Pectoral Fin Buds Research Articles

Cyp1a reporter zebrafish reveals target tissues for dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the unintentional byproduct of various industrial processes, is classified as human carcinogen and could disrupt reproductive, developmental and endocrine systems. Induction of cyp1a1 is used as an indicator of TCDD exposure. We sought to determine tissues that are vulnerable to TCDD toxicity using a transgenic zebrafish (Danio rerio) model. We inserted a nuclear enhanced green fluorescent protein gene (EGFP) into the start codon of a zebrafish cyp1a gene in a fosmid clone using DNA recombineering. The resulting recombineered fosmid was then used to generate cyp1a reporter zebrafish, embryos of which were exposed to TCDD. Expression pattern of EGFP in the reporter zebrafish mirrored that of endogenous cyp1a mRNA. In addition, exposure of the embryos to TCDD at as low as 10pM for 72h, which does not elicit morphological abnormalities of embryos, markedly increased GFP expression. Furthermore, the reporter embryos responded to other AhR ligands as well. Exposure of the embryos to TCDD revealed previously reported (the cardiovascular system, liver, pancreas, kidney, swim bladder and skin) and unreported target tissues (retinal bipolar cells, otic vesicle, lateral line, cloaca and pectoral fin bud) for TCDD. Transgenic cyp1a reporter zebrafish we have developed can further understanding of ecotoxicological relevance and human health risks by TCDD. In addition, they could be used to identify agonists of AhR and antidotes to TCDD toxicity.

Opa1 Is Required for Proper Mitochondrial Metabolism in Early Development

Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. OPA1 mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, pgc1a, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.

Expression analysis of zebrafish membrane type-2 matrix metalloproteinases during embryonic development

Membrane tethered matrix metalloproteinases (MMPs) cleave a variety of extracellular matrix (ECM) and non-ECM targets and play important roles during embryonic development and tumor progression. Membrane tethered MMPs in particular are important regulators of both tissue invasion and morphogenesis. Much attention has been given to understanding the function of human and mouse MMP14 (also called membrane type-1 MMP, MT1-MMP) and our own data have linked zebrafish Mmp14 to the regulation of gastrulation cell movements. However, less is known regarding the expression and function of other membrane tethered MMPs. We report the cloning and gene expression analysis of zebrafish mmp15a and mmp15b (MT2-MMP) during early embryonic and larval development. Our data show that mmp15a exhibits limited expression prior to segmentation stages and is first detected in the tectum and posterior tailbud. At 24hours post-fertilization (hpf) mmp15a localizes to the caudal hematopoietic tissue, pectoral fin buds, and mandibular arch. By contrast, mmp15b is strongly expressed during gastrula stages before becoming restricted to the polster and anterior neural plate. From 24 to 48hpf, mmp15b expression is detected in the pharyngeal arches, fin buds, otic vesicle, pronephric ducts, proctodeum, tail epidermis, posterior lateral line primordia, and caudal notochord. During the larval period beginning at 72hpf, mmp15b expression becomes restricted to the brain ventricular zone, pharyngeal arches, pectoral fins, and the proctodeum. Many of the mmp15-expressing tissues have been shown to express genes encoding components of the ECM including collagens, fibronectin, and laminins. Our data thus provide a foundation for uncovering the role of Mmp15-dependent pericellular proteolysis during zebrafish embryonic development.

Spawning habitat and early development of Luciogobius ryukyuensis (Gobiidae)

Egg masses of Luciogobius ryukyuensis were found in spawning grounds around the lowest reach of the adult’s habitat in the tidally influenced area of streams on Okinawa Island. The eggs were attached to the underside of stones and were cared for by a solitary male fish. The number of eggs within an egg mass was 191–1368. The eggs were elliptical, measuring 2.0–2.6 mm in length, and 0.6–0.8 mm in width. Early development of L. ryukyuensis was described from laboratory-reared specimens. Newly hatched larvae, measuring 2.8–3.3 mm in body length, had an open mouth, pigmented eyes, pectoral fin buds, an orange-colored yolk sac, and characteristic melanophores along the dorsal and ventral midlines of the body. The yolk was completely absorbed at days 2–3. Notochord flexion began at day 10 and was completed at day 15. The fish started settling on the bottom of the tank at day 34 (14.1 mm in body length) when the body surface started to be covered by intense pigmentation. The eggs and newly hatched larvae of L. ryukyuensis were of standard size of the genus and their morphologies closely resemble those of L. guttatus.

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.

Theosr1andosr2genes act in the pronephric anlage downstream of retinoic acid signaling and upstream ofwnt2bto 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.

Single and joint action toxicity of heavy metals on early developmental stages of Chinese rare minnow ( Gobiocypris rarus)

In this work, acute toxicities of heavy metals (Cu, Zn and Cd) were evaluated singly or in mixtures on Chinese rare minnow ( Gobiocypris rarus) in its early stages of development (embryos, larvae). Normal embryos not later than 3 h post-fertilization and newly hatched larvae were selected for the tests. The embryos were exposed to the metal solutions studied until developing to the stages of somite formation (15 h), tail detachment (25 h 10 min), heart-beat visible (34 h 10 min), pectoral fin bud appearance (47 h 40 min) and hatching (75 h), respectively. Exposures of the larvae were continued for 24 h. Results from the single toxicity tests revealed that the mortality of embryos increases obviously with increasing exposure duration. G. rarus appears to be more sensitive to heavy metal exposures at larval stage than at embryonic stage. The toxicity order of the tested metals was Cu>Cd>Zn for the embryos and Cu>Zn>Cd for the larvae with LC 50 as the toxicity criterion. These data suggest that the sensitivity of G. rarus to heavy metals (Cu, Zn and Cd) is different and it depends on the exposition duration and the stages of fish development. Among the metals tested, Cu is the one most toxic to G. rarus at its early development stages. In addition, results of the mixture experiments showed that binary combinations of Cu–Zn and Cu–Cd had strictly synergistic lethal effects on the larvae. The enhanced toxicity suggested inclusion of mixture considerations in the risk assessment of heavy metals might be recommended.

MiR-196 regulates axial patterning and pectoral appendage initiation

Vertebrate Hox clusters contain protein-coding genes that regulate body axis development and microRNA (miRNA) genes whose functions are not yet well understood. We overexpressed the Hox cluster microRNA miR-196 in zebrafish embryos and found four specific, viable phenotypes: failure of pectoral fin bud initiation, deletion of the 6th pharyngeal arch, homeotic aberration and loss of rostral vertebrae, and reduced number of ribs and somites. Reciprocally, miR-196 knockdown evoked an extra pharyngeal arch, extra ribs, and extra somites, confirming endogenous roles of miR-196. miR-196 injection altered expression of hox genes and the signaling of retinoic acid through the retinoic acid receptor gene rarab. Knocking down rarab mimicked the pectoral fin phenotype of miR-196 overexpression, and reporter constructs tested in tissue culture and in embryos showed that the rarab 3′UTR is a miR-196 target for pectoral fin bud initiation. These results show that a Hox cluster microRNA modulates development of axial patterning similar to nearby protein-coding Hox genes, and acts on appendicular patterning at least in part by modulating retinoic acid signaling.

Characterization of the expression, promoter activity and molecular architecture of fibin

BackgroundFibin was initially discovered as a secreted signal molecule essential for pectoral fin bud initiation in zebrafish. Currently, there is little information about the molecular architecture and biological relevance of fibin in humans and other mammals.ResultsFibin is expressed in cerebellum, skeletal muscle and many other embryonic and adult mouse tissues suggesting not only a role during embryonic development but also in adult functions. A 2.5-kbp genomic sequence fragment upstream of the coding sequence is sufficient to drive and regulate fibin expression through stimulation by glucocorticoids, activators of the protein kinase C signalling pathways and manganese ions. Fibin is an evolutionarily conserved protein, carries a cleavable signal peptide (amino acids 1-18) and is glycosylated at Asn30. The two conserved cysteines participate in intermolecular disulfide bond and multimer formation. Although fibin displays all features of a secretory protein, it is mostly retained in the endoplasmic reticulum when heterologously expressed.ConclusionFibin is functionally relevant during embryogenesis and adult life. Its expression is regulated by a number of cellular signalling pathways and the protein is routed via the secretory pathway. However, proper secretion presumably requires an unknown covalently-linked or associated co-factor.

Growth and morphological development of laboratory-reared larval and juvenile Hemibagrus filamentus (Siluriformes: Bagridae)

Morphological development in laboratory-reared larval and juvenile Hemibagrus filamentus, and behavioral features observed under rearing conditions are described. Body lengths (BL) of larvae and juveniles were 3.8 ± 0.2 (mean ± SD) mm just after hatching and 11.7 ± 1.6 mm on day 15, reaching 26.5 ± 5.4 mm on day 30 after hatching. Aggregate fin ray numbers (for caudal fin, except for procurrent rays) attained full complements in specimens larger than 12.9 mm BL. A maxillary barbel bud appeared on day 0, and all larvae initiated feeding on day 3 with the development of mandibular barbels and conical teeth. Pectoral fin buds and primordial nostrils were present on day 1. Notochord flexion began on day 3, and the yolk was completely absorbed by day 4. Melanophores were scarce at hatching, but increased with growth to cover almost the entire body except the ventral surface of the head and body. Body proportions became relatively constant in juveniles, excepting maxillary barbel length that continued to increase, reaching over 40% BL. Fish were negatively phototactic from day 1. Cannibalism was observed from day 6, continuing to the juvenile stage.

Expression patterns of lgr4 and lgr6 during zebrafish development

Leucine-rich repeat (LRR)-containing G protein-coupled receptors (LGRs) belong to the superfamily of G protein-coupled receptors, and are characterized by the presence of seven transmembrane domains and an extracellular domain that contains a series of LRR motifs. Three Lgr proteins – Lgr4, Lgr5, and Lgr6 – were identified as members of the LGR subfamily. Mouse Lgr4 has been implicated in the formation of various organs through regulation of cell proliferation during development, and Lgr5 and Lgr6 are stem cell markers in the intestine or skin. Although the expression of these three genes has already been characterized in adult mice, their expression profiles during the embryonic and larval development of the organism have not yet been defined. We cloned two zebrafish lgr genes using the zebrafish genomic database. Phylogenetic analyses showed that these two genes are orthologs of mammalian Lgr4 and Lgr6. Zebrafish lgr4 is expressed in the neural plate border, Kupffer’s vesicle, neural tube, otic vesicles, midbrain, eyes, forebrain, and brain ventricular zone by 24 h post-fertilization (hpf). From 36 to 96 hpf, lgr4 expression is detected in the midbrain–hindbrain boundary, otic vesicles, pharyngeal arches, cranial cartilages such as Meckel’s cartilages, palatoquadrates, and ceratohyals, cranial cavity, pectoral fin buds, brain ventricular zone, ciliary marginal zone, and digestive organs such as the intestine, liver, and pancreas. In contrast, zebrafish lgr6 is expressed in the notochord, Kupffer’s vesicle, the most anterior region of diencephalon, otic vesicles, and the anterior and posterior lateral line primordia by 24 hpf. From 48 to 72 hpf, lgr6 expression is confined to the anterior and posterior neuromasts, otic vesicles, pharyngeal arches, pectoral fin buds, and cranial cartilages such as Meckel’s cartilages, ceratohyals, and trabeculae. Our results provide a basis for future studies aimed at analyzing the functions of zebrafish Lgr4 and Lgr6 in cell differentiation and proliferation during organ development.

Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). We previously reported a sensitive and useful endpoint of TCDD developmental toxicity in zebrafish, namely a decrease in blood flow in the dorsal midbrain, but downstream genes involved in the effect are not known. The present study addressed the role of zebrafish cytochrome P450 1C (CYP1C) genes in association with a decrease in mesencephalic vein (MsV) blood flow. The CYP1C subfamily was recently discovered in fish and includes the paralogues CYP1C1 and CYP1C2, both of which are induced via AHR2 in zebrafish embryos. We used morpholino antisense oligonucleotides (MO or morpholino) to block initiation of translation of the target genes. TCDD-induced mRNA expression of CYP1Cs and a decrease in MsV blood flow were both blocked by gene knockdown of AHR2. Gene knockdown of CYP1C1 by two different morpholinos and CYP1C2 by two different morpholinos, but not by their 5 nucleotide-mismatch controls, was effective in blocking reduced MsV blood flow caused by TCDD. The same CYP1C-MOs prevented reduction of blood flow in the MsV caused by β-naphthoflavone (BNF), representing another class of AHR agonists. Whole-mount in situ hybridization revealed that mRNA expression of CYP1C1 and CYP1C2 was induced by TCDD most strongly in branchiogenic primordia and pectoral fin buds. In situ hybridization using head transverse sections showed that TCDD increased the expression of both CYP1Cs in endothelial cells of blood vessels, including the MsV. These results indicate a potential role of CYP1C1 and CYP1C2 in the local circulation failure induced by AHR2 activation in the dorsal midbrain of the zebrafish embryo.

HMGB factors are required for posterior digit development through integrating signaling pathway activities

The chromatin factors Hmgb1 and Hmgb2 have critical roles in cellular processes, including transcription and DNA modification. To identify the function of Hmgb genes in embryonic development, we generated double mutants of Hmgb1;Hmgb2 in mice. While double null embryos arrest at E9.5, Hmgb1(-/-) ; Hmgb2(+/-) embryos exhibit a loss of digit5, the most posterior digit, in the forelimb. We show that Hmgb1(-/-) ; Hmgb2(+/-) forelimbs have a reduced level of Shh signaling, as well as a clear downregulation of Wnt and BMP target genes in the posterior region. Moreover, we demonstrate that hmgb1 and hmgb2 in zebrafish embryos enhance Wnt signaling in a variety of tissues, and that double knockdown embryos have reduced Wnt signaling and shh expression in pectoral fin buds. Our data show that Hmgb1 and Hmgb2 function redundantly to enhance Wnt signaling in embryos, and further suggest that integrating Wnt, Shh, and BMP signaling regulates the development of digit5 in forelimbs.

Carboxypeptidase A6 in Zebrafish Development and Implications for VIth Cranial Nerve Pathfinding

Carboxypeptidase A6 (CPA6) is an extracellular protease that cleaves carboxy-terminal hydrophobic amino acids and has been implicated in the defective innervation of the lateral rectus muscle by the VIth cranial nerve in Duane syndrome. In order to investigate the role of CPA6 in development, in particular its potential role in axon guidance, the zebrafish ortholog was identified and cloned. Zebrafish CPA6 was secreted and interacted with the extracellular matrix where it had a neutral pH optimum and specificity for C-terminal hydrophobic amino acids. Transient mRNA expression was found in newly formed somites, pectoral fin buds, the stomodeum and a conspicuous condensation posterior to the eye. Markers showed this tissue was not myogenic in nature. Rather, the CPA6 localization overlapped with a chondrogenic site which subsequently forms the walls of a myodome surrounding the lateral rectus muscle. No other zebrafish CPA gene exhibited a similar expression profile. Morpholino-mediated knockdown of CPA6 combined with retrograde labeling and horizontal eye movement analyses demonstrated that deficiency of CPA6 alone did not affect either VIth nerve development or function in the zebrafish. We suggest that mutations in other genes and/or enhancer elements, together with defective CPA6 expression, may be required for altered VIth nerve pathfinding. If mutations in CPA6 contribute to Duane syndrome, our results also suggest that Duane syndrome can be a chondrogenic rather than a myogenic or neurogenic developmental disorder.

Zebrafish Wnt9a,9b paralog comparisons suggest ancestral roles for Wnt9 in neural, oral–pharyngeal ectoderm and mesendoderm

The Wnts are a highly conserved family of secreted glycoproteins involved in cell–cell signaling and pattern formation during early embryonic development. Teasing out the role of individual Wnt molecules through development is challenging. Gene duplications are one of the most important mechanisms for generating evolutionary variations. The current consensus suggests that most anatomical variation is generated by divergence of regulatory control regions rather than by coding sequence divergence. Thus phylogenetic comparisons of divergent gene expression patterns are essential to understanding ancestral morphogenetic patterns from which subsequent anatomy diversified in modern lineages. We previously demonstrated strongest expression of zebrafish wnt9b within its heart tube, limb bud and ventral/anterior ectoderm during oral and pharyngeal arch patterning. Our goal is to compare and contrast zwnt9b to its closest paralog, zwnt9a. Sequenced, fulllength zebrafish wnt9a and wnt9b cDNA clones were used for phylogenetic analysis, which suggests their derivation from a common pre-vertebrate archeolog by gene duplication and divergence. Here we demonstrate that zwnt9a expression is found within unique (CNS, pronephric ducts, sensory organs) and overlapping (pectoral fin buds) expression domains relative to zwnt9b. Apparently, Wnt9 paralogs differentially parsed common ancestral expression domains during their subsequent rounds of gene duplication, divergence and loss in different vertebrate lineages. This expression data suggests ancestral roles for Wnt9s in early patterning of neural/oral–pharyngeal ectoderm and mesendoderm derivatives.

A Sox5 gene is expressed in the myogenic lineage during trout embryonic development

Sox proteins form a family of transcription factors characterized by the presence of a DNA-binding domain called the high-mobility-group domain (HMG). The presence of a large number of potential Sox5 binding sites in the trout promoter of Pax7, a gene which has emerged as an important regulator of neural and somite development, prompted us to clone trout Sox5 and to examine its expression pattern in the developing trout embryo. Using whole mount in situ hybridisation, we show here that the Sox5 transcript is first expressed before segmentation in the whole presomitic mesoderm. In newly formed somites, Sox5 labelling was observed in myogenic progenitor cells of the posterior and anterior walls. As the somite matured rostrocaudally, Sox5 expression disappeared from the differentiating embryonic myotome, deep in the somite, to become restricted to the undifferentiated myogenic precursors forming the dermomyotome-like epithelium at the surface of the embryonic myotome. Sox5 was also expressed in the developing nervous system and in pectoral fin buds. On the whole, this work suggests a hitherto unappreciated role for Sox5 in regulating myogenic cells destined to muscle formation and growth.

Morphological development of Anchoviella vaillanti (Steindachner, 1908) (Clupeiformes: Engraulidae) larvae and early juveniles

The considerable similarity in the early life stages of different fish species makes egg and larvae identification in fishery biology and ichthyoplankton surveys a difficult task. Knowledge on early larval development of morphologically similar taxa and species-rich orders, such as Clupeiformes, mainly in the Neotropical fresh waters is rather limited. The aim of the present study was to describe morphological and meristic aspects of the larvae and early juveniles of Anchoviella vaillanti, an endemic species of the São Francisco River basin in Brazil. The characterization was based on an ontogenetic series of 132 individuals (1.3-51.0 mm SL). In the larval period, body varies from elongated to very elongated and the head is small, which is typical of Clupeiformes. The finfold is present beginning in the yolk-sac stage, when larvae have a large yolk sac, until the flexion stage. Pectoral fin buds are the first to form during the preflexion stage, when dorsal- and anal-fin pterygiophores and hypural bones are first visible. The total vertebra count ranges from 36 to 39 and the myomere number ranges from 31 to 45. Complete fin formation obeys the following sequence: anal and dorsal fins during flexion stage; and pectoral, pelvic and caudal fins during postflexion stage. Despite being the only freshwater clupeiform representative in the São Francisco River, A. vaillanti may occur sympatrically with A. lepidentostole in the lower stretches of the river basin. Although early larvae characteristics of A. lepidentostole are not known, its late larvae and early juveniles may be distinguished from those of A. vaillanti, by the higher number of dorsal-fin rays (15 or 16 vs. 12 or 13 in A. vaillanti), higher total vertebra count (40 vs. 37 to 40) and shorter pre-pectoral length (14 to 16 vs. 22.8 to 28.9% SL).

A gain of function mutation causing skeletal overgrowth in the rapunzel mutant

Mechanisms that regulate the growth and form of the vertebrate skeleton are largely unknown. The zebrafish mutant rapunzel has heterozygous defects in bone development, resulting in skeletal overgrowth, thus identification of the genetic lesion underlying rapunzel might provide insight into the molecular basis of skeletogenesis. In this report, we demonstrate that the rapunzel mutant results from a missense mutation in the previously uncharacterized rpz gene. This conclusion is supported by genetic mapping, identification of a missense mutation in rapunzel(c14) in a highly conserved region of the rpz gene, and suppression of the rapunzel homozygous embryonic phenotype with morpholino knockdown of rpz. In addition, rpz transcripts are identified in regions correlating with the homozygous embryonic phenotype (head, pectoral fin buds, somites and fin fold). This report provides the first gene identification for a mutation affecting segment number in the zebrafish fin and development of both the fin ray (dermal) and the axial skeleton.

The accumulation of substances in Recirculating Aquaculture Systems (RAS) affects embryonic and larval development in common carp Cyprinus carpio

The accumulation of substances in Recirculating Aquaculture Systems (RAS) may impair the growth and welfare of fish. To test the severity of contaminants accumulated in RAS, early-life stages of fish were used. Ultrafiltered water from two Recirculating Aquaculture Systems (RAS), one RAS with a high accumulation of substances (water exchange rate 30 L/kg feed/day) and one RAS with a low accumulation of substances (water exchange rate 1500 L/kg feed/day), was used to incubate eggs and rear larvae of common carp Cyprinus carpio. A broad range of read-out parameters was used to determine the effect of accumulation level on the development of the early-life stages; from hatching dynamics to larvae length and dry weight. The water quality (temperature, pH, dissolved O 2, conductivity, total bicarbonate, ortho-phosphate-P, TAN, NO 2 −–N, NO 3 −–N and minerals) was compared between the 2 treatments. Carp eggs developing in the high-accumulation water had higher mortality percentages (both for eggs and larvae), reduced hatching percentages, delayed hatching dynamics and reduced larvae length and body weight. However, these larvae exhibited fewer deformities than larvae incubated in the low-accumulation water. Furthermore, an accelerated development both of the embryo (appearance of heart beat, pectoral fin bud and tail movement) and yolk-sac larvae (depletion of the yolk sac) was observed in the high-accumulation water. The high-accumulation water had significantly lower pH and higher conductivity, NO 2 −–N, NO 3 −–N and ortho-phosphate-P. Most of the minerals (As, Cu, Mn, Ni, Zn, K, Mg, Na, P and S) including heavy metals, were present at a higher concentration in the high-accumulation water. The influence of these parameters on the embryonic and larval development of fish is discussed. It is suggested that in the high-accumulation water, the concentration of ortho-phosphate-P, nitrate and of the heavy metals arsenic and copper is likely to have impaired the embryonic and larval development and therefore deserves further research as potential growth inhibiting factors in RAS.

Expression profiling of zebrafish sox9 mutants reveals that Sox9 is required for retinal differentiation

The transcription factor gene Sox9 plays various roles in development, including differentiation of the skeleton, gonads, glia, and heart. Other functions of Sox9 remain enigmatic. Because Sox9 protein regulates expression of target genes, the identification of Sox9 targets should facilitate an understanding of the mechanisms of Sox9 action. To help identify Sox9 targets, we used microarray expression profiling to compare wild-type embryos to mutant embryos lacking activity for both sox9a and sox9b, the zebrafish co-orthologs of Sox9. Candidate genes were further evaluated by whole-mount in situ hybridization in wild-type and sox9 single and double mutant embryos. Results identified genes expressed in cartilage ( col2a1a and col11a2), retina ( calb2a, calb2b, crx, neurod, rs1, sox4a and vsx1) and pectoral fin bud ( klf2b and EST AI722369) as candidate targets for Sox9. Cartilage is a well-characterized Sox9 target, which validates this strategy, whereas retina represents a novel Sox9 function. Analysis of mutant phenotypes confirmed that Sox9 helps regulate the number of Müller glia and photoreceptor cells and helps organize the neural retina. These roles in eye development were previously unrecognized and reinforce the multiple functions that Sox9 plays in vertebrate development.