Nikishin DA et al. (2012), Expression of serotonergic system components du...

XB-ART-45657

Int J Dev Biol 2012 Jan 01;565:385-91. doi: 10.1387/ijdb.113475dn.

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Expression of serotonergic system components during early Xenopus embryogenesis.

Nikishin DA , Kremnyov SV , Konduktorova VV , Shmukler YB .

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Despite abundant research studies on the physiological and biochemical nature of embryonic neurotransmitter function, little is known about the molecular genetic mechanisms involved. The expression of the main components of the serotonergic system during early Xenopus embryogenesis was investigated using RT-PCR, real time PCR and in situ hybridization. Transcripts encoding the serotonin receptors HTR2C and HTR7, as well as the vesicular monoamine transporter VMAT2, the serotonin transporter (SERT) and the serotonin synthesis enzymes tryptophan hydroxylase (TPH2) and aromatic amino acid decarboxylase (AAAD) were found to be expressed during the cleavage division stages, whereas the degradation enzyme monoamine oxidase A (MAOA) was absent. The main components of the serotonergic system were found to be expressed during the earliest stages of embryonic development. The embryonic transmitter mechanism, its complexity, and its variability among various species are discussed.

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Species referenced: Xenopus laevis
Genes referenced: htr1a htr2b htr2c htr3a htr4 htr7 htr7l maoa odc1 slc18a2 slc6a4l tbx2 tph1 tph2

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	Fig. 1. Temporal expression of serotonergic system components during Xenopus laevis development. The serotonin receptors HTR2C and HTR7, vesicular transporter VMAT2, sodium-dependent transporter SERT, enzymes of synthesis TPH2 and AAAD are expressed during the early stages of development. The MAOA expression detected in oocytes is due to the jelly coats (see Fig. 8 and Discussion). Abbreviations: Oo, oocyte; 2, 2-cell embryo (stage 2); 8, midblastula (stage 8); 9, late blastula (stage 9); 10, early gastrula (stage 10); N, neurula (stage 15); H, hatched larvae (stage 33-35) and T, tadpoles (stage 57). ODC is the endogenous control.
	Fig. 2 (left). Relative expression levels of HTR2C, HTR7 and VMAT2 compared with ODC at the two-cell embryonic stage. Real-time PCR was performed using different primers for the same probe. The Ct values were calculated via equal threshold levels. The relative expression levels (RQ) were calculated via the efficiency-corrected δCt method (Bookout et al., 2005). The ODC expression level was considered to be 100%. The RQ for VMAT2 was approximately 43.9%, HTR7 approximately 4.9%, and HTR2C 0.002%.
	Fig. 3 (right). The mRNA expression profiles of HTR2C, HTR7 and VMAT2 during X. laevis embryogenesis. The relative expression levels (RQ) were normalized to ODC expression at the 2-cell embryonic stage (SD). Abbreviations: 2, 2-cell embryo (stage 2); 8, midblastula (stage 8); 9, late blastula (stage 9); 10, early gastrula (stage 10); N, neurula (stage 15); H, hatched larvae (stage 33-35).
	Fig. 4. The spatial distribution of the mRNA expression of HTR2C, HTR7 and VMAT2 within the Xenopus blastula-stage embryo. The relative expression levels (RQ) were normalized to ODC of the AC sample (SD). VMAT2 expression has pronounced animal-vegetal gradient, whereas HTR2C and HTR7 expression have not. AC, animal cap; MZ, marginal zone; VP, vegetal portion of the embryo.
	Fig. 5. Pattern of expression of HTR7 and VMAT2 mRNA during early Xenopus embryogenesis. In situ hybridization for (A-C) HTR7 and (D-F) VMAT2 to oocytes (A,D), cleavage embryos (B,E) and blastula (C,F). The animal-vegetal gradient of distribution was detected both for the VMAT2 and HTR7 transcripts. During the blastula stage, the VMAT2 transcripts were localized mainly to the animal hemisphere, whereas the HTR7 transcripts were also present in the vegetal hemisphere.
	Fig. 7 (left). Expression of serotonin synthesis enzymes in Xenopus ovaries. I-III, immature oocytes (stages I-III); AAAD, aromatic aminoacid decarboxylase; F, follicular envelope; Ov, total ovary; TPH, tryptophan hydroxylase.
	Fig. 8 (right). Expression of monoamine oxidase A (MAOA) in dejelled Xenopus oocytes and jelly coats. JC, jelly coat (which can comprise the cells from the female genital tract); Oo, dejellied oocytes. MAOA is not expressed in the dejellied oocytes but is present in the material obtained upon dejelling.
	slc18a2 (solute carrier family 18 (vesicular monoamine transporter), member 1) gene expression in Xenopus laevis ooctye, assayed via in situ hybridization, lateral view, animal pole up.
	slc18a2 (solute carrier family 18 (vesicular monoamine transporter), member 2 gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3 (4 cell), lateral view, animal pole up.
	slc18a2 (solute carrier family 18 (vesicular monoamine transporter), member 2) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10, dorsal right, animal hemisphere up.
	htr7l (5-hydroxytryptamine (serotonin) receptor 7, adenylate cyclase-coupled like) gene expression in Xenopus laevis oocyte, assayed via in situ hybridization, lateral view, animal pole up.
	htr7l (5-hydroxytryptamine (serotonin) receptor 7, adenylate cyclase-coupled like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3 (4-cell), mid sagittal section, lateral view, animal pole up.
	htr7l (5-hydroxytryptamine (serotonin) receptor 7, adenylate cyclase-coupled like) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10, dorsal right, animal hemisphere up.