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Dev Growth Differ
2015 Dec 01;579:601-13. doi: 10.1111/dgd.12241.
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Application of local gene induction by infrared laser-mediated microscope and temperature stimulator to amphibian regeneration study.
Kawasumi-Kita A
,
Hayashi T
,
Kobayashi T
,
Nagayama C
,
Hayashi S
,
Kamei Y
,
Morishita Y
,
Takeuchi T
,
Tamura K
,
Yokoyama H
.
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Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three-dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the single-cell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR-LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature-controlled metal probe (temperature stimulator). The local gene manipulation systems, the IR-LEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre-LoxP system as well as to analyze gene function in a target region or cells with less off-target effects in the study of amphibian regeneration.
Figure 1.
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Constructs for transgenesis and transgenic animals used for local gene induction. (A) Map of the heat-shock-inducible Dkk1GFP transgene with lens labeling for Xenopus laevis. Dkk1GFP is located under control of a Xenopus laevis heat-shock promoter (hsp70). (B) Map of the heat-shock-inducible GFP transgene for the newt Pleurodeles waltl. (C) and (D) Prior to heat shock, a X. laevis F1 tadpole at stage 55 containing the transgene could be recognized by the tdTomato fluorescence in its lenses. However no GFP fluorescence could be recognized in the tadpole body. (G) and (H) After whole-body heat shock, ubiquitous Dkk1GFP expression was induced in the tadpole body. This tadpole was photographed at 4 h after heat shock. (E) and (F) Prior to heat shock, a P. waltl F2 two-week-old larva had no obvious GFP fluorescence but had a few background spots around the eye and tail. (I) and (J) After whole-body heat shock, ubiquitous GFP expression was induced in the body. This larva was photographed 72 h after heat shock. Scale bar = 1 mm.
Figure 2.
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Infrared laser irradiation (IR-LEGO) operation in Xenopus laevis. (A) Schematic diagram of IR laser irradiation to a transgenic animal. The IR laser was irradiated through the objective lens of an inverted microscope. An anesthetized Dkk1GFP F1 Tg animal is put in the slit of agarose gel on a glass-bottom dish. The upper panels (B, E, H) show the irradiated areas marked by red circles. The middle panels (C, F, I) are fluorescent images and the lower panels (D, G, J) are bright field images. (B) Skeletal muscles in the tail of a tadpole at stage 54 were irradiated. (C, D) Pictures were taken 18 h after irradiation. (E) A lefthindlimb bud of a tadpole at stage 54 was irradiated from the dorsal side. (F, G) Pictures were taken 18 h after irradiation. (H) A forelimbblastema was irradiated from the ventral side at 11 dpa (days post amputation). (I, J) Pictures were taken 18 h after irradiation. (C, D, F, G) Scale bar = 500 μm; (I, J) Scale bar = 200 μm.
Figure 3.
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Infrared laser irradiation (IR-LEGO) operation in Pleurodeles waltl. The upper panels (A, D) show the irradiated areas marked by red circles. The middle panels (B, E) are fluorescent images and the lower panels (C, F) are bright field images. (A) Skeletal muscles in a larval newt (2 weeks old) were irradiated. (B, C) Pictures were taken 16 h after irradiation. (D) A left forelimb bud (7 days post-amputation) of a larva (7 weeks old) was irradiated from the dorsal side. (E, F) Pictures were taken 16 h after irradiation. Scale bar = 500 μm.
Figure 4.
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3D imaging of a gene product induced by Infrared laser irradiation (IR-LEGO). (A) A bright field image of a lefthindlimb bud of a Dkk1GFP F2 Tg X. laevis tadpole at stage 55. This tadpole was irradiated and then photographed from the dorsal side. Irradiation was done with 20× objective lens at 60 mW for 1.0 s (arrow), 80 mW for 1.0 s (arrowhead) and 50 mW for for 1.0 s (open arrowhead). (B) A fluorescent (GFP) image of the same sample as that shown in (A). (C) Image stacks of 334 μm by 144 μm by 90 μm (xyz) were acquired from the dashed square in (A) and (B) and then converted into 3D images with ImageJ 3D Viewer. (A)–(C) Pictures were taken 18 h after irradiation. (D) Representative images from image stacks acquired in (C). The numbers in the upper left corner indicate the depth from the dorsal surface of the hindlimb bud. Scale bar = 100 μm.
Figure 5.
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Temperature stimulator application in Xenopus laevis. (A) Schematic diagram of temperature stimulator application to a transgenic animal. An anesthetized Tg animal is locally heat-shocked by the metal probe of the temperature stimulator. (B) and (C) The most distal side of a rightforelimbblastema of an hsp70-Dkk1GFP F1 Tg froglet at 6 dpa was heat-shocked at 37°C for 10 min, and the posterior-ventral side of the same blastema was heat-shocked at 37°C for 10 min again and then photographed at 7 dpa from the ventral side. (D) and (E) A section of the blastema shown in (B) and (C) was fixed at 8 dpa and stained with anti-GFP antibody (D) and DAPI (E). (F) and (G) Posterior-dorsal side of a rightforelimbblastema of an hsp70-Dkk1GFP F1 Tg froglet at 7 dpa was heat-shocked at 37 °C for 10 min and then photographed at 8 dpa from the dorsal side. (H) and (I) A lefthindlimb bud of an hsp70-dnTead4 F1 Tg tadpole at stage 50 was heat-shocked. The upper panels (B, F, H) are fluorescent (GFP) images and the lower panels (C, G, I) are bright field images. (B, C, F, G, H, I) Scale bar = 1 mm; (D, E) Scale bar = 100 μm. a, anterior; p, posterior; d, dorsal; v, ventral.
