The expression of gbx-2 during zebrafish embryogenesis

Gbx-2 is a homeobox gene essential for normal development of the midbrain and the anterior hindbrain. Zebrafish gbx-2 shares an overall similarity of 67.8, 68.1, 60.6 and 66.5% in amino acid sequence to human, mouse, chick and Xenopus Gbx-2, respectively. The expression of zebrafish gbx-2 is initially, before the completion of epiboly, restricted to the prospective posterior midbrain. The expression remains detectable until the end of pharyngula period. The gbx-2 mRNA is also detected in the otic vesicles, the dorsoposterior telencephalon, the rostral branchial arches, the pronephric duct and median fin fold.     

Heat shock gene expression and function during zebrafish embryogenesis

Recent work in the zebrafish, Danio rerio, indicates that heat shock genes are expressed in unique spatial patterns under non-stress conditions. In particular, hsp90alpha is expressed during the normal differentiation of striated muscle fibres, and hsp70-4 is expressed during normal lens development in the eye. Furthermore, disruption of the activity of either of these genes or their protein products gives rise to unique embryonic phenotypes that result from failures in proper somitic muscle development and lens development, respectively. Embryonic hsp70-4 expression is also activated in a cell-specific manner following heavy metal exposure. This has allowed for the development of a hsp70-4/eGFP reporter gene system in stable transgenic zebrafish that serves as a reliable yet extremely quick indicator of cell-specific toxicity in the context of the multicellular, living embryo.     

Developmental expression of zebrafish emx1 during early embryogenesis

Emx family homeobox genes, Emx1 and Emx2, play an essential role in rostral brain development in mammalian embryos. Here we report a zebrafish emx family gene, emx1, which is more similar to the mouse Emx1 gene than the previously reported zebrafish emx1 gene; we propose to rename that gene emx3. The expression of emx1 is first detected around the 10-somite stage in the pineal gland (epiphysis) primodium in the developing anterior brain and in the pronephric primodium within the intermediate mesoderm. emx1 expression in the epiphysis has not been reported in other species. Expression in the epiphysis is suppressed at 23 h post-fertilization (hpf) in the floating head (flh) mutant, in which development of the epiphysis is impaired. Subsequently, emx1 is expressed in the telencephalon, as reported in mammals, and can be detected in the olfactory placode and in a small group of cells in the forebrain at 25 hpf. In the mesoderm, emx1 expression is gradually concentrated in the posterior pronephric duct during somitogenesis, and becomes expressed predominantly in the urogenital opening at 25 hpf. Thus, emx1 displays a unique expression pattern that is distinct from the patterns of emx2 and emx3.     

Spatially and temporally-restricted expression of two T-box genes during zebrafish embryogenesis

T-box genes are conserved in all animal species. We have identified two members of the T-box gene family from the zebrafish, Danio rerio. Zf-tbr1 and zf-tbx3 share high amino acid identity with human, murine, chick and Xenopus orthologs and are expressed in specific regions during zebrafish development.     

Expression of Frizzled10 in mouse central nervous system

Frizzled transmembrane proteins (Fzd) are receptors of Wnts, and they play key roles during central nervous system (CNS) development in vertebrates. Here we report the expression pattern of Frizzled10 in mouse CNS from embryonic stages to adulthood. Frizzled10 is expressed strongly at embryonic days E8.5 and E9.5 in the neural tube and tail bud. At E10.5, Frizzled10 is expressed in the forebrain vesicle, the fourth ventricle and the dorsal spinal cord. From E12.5 to E16.5, Frizzled10 expression is mainly observed in the cortical hem/fimbria, the neuroepithelium of the third ventricular zone, midbrain, developing cerebellum, and dorsal spinal cord. At P0, with the exception of expression in the fimbria, Frizzled10 mRNA expression is limited to specific nuclei including the ventral posterior thalamic nucleus (VP) and the dorsal lateral geniculate nucleus (DLG) in the developing thalamus as well as in the proliferative ventricular zone of the developing cerebellum. From P20 to adult, Frizzled10 mRNA is detected only in the internal capsule (ic). Our data show that expression of Frizzled10 is very strong during embryonic development of the CNS and suggest that Frizzled10 may play an essential role in spatial and temporal regulation during neural development.     

Nephrotoxicity assessments of acetaminophen during zebrafish embryogenesis

We used a green fluorescent kidney line, Tg(wt1b:GFP), as a model to access the acetaminophen (AAP)-induced nephrotoxicity dynamically. Zebrafish (Danio rerio) embryos at different developmental stages (12–60 hpf) were treated with different dosages of AAP (0–45 mM) for different time courses (12–60 h). Results showed that zebrafish embryos exhibited no evident differences in survival rates and morphological changes between the mock-treated control (0 mM) and 2.25 mM AAP-exposure (12–72 hpf) groups. In contrast, after higher doses (22.5 and 45 mM) of exposure, embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tube, pronephric duct, and a cystic and atrophic glomerulus. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AAP increased. Interestingly, under the same exposure time course (12 h) and dose (22.5 mM), embryos displayed higher percentages of severe defects at earlier developmental stage of exposure (12–24 hpf), whereas embryos displayed higher percentages of mild defects at later exposure (60–72 hpf). With an exposure time course less than 24 h of 45 mM AAP, no embryo survived by the developmental stage of 72 hpf. These results indicated that AAP-induced nephrotoxicity depended on the exposure dose, time course and developmental stages. Immunohistochemical experiments showed that the cells' morphologies of the pronephric tube, pronephric duct and glomerulus were disrupted by AAP, and consequently caused cell death. Real-time RT-PCR revealed embryos after AAP treatment decreased the expression of cox2 and bcl2, but increased p53 expression. In conclusion, AAP-induced defects on glomerulus, pronephric tube and pronephric duct could be easily and dynamically observed in vivo during kidney development in this present model.     

Cytoplasmic expression of ribozyme in zebrafish using a T7 autogene system

A cytoplasmic ribozyme expression system, based on codelivery of a ribozyme vector, a T7 autogene vector, and T7 RNA polymerase (RNAP), has been developed and used to generate a specific phenotype in zebrafish by targeting a no tail (ntl) mRNA. The expression of the no tail ribozyme sequence is under the control of a tandem of two promoters: The T7 promoter and an adenoviral va 1 (pol III) promoter. The coinjection of the ribozyme vector pT7vaRz, the T7 autogene vector pT7T7, and the T7 RNAP resulted in rapid synthesis of the ribozyme against the ntl mRNA in the cytoplasm of the injected zebrafish embryos, generating no tail phenotypes in up to 10-20% of the injected embryos. The phenotypic change rates have been found to be related to the concentrations of the plasmid vectors and T7 RNAP injected and to the ratios of the three injected components. This cytoplasmic ribozyme expression system may be useful for efficiently targeting other mRNA and for various biomedical applications. These potential applications may include rapid identification of biological functions of novel genes from zebrafish and humans based on partial gene sequence information and gene therapy of genetic and acquired diseases.     

Neuron navigator 3a regulates liver organogenesis during zebrafish embryogenesis

Endodermal organogenesis requires a precise orchestration of cell fate specification and cell movements, collectively coordinating organ size and shape. In Caenorhabditis elegans, uncoordinated-53 (unc-53) encodes a neural guidance molecule that directs axonal growth. One of the vertebrate homologs of unc-53 is neuron navigator 3 (Nav3). Here, we identified a novel vertebrate neuron navigator 3 isoform in zebrafish, nav3a, and we provide genetic evidence in loss- and gain-of-function experiments showing its functional role in endodermal organogenesis during zebrafish embryogenesis. In zebrafish embryos, nav3a expression was initiated at 22 hpf in the gut endoderm and at 40 hpf expanded to the newly formed liver bud. Endodermal nav3a expression was controlled by Wnt2bb signaling and was independent of FGF and BMP signaling. Morpholino-mediated knockdown of nav3a resulted in a significantly reduced liver size, and impaired development of pancreas and swim bladder. In vivo time-lapse imaging of liver development in nav3a morphants revealed a failure of hepatoblast movement out from the gut endoderm during the liver budding stage, with hepatoblasts being retained in the intestinal endoderm. In hepatocytes in vitro, nav3a acts as a positive modulator of actin assembly in lamellipodia and filipodia extensions, allowing cellular movement. Knockdown of nav3a in vitro impeded hepatocyte movement. Endodermal-specific overexpression of nav3a in vivo resulted in additional ectopic endodermal budding beyond the normal liver and pancreatic budding sites. We conclude that nav3a is required for directing endodermal organogenesis involving coordination of endodermal cell behavior.     

Expression of zebrafish btg-b, an anti-proliferative cofactor, during early embryogenesis

BTG/tob family proteins are thought to be a potential tumor suppressor due to their anti-proliferative activity. We cloned zebrafish btg-b, a member of the BTG1/2 subfamily, using in situ hybridization screening. The tissue-specific expression of btg-b is first observed in the organizer region at the early gastrula stage. Later in development, the forebrain, the hindbrain, the polster and the paraxial mesoderm transiently express btg-b. Recently, mouse Btg1 and Btg2 have been shown to be a cofactor for Hoxb9. Double in situ hybridization with zebrafish btg-b and hoxb9a indicates that the expression domains of these two genes overlap in the posterior paraxial mesoderm.