Specific expression in mouse mesoderm- and neural crest-derived tissues of a human PDGFRA promoter/lacZ transgene

The platelet-derived growth factor -receptor (PDGFR-) displays a lineage-specific expression pattern in the mouse embryo and is required for normal development of mesoderm and cephalic neural crest derivatives. The purpose of the present study was to demonstrate the in vivo promoter function of genomic DNA fragments representing the 5′-flanking part of the human PDGFRA gene. 2.2, 0.9 and 0.4 kb PDGFRA promoter fragments, ligated to a lacZ reporter gene, were microinjected into fertilized mouse eggs and transgenic mouse lines were established. The expression patterns were basically similar in the 2.2 and 0.9 kb lines and overlapped grossly the endogenous Pdgfra gene expression pattern. The transgenic line with the highest expression level was chosen for detailed analysis. Expression was, as expected, mainly confined to tissues of mesodermal and neural crest origin. No expression was found in epithelial tissues of endo- or ectodermal origin. The promoter fragments were also active in neuroepithelium and in certain neuronal cell types that did not faithfully express PDGFR- mRNA, while they failed to specify reporter expression in PDGFR- expressing O-2A progenitor cells and other glial elements of the central nervous system. Thus, the isolated human PDGFRA promoter contains most but not all of the regulatory elements that are necessary to establish tissue specific gene expression during development.     

Increased transgene expression by the mouse tyrosinase enhancer is restricted to neural crest-derived pigment cells

In this study, we have addressed the impact of the mouse tyrosinase enhancer on regulated expression from the mouse tyrosinase promoter during embryonic development. Stable and transient transgenic experiments using the reporter gene lacZ reveal that (1) expression is detected in neural crest-derived melanoblasts from E11.5 onward, (2) the enhancer does not increase transgenic expression in optic cup-derived pigment cells of the retinal pigment epithelium (RPE), and (3) expression in the telencephalon is not any longer detected. The importance of the enhancer for expression in pigment cells of the eye was further investigated in adult mice using an attenuated diphtheria toxin A gene. This demonstrated that in presence of the enhancer the transgene expression is specifically targeted to neural crest-derived melanocytes of the choroid and not, or slightly, to the RPE. This suggests that tyrosinase is differentially regulated in the two pigment cell lineages, and that this promoter can be used to target expression preferentially to the neural crest-derived melanocyte lineage.     

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.     

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest-derived melanophores

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest-derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell-type-specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin-synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.     

Neurofilament expression in vagal neural crest-derived precursors of enteric neurons

In order to gain insight into the potential role of the enteric microenvironment in the neuronal determination of the neural crest-derived precursor cells of enteric neurons, an attempt was made to ascertain when and where along the migratory route of these cells that they first express neuronal properties. The immunocytochemical detection of the 160-kDa component of the triplet of the chick neurofilament peptides served as a neuronal marker. In addition, neurogenic potential was assessed by growing explants of tissue suspected of containing presumptive neuroblasts in culture or as grafts on the chorioallantoic membrane of chick embryonic hosts. Neurofilament immunoreactivity was first detected in the foregut by Day 4 of development and spread to the hindgut by Day 7. Within the hindgut, development was more advanced within the colorectum than within the more proximal terminal ileum and caecal appendages. This probably reflects the distal-proximal migration of sacral neural crest cells in the postumbilical bowel. The ability of enteric explants to show neuronal development in vitro correlated with whether or not cells containing neurofilament immunoreactivity had reached that segment of gut at the age of explantation. These data suggest that enteric neuronal precursors have already begun to differentiate as neurons by the time they colonize the gut. Prior to the appearance of fibrillar neurofilament immunoreactivity in the foregut, cells that express this marker were found transiently within the mesenchyme of branchial arches 3, 4, and 5. These cells had disappeared from this region by developmental Day 6. The neurogenic potential of branchial arches 3 and 4 was demonstrated by the correlation that was found between the ability of explants of these arches to show neuronal development in vitro and the presence within them of cells that display neurofilament immunoreactivity. No similar neurogenic potential was found in the more rostral branchial arches which lacked the masses of neurofilament-immunoreactive cells. The location of the caudal branchial arches below the migrating vagal neural crest, the transience of the neurofilament immunoreactivity in them, and the coincident transience of their neurogenic potential in vitro, suggested that the masses of neurofilament immunoreactive cells in the caudal branchial arches might be vagal neural crest-derived neuronal precursor cells en route to the pharynx and the rest of the gut. This possibility was supported by the observation of neurofilament immunoreactivity in a subset of cells of the premigratory and early migratory neural crest in the vagal, but not other, regions of the neuraxis prior to the appearance of neurofilament immunoreactivity in the branchial arches. Proliferative expansion of cells with neurofilament immunoreactivity was indicated by the observation of mitotic figures in them. It is suggested that the vagal neural crest cells that populate the ENS are already committed to the neuronal lineage while still in the vagal region of the neuraxis. It is therefore not likely that the enteric microenvironment plays a role in this process.     

Germ cell specific promoter drives ectopic transgene expression during embryogenesis

In this study, we used the male germ cell-specific phosphoglycerate kinase 2 (Pgk2) promoter to generate Pgk2Cre transgenic mice to allow investigation of genes critically involved in meiosis. The Pgk2 promoter had been used previously to target transgene expression to spermatocytes and spermatids in several laboratories including ours. In several Cre targeting experiments using other promoters, ectopic Cre expression had been observed, but the timing and extent of this expression was not analyzed. We demonstrate that in adult mice the Pgk2Cre transgene is expressed specifically in spermatocytes and spermatids, as expected. However, in offspring from matings of Pgk2Cre mice and an H19loxP indicator strain, we discovered that recombination events had occurred in several, but not all, tissues to varying extents. The lacZ-loxP transgenic indicator strain was next used to uncover ectopic Cre expression even in single cells, which indicated that the Pgk2Cre transgene is expressed between days 11 and 15 during embryogenesis in several tissues and organs. Using an RT PCR assay we were unable to detect endogenous Pgk2 mRNA during embryogenesis or in adult tissues other than testis. In conclusion, the Pgk2 promoter is a valid choice for targeting gene expression to meiotic male germ cells, since transient ectopic expression is unlikely to have a discernable effect in most studies, but it may be inappropriate for utilization with Cre recombinase.     

Prenatal alcohol exposure triggers ceramide-induced apoptosis in neural crest-derived tissues concurrent with defective cranial development

Fetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy. The reason why specific embryonic tissues are sensitive toward ethanol is not understood. We found that in neural crest-derived cell (NCC) cultures from the first branchial arch of E10 mouse embryos, incubation with ethanol increases the number of apoptotic cells by fivefold. Apoptotic cells stain intensely for ceramide, suggesting that ceramide-induced apoptosis mediates ethanol damage to NCCs. Apoptosis is reduced by incubation with CDP-choline (citicoline), a precursor for the conversion of ceramide to sphingomyelin. Consistent with NCC cultures, ethanol intubation of pregnant mice results in ceramide elevation and increased apoptosis of NCCs in vivo. Ethanol also increases the protein level of prostate apoptosis response 4 (PAR-4), a sensitizer to ceramide-induced apoptosis. Prenatal ethanol exposure is concurrent with malformation of parietal bones in 20% of embryos at day E18. Meninges, a tissue complex derived from NCCs, is disrupted and generates reduced levels of TGF-β1, a growth factor critical for bone and brain development. Ethanol-induced apoptosis of NCCs leading to defects in the meninges may explain the simultaneous presence of cranial bone malformation and cognitive retardation in FAS. In addition, our data suggest that treatment with CDP-choline may alleviate the tissue damage caused by alcohol.     

Cytoplasmic β-actin promoter produces germ cell and preimplantation embryonic transgene expression

The cytoplasmic β-actin promoter, commonly used as strong promoter in many gene regulation studies, produces a pattern of male germ cell and preimplantation, embryonic gene expression in transgenic mice. In seven of ten expressing transgenic lines, a chicken β-actin-lacZ fusion gene was expressed in adult testes. In addition, five of the ten lines demonstrated transgene expression in the preimplantation mouse embryo. This is the first example of transgene expression at the stages of both gamete and early embryo. Overall, the site or transgene integration appeared to influence transgene expression in adult tissues. © 1993 Wiley-Liss, Inc.     

High expression of transgene protein in Spirodela

The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2 days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18 months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA–DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant.     

Rat synapsin 1 promoter mediated transgene expression in testicular cell types

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.     

Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.     

Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish

Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:cre(ERt2) transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish.     

Tubulin superfamily genes in Tribolium castaneum and the use of a Tubulin promoter to drive transgene expression

The use of native promoters to drive transgene expression has facilitated overexpression studies in Drosophila and other insects. We identified 12 Tubulin family members from the genome sequence of the red flour beetle, Tribolium castaneum, and used the promoter from one of these to drive constitutive expression of a transgene. The activity of the T. castaneum alpha-Tubulin1 (TcalphaTub1) putative promoter was pre-tested in conjunction with an eye-color gene, T. castaneum vermilion (Tcv), by transient expression in Tcv-deficient embryos. Such embryos showed complete rescue of larval eyespot pigmentation. We also examined the TcalphaTub1 expression pattern in germline transformants using the enhanced green fluorescent protein (EGFP) reporter. Beetles transformed with this piggyBac-based reporter ubiquitously expressed EGFP at all stages.     

A sterol biosynthetic geneAtCYP51A2 promoter for constitutive and ectopic expression of a transgene in plants

Arabidopsis CYP51A2 (AtCYP51A2) mediates the sterol 14α-demethylation step inde novo sterol biosynthesis, and is constitutively and highly expressed in all plant tissues (Kim et al., 2005). We exploited the molecular features of its expression and the fundamental role of sterol biosynthesis in cells to develop a plant-derived promoter. Our GUS expression analysis between transgenicArabidopsis lines forAtCYP51A2::GUS and35S::GUS revealed that activity of theAtCYP51A2 promoter was comparable to that of the35S promoter, based on enzymatic activities and protein levels. TheAtCYP51A2 promoter was also constitutively active in transgenic tobacco, indicating that 5′ regulatory elements could be conserved amongCYP51 promoters in dicot plants. A homologue ofAtCYP51A2 was identified from rape seed, a crop species closely related toArabidopsis. Its constitutive tissue expression pattern implies that the application of thisAtCYP51A2 promoter is possible for that species. Based on these results, we present a new binary vector system with the plant-derivedAtCYP51A2 promoter, which is able to constitutively and ectopically drive a transgene in various dicotyledonous plants. These two authors are equally contributed to this work.