Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers.

The swimbladder is a hydrostatic organ in fish postulated as a homolog of the tetrapod lung. While lung development has been well studied, the molecular mechanism of swimbladder development is essentially uncharacterized. In the present study, swimbladder development in zebrafish was analyzed by using several molecular markers: hb9 (epithelium), fgf10a and acta2 (mesenchyme), and anxa5 (mesothelium), as well as in vivo through enhancer trap transgenic lines Et(krt4:EGFP)(sq33-2) and Et(krt4:EGFP)(sqet3) that showed strong EGFP expression in the swimbladder epithelium and outer mesothelium respectively. We defined three phases of swimbladder development: epithelial budding between 36 and 48 hpf, growth with the formation of two additional mesodermal layers up to 4.5 dpf, and inflation of posterior and anterior chambers at 4.5 and 21 dpf respectively. Similar to those in early lung development, conserved expression of Hedgehog (Hh) genes, shha and ihha, in the epithelia, and Hh receptor genes, ptc1 and ptc2, as well as fgf10a in mesenchyme was observed. By analyzing several mutants affecting Hh signaling and Ihha morphants, we demonstrated an essential role of Hh signaling in swimbladder development. Furthermore, time-specific Hh inhibition by cyclopamine revealed different requirements of Hh signaling in the formation and organization of all three tissue layers of swimbladder.

The role of vasculature and blood circulation in zebrafish swimbladder development.

BACKGROUND: Recently we have performed a detailed analysis of early development of zebrafish swimbladder, a homologous organ of tetrapod lung; however, the events of swimbladder development are still poorly characterized. Many studies have implicated the role of vascular system in development of many organs in vertebrates. As the swimbladder is lined with an intricate network of blood capillaries, it is of interest to investigate the role of the vascular system during early development of swimbladder. RESULTS: To investigate the role of endothelial cells (ECs) and blood circulation during development of the swimbladder, phenotypes of swimbladder were analysed at three different stages (approximately 2, 3 and 5 dpf [day postfertilization]) in cloche (clo) mutant and Tnnt2 morphants, in the background of transgenic lines Et(krt4:EGFP)sq33-2 and Et(krt4:EGFP)sqet3 which express EGFP in the swimbladder epithelium and outer mesothelium respectively. Analyses of the three tissue layers of the swimbladder were performed using molecular markers hb9, fgf10a, acta2, and anxa5 to distinguish epithelium, mesenchyme, and outer mesothelium. We showed that the budding stage was independent of ECs and blood flow, while early epithelial growth, mesenchymal organization and its differentiation into smooth muscle, as well as outer mesothelial organization, were dependent on ECs. Blood circulation contributed to later stage of epithelial growth, smooth muscle differentiation, and organization of the outer mesothelium. Inflation of the swimbladder was also affected as a result of absence of ECs and blood flow. CONCLUSION: Our data demonstrated that the vascular system, though not essential in swimbladder budding, plays an important role in the development of the swimbladder starting from the early growth stage, including mesenchyme organization and smooth muscle differentiation, and outer mesothelial organization, which in turn may be essential for the function of the swimbladder as reflected in its eventual inflation.

Mercury-induced hepatotoxicity in zebrafish: in vivo mechanistic insights from transcriptome analysis, phenotype anchoring and targeted gene expression validation.

BACKGROUND: Mercury is a prominent environmental contaminant that causes detrimental effects to human health. Although the liver has been known to be a main target organ, there is limited information on in vivo molecular mechanism of mercury-induced toxicity in the liver. By using transcriptome analysis, phenotypic anchoring and validation of targeted gene expression in zebrafish, mercury-induced hepatotoxicity was investigated and a number of perturbed cellular processes were identified and compared with those captured in the in vitro human cell line studies. RESULTS: Hepato-transcriptome analysis of mercury-exposed zebrafish revealed that the earliest deregulated genes were associated with electron transport chain, mitochondrial fatty acid beta-oxidation, nuclear receptor signaling and apoptotic pathway, followed by complement system and proteasome pathway, and thereafter DNA damage, hypoxia, Wnt signaling, fatty acid synthesis, gluconeogenesis, cell cycle and motility. Comparative meta-analysis of microarray data between zebrafish liver and human HepG2 cells exposed to mercury identified some common toxicological effects of mercury-induced hepatotoxicity in both models. Histological analyses of liver from mercury-exposed fish revealed morphological changes of liver parenchyma, decreased nucleated cell count, increased lipid vesicles, glycogen and apoptotic bodies, thus providing phenotypic evidence for anchoring of the transcriptome analysis. Validation of targeted gene expression confirmed deregulated gene-pathways from enrichment analysis. Some of these genes responding to low concentrations of mercury may serve as toxicogenomic-based markers for detection and health risk assessment of environmental mercury contaminations. CONCLUSION: Mercury-induced hepatotoxicity was triggered by oxidative stresses, intrinsic apoptotic pathway, deregulation of nuclear receptor and kinase activities including Gsk3 that deregulates Wnt signaling pathway, gluconeogenesis, and adipogenesis, leading to mitochondrial dysfunction, endocrine disruption and metabolic disorders. This study provides important mechanistic insights into mercury-induced liver toxicity in a whole-animal physiology context, which will help in understanding the syndromes caused by mercury poisoning. The molecular conservation of mercury-induced hepatotoxicity between zebrafish and human cell line reveals the feasibility of using zebrafish to model molecular toxicity in human for toxicant risk assessments.

Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish.

BACKGROUND: Early events in vertebrate liver development have been the major focus in previous studies, however, late events of liver organogenesis remain poorly understood. Liver vasculogenesis in vertebrates occurs through the interaction of endoderm-derived liver epithelium and mesoderm-derived endothelial cells (ECs). In zebrafish, although it has been found that ECs are not required for liver budding, how and when the spatio-temporal pattern of liver growth is coordinated with ECs remains to be elucidated. RESULTS: To study the process of liver development and vasculogenesis in vivo, a two-color transgenic zebrafish line Tg(lfabf:dsRed; elaA:EGFP) was generated and named LiPan for liver-specific expression of DsRed RFP and exocrine pancreas-specific expression of GFP. Using the LiPan line, we first followed the dynamic development of liver from live embryos to adult and showed the formation of three distinct yet connected liver lobes during development. The LiPan line was then crossed with Tg(fli1:EGFP)y1 and vascular development in the liver was traced in vivo. Liver vasculogenesis started at 55-58 hpf when ECs first surrounded hepatocytes from the liver bud surface and then invaded the liver to form sinusoids and later the vascular network. Using a novel non-invasive and label-free fluorescence correction spectroscopy, we detected blood circulation in the liver starting at approximately 72 hpf. To analyze the roles of ECs and blood circulation in liver development, both cloche mutants (lacking ECs) and Tnnt2 morphants (no blood circulation) were employed. We found that until 70 hpf liver growth and morphogenesis depended on ECs and nascent sinusoids. After 72 hpf, a functional sinusoidal network was essential for continued liver growth. An absence of blood circulation in Tnnt2 morphants caused defects in liver vasculature and small liver. CONCLUSION: There are two phases of liver development in zebrafish, budding and growth. In the growth phase, there are three distinct stages: avascular growth between 50-55 hpf, where ECs are not required; endothelium-dependent growth, where ECs or sinusoids are required for liver growth between 55-72 hpf before blood circulation in liver sinusoids; and circulation-dependent growth, where the circulation is essential to maintain vascular network and to support continued liver growth after 72 hpf.

Transcriptome kinetics of arsenic-induced adaptive response in zebrafish liver.

Arsenic is a prominent environmental toxicant and carcinogen; however, its molecular mechanism of toxicity and carcinogenicity remains poorly understood. In this study, we performed microarray-based expression profiling on liver of zebrafish exposed to 15 parts/million (ppm) arsenic [As(V)] for 8-96 h to identify global transcriptional changes and biological networks involved in arsenic-induced adaptive responses in vivo. We found that there was an increase of transcriptional activity associated with metabolism, especially for biosyntheses, membrane transporter activities, cytoplasm, and endoplasmic reticulum in the 96 h of arsenic treatment, while transcriptional programs for proteins in catabolism, energy derivation, and stress response remained active throughout the arsenic treatment. Many differentially expressed genes encoding proteins involved in heat shock proteins, DNA damage/repair, antioxidant activity, hypoxia induction, iron homeostasis, arsenic metabolism, and ubiquitin-dependent protein degradation were identified, suggesting strongly that DNA and protein damage as a result of arsenic metabolism and oxidative stress caused major cellular injury. These findings were comparable with those reported in mammalian systems, suggesting that the zebrafish liver coupled with the available microarray technology present an excellent in vivo toxicogenomic model for investigating arsenic toxicity. We proposed an in vivo, acute arsenic-induced adaptive response model of the zebrafish liver illustrating the relevance of many transcriptional activities that provide both global and specific information of a coordinated adaptive response to arsenic in the liver.

Fish and Chips: a microfluidic perfusion platform for monitoring zebrafish development.

We have developed a multi-channel microfluidic perfusion platform for culturing zebrafish embryos and capturing live images of various tissues and organs inside the embryo. The Fish and Chips was micro-fabricated in silicon and glass for reproducibility and accuracy of the microfluidic structure. The microfluidic platform consists of three parts: a microfluidic gradient generator, a row of eight fish tanks, in which the fish embryos are individually placed, and eight output channels. The fluidic gradient generator supports dose-dependent drug and chemical studies. A unique perfusion system ensures a uniform and constant flow of media to the fish tank while the wastes are efficiently removed. The fish tanks restrict the embryo movements, except rotationally, for live imaging of internal tissues and organs. The embryos showed developmental abnormalities under the influence of the drug valproic acid (VPA).

Depletion of Bhmt elevates sonic hedgehog transcript level and increases ß-cell number in zebrafish.

Betaine homocysteine S-methyltransferase (BHMT, EC 2.1.1.5) is a key enzyme in the methionine cycle and is highly expressed in the liver. Despite its important biochemical function, it is not known whether BHMT plays a role during organ development. In this report, we showed that early in development of zebrafish before endoderm organogenesis, bhmt is first expressed in the yolk syncytial layer and then after liver formation becomes a liver-enriched gene. By using the anti-bhmt morpholinos that deplete the Bhmt, we found that in morphant embryos, several endoderm-derived organs, including liver, exocrine pancreas, and intestine are hypoplastic. Strikingly, the number of ß-cells in the pancreatic islet was increased rather than reduced in the morphant. Additional studies showed that Bhmt depletion elevates the sonic hedgehog (shh) transcript level in the morphant, whereas Bhmt-depletion in the Shh-deficient mutant syu failed to rescue the isletless phenotype. These molecular and genetic data strongly suggest that Shh functions downstream of Bhmt to promote ß-cell development. Therefore, although there are still many intriguing questions to be answered, our finding may identify a novel function for Bhmt involving modulation of Shh signaling to control ß-cell development.

Zebrafish cdx1b regulates expression of downstream factors of Nodal signaling during early endoderm formation.

We identified a zebrafish caudal-related homeobox (cdx1b) gene, which shares syntenic conservation with both human and mouse Cdx1. Zebrafish cdx1b transcripts are maternally deposited. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to the 1-2s stages and can be identified in the retinas, brain and somites during 18-22 hpf stages. After 28 hours of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino oligonucleotide-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function. Hypoplastic development of the liver and pancreas and intestinal abnormalities were observed in 96 hpf cdx1b morphants. In 85% epiboly cdx1b morphants, twofold decreases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1-binding motifs were identified in both gata5 and foxa2 genes by interspecific sequence comparisons. Cdx1b can bind to the Cdx1-binding motif located in intron 1 of the foxa2 gene based on an electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with the cdx1b MO rescued the expression domains of ceruloplasmin in the liver of 53 hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression, thus affecting early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs compared with its mammalian homologs.