP4 missense mutations or by a knockdown of LRP4. Our finding underscore that LRP4 is required for limiting Jagged-Notch signaling throughout the fin/limb and kidney development, whose perturbation representing a novel mechanism for LRP4-related diseases. Moreover, our study reveals an evolutionarily conserved relationship between LRP4 and Jagged-Notch signaling, which may shed light on how the Notch signaling is fine-tuned during fin/limb development.

Gambogic acid causes fin developmental defect in zebrafish embryo partially via retinoic acid signaling.

Gambogic acid (GA), the major active ingredient of gamboge, has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients due to its strong anticancer activity. However, our previous research showed that GA was teratogenic against zebrafish fin development. To explore the teratogenicity and the underlying mechanisms, zebrafish (Danio rerio) embryos were used. The morphological observations revealed that GA caused fin defects in zebrafish embryos in a concentration-dependent manner. The critical exposure time of GA to reveal teratogenicity was before 8 hpf (hours post fertilization). LC/MS/MS analysis revealed that a maximum bioconcentration of GA was occurred at 4 hpf. Q-PCR data showed that GA treatment resulted in significant inactivation of RA signaling which could be partially rescued by the exogenous supply of RA. These results indicate the potential teratogenicity of GA and provide evidence for a caution in its future clinic use.

Ptbp1 and Exosc9 knockdowns trigger skin stability defects through different pathways.

In humans, genetic diseases affecting skin integrity (genodermatoses) are generally caused by mutations in a small number of genes that encode structural components of the dermal-epidermal junctions. In this article, we first show that inactivation of both exosc9, which encodes a component of the RNA exosome, and ptbp1, which encodes an RNA-binding protein abundant in Xenopus embryonic skin, impairs embryonic Xenopus skin development, with the appearance of dorsal blisters along the anterior part of the fin. However, histological and electron microscopy analyses revealed that the two phenotypes are distinct. Exosc9 morphants are characterized by an increase in the apical surface of the goblet cells, loss of adhesion between the sensorial and peridermal layers, and a decrease in the number of ciliated cells within the blisters. Ptbp1 morphants are characterized by an altered goblet cell morphology. Gene expression profiling by deep RNA sequencing showed that the expression of epidermal and genodermatosis-related genes is also differentially affected in the two morphants, indicating that alterations in post-transcriptional regulations can lead to skin developmental defects through different routes. Therefore, the developing larval epidermis of Xenopus will prove to be a useful model for dissecting the post-transcriptional regulatory network involved in skin development and stability with significant implications for human diseases.

Transcriptional and morphological effects of tamoxifen on the early development of zebrafish (Danio rerio).

Tamoxifen is a widely used anticancer drug with both an estrogen agonist and antagonist effect. This study focused on its endocrine disrupting effect, and overall environmental significance. Zebrafish embryos were exposed to different concentrations (0.5, 5, 50 and 500 µg l(-1) ) of tamoxifen for 96 h. The results showed a complex effect of tamoxifen on zebrafish embryo development. For the 500 µg l(-1) exposure group, the heart rate was decreased by 20% and mild defects in caudal fin and skin were observed. Expressions of a series of genes related to endocrine and morphological changes were subsequently tested through quantitative real-time polymerase chain reaction. Bisphenol A as a known estrogen was also tested as an endocrine-related comparison. Among the expression of endocrine-related genes, esr1, ar, cyp19a1b, hsd3b1 and ugt1a1 were all increased by tamoxifen exposure, similar to bisphenol A. The cyp19a1b is a key gene that controls estrogen synthesis. Exposure to 0.5, 5, 50 and 500 µg l(-1) of tamoxifen caused upregulation of cyp19a1b expression to 152%, 568%, 953% and 2024% compared to controls, higher than the effects from the same concentrations of bisphenol A treatment, yet vtg1 was suppressed by 24% from exposure to 500 µg l(-1) tamoxifen. The expression of metabolic-related genes such as cyp1a, cyp1c2, cyp3a65, gpx1a, gstp1, gsr and genes related to observed morphological changes such as krt17 were also found to be upregulated by high concentrations of tamoxifen. These findings indicated the potential environmental effect of tamoxifen on teleost early development. Copyright © 2015 John Wiley & Sons, Ltd.

Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development, Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis.

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes--fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a--had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a)-ErbB2/3-AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a-ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.

Desarrollo osteológico de la columna vertebral y del complejo caudal de larvas de Lutjanus guttatus (Perciformes: Lutjanidae) en condiciones de cultivo / Osteological development of the vertebral column and caudal complex of Lutjanus guttatus (Perciformes: Lutjanidae) larvae under rearing conditions

The spotted rose snapper (Lutjanus guttatus) is an important commercial species in Mexico with good culture potential. The osteological study at early stages in this species is an important tool to confirm normal bone structure and for the detection of malformations that may occur during early development. This study was carried out in order to evaluate and describe the normal osteological development of the vertebral column and caudal complex of this species grown under controlled conditions. For this, a total of 540 larvae of L. guttatus, between 2.1 and 17.5mm of total length (TL), were cultured during 36 days; culture conditions were 28ºC, 5.74mg/L oxygen and 32.2ups salinity with standard feeding rates. To detect growth changes, a sample of 15 organisms was daily taken from day one until day 36 of post-hatch (DPH). Samples were processed following standard techniques of clearing, and cartilage (alcian blue) and bone staining (alizarin red). Results showed that the vertebral column is composed of ten vertebrae in the abdominal region, and 14 vertebrae including the urostyle in the caudal region. The development of the axial skeleton starts with the neural arches and haemal arches at 3.8mm TL. Caudal elements such as the hypurals and parahypural began to develop at 4.1mm TL. Pre-flexion and flexion of the notochord and the formation of all hypurals were observed between 5.3 and 5.8mm TL. Ossification of the vertebrae in the abdominal region and in some neural arches initiated at 9.5mm TL. In the caudal region, all the neural and haemal arches ossified at 10.2mm TL. All the abdominal vertebrae and their respective neural arches and parapophyses ossified at 11.2mm TL, while the elements of the caudal complex that ossified were the hypurals, parahypurals and modified haemal spines. All caudal fin rays, 12 neural spines and 3 haemal arches were ossified by 15.5mm. The complete ossification process of this specie under laboratory culture conditions was observed when larvae reached 17.3mm TL on 36 DPH. Detailed analysis of the osteological structures will allow a reference description to evaluate and detect malformations that may occur during the larval culture of the spotted rose snapper.

El pargo flamenco (Lutjanus guttatus) es una especie de importancia comercial en México con un gran potencial para su cultivo. El estudio osteológico en estadios tempranos de esta especie bajo condiciones controladas, es una herramienta importante para el conocimiento de su estructura ósea normal y poder detectar las malformaciones que se puedan presentar. El objetivo del presente trabajo se realizó para conocer y describir el desarrollo osteológico normal de la columna vertebral y el complejo caudal de 540 larvas de 2.1 a 17.5mm de longitud total (LT) bajo condiciones de cultivo a 28°C, 5.74mg/L de oxígeno y 32.2UPS de salinidad. Diariamente se tomó una muestra de 15 organismos desde el día uno hasta el 36 después de la eclosión (DDE) y se procesaron con las técnicas de clareado y tinción de cartílago (azul aciano) y hueso (rojo alizarina) para llevar a cabo la descripción de las estructuras. La columna vertebral se divide en región abdominal con diez vértebras y región caudal compuesta por 14 vértebras incluido el urostilo. El desarrollo del esqueleto axial inicia con la formación de los arcos neurales y hemales a los 3.8mm de LT. A los 4.1mm de LT empieza la formación de los hipurales y parahipural que son elementos caudales. Entre los 5.3 y 5.8mm de LT se observó en pre-flexión y flexión del notocordio y la formación de todos los hipurales. La osificación de las vértebras en la región abdominal y en algunos arcos neurales inició a los 9.5mm de LT. A los 10.2mm de LT se osificó la región caudal y todos los arcos neurales y hemales. A los 11.2mm LT se osificaron todas las vértebras abdominales con sus respectivos arcos neurales y los parapófisis, mientras que los elementos del esqueleto caudal que se osificaron fueron los hipurales, parahipurales y las espinas hemales modificadas. A los 15.5mm de LT se osificaron los radios de la aleta caudal y 12 espinas neurales y 3 hemales. El proceso de osificación de las larvas de esta especie en condiciones de cultivo se completó a los 17.3mm LT o 36 DDE. El análisis detallado de las estructuras osteológicas, permitirá una descripción de referencia para evaluar y detectar las malformaciones que se puedan presentar durante el cultivo larvario.

Characterization of zebrafish Pax1b and Pax9 in fin bud development.

Both Pax1 and Pax9 belong to the important paired box gene family (PAX), which mainly participates in animal development and sclerotome differentiation. To date, the precise molecular mechanism and related signaling pathway of Pax1 remain unclear. In our study, microinjection of morpholino- (MO-) modified antisense oligonucleotides against pax1b induced pectoral fin bud defects. Furthermore, we demonstrate that the phenotypes caused by the knockdown of Pax1b in zebrafish could not be phenocopied by pax9 MO and could not be rescued by either Pax1a or Pax9 overexpression. We further find that Pax1b affects the expression of col2a1, Uncx4.1, Noggin3, and aggrecan, confirming the role of Pax1b in chondrocyte differentiation and bone maturation. Moreover, we identify an interaction between PAX1 and FOXO1 and find that the interaction was enhanced under hypoxia stress. Together, this evidence for cell death caused by pax1b knockdown provides new insight into the role of the Pax protein family in cell fate determination and tissue specification.

In vivo imaging of Hedgehog pathway activation with a nuclear fluorescent reporter.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue regeneration, and its dysregulation can lead to birth defects and tumorigenesis. Understanding how this signaling mechanism contributes to these processes would benefit from an ability to visualize Hedgehog pathway activity in live organisms, in real time, and with single-cell resolution. We report here the generation of transgenic zebrafish lines that express nuclear-localized mCherry fluorescent protein in a Gli transcription factor-dependent manner. As demonstrated by chemical and genetic perturbations, these lines faithfully report Hedgehog pathway state in individual cells and with high detection sensitivity. They will be valuable tools for studying dynamic Gli-dependent processes in vertebrates and for identifying new chemical and genetic regulators of the Hh pathway.

Eda/Edar signaling guides fin ray formation with preceding osteoblast differentiation, as revealed by analyses of the medaka all-fin less mutant afl.

BACKGROUND: Ectodysplasin (Eda) signaling is essential for the morphogenesis of several ectodermal appendages. RESULTS: Here, we report a medaka mutant, all-fin less (afl), which has a nonsense mutation in its eda gene. The adult afl fish displayed various abnormalities of its dermal skeleton, such as short and twisted fin rays, missing and abnormally shaped scales and teeth, and skull deformation. Focusing on the developing fin rays in the caudal region of afl larvae, we found that the fin rays did not elongate; although the initial formation of fin rays proceeded normally. Additionally, eda expression was lost, and the expression pattern of edar, the gene for the receptor of Eda, was different from wild-type one. In vivo imaging of the double-transgenic medaka expressing enhanced green fluorescent protein under control of the edar promoter and DsRed under control of the osterix promoter revealed that edar expression preceded that of osterix and that the edar-expressing cells migrated in the direction of fin ray elongation, indicating that the Eda/Edar signaling event precedes osteoblast differentiation. CONCLUSIONS: Our findings provide evidence that Eda signaling accompanied with the binding of Eda to Edar are essential for fin ray formation guided by cell migration.

Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration.

Zebrafish have the capacity to regenerate several organs, including the heart and fins. Fin regeneration is epimorphic, involving the formation at the amputation plane of a mass of undifferentiated, proliferating mesenchymal progenitor-like cells, called blastema. This tissue provides all the cell types that form the fin, so that after damage or amputation the fin pattern and structure are fully restored. How blastema cells remain in this progenitor-like state is poorly understood. Here, we show that the Notch pathway plays an essential role during fin regeneration. Notch signalling is activated during blastema formation and remains active throughout the regeneration process. Chemical inhibition or morpholino-mediated knockdown of Notch signalling impairs fin regeneration via decreased proliferation accompanied by reduced expression of Notch target genes in the blastema. Conversely, overexpression of a constitutively active form of the Notch1 receptor (N1ICD) in the regenerating fin leads to increased proliferation and to the expansion of the blastema cell markers msxe and msxb, as well as increased expression of the proliferation regulator aldh1a2. This blastema expansion prevents regenerative fin outgrowth, as indicated by the reduction in differentiating osteoblasts and the inhibition of bone regeneration. We conclude that Notch signalling maintains blastema cells in a plastic, undifferentiated and proliferative state, an essential requirement for fin regeneration.

The Polycomb group protein Ring1b is essential for pectoral fin development.

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.

Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Role of lbx2 in the noncanonical Wnt signaling pathway for convergence and extension movements and hypaxial myogenesis in zebrafish.

It has been suggested that mouse lbx1 is essential for directing hypaxial myogenic precursor cell migration. In zebrafish, the expression of lbx1a, lbx1b, and lbx2 has been observed in pectoral fin buds. It has also been shown that knocking down endogenous lbx2 in zebrafish embryos diminishes myoD expression in the pectoral fin bud. However, downstream lbxs signals remain largely unexplored. Here, we describe a previously unknown function of zebrafish lbx2 (lbx2) during convergent extension (CE) movements. The abrogation of the lbx2 function by two non-overlapping morpholino oligonucleotides (MOs) resulted in the defective convergence and extension movements in morphants during gastrulation. Our transplantation studies further demonstrated that the overexpression of lbx2 autonomously promotes CE movements. Expression of wnt5b is significantly reduced in lbx2 morphants. We have demonstrated that application of the wnt5b MO, a dominant-negative form of disheveled (Dvl) and a chemical inhibitor of Rho-associated kinase Y27632 in zebrafish embryos have effects reminiscent that are of the CE and hypaxial myogenesis defects observed in lbx2 morphants. Moreover, the CE and hypaxial mesoderm defects seen in lbx2 morphants can be rescued by co-injection with wnt5b or RhoA mRNA. However, this reduced level of active RhoA and hypaxial myogenesis defects in the embryos injected with the dominant-negative form of Dvl mRNA cannot be effectively restored by co-injection with lbx2 mRNA. Our results suggest that the key noncanonical Wnt signaling components Wnt5, Dvl, and RhoA are downstream effectors involved in the regulative roles of lbx2 in CE movement and hypaxial myogenesis during zebrafish embryogenesis.

The medaka zic1/zic4 mutant provides molecular insights into teleost caudal fin evolution.

Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.

The Osr1 and Osr2 genes act in the pronephric anlage downstream of retinoic acid signaling and upstream of Wnt2b to maintain pectoral fin development.

Vertebrate odd-skipped related genes (Osr) have an essential function during the formation of the intermediate mesoderm (IM) and the kidney structures derived from it. Here, we show that these genes are also crucial for limb bud formation in the adjacent lateral plate mesoderm (LPM). Reduction of zebrafish Osr function impairs fin development by the failure of tbx5a maintenance in the developing pectoral fin bud. Osr morphant embryos show reduced wnt2b expression, and increasing Wnt signaling in Osr morphant embryos partially rescues tbx5a expression. Thus, Osr genes control limb bud development in a non-cell-autonomous manner, probably through the activation of Wnt2b. Finally, we demonstrate that Osr genes are downstream targets of retinoic acid (RA) signaling. Therefore, Osr genes act as a relay within the genetic cascade of fin bud formation: by controlling the expression of the signaling molecule Wnt2ba in the IM they play an essential function transmitting the RA signaling originated in the somites to the LPM.

Lef1-dependent Wnt/ß-catenin signalling drives the proliferative engine that maintains tissue homeostasis during lateral line development.

During tissue morphogenesis and differentiation, cells must self-renew while contemporaneously generating daughters that contribute to the growing tissue. How tissues achieve this precise balance between proliferation and differentiation is, in most instances, poorly understood. This is in part due to the difficulties in dissociating the mechanisms that underlie tissue patterning from those that regulate proliferation. In the migrating posterior lateral line primordium (PLLP), proliferation is predominantly localised to the leading zone. As cells emerge from this zone, they periodically organise into rosettes that subsequently dissociate from the primordium and differentiate as neuromasts. Despite this reiterative loss of cells, the primordium maintains its size through regenerative cell proliferation until it reaches the tail. In this study, we identify a null mutation in the Wnt-pathway transcription factor Lef1 and show that its activity is required to maintain proliferation in the progenitor pool of cells that sustains the PLLP as it undergoes migration, morphogenesis and differentiation. In absence of Lef1, the leading zone becomes depleted of cells during its migration leading to the collapse of the primordium into a couple of terminal neuromasts. We show that this behaviour resembles the process by which the PLLP normally ends its migration, suggesting that suppression of Wnt signalling is required for termination of neuromast production in the tail. Our data support a model in which Lef1 sustains proliferation of leading zone progenitors, maintaining the primordium size and defining neuromast deposition rate.

Tissue regeneration after injury in adult zebrafish: the regenerative potential of the caudal fin.

The zebrafish has the potential to regenerate many of its tissues. In this study, we examined caudal fin regeneration in zebrafish that received repeated injuries (fin amputation) at different ages. In zebrafish that received repeated injuries, the potential for caudal fin regeneration, such as tissue growth and the expression of regeneration marker genes (msxb, fgf20a, bmp2b), did not decline in comparison to zebrafish that received only one amputation surgery. The process of initial fin regeneration (e.g., tissue outgrowth and the expression of regeneration marker genes at 7 days post-amputation) did not seem to correlate with age. However, slight differences in fin outgrowth were observed between young and old animals when examined in the late regeneration stages (e.g., 20 and 30 days post-amputation). Together, the data suggest that zebrafish has unlimited regenerative potential in the injured caudal fin.

Amphioxus Tbx6/16 and Tbx20 embryonic expression patterns reveal ancestral functions in chordates.

T-box transcription factors are found in all metazoans and play diverse roles during embryogenesis. In the cephalochordate amphioxus, nine T-box genes were previously identified. In this work we undertook the analysis of the embryonic expression pattern of Tbx6/16 and Tbx20, the last two T-box genes for which no such data are available. We found that Tbx6/16 is expressed in the unsegmented paraxial mesoderm, in a subpopulation of neurons, and in the tail epidermis. Comparison with the expression patterns of the different vertebrate orthologues indicates a conserved role of those genes in posterior mesoderm formation in chordates. Tbx20 expression is detected in the ventral mesoderm of amphioxus embryos, in cells that are proposed to be precursors of the amphioxus myocardium, in some neurons of the neural tube, and in the pre-oral pit which is thought to be the homologue of the vertebrate adenohypophysis. In vertebrates, Tbx20 is also one of the first genes expressed in the embryonic heart field, suggesting that the function of this gene in heart development has been conserved during chordate evolution.