XB-ART-35373Dev Biol 2007 Apr 15;3042:675-86. doi: 10.1016/j.ydbio.2007.01.019.
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Transgenic Xenopus with prx1 limb enhancer reveals crucial contribution of MEK/ERK and PI3K/AKT pathways in blastema formation during limb regeneration.
Understanding the mechanisms that control amphibian limb regeneration should allow us to decipher the critical differences between amphibians and humans, which have the limited ability of organ regeneration. However, many issues at the cellular and molecular levels still remain unresolved. We have generated a transgenic Xenopus laevis line that expresses green fluorescent protein (GFP) under the control of mouse prx1 limb enhancer, which directs reporter gene expression in limb mesenchyme in mice, and found that GFP accumulated in blastemal mesenchymal cells of the transgenic froglets after limb amputation. Thus, this transgenic line should provide a new approach to gain insights into the cellular dynamics and signaling pathways involved in limb blastema formation. We have also developed a culture system for forelimb explants of froglets and found that treatment with inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase 1/2 (MEK1/2) and phosphatidylinositol 3-kinase (PI3K) repressed GFP expression. These effects were partially reversible, and down-regulation of GFP was associated with inhibition of cell-cycle progression and induction of ectopic apoptosis. In addition, we found that ERK1/2 and AKT, downstream mediators of MEK1/2 and PI3K pathways, were activated in amputated forelimb stumps. These results demonstrate that MEK/ERK and PI3K/AKT pathways regulate limb blastema formation in the X. laevis froglet.
PubMed ID: 17303106
Article link: Dev Biol
Species referenced: Xenopus laevis
Genes referenced: akt1 map2k1 mapk1 msx1 myh1 pik3ca pik3cb pik3cg prrx1
Antibodies: Akt1 Ab5 BrdU Ab7 Casp3 Ab6 GFP Ab12 H3f3a Ab9 Mapk1 Ab3 Msx1/2 Ab1 Myh1 Ab1
Article Images: [+] show captions
|Fig. 1. Expression pattern of Xprx1 during hindlimb development. (A) Lateral view of a stage 45 tadpole. Transcripts were restricted to the prospective limb-forming region (arrow). (B) A longitudinal section of a limb bud at stage 50. Note that mesenchymal expression is restricted to the limb bud. An arrow indicates the boundary between the limb bud and trunk mesodermal tissues. (C) A longitudinal section of a limb bud blastema at stage 53 shows expression of Xprx1 similar to that in the limb bud. Dotted lines indicate the amputation plane. (D) A longitudinal section of a thigh region at stage 56. Transcripts were preferentially detected in the perichondrium and undifferentiated mesenchyme. (E and F) Serial sections of panel D stained with Alcian blue (cartilage) and myosin heavy chain (muscle), respectively. Scale bars: 200 μm. ab, abdomen; ca, presumptive cartilage-forming region; mf, medial fin fold; ms, muscle; tr, trunk.|
|Fig. 2. Expression pattern of GFP in Mprx1-GFP transgenic Xenopus. (A–G) are F0 and (I–M) are F2 animals of line 1. (A, B) Lateral view of a stage 53 tadpole shows GFP expression in the forelimb bud (arrow) and hindlimb bud (arrowhead). (B) Bright-field image of panel A. (C, D) High power views of fore- and hindlimb buds of panel A, respectively. (E) Lateral view of limb bud blastema of a stage 54 tadpole shows uniform GFP expression in the blastema. Lines indicate the amputation plane. (F, G) Dorsal view of froglet forelimb blastema shows GFP expression in the blastema and proximal region (bracket). (G) Bright-field image of panel F. (H) Dorsal views of froglet blastema of wild-type animal. (I–M) Longitudinal sections of amputated froglet forelimbs. (I) In the intact limb, GFP was detectable in the periosteum and ectoderm. (J) At 2 days, extensive up-regulation was observed in the mesodermal region. A high-power view of the boxed region (L) shows a number of fibroblastic cells expressing GFP. Yellow staining of the right distal region (asterisk in panel J) is autofluorescence of the tissue debris. (K) A limb stump at 4 days after amputation. (M) A high-power view of the boxed region in panel K shows that the majority of mesenchymal cells in the early blastema expressed GFP. Scale bars: in panel B, 2 mm for panels A, B; in panels I, J, K 200 μm.|
|Fig. 3. Characterization of GFP-positive cells in limb stumps. (A) A longitudinal section of a limb blastema at 11 days stained with anti-GFP and anti-MSX1/2. The merge shows that MSX1/2-positive blastemal cells were GFP positive. (B) A longitudinal section of a limb blastema at 4 days stained with anti-GFP and anti-BrdU. The merge shows that cycling cells incorporating BrdU were GFP positive. (C, D) Numbers of BrdU- and phospho-Histone H3-positive cells in limb stumps. Note that reentry of mesodermal cells into the cell cycle became evident at 2 days after limb amputation. (E) Temporal transition of the percentage of GFP-positive cells in BrdU-positive cells, showing that most of the cycling cells were GFP positive. In all cases, points represent the average of at least four stumps. Scale bars: 200 μm.|
|Fig. 5. Treatment with LY294002 induces ectopic apoptosis in limb explants. (A) Activity of caspase-3 in control explants. Arrowheads indicate non-specific staining within bones. (B, C) Treatment with LY294002, but not treatment with U0126, increased the number of apoptotic cells. (D) Numbers of active-caspase 3-positive cells in the explants. Treatment with LY294002 significantly increased the number of apoptotic cells. In all cases, bars represent the average of at least four explants. Scale bars: 200 μm.|
|Fig. 6. Activities of ERK1/2 and AKT in limb stumps. Immunostaining was performed with anti-diphospho-ERK1/2 (A–C) and anti-phospho AKT substrate (D–E) at 1 day (A, D), 2 days (B, E) and 4 days (C, F). All sections are longitudinal sections. (A, B) At 2 days after amputation, activity of ERK1/2 was detected in bundles of axons in the stump (arrowheads). (C) At 4 days, however, diphosphorylation of ERK1/2 was evident in mesenchymal cells under the WE as well as regenerating axons. (D) Activity of AKT was extensively detectable in the mesodermal region of the stump at 1 day. Activity level of AKT in the distal region was low (bracket). (E) Activity of AKT was more evident at 2 days. (F) At 4 days, activated AKT-positive cells became restricted to the distal region where the majority of mesodermal cells are GFP positive. Scale bars: 200 μm.|
|Fig. 4. Organ culture of Mprx1-GFP froglet forelimbs and effects of treatment with U0126 and LY294002. (A) Schematic representation of organ culture system. See text for details. (B) GFP expression in a control explant cultured for 2 days with DMSO, suggesting that the culture condition recapitulates the process of dedifferentiation. Treatment with U0126 at 20 μM (C) and 100 μM (D) and treatment with LY294002 at 20 μM (F) and 100 μM (G) inhibited up-regulation of GFP. Residual expression of GFP in the periosteum shows that these explants were derived from transgenic animals. (E, H) Additional incubation for 2 days with DMSO after treatment for 2 days with U0126 or LY294002 at higher concentrations induced up-regulation of GFP. (I) Numbers of BrdU- and phospho-Histone H3-positive cells in the explants. Treatments with U0126 and LY294002 repressed cell cycle progression. Asterisks represent 4 days of incubation (additional incubation for 2 days with DMSO). In all cases, bars represent the average of at least four explants. Scale bars: 200 μm.|
|prrx1 ( paired related homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 45, lateral view of trunk region only, anterior left, dorsal up.|