Fin Buds Research Articles

Waste Nitrogen Metabolism and Excretion in Zebrafish Embryos: Effects of Light, Ammonia, and Nicotinamide

Bony fish primarily excrete ammonia as adults however the persistence of urea cycle genes may reflect a beneficial role for urea production during embryonic stages in protecting the embryo from toxic effects of ammonia produced from a highly nitrogenous yolk. This study aimed to examine the dynamic scope for changes in rates of urea synthesis and excretion in one such species (zebrafish, Danio rerio) by manipulating the intrinsic developmental rate (by alteration of light:dark cycles), as well as by direct chemical manipulation via ammonia injection (to potentially activate urea production) and nicotinamide exposure (to potentially inhibit urea production). Continuous dark exposure delayed development in embryos as evidenced by delayed appearance of hallmark anatomical features (heartbeat, eye pigmentation, body pigmentation, lateral line, fin buds) at 30 and 48 hr post-fertilization, as well by a lower hatching rate compared to embryos reared in continuous light. Both ammonia and urea excretion were similarly effected and were generally higher in embryos continuously exposed to light. Ammonia injection resulted in significant increases (up to fourfold) of urea N excretion and no changes to ammonia excretion rates along with modest increases in yolk ammonia content during 2-6 hr post-injection. Nicotinamide (an inhibitor of urea synthesis in mammals) reduced the ammonia-induced increase in urea excretion and led to retention of ammonia in the yolk and body of the embryo. Our results indicate that there is a relatively rapid and large scope for increases in urea production/excretion rates in developing embryos. Potential mechanisms for these increases are discussed.

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.

Cloning and expression of <I>Tbx3</I> gene in Siberian sturgeon, <I>Acipenser baerii</I>

Tbx3, a member of the TBX2 subfamily of T-box gene family, encodes a transcription factor with a highly conserved DNA-binding domain, which called T-domain. Tbx3 is involved in morphogenesis and organogenesis in vertebrates, such as limb development, heart remodeling, and neural placode differentiation. In the present study, a full-length 2 908 bp Tbx3 cDNA from Acipenser baerii (AbTbx3) was obtained using RT-PCR and RACE technique, which includes a 2 166 bp complete open reading frame encoding a putative peptide of 721 amino acids. AbTbx3 shares 73.5% identity with its human homolog. Particularly, the DNA-binding domain of AbTbx3 shared 95.2% identity with human Tbx3. Phylogenetic analysis revealed that AbTbx3 was grouped with Tbx3s in other vertebrates, which were clustered with Tbx2s and separated from Tbx4/5s. The predicted secondary and three-dimensional structures of the T-domain of AbTbx3 were remarkably similar to human Tbx3. Through semi-quantity RT-PCR, the expression of AbTbx3 was first detected at blastula stage during Siberian sturgeon embryonic development, increased gradually, reached its peak at early tail-bud stage and then decreased slightly. In adult sturgeon, AbTbx3 was strongly expressed in eye, brain, gill, intestines, pectoral fin and pelvic fin, but not in liver, blood, heart, kidney and muscle. The whole mount in situ hybridization showed that AbTbx3 was mainly expressed in the otic vesicle, hindbrain, dorsal notochord, pineal organ and dorsal fin bud in the larvae of stage 37 and 43.

The function of <i>DrPax1b</i> gene in the embryonic development of zebrafish

Vertebrate Pax1 gene is a member of Pax gene family and encodes a transcription factor associated with crucial roles in the development of pharyngeal pouch, scletrotome and limb bud. In zebrafish, the genome contains two Pax1 paralogs, DrPax1a and DrPax1b, which share high sequence similarity with other Pax1 genes. To elucidate the function of zebrafish DrPax1b gene, we first examined the gene expression pattern and found that it was mainly expressed in the endodermal pharyngeal pouch, caudal somites, notochord, and fin bud. Then, we performed knockdown experiments using antisense morpholino oligonucleotides, which lead to the defects in the vertebral column, tail, pharyngeal skeleton, and pectoral fin. Additionally, we also found that the mouse MmPax1 mRNA, but not the amphioxus AmphiPax1/9 mRNA, could rescue the MO-induced defects. Furthermore, sequence alignment revealed that the N-terminal region of vertebrate Pax1 and amphioxus Pax1/9 were highly conserved, whereas their C-terminal regions were relatively divergent. However, the chimeric Am(N)Dr(C)Pax1, Mm(N)Dr(C)Pax1 and Dr(N)Mm(C)Pax1 mRNA could partially rescue the defects, while the Dr(N)Am(C)Pax1 mRNA could not. In conclusion, our data demonstrate a conserved function of DrPax1b in the development of the vertebral column, pectoral fin and pharyngeal skeleton formation in zebrafish and also provide critical insight into the functional evolution of Pax1 gene by changing its C-terminal sequence.

Effect of lead exposure on gene expression of Fgf3 in zebrafish embryonic development

To investigate the effect of lead exposure on the gene expression of fibroblast growth factor 3 (Fgf3) in zebrafish embryonic development and the mechanism of lead-induced embryonic developmental toxicity. The embryos of zebrafish (wild types A and B) were exposed to lead acetate (PbAc) at the doses of 0, 0.1, 0.5, 2.5, and 12.5 µmol/L separately. Total RNA was extracted from each treatment group of zebrafish embryos at 8, 12, 16, 24, 36, 48, and 72 hours post fertilization (hpf). The total mRNA expression of Fgf3 was measured by real-time quantitative PCR. The spatial expression of Fgf3 in zebrafish embryos was determined by whole-mount in situ hybridization using synthesized Fgf3 RNA probe. The mRNA expression of Fgf3 in each group peaked at 12 hpf (P < 0.01). With the increase in PbAc concentration, the mRNA expression of Fgf3 rose. Compared with the mRNA expression level of Fgf3 in the control group, the relative mRNA expression levels of Fgf3 in the 0.1, 0.5, 2.5, and 12.5 µmol/L PbAc exposure groups were 1.02 ± 0.24, 1.05 ± 0.26, 1.22 ± 0.46, and 1.25 ± 0.38, respectively, and the 2.5 and 12.5 µmol/L PbAc exposure groups showed significantly higher Fgf3 expression than the control group (P < 0.05). The whole-mount in situ hybridization results showed that Fgf3 expression occurred mainly in the head and tail in the early stage of embryonic development and in the midbrain, fin bud, and pharyngeal arch in the middle/late stage of embryonic development; there were the most significant regions and intensities of positive hybridization signals at 12 hpf; but no significant differences were found between the control group and exposure groups in the location and intensity of Fgf3 expression Lead exposure can result in the upregulation of Fgf3 expression in zebrafish embryonic development, which might contribute to lead-induced embryonic developmental toxicity.

Mechanism of pectoral fin outgrowth in zebrafish development

Fins and limbs, which are considered to be homologous paired vertebrate appendages, have obvious morphological differences that arise during development. One major difference in their development is that the AER (apical ectodermal ridge), which organizes fin/limb development, transitions into a different, elongated organizing structure in the fin bud, the AF (apical fold). Although the role of AER in limb development has been clarified in many studies, little is known about the role of AF in fin development. Here, we investigated AF-driven morphogenesis in the pectoral fin of zebrafish. After the AER-AF transition at ∼36 hours post-fertilization, the AF was identifiable distal to the circumferential blood vessel of the fin bud. Moreover, the AF was divisible into two regions: the proximal AF (pAF) and the distal AF (dAF). Removing the AF caused the AER and a new AF to re-form. Interestingly, repeatedly removing the AF led to excessive elongation of the fin mesenchyme, suggesting that prolonged exposure to AER signals results in elongation of mesenchyme region for endoskeleton. Removal of the dAF affected outgrowth of the pAF region, suggesting that dAF signals act on the pAF. We also found that the elongation of the AF was caused by morphological changes in ectodermal cells. Our results suggest that the timing of the AER-AF transition mediates the differences between fins and limbs, and that the acquisition of a mechanism to maintain the AER was a crucial evolutionary step in the development of tetrapod limbs.

Duplicated zebrafish co-orthologs of parathyroid hormone-related peptide (PTHrP, Pthlh) play different roles in craniofacial skeletogenesis

In mammals, parathyroid hormone-related peptide (PTHrP, alias PTH-like hormone (Pthlh)) acts as a paracrine hormone that regulates the patterning of cartilage, bone, teeth, pancreas, and thymus. Beyond mammals, however, little is known about the molecular genetic mechanisms by which Pthlh regulates early development. To evaluate conserved pathways of craniofacial skeletogenesis, we isolated two Pthlh co-orthologs from the zebrafish (Danio rerio) and investigated their structural, phylogenetic, and syntenic relationships, expression, and function. Results showed that pthlh duplicates originated in the teleost genome duplication. Zebrafish pthlha and pthlhb were maternally expressed and showed overlapping and distinct zygotic expression patterns during skeletal development that mirrored mammalian expression domains. To explore the regulation of duplicated pthlh genes, we studied their expression patterns in mutants and found that both sox9a and sox9b are upstream of pthlha in arch and fin bud cartilages, but only sox9b is upstream of pthlha in the pancreas. Morpholino antisense knockdown showed that pthlha regulates both sox9a and sox9b in the pharyngeal arches but not in the brain or otic vesicles and that pthlhb does not regulate either sox9 gene, which is likely related to its highly degraded nuclear localization signal. Knockdown of pthlha but not pthlhb caused runx2b overexpression in craniofacial cartilages and premature bone mineralization. We conclude that in normal cartilage development, sox9 upregulates pthlh, which downregulates runx2, and that the duplicated nature of all three of these genes in zebrafish creates a network of regulation by different co-orthologs in different tissues.

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.

Transient downregulation of Bmp signalling induces extra limbs in vertebrates

Bone morphogenetic protein (Bmp) signalling has been implicated in setting up dorsoventral patterning of the vertebrate limb and in its outgrowth. Here, we present evidence that Bmp signalling or, more precisely, its inhibition also plays a role in limb and fin bud initiation. Temporary inhibition of Bmp signalling either by overexpression of noggin or using a synthetic Bmp inhibitor is sufficient to induce extra limbs in the Xenopus tadpole or exogenous fins in the Danio rerio embryo, respectively. We further show that Bmp signalling acts in parallel with retinoic acid signalling, possibly by inhibiting the known limb-inducing gene wnt2ba.

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.

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.

Functional Analysis of Conserved Non-Coding Regions Around the Short Stature hox Gene (shox) in Whole Zebrafish Embryos

BackgroundMutations in the SHOX gene are responsible for Leri-Weill Dyschondrosteosis, a disorder characterised by mesomelic limb shortening. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from Leri-Weill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox gene across the whole embryo and characterise the enhancer domains of different CNEs associated with this gene.Methodology/Principal FindingsExpression of the shox gene in zebrafish was identified using in situ hybridization, with embryos showing expression in the blood, putative heart, hatching gland, brain pharyngeal arch, olfactory epithelium, and fin bud apical ectodermal ridge. By identifying sequences showing 65% identity over at least 40 nucleotides between Fugu, human, dog and opossum we uncovered 35 CNEs around the shox gene. These CNEs were compared with CNEs previously discovered by Sabherwal et al., resulting in the identification of smaller more deeply conserved sub-sequence. Sabherwal et al.'s CNEs were assayed for regulatory function in whole zebrafish embryos resulting in the identification of additional tissues under the regulatory control of these CNEs.Conclusion/SignificanceOur results using whole zebrafish embryos have provided a more comprehensive picture of the expression pattern of the shox gene, and a better understanding of its regulation via deeply conserved noncoding elements. In particular, we identify additional tissues under the regulatory control of previously identified SHOX CNEs. We also demonstrate the importance of these CNEs in evolution by identifying duplicated shox CNEs and more deeply conserved sub-sequences within already identified CNEs.

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.