Zhiyuan Gong

Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.

The development and maturation of the immune system in zebrafish was investigated using immune-related gene expression profiling by quantitative real-time polymerase chain reaction, in situ hybridization (ISH), immunoglobulin (Ig) detection by immuno-affinity purification and Western blotting as well as immersion immunization experiments. Ikaros expression was first detected at 1 day post-fertilization (dpf) and thereafter increased gradually to more than two-fold between 28 and 42dpf before decreasing to less than the initial 1dpf expression level in adult fish (aged 105dpf). Recombination activating gene-1 (Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17dpf, reaching a maximum between 21 and 28dpf before decreasing gradually. However, in adult fish aged 105dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3dpf. T-cell receptor alpha constant region and immunoglobulin light chain constant region (IgLC) isotype-1, 2 and 3 mRNAs were detected at low levels by 3dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-section ISH, Rag-1 expression was detected in head kidney by 2wpf while IgLC-1, 2 and 3 were detected in the head kidney and the thymus by 3wpf onwards. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4wpf. Humoral response to T-independent antigen (formalin-killed Aeromonas hydrophila) and T-dependent antigen (human gamma globulin) was observed in zebrafish immunized at 4 and 6wpf, respectively, indicating that immunocompetence was achieved. The findings reveal that the zebrafish immune system is morphologically and functionally mature by 4-6wpf.

Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation.

Two zebrafish bHLH genes, neurogenin‐related gene I (ngn1) and neuroD (nrd), have been isolated. ngn1expression is initiated at the end of gastrulation in the neural plate and defines broad domains of cells that probably possess an ability to develop as neurons. This finding suggests that ngn1 may play a role during determination of cell fate in neuroblasts. ngn1 and pax‐b are expressed in a mutually exclusive manner. nrd expression follows that of ngn1 in restricted populations of cells selected from ngn1‐positive clusters of cells. The earliest nrd‐positive cells in the brain and the trunk are a subset of the primary neurons. ngn1 is not expressed in the eye. Here, nrd transcription is activated at 25 hours postfertilization in the ventral retina. Expression of islet‐1 occurs in nrd‐positive cells after expression of nrd, and the expression of the two genes partially overlaps in time. These observations suggest that during eye development nrd expression may follow expression of some other neurodetermination gene(s). This supports the idea that expression of nrd is a necessary step leading toward overt neuronal differentiation. Dev. Dyn. 1998;213:92–104.

Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression.

The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.

Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition

Maternally deposited mRNAs direct early development before the initiation of zygotic transcription during mid-blastula transition (MBT). To study mechanisms regulating this developmental event in zebrafish, we applied mRNA deep sequencing technology and generated comprehensive information and valuable resources on transcriptome dynamics during early embryonic (egg to early gastrulation) stages. Genome-wide transcriptome analysis documented at least 8000 maternal genes and identified the earliest cohort of zygotic transcripts. We determined expression levels of maternal and zygotic transcripts with the highest resolution possible using mRNA-seq and clustered them based on their expression pattern. We unravel delayed polyadenylation in a large cohort of maternal transcripts prior to the MBT for the first time in zebrafish. Blocking polyadenylation of these transcripts confirms their role in regulating development from the MBT onward. Our study also identified a large number of novel transcribed regions in annotated and unannotated regions of the genome, which will facilitate reannotation of the zebrafish genome. We also identified splice variants with an estimated frequency of 50%-60%. Taken together, our data constitute a useful genomic information and valuable transcriptome resource for gene discovery and for understanding the mechanisms of early embryogenesis in zebrafish.

Fgf10 regulates hepatopancreatic ductal system patterning and differentiation

During organogenesis, the foregut endoderm gives rise to the many different cell types that comprise the hepatopancreatic system, including hepatic, pancreatic and gallbladder cells, as well as the epithelial cells of the hepatopancreatic ductal system that connects these organs together and with the intestine. However, the mechanisms responsible for demarcating ducts versus organs are poorly understood. Here, we show that Fgf10 signaling from the adjacent mesenchyme is responsible for refining the boundaries between the hepatopancreatic duct and organs. In zebrafish fgf10 mutants, the hepatopancreatic ductal epithelium is severely dysmorphic, and cells of the hepatopancreatic ductal system and adjacent intestine misdifferentiate toward hepatic and pancreatic fates. Furthermore, Fgf10 also functions to prevent the differentiation of the proximal pancreas and liver into hepatic and pancreatic cells, respectively. These data shed light onto how the multipotent cells of the foregut endoderm, and subsequently those of the hepatopancreatic duct, are directed toward different organ fates.

Asynchronous activation of 10 muscle-specific protein (MSP) genes during zebrafish somitogenesis

In the present study, 10 zebrafish cDNA clones coding for muscle‐specific proteins (MSPs) were characterized and most of them encode fast skeletal muscle isoforms. They are skeletal muscle α‐actin (acta1), fast skeletal muscle a‐tropomyosin (tpma), fast skeletal muscle troponin C (tnnc), fast skeletal muscle troponin T (tnnt), fast skeletal muscle myosin heavy chain (myhz1), fast skeletal muscle myosin light chain 2 (mylz2), fast skeletal muscle myosin light chain 3 (mylz3), muscle creatine kinase (ckm), parvalbumin (pvalb), and desmin (desm). Using these cDNA probes, their expression patterns in developing embryos and adults were compared by Northern blot hybridization and whole‐mount in situ hybridization. All of the 10 genes are expressed in both embryos and adult fish, and the expression is highly abundant in skeletal muscle. Among them, acta1, tpma, tnnc, tnnt, myhz1, mylz2, mylz3 and pvalb, are expressed specifically in fast skeletal muscle while ckm and desm are expressed in both fast and slow skeletal muscles. In addition, tpma, ckm, and desm are also expressed in the heart. Ontogenetically, the onset of expression of these MSP genes in zebrafish skeletal muscle varies and the expression occurs rostral‐caudally in developing somites. Shortly after the expression of myoD, desm is the first to be activated at ∼9 hpf, followed by tpma (∼10 hpf), tnnc (∼12 hpf), acta1 (∼12 hpf), ckm (∼14 hpf), myhz1 (∼14 hpf), mylz2 (∼16 hpf), mylz3 (∼16.5 hpf), tnnt (∼16.5 hpf), and pvalb (∼16.5 hpf). At later stages (after 48 hpf), these MSP genes are also expressed in fin buds and head muscles including eye, jaw, and gill muscles. Thus, our experiment demonstrated the order of expression of the 10 MSP genes, which may reflect the sequence of muscle filament assembly. In spite of the asynchrony in activation of these MSP genes, the timing of expression for each individual MSP gene appears to be synchronous to somite development as each somite has an identical timetable to express the set of MSP genes.

Rapid identification and isolation of zebrafish cDNA clones

A fast and economical approach, referred to as cDNA clone tagging, was adapted to identify and isolate zebrafish cDNA clones. The basic approach was to partially sequence the coding region of size selected cDNA clones and the partial sequences were then used as tags for identifying the clones through homology search. To benefit maximally from the tagging approach, two cDNA libraries, derived from embryonic and adult fish poly(A)+ RNAs, respectively, were constructed by unidirectional cloning; conceptually, they have the potential to represent all expressed zebrafish genes. A total of 1084 clones were sequenced from the two libraries, and 511 clones were identified, based on sequence homology. These identified clones were derived from at least 261 genes, encoding 48 translational machinery proteins, 47 cytosolic proteins, 43 cytoskeletal proteins, 41 nuclear proteins, 32 membrane proteins, 22 secreted proteins, 20 mitochondrial proteins and 8 proteins with an unknown location. Of the 261 distinct cDNA clones identified, 254 were isolated for the first time in the zebrafish. These tagged cDNA clones, identified and unidentified, provide rich resources for developmental analysis as well as mapping of zebrafish genome. The long-term objective of this study is to establish a tagged zebrafish gene library that can be accessed both by hybridization screening against the plasmid DNAs and by electronic screening using the sequence information.

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.

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

BackgroundEarly 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.ResultsTo 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 ~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.ConclusionThere 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.

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

BackgroundMercury 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.ResultsHepato-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.ConclusionMercury-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.