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Int J Dev Biol
2019 Jan 01;631-2:37-43. doi: 10.1387/ijdb.180384hk.
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Axis elongation during Xenopus tail-bud stage is regulated by GABA expressed in the anterior-to-mid neural tube.
Furukawa T
,
Yamasaki Y
,
Hara Y
,
Otsuki C
,
Maki H
,
Soga T
,
Moriyama Y
,
Kuroda H
.
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The receptors of gamma-aminobutyric acid (GABA), which is a well-known neurotransmitter, are expressed in the anterior-to-mid neural tube at an early stage of Xenopus development, but there has been no report on the role of GABA in the presumptive central nervous system. Therefore, we tried to reveal the function of GABA for Xenopus early embryogenesis. We first confirmed that the region expressing a gene encoding glutamate decarboxylase 1 (gad1), which is an enzyme that catalyzes the decarboxylation of L-glutamate to GABA, overlapped with that of several genes encoding GABA receptors (gabr) in the neural tube. Metabolome analysis of culture medium of dorsal tail-bud explants containing the neural tube region of tail-bud stage embryos also revealed that GABA was expressed at this stage. Then, we examined the treatment of pentylenetetrazole (PTZ) and picrotoxin (PTX), which are known as inhibitors of GABA receptors (GABA-R), on the early stages of Xenopus embryogenesis, and found that axis elongation in the tail-bud was inhibited by both treatments, and these phenotypic effects were rescued by co-treatment with GABA. Moreover, our spatial- and temporal-specific inhibitor treatments revealed that the gabr- and gad1-overlapped region, which presents at the anterior-to-mid neural tube during the tail-bud stages, was much more sensitive to PTZ and thus caused severe inhibition of axis elongation. Taken together, our results indicate that the small ligand molecule GABA functions as a regulator to induce the axis elongation event in the tail-bud during early embryogenesis via direct stimulation of the neural tube and indirect stimulation of the surrounding area.
Fig. 1. gad1 and GABA are expressed in the tail-bud stages. (A) Schematic representation of the GABA pathway. GABA, gamma-aminobutyric acid; GABA-R, GABA receptors; GAD, glutamic acid decarboxylase; PTZ, pentylenetetrazole; PTX, picrotoxin. (B-G) Whole-mount in situ hybridization analysis of gad1 of embryos at stages 22 (B), 26 (C), 30 (D, E), and 36 (F, G). nt, neural tube; sc, spinal cord; mb, midbrain; hb, hindbrain. Scale bars represent 1 mm. (H) Schematic diagram of the sampling method for metabolome analysis. Yolk-rich and most ventral regions were removed from embryos at stages 25 26. The remaining area was called the dorsal tail-bud explant (DTE). 10 DTEs were cultured in 0.1x Steinberg’s solution for 8 h, and the supernatant was used for metabolome analysis. SS, Steinberg’s solution. (I) Concentration of cations detected by CE-TOF-MS analysis in the control sample (upper; n=1) and DTEs (bottom; n=3, obtained from 2 female frogs). The m/z represents mass divided by charge number. (J) Concentrations of L-glutamate (m/z = 148.063) and GABA (m/z = 104.072) in the control and DTEs. Data represent mean±S.D. N.D., not detected. Data sets are the same as those in Fig. 1I.
Fig. 2. Elongation of body axis is inhibited by pentylenetetrazole (PTZ) treatment. (A-F) Results of PTZ treatment assay from stages 8 - 38. Scale bars represent 1 mm. (A) Control embryo. (B) Embryo treated with 20 mM GABA. (C) 5 mM PTZ. (D) 10 mM PTZ. (E) 10 mM PTZ and 10 mM GABA. (F) 10 mM PTZ and 20 mM GABA. (G,H) Sagittal histological section of control embryo at stage 38 (G) and embryo treated with 10 mM PTZ (H). A red region in a right-bottom small panel indicates the neural region in G and H. br, brain; nc, notochord; nt, neural tube. (I) The body-length index table to quantify inhibitory action in treated embryos. We categorized embryos into 3 groups: embryos had >90% body length (Normal), 75-90% body length (Level 1), and <75% body length (Level 2) referring to the average length of control embryos. (J) Quanti cation of inhibitory action by the PTZ treat- ment. See (I) for the degree of defects. Sample number is indicated at the upper side of each bar graph.
Fig. 3. Elongation of body axis is inhibited by picrotoxin (PTX) treat- ment. (A-D) Results of PTX treatment assay from stages 8838. Scale bars represent 1 mm. (A) Embryo treated with 1% DMSO. (B) 0.5 mM PTX. (C) 0.5 mM PTX and 10 mM GABA. (D) 0.5 mM PTX and 20 mM GABA. (E) Quanti cation of the PTX treatment. See Fig. 2I for the degree of defects. Sample number is indicated at the upper side of each bar graph.
Fig. 4. Pentylenetetrazole (PTZ) treatment is effective at the tail-bud stage. (A-F) Results of PTZ treatment assay in different time windows: control (A), from stages 3-13 (B), from stages 13-20 (C), from stages 20-28 (D), from stages 28-38 (E), and from stages 3-38 (F). Scale bars represent 1 mm. (G) Quanti cation of the PTZ treatment. See Fig. 2I for the degree of defects. Sample number is indicated at the upper side of each bar graph.
gad1.1 (glutamate decarboxylase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anteriorleft.
gad1.1 (glutamate decarboxylase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anteriorleft, dorsal up.
