Hirsch N et al. (2002), Xenopus tropicalis transgenic lines and their u...

XB-ART-6122

Dev Dyn 2002 Dec 01;2254:522-35. doi: 10.1002/dvdy.10188.

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Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.

Hirsch N , Zimmerman LB , Gray J , Chae J , Curran KL , Fisher M , Ogino H , Grainger RM .

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For over a century, amphibian embryos have been a source of significant insight into developmental mechanisms, including fundamental discoveries about the process of induction. The recently developed transgenesis for Xenopus offers new approaches to these poorly understood processes, particularly when undertaken in the quickly maturing species Xenopus tropicalis, which greatly facilitates establishment of permanent transgenic lines. Several X. tropicalis transgenic lines have now been generated, and experiments demonstrating the value of these lines to study induction in embryonic tissue recombinants and explants are presented here. A revised protocol for transgenesis in X. tropicalis resulting in a significant increase in the percentage of transgenic animals that reach adulthood is presented, as well as improvements in tadpole and froglet husbandry, which have facilitated the raising of large numbers of adults. Working transgenic populations have been rapidly expanded, and some transgenes have been bred to homozygosity. Established lines include those bearing the promoter regions of Pax-6, Otx-2, Rx, and EF1alpha coupled to fluorescent reporter genes. Multireporter lines combining, in a single animal, up to three gene promoters coupled to different fluorescent reporters have also been established. The value of X. tropicalis transgenic lines for the study of induction is demonstrated by showing activation of Pax-6 by noggin treatment of Pax-6/GFP transgenic animal caps, illustrating how reporter lines allow a rapid, in vivo assay for an inductive response. An experiment showing lens induction in gamma-crystallin/GFP transgenic lens ectoderm when it is recombined with mouse optic vesicle demonstrates conservation of inducing signals from amphibians and mammals. It also shows how the warmer culture temperatures tolerated by X. tropicalis embryos can be used in assays of factors produced by mammalian cells and tissues. The many applications of transgenic reporter lines and other lines designed to target gene expression in particular tissues promise to bring significant new insights to the classic issues first defined in amphibian systems.

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Species referenced: Xenopus tropicalis
Genes referenced: eef1a1 nog otx2 pax6

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	Figure 1. Karyotyping Xenopus tropicalis and Xenopus laevis tadpoles and the effects of vector sequences on transgene expression. A: A typical diploid karyotype from an X. tropicalis tadpole. Note the small, submetacentric pair of chromosomes (arrows) that is characteristic of X. tropicalis. B: A typical karyotype from X. laevis, provided for comparison. C: Stage 32 embryos transgenic for a Rx/green fluorescent protein (GFP) construct including the pEGFP-1 plasmid vector sequence. Normal expression is detected in the eyes (arrow), but ectopic expression in the axial muscle (arrowheads) is quite strong. D: Stage 32 embryos transgenic for Rx/GFP using the Rx promoter/GFP/polyA cassette only. Note that normal Rx expression is unaffected (arrows), but the ectopic expression is eliminated.
	Figure 2. Improvements in the transgenesis technique enhance long-term survival of transgenic embryos. A–D: Phenotypes of nuclear-injected embryos that are selected at the four-cell stage, assayed after an overnight incubation at 22°C. Examples are shown: A, dead embryo; B, exogastrulae; C, “imperfect” (arrows show yolky extrusions); and D, “perfect” embryos. E: A field of X. tropicalis sperm nuclei after dilution in SDB (see Methods section). A single, diploid red blood cell nucleus is present (arrowhead). F: A typical field of nuclear-injected embryos at four-cell stage, the time when they are first sorted. Embryos showing a canonical four-cell animal pattern (arrows, cleavage furrows forming right angles) are selected. Embryos showing irregular cleavages (arrowheads) are likely to be polyploid and are rejected, as are noncleaving embryos.
	Figure 3. Developmental profile of the expression of Pax-6/green fluorescent protein (GFP), Rx/GFP, and Otx2/GFP transgenes in F1 embryos. A–C:Pax-6/GFP expression at stage 16 (A, anterodorsal view), stage 23 (B, lateral view), and stage 37/8 (C, lateral view). The bracket marks a characteristic lack of Pax-6 expression in the midbrain region. Arrows show expression in the eye at all stages, although it weakens by stage 37/8 (I). The asterisk in A marks the anterior neural tube, whereas the small arrowhead in C shows Pax-6/GFP expression in the spinal cord. D–F:Rx/GFP expression at stage 16 (D, anterodorsal view), stage 23 (E, lateral view), and stage 35/6 (F, lateral view). Arrows mark the developing eye, whereas the asterisk in D marks the anterior neuropore. G:Otx2/GFP expression at stage 13 in the notochord (dorsal view), neural tissue layers have been removed from the embryo to highlight expression in the dorsal mesoderm. H: Expression of Otx2/GFP in anterior neural tissue (arrowheads) at stage 19 (anterior view). I: stage 37/8 (lateral view). Arrowheads indicate persistent GFP expression in the notochord area, whereas arrows highlight GFP expression in the eye.
	Figure 4. Late-stage tadpoles reveal complex patterns of transgene expression. Dorsal view, anterior to the left in all panels. A:Pax-6/green fluorescent protein (GFP) transgenic tadpole at stage 47. GFP expression is strong in the brain and in other nervous tissue. The arrowhead with asterisk shows the olfactory placodes, which are strongly GFP positive. Arrows indicate the olfactory and optic nerves, whereas the arrowheads indicate neurons of the lateral line. B:Otx2/GFP transgenic tadpole at stage 45, showing complex, segmental expression in the hindbrain (arrowhead with asterisk) as well as in the forebrain (arrowhead). The eye still strongly expresses GFP even at this late stage (arrow). C: An EF1α/GFP transgenic tadpole at stage 45. Expression is strong in the axial muscle (posterior arrowhead) and in muscles of the pharynx (anterior arrowhead). Brain expression appears regionalized (arrows), although that is likely an effect of the thickness of different brain tissues.
	Figure 6. Multiple-reporter transgenics simultaneously show different promoter activities. A: A double transgenic F0 tadpole, Pax-6/green fluorescent protein (GFP) plus crystallin/red fluorescent protein (RFP). B: A stage 42 triple transgenic (Pax-6/GFP, crystallin/RFP, and cardiac actin/RFP) F0 tadpole. The arrows in A,B indicate the yellow appearance of the lens, produced by green from Pax-6/GFP in the neural retina and red from crystallin/RFP in the lens. C: A group of F1 transgenic triples show similar levels of expression of all three transgenes. A,B are lateral views, anterior to the right. C shows both dorsal and lateral aspects of the tadpoles, heads are toward the center.
	Figure 7. Assaying the results of neural and lens induction experiments is simplified when using transgenic lines. A,B:Pax-6/green fluorescent protein (GFP) transgenic animal caps treated either with control CHO medium (A) or 10% noggin-containing CHO cell medium (B). Arrowhead in A indicates yolky extrusion that is not GFP positive. Arrows in B show positive GFP responses in animal cap tissues. C,D:gamma-crystallin/GFP transgenic X. tropicalis lens ectoderm recombined with mouse optic vesicle. C, bright field; D, fluorescence. Tissue from the mouse optic vesicle is marked by the dotted line in C; In both C and D, the arrows indicate the primary, GPF-positive lens response of the X. tropicalis transgenic tissue, whereas the arrowheads indicate a smaller, secondary GFP-positive lens response. Scale bars = 200 μm in A (applies to A,B), in C (applies to C,D).
	The ubiquitously expressed EF1α/green fluorescent protein (GFP) transgene can be used as a host/donor marker in transplant and recombinant experiments. A:EF1α/GFP-expressing transgenic tadpole at stage 32, highlighting the widespread expression. B: A wild-type host embryo with an EF1α/GFP ectodermal transplant ventrally. The boundaries of the transplant are marked by arrowheads. C,D: A transplant of the presumptive lens ectoderm from a stage 14 EF1α/GFP transgenic donor to the lens region of a stage 14 wild-type host. C, brightfield; D, fluorescence. The arrows in C,D indicate the lens induced from donor tissue. All panels are lateral views; anterior is to the right.