XB-ART-35877Int J Dev Biol 2007 Jan 01;513:183-90. doi: 10.1387/ijdb.062230ks.
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Xenopus glucose transporter 1 (xGLUT1) is required for gastrulation movement in Xenopus laevis.
Glucose transporters (GLUTs) are transmembrane proteins that play an essential role in sugar uptake and energy supply. Thirteen GLUT genes have been described and GLUT1 is the most abundantly expressed member of the family in animal tissues. Deficiencies in human GLUT1 are associated with many diseases, such as metabolic abnormalities, congenital brain defects and oncogenesis. It was suggested recently that Xenopus GLUT1 (xGLUT1) is upregulated by Activin/Nodal signaling, although the developmental role of xGLUT1 remains unclear. Here, we investigated the expression pattern and function of xGLUT1 during Xenopus development. Whole-mount in situ hybridization analysis showed expression of xGLUT1 in the mesodermal region of Xenopus embryos, especially in the dorsal blastopore lip at the gastrula stage. From the neurula stage, it was expressed in the neural plate, eye field, cement gland and somites. Loss-of-function analyses using morpholino antisense oligonucleotides against xGLUT1 (xGLUT1MO) caused microcephaly and axis elongation error. This elongation defect of activin-treated animal caps occurred without downregulation of early mesodermal markers. Moreover, dorsal-marginal explant analysis revealed that cell movement was suppressed in dorsal marginal zones injected with xGLUT1MO. These findings implicate xGLUT1 as an important player during gastrulation cell movement in Xenopus.
PubMed ID: 17486538
Article link: Int J Dev Biol
Species referenced: Xenopus laevis
Genes referenced: chrd.1 gal.2 gsc mix1 mixer myc nodal nodal1 odc1 slc2a1 tbxt
Morpholinos: slc2a1 MO1
Article Images: [+] show captions
|Fig. 1. xGLUT1 expression in activin-treated animal caps. Animal caps were treated with 0-10 ng/ml of activin for 5 hours. A low level of xGLUT1 expression was detected in animal caps treated with 1 ng/ml activin, whereas high levels of xGLUT1 expression could be seen in animal caps treated with 5 or 10 ng/ml activin. ODC was used as a loading control.|
|Fig. 2. Expression patterns of xGLUT1. (A) Temporal expression of xGLUT1 was analyzed by RT-PCR. xGLUT1 expression was not detected maternally. At the blastula stage, low levels of xGLUT1 expression were detected. Expression levels increased at stage 9.5, with high levels of expression being maintained by stage 31. Spatial patterns of xGLUT1 expression were analyzed by in situ hybridization. (B-N) Spatial expression patterns of xGLUT1 analyzed by whole mount in situ hybridization. (B-H) Outer view of albino embryos. Left column, lateral view; middle column, vegetal view; right column, anterior view. (I-N) Sagittal sections of wild-type embryos. Signal was detected as blue staining. (C,D,J,K) From late blastula stage, xGLUT1 mRNA expression was seen in the mesodermal region, especially in the dorsal lip (DL, white arrowhead). (E,L) At the late gastrula, xGLUT1 expression was extended to the ventral lip (VL, white arrowhead). (M) At stage 13, ventral expression had disappeared, but intense expression was maintained in the deeply involuted dorsal-mesodermal region. (F-H, N) From the neurula stage, expression was detected mainly in the cement gland, eye fields and somite. DL, dorsal lip; VL, ventral lip; yp, yolk plug; me, mesoderm; ce, cement gland; so, somite; ey, eye field.|
|Fig. 3. xGLUT1MO caused gastrulation error and loss of head structure. (A) Nucleotide sequence of xGLUT1 mRNA and 5 miss-N- GLUT1-myc mRNA. xGLUT1MO recognized the sequence indicated by the black line in the upper column. 5 silent mutations in 5miss-N-xGLUT1- myc mRNA are shown as pink letters in the lower column. (B) Western blot analysis of myc-tagged xGLUT1 fusion protein. Either 1 ng of N- xGLUT1-myc or 5miss-N-xGLUT1-myc mRNA was coinjected with 20 ng of xGLUT1MO or standard control MO into four blastomeres of a 4-cell stage embryo. Western blot analysis was performed with anti-myc antibody. Anti-α-tubulin antibody was used as a loading control. The translation of N-xGLUT1-myc mRNA was inhibited by the xGLUT1MO. (C) Phenotype of embryos injected with 20 ng of xGLUT1MO. (C.a-c) Two dorsal blastomeres were injected. (C.d-f) Two ventral blastomeres were injected. (a) xGLUT1MO injection into dorsal animal pole caused short axis and head disruption. (b) Dorsal marginal zone injection caused severe axis defect and neural tube closure defect. (c) Dorsal vegetal injection showed a weaker phenotype. (d-f) Ventral injection also showed an axis defect but the level of severity was weak. (D) Rescue of xGLUT1MO-derived defect by xGLUT1 mRNA. 10 ng of xGLUT1MO and 500 pg of beta-galactosidase (β-gal) mRNA (D.a) or xGLUT1 mRNA (D.b) was coinjected into two dorsal marginal zones of a 4-cell stage embryo. Obvious rescue was observed by addition of xGLUT1 mRNA.|
|Fig. 4 (Left). xGLUT1MO caused gastrulation defect. (A) Blastopore closure was delayed by xGLUT1MO injection compared with normal embryos. (A.a) 10 ng of xGLUT1MO was injected into the dorsal marginal zone. (A.b) Control embryo. (B) Sagittal sections of the gastrula. (a,c) stage 10. (b,bd,d stage 11. (a-b 10 ng of xGLUT1MO was injected into the dorsal marginal zone. (c-d Control embryo. (b and (d are high magnification images of the white boxes in (b) and (d), respectively. In xGLUT1MO-injected embryos, the involuting marginal zone and inner surface of the deep layer remained thick (b yellow arrowheads).|
|Fig. 5 (Right). Mesodermal genes were not influenced by inhibition of xGLUT1. (A) xGLUT1MO inhibited elongation of animal caps. 20 ng of xGLUT1MO or control MO was injected into the animal pole of an 8-cell stage embryo. At late blastula, animal caps were dissected and treated with 10 ng/ml of activin and cultured for 10 hours. Without activin treatment, caps remained round (A.a-c) and activin-treated caps dissected from control embryos (d) or standard control MO-injected embryos (e) became elongated in response to activin. However, the elongation of xGLUT1MO-injected caps was inhibited (A.f). (B) Expression of mesodermal marker genes in animal caps injected with xGLUT1MO. Animal caps were treated with activin for 3 hours and sampled for RT-PCR. Xbra, mix2 and Chd transcript levels were not influenced by xGLUT1MO injection. ODC was used as a loading control. (C) Whole-mount in situ hybridization (WISH) with late gastrula. 10 ng of xGLUT1MO and 100 pg of β-gal mRNA was coinjected into one side of the dorsal marginal zone of 4-cell stage embryos. Before WISH, the embryos were stained with Red-gal for cell lineage tracing. xGLUT1MO injection caused no change in xbra (a), chd (b), or gsc (c) expression.|
|Fig. 6. Dorsal sandwich explant analysis on xGLUT1MO-injected embryos. (A) Schematic diagrams of making dorsal sandwich explants. Four-cell stage embryos were injected with 40 ng of xGLUT1MO or control MO into the dorsal marginal zone of two dorsal blastomeres. At stage 10, the dorsal marginal zones were dissected and two explants were put together to make a sandwich, which was cultured for 12 hours. (B) Dorsal sandwich explants from normal embryo (a) or standard control MO-injected embryo (b) showed elongation, whereas in explants in- jected with xGLUT1MO, elongation was inhibited (c).|
|slc2a1 ( solute carrier family 2 (facilitated glucose transporter), member 1) gene expression in Xenopus laevis embryos, NF stage 30, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.|