Eye defects in receptor protein-tyrosine phosphatase alpha knock-down zebrafish.

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is highly expressed in the developing retina of different species, but little is known about its function there. Here, we report that injection of antisense morpholinos in zebrafish embryos reduced RPTP alpha expression to almost nondetectable levels up to 3 days postfertilization (dpf). RPTP alpha was detectable again from 4 dpf onward. RPTP alpha knock-down resulted in smaller eyes. Examination of sections of the retina at different developmental stages demonstrated that already at 28 hours postfertilization (hpf) fewer cells were present in the retina of RPTP alpha-morpholino-injected embryos. At 3 dpf, the layered organization of the retina was absent. In addition, the morphology and labeling with an axon specific antibody, acetylated tubulin, demonstrated that most cells appeared to be undifferentiated. Strikingly, at 5 dpf the lamination of the retina was partially restored, concomitant with re-expression of RPTP alpha protein. Although cells in the retina were now differentiated, the layering of the retina remained disrupted and significant gaps were observed in the amacrine cell layer. Therefore, knock-down of RPTP alpha protein provides evidence that RPTP alpha is essential for normal retinal development.     

Zebrafish tiggy-winkle hedgehog promoter directs notochord and floor plate green fluorescence protein expression in transgenic zebrafish embryos.

Zebrafish tiggy-winkle hedgehog (twhh) is a member of the hedgehog gene family that plays an important role in patterning brain, neural tube, somites, and eyes. To better understand the regulation of its tissue-specific expression, the activity of the twhh promoter was determined in zebrafish embryos by transient and transgenic expression analysis. Transient expression studies revealed that the 5.2-kb twhh promoter drove green fluorescence protein (GFP) expression in the notochord, floor plate, and branchial arches. Deletion analysis showed that distinct regions of the twhh promoter regulated the respective notochord or floor plate specific expression. To confirm the tissue specificity of the twhh promoter, transgenic zebrafish containing the twhh-GFP transgene were generated. GFP expression was analyzed in the F1, F2, and F3 generations of the transgenic embryos. The results confirmed the tissue-specific expression of the transgene in the notochord, floor plate, and branchial arches. In addition, GFP expression was also found in the pectoral fin buds, retina, and epithelial lining cells of the Kupffer's vesicle in the transgenic fish embryos. The expression pattern of the twhh-GFP transgene mimicked the expression of the endogenous twhh mRNAs in the floor plate, fin buds, branchial arches, retina, and epithelial lining cells of the Kupffer's vesicle. The expression in the notochord, however, did not mimic the pattern of the endogenous twhh expression. To determine whether no tail (ntl) or floating head (flh) mutants that have developmental defect in the notochord or the Kupffer's vesicle may affect the GFP expression in these regions, GFP expression was analyzed in ntl or flh transgenic embryos. No GFP expression could be detected in the midline region of the ntl transgenic embryos. However, in flh transgenic embryos, although GFP expression was affected in the midline region, its expression in the Kupffer's vesicle appeared normal. Together, these data indicated that the 5.2-kb twhh promoter contains regulatory elements for tissue-specific expression of twhh in the floor plate, pectoral fin bud, branchial arches, retina, and Kupffer's vesicle.     

Molecular characterization and embryonic expression of the family of N-methyl-D-aspartate receptor subunit genes in the zebrafish.

We present the cloning of 10 N-methyl-D-aspartate (NMDA) receptor subunits from the zebrafish. These subunits fall into five subtypes, each containing two paralogous genes. Thus, we report two NMDAR1 genes (NR1.1 and NR1.2), and eight NMDAR2 genes, designated NR2A.1 and NR2A.2, NR2B.1 and NR2B.2, NR2C.1 and NR2C.2, and NR2D.1 and NR2D.2. The predicted sequences of the NR1 paralogs display 90% identity to the human protein. The NR2 subunits show less identity, differing most at the N- and C-termini. The NR1 genes are both expressed embryonically, although in a nonidentical manner. NR1.1 is found in brain, retina, and spinal cord at 24 hours postfertilization (hpf). NR1.2 is expressed in the brain at 48 hpf but not in the spinal cord. NR2 developmental gene expression varies: both paralogs of the NR2A are expressed at 48 hpf in the retina, only one paralog of the NR2B is expressed at low levels in the heart at 48 hpf. Neither of the NR2C is expressed embryonically. Both paralogs of the NR2D are expressed: 2D.1 is in the forebrain, retina, and spinal cord at 24 hpf, whereas the 2D.2 is only found in the retina. Our findings demonstrate that the zebrafish can serve as a useful model system for investigating the role of NMDA receptors in the development of the nervous system.     

Transgene driving GFP expression from the promoter of the zona pellucida gene zpc is expressed in oocytes and provides an early marker for gonad differentiation in zebrafish.

Although mechanisms of sex differentiation have been studied intensely in mammals, insects, and worms, little is known about this process in lower vertebrates. To establish a marker for female gonad differentiation in zebrafish, we generated a transgenic line in which 412 bp from the promoter and 5' mRNA leader of the female-specific zebrafish zona pellucida gene zpc are fused to the coding region of green fluorescent protein (GFP). The zpc0.5:GFP transgene is expressed exclusively in oocytes, starting from the onset of female-specific differentiation, and closely resembles the expression pattern of the wild-type zpc. Strong GFP expression persists throughout oogenesis and is visible through the body wall of females. We have also characterized a putative upstream factor of zpc, FIGalpha, and show that distribution of FIGalpha RNA is compatible with its postulated role in the regulation of zpc. The zpc0.5:GFP transgenic line described here will be useful for studying oocyte development and the mechanisms that determine sex-specific gene expression in the zebrafish. It is also the first promoter characterized to date to drive stable and efficient expression specifically in the zebrafish female germline.     

Muscle-specific expression of the smyd1 gene is controlled by its 5.3-kb promoter and 5'-flanking sequence in zebrafish embryos.

Zebrafish SmyD1 is a SET and MYND domain-containing protein that plays an important role in myofiber maturation and muscle contraction. SmyD1 is required for myofibril organization and sarcomere assembly during myofiber maturation. Whole-mount in situ hybridization revealed that smyd1 mRNAs are specifically expressed in skeletal and cardiac muscles in zebrafish embryos. However, it is unknown if smyd1 is expressed in other striated muscles, such as cranial and fin muscles, and moreover, the regulatory elements required for its muscle-specific expression. We report here the analyses of smyd1 expression using smyd1-gfp transgenic zebrafish. smyd1-gfp transgenic zebrafish were generated using the 5.3-kb smyd1 promoter and its 5'-flanking sequence. GFP expression was found in the skeletal and cardiac muscles of smyd1-gfp transgenic embryos. GFP expression appeared stronger in slow muscles than fast muscles in transgenic zebrafish larvae. In addition, GFP expression was also detected in cranial and fin muscles of smyd1-gfp transgenic zebrafish larvae. In situ hybridization confirmed smyd1 mRNA expression in these tissues, suggesting that the expression of the smyd1-gfp transgene recapitulated that of the endogenous smyd1 gene. Deletion analysis revealed that the 0.5-kb sequence in the proximal promoter of smyd1 was essential for its muscle specificity. Together, these data indicate that smyd1 is specifically expressed in most, if not all, striated muscles, and the muscle specificity is controlled by the 5.3-kb promoter and flanking sequences.     

Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish.

Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.     

Faithful expression of living color reporter genes in transgenic medaka under two tissue-specific zebrafish promoters.

To test tissue specificity of zebrafish gene promoters in a heterologous fish species, two transgenic medaka lines under two zebrafish promoters were generated. Under the zebrafish skeletal muscle-specific mylz2 promoter, transgenic medaka expressed green fluorescent protein (GFP) exclusively in skeletal muscles, mimicking the endogenous medaka mylz2 mRNA expression and also identical to GFP expression in mylz2:gfp transgenic zebrafish. A madaka mylz2 promoter was also capable of directing skeletal muscle-specific GFP expression in transient transgenic zebrafish embryos. In the krt8:rfp transgenic medaka line with the zebrafish epithelial krt8 promoter, red fluorescent protein was specifically expressed in the skin epithelia as well as the epithelial lining cells of the anterior digestive tract, which was also identical to GFP expression in krt8:gfp transgenic zebrafish. Therefore, the two zebrafish promoters faithfully function in a heterologous fish species, and it is likely that the mechanisms of tissue-specific expression are largely conserved among fish species.     

Characterization of green fluorescent protein–expressing retinal cone bipolar cells in a 5-hydroxytryptamine receptor 2a transgenic mouse line

Retinal bipolar cells relay visual information from photoreceptors to third-order retinal neurons. Bipolar cells, comprising multiple types, play an essential role in segregating visual information into multiple parallel pathways in the retina. The identification of molecular markers that can label specific retinal bipolar cells could facilitate the investigation of bipolar cell functions in the retina. Transgenic mice with specific cell type(s) labeled with green fluorescent protein (GFP) have become a powerful tool for morphological and functional studies of neurons in the CNS, including the retina. In this study, we report a 5-hydroxytryptamine receptor 2a (5-HTR2a) transgenic mouse line in which expression of GFP was observed in two populations of bipolar cells in the retina. Based on the terminal stratification and immunostaining, all the strongly GFP-labeled bipolar cells were found to be type 4 cone bipolar cells. A small population of weakly labeled bipolar cells was also observed, which may represent type 8 or 9 cone bipolar cells. GFP expression in retinal cone bipolar cells was seen as early as postnatal day 5. In addition, despite severe retinal degeneration due to the presence of the rd1 mutation in this transgenic line, the density of GFP-labeled cone bipolar cells remained stable up to at least 6 months of age. This transgenic mouse line will be a useful tool for the study of type 4 cone bipolar cells in the retina under both normal and disease conditions.     

Defining the integration capacity of embryonic stem cell-derived photoreceptor precursors

Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), potentially provide a renewable source of cells for retinal transplantation. Previously, we have shown both the functional integration of transplanted rod photoreceptor precursors, isolated from the postnatal retina, in the adult murine retina, and photoreceptor cell generation by stepwise treatment of ESCs with defined factors. In this study, we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ESC-derived retinal cells following transplantation using our established procedures. Optimized retinal differentiation via isolation of Rax.GFP retinal progenitors recreated a retinal niche and increased the yield of Crx(+) and Rhodopsin(+) photoreceptors. Rod birth peaked at day 20 of culture and expression of the early photoreceptor markers Crx and Nrl increased until day 28. Nrl levels were low in ESC-derived populations compared with developing retinae. Transplantation of early stage retinal cultures produced large tumors, which were avoided by prolonged retinal differentiation (up to day 28) prior to transplantation. Integrated mature photoreceptors were not observed in the adult retina, even when more than 60% of transplanted ESC-derived cells expressed Crx. We conclude that exclusion of proliferative cells from ESC-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ESC-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina.