Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
TNF receptor-associated protein1 (TRAP1) is a mitochondrial molecular chaperon with high homology with a cytosolic chaperon HSP90. It has been shown that TRAP1 functions as an inhibitor for apoptosis by preventing cytochrome-c release from mitochondria. In addition, TRAP1 seems to play critical roles in metabolic processes for energy production, such as glycolysis and β-oxidation. It has also been reported that TRAP1 is a direct target of PTEN-induced kinase 1 (PINK1) and may be a cause of Parkinson's disease (PD) in humans. Although the biochemical functions of TRAP1 are under intense study for the physiology and treatment of various cancers, its roles in vertebrate development have not been reported. This study shows that Xenopus TRAP1 is strongly expressed in the developing muscle, kidney, and brain tissues. Perturbation of TRAP1 function by treating TRAP1 inhibiter GTPP or microinjection of antisense-morpholino oligo (MO) caused mild defects in striated muscle fiber formation. Furthermore, the looping patterns of developing kidney tubules were perturbed, indicating that TRAP1 function is necessary for proper kidney development.
Figure 1. TRAP1 is expressed at developing neural crest cells, somite, kidney, and pharyngeal arches. TRAP1 expression in Xenopus embryos was analyzed by performing an RNA whole-mount in situ hybridization (WISH) assay. (A) TRAP1 is predominantly expressed in the presomitic tissues and neural crest cells. Scale bar = 500 μm. (B) At early tailbud stages, TRAP1 is expressed at somite and pharyngeal arches. Scale bar = 500 μm. (C) At late tailbud stages, TRAP1 is expressed at somite, pharyngeal arches, and kidney Scale bar = 500 μm. c′, c″. Expression pattern of TRAP1 in sectioned embryos. The sectioning plane is indicated in the C as yellow and red lines. Scale bar = 200 μm. NC; Neural crest cells, Kd; Kidney, Sm; Somite, PAs; Pharyngeal arches.
Figure 2. TRAP1 inhibition causes severe defects in craniofacial and cartilage and muscles. The embryos were grown in various concentrations of TRAP1 inhibitor, GTPP, until Stage 45, and the embryonic phenotypes were analyzed. (A–C) The craniofacial cartilages were analyzed using alcian blue staining. 20μM of GTPP did not cause any noticeable phenotypes. 50μM of GTPP severely disrupted normal facial morphogenesis (red rectangles). Scale bar = 500 μm. (D–F) The embryonic muscles were analyzed by immunostaining muscle myosin heavy chain. The interhyoideus and body wall muscles were unaffected at the embryos in 20μM of GTPP. However, in 50μM of GTPP, embryos were significantly affected and noticeably hypomorphic compared to control embryos. Scale bar = 500 μm.
Figure 3. TRAP1 knockdown did not cause severe damage to skeletal muscles and cartilage. TRAP1 expression was depleted by micro-injection of the antisense morpholino oligo (MO). (A–B) RT-PCR and western blot analysis showed that the antisense MO injection efficiently decreased TRAP1 expression. (C–D) The craniofacial cartilages were analyzed using alcian blue staining. TRAP1 knockdown did not severely disrupt cartilage development. Scale bar = 500 μm. (E–F) The embryonic muscles were analyzed by immunostaining muscle myosin heavy chain. The areas highlighted by yellow rectangles were displayed in e’ and f’ with higher magnification. We observed mild defects in body wall muscles. Scale bar = 500 μm. (G) TRAP1-MO was injected into half of the embryos by Unilateral injection. The TRAP1-MO efficiently blocked the expression of TRAP1 in the targeted sites of the embryos (g’), but apoptotic cell death was not increased. Scale bar = 100 μm.
Figure 4. TRAP1 knockdown did not affect the expression of early kidney genes. Pax8 and Nephrin expression were analyzed by WISH during kidney development. (A, B) TRAP1 knockdown did not significantly change the expression of Pax8 and Nephrin in developing kidneys. Scale bar = 200 μm.
Figure 5. TRAP1 function is necessary for the proper looping of kidney tubules. Xemx1 and Lim1 expression were analyzed by WISH to visualize kidney tubules. (A, B) The area of Xemx1 expression in proximal kidney tubules was measured, and the Gaussian distribution of kidney areas was analyzed by F-test. The average of the kidney area was not distinguishable. However, in TRAP1 morphants, the distribution of the value of the total kidney tubule area was considerably broader than that of control embryos. Scale bar = 200 μm. (CTL; n = 19, TRAP1-MO; n = 21, TRAP1-MO + mRNA; n = 18) (C) The expression of Lim1 in developing kidneys was analyzed by WISH, and the Lim1 signals of embryos were overlayed according to the first anterior-most nephrostome and distal kidney tubules. Scale bar = 200 μm. (D) Image of overlayed Lim1 positive nephrostomes.
Supplementary informationSupplementary figure 1. The brightfield images of the embryos grown in various concentrations of TRAP1 inhibitor, GTPP. The embryo grown in 50 μM of GTPP showed tail-bending phenotypes.
