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Dev Biol
2018 Jan 15;4332:404-415. doi: 10.1016/j.ydbio.2017.08.012.
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A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole.
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Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp). The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpoletail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC. We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-β or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation. These findings showed that es1:egfp Xenopus laevis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration.
Fig. 1. Expression of es1:EGFP and endogenous es1 in embryos. (A) Structure of Xenopus laevis es1 gene (upper) and the construct used for transgenesis (lower). Open squares represent exons. pA, poly adenylation signals. (B-E) Representative images of es1: egfp transgenic embryos. C, D and E show EGFP fluorescence images. B shows a normal light image of the same field of C. Asterisks in B and C show an EGFP-negative embryo. Arrowheads in D indicate cells expressing EGFP in dorsal midline. Arrowheads in E indicate EGFP expression in oral primodium, pharyngeal arches and anus region. (F, G) Representative images of WISH showing endogenous es1 expression in embryos. Arrowheads in G indicate es1 expression in oral primordium, pharyngeal arches and anus region. B, C, D and F are dorsal images. Embryos in E and G are anteriorleft and dorsal up. Embryonic stages are indicated. Bars, 500 µm.
Fig. 2. Expression of es1:EGFP during tail regeneration. (A) Successive images of an F1 tadpole (line 1) after tail amputation. Upper and lower panels show EGFP fluorescence and normal light images in the same fields. Images were captured in the same condition. Arrowheads indicate the same region. Time points after amputation are indicated. Anteriorleft, dorsal up. Bar, 500 µm. (B) EGFP expression in another es1:egfp tadpole (line 1) from 3 dpa to 13 dpa after tail amputation. Lower panels show the normal light images in the same fields as the upper images. Images were captured in the same conditions. Insets are magnified and brightened fluorescence images of boxed regions. Anteriorleft, dorsal up. Bar, 500 µm.
Fig. 3. Confocal images showing expression of es1:EGFP in epidermal cells at 1 dpa. Tadpole tails of es1:egfp (line 2) were fixed at 1 dpa and stained with rhodamine-phalloidin and DAPI. Positions and focal planes are indicated by red lines in illustrations on the left. (A) Superficial epidermal layer of lateral side near the amputation plane. A few superficial cells express EGFP (arrows). (B) Images of basal epidermal layer of the same field as (A). Most cells express EGFP, at various levels. (C) Sagittal images of the amputated stump at the edge region. Amputation plane is covered with two or three cell layers of WE (brackets). Cells expressing high level of EGFP are not located in the superficial layer. Anteriorleft. Bars, 10 µm.
Fig. 4. Confocal images showing expression of es1:EGFP at 3 dpa. Tadpole tails of es1:egfp (line 2) were fixed at 3 dpa and stained with rhodamine-phalloidin and DAPI. (A) Sagittal midline images of the regenerating tail. Intense EGFP signals were observed in epidermal cells, especially at the most distal multilayered region, the AEC (arrowheads). Yellow broken lines indicate regenerating spinal cord and notochord. nc, notochord. na, neural ampula of regenerating spinal cord. Bar, 50 µm. (B) Sagittal images of regenerating tail at muscle level. EGFP signals were exclusively observed in epidermis. m, muscle tissues. Bars, 75 µm. Anteriorleft, dorsal up.
Fig. 5. Expression of es1:EGFP after wounding. Successive images of es1:EGFP expression in tailskin of an F1 tadpole (line 2) after injuring by pricking with a tungsten needle. Upper and lower panels show EGFP fluorescence and normal light images in the same fields. Images were captured in the same condition. Arrows indicate the injured site. Insets are magnified and brightened fluorescence images of the injured site. Time points after injuring are indicated. Anteriorleft, dorsal up. Bar, 500 µm.
Fig. 6. Effect of chemicals on wound healing. (A) Tail-amputated wild-type tadpoles were treated with DMSO (Control), or inhibitor for TGF-β (TGFβ i, SB431542), ROS (ROS i, DPI) or ERK (ERK i, U0126) signaling for 24 h. Surface epidermal cells were labeled with Alexa488-WGA and amputation planes were observed under fluorescent light as illustrated. Asterisks indicate the dark open areas not covered with WE. Number of tadpoles with complete wound closure / number of observed tadpoles is indicated. Dorsal up. Bar, 100 µm. (B) Tadpoles were treated with DMSO (Control), or inhibitor for ROS (ROS i, DPI), Calcium (Ca i, U73122), JNK (JNK i, SP600126) or V-ATPase (VATP i, bafilomycin) signaling, and observed as indicated in (A). Bar, 100 µm.
Fig. 7. Effect of chemicals on es1:EGFP and endogenous es1 expression. (A, B) Successive fluorescence images of tail–amputated line 1 tadpoles which were treated with DMSO (Control) or TGF-β signal inhibitor (TGFβ i, SB431542). Time points after tail amputation are indicated. (C-F) Representative EGFP fluorescence images at 24 hpa in tadpoles treated with DMSO (Control), or inhibitor for ERK (ERK i, U0126), ROS (ROS i, DPI) or JNK (JNK i, SP600126) signal. A pair of arrows indicates the amputation plane. Arrowheads indicate the posterior margins of the amputated stump. Anteriorleft, dorsal up. Bars, 500 µm. (G-I) Expression level of es1:EGFP at 12 hpa in tail stump was quantified with ImageJ software. Relative mean values are shown with standard errors. ***P<0.001; Student's t-test. (J, K) Relative expression level of es1 compared to that in wild-type tadpoles at 12 hpa was determined by qRT-PCR after tail amputation. Tail-amputated tadpoles were treated with the indicated chemicals. Mean value of four (J) or six (K) biological replicates is shown with standard error. *p<0.05; Student's t-test.
Fig. 8. Effects of chemicals on signal activation of TGF-β, ERK and ROS. Tail-amputated wild-type tadpoles were treated with DMSO (Control), or inhibitors for ERK (ERK i, U0126), ROS (ROS i, DPI) or TGF-β (TGFβ i, SB431542) signal. Upper and lower panels show fluorescence and normal light images in the same fields. (A, B) Representative images stained with anti-phospho Smad2 at 6 hpa (A) and anti-phospho ERK at 6 hpa (B). “–Ab†indicates control without 1st antibody. Arrowheads indicate specific signals in the stump epidermis. Arrows indicate non-specific fluorescence in deep region. (C) Representative images stained with H2DCFDA at 9 hpa. “–Dye†indicates control without incubation in H2DCFDA. Arrowheads indicate sites of ROS signaling activation. Anteriorleft, dorsal up. Bars, 100 µm.
Supplementary figure S1.Expression of es1:EGFP during tail regeneration in line 2 tadpoles.
(A) Successive images of an F1 tadpole (line 2) a9er tail amputa;on. Upper
and lower panels show EGFP fluorescence and normal light images in the
same fields. Images were captured in the same condi;on. Arrowheads
indicate the same region. Time points a9er amputa;on are indicated.
Anterior le9, dorsal up. Bars, 500 µm. (B) EGFP expression in another
es1:egfp tadpole (line 2) from 3 dpa to 5 dpa. Lower panels show the
normal light images in the same fields as the upper images. Images were
captured in the same condi;on. Arrowheads indicate the AEC region with
intense EGFP signal. Bars, 500 µm.
Supplementary figure S2. Effect of inhibitors for Ca and vATP
signalings on es1:EGFP.
(A) Representa;ve images at 12 hpa in tadpoles treated with DMSO
(Control), or inhibitor for ROS (ROS i), Calcium (Ca i) or V-ATPase (VATP
i) signaling. Upper and lower panels show EGFP fluorescence and
normal light images in the same fields. Bar, 100 µm. (B) Expression
level of es1:EGFP at 12 hpa in tail stump was quan;fied with ImageJ
so9ware. Rela;ve mean values are shown with standard errors. ***P <
0.001; Studentâs t-test.
