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In amphibians, thyroid hormones (THs) are the primary regulators of metamorphosis; however, their physiological role during embryogenesis remains unclear. First, we established complete developmental profiles for TH receptors (tr alpha and tr beta), deiodinases (dio; types 1, 2, 3), estrogen receptors (er alpha and er beta) and androgen receptor (ar) mRNA levels during embryogenesis and early larval stages in Silurana (Xenopus) tropicalis (from Nieuwkoop and Faber (NF) stage 2 until NF 46). Real-time RT-PCR analyses in whole embryos and larvae revealed that all transcripts except tr alpha were detected throughout development; tr alpha only appears after gastrulation. The first significant increase in the expression of tralpha and tr beta was observed before hatching, between NF 21 and NF 27 (2.5- and 11-fold, respectively). In order to test if these genes could be regulated by THs during early larval development, embryos were exposed to triiodothyronine (T3; 0.5, 5.0, 50 nM) from NF 27 to NF 46. T3 exposure caused a dose-dependent increase relative to control in the expression of tr alpha, tr beta, dio (types 2 and 3), ar, and 5 alpha-reductase type 1 in whole larvae. These results indicate that in S. tropicalis, tr and dio can be induced by T3 as early as NF 46, a response that had only been characterized later during frog metamorphosis. In addition, T3 also affected androgen-related gene expression, supporting our hypothesis that THs are involved in male development in frogs.
Fig. 1.
Developmental profiles of TH-related genes during S. tropicalis embryogenesis and larval development. Transcript levels of tralpha (A), trbeta (B), dio1 (C), dio2 (D) and dio3 (E) were measured in whole embryos and larvae from NF 2 (two-cell stage) to NF 46 (beginning of feeding). Levels of mRNA are expressed relative to NF 2 except for tralpha (relative to NF 16) and are normalized to RNA content. Results for the reference gene odc (F) are also presented. Bars represent the mean + SEM. Different letters indicate statistically significant differences between stages (n = 6–8 pools; p < 0.05). Note that the scales of the y-axis vary between genes. ND, not detected.
Fig. 2.
Developmental profiles of sex steroid-related genes during S. tropicalis embryogenesis and larval development. Transcript levels of eralpha (A), erbeta (B) and ar (C), were measured in whole embryos and larvae from NF 2 (two-cell stage) to NF 46 (beginning of feeding). Levels of mRNA are expressed relative to NF 2 and are normalized to RNA content. Bars represent the mean + SEM. Different letters indicate statistically significant differences between stages (n = 6â8 pools; p < 0.05). Note that the scales of the y-axis vary between genes.
Fig. 3.
Effects of T3 exposure on the expression of TH-related genes during S. tropicalis larval development. Silurana tropicalis were exposed from NF 26â27 to NF 46 to T3 (0, 0.5, 5, 50 nM). Effects of T3 on tralpha (A), trbeta (B), dio1 (C), dio2 (D) and dio3 (E) are presented. Data are presented as fold changes relative to control and are normalized to RNA content. Results for the reference gene rpl8 (F) are also presented. Bars represent the mean + SEM. Asterisks represent significant differences from the control group (n = 8 pools; âp < 0.05; âââp < 0.001). Note that the scales of the y-axis vary between genes.
Fig. 4.
Effects of T3 exposure on the expression of sex steroid-related genes during S. tropicalis larval development. Silurana tropicalis were exposed from NF 26â27 to NF 46 to T3 (0, 0.5, 5, 50 nM). Effects of T3 on eralpha (A), cyp19 (B), erbeta (C), srd5alpha1 (D), ar (E), and srd5alpha2 (F) are presented. Data are presented as fold changes relative to control and are normalized to RNA content. Bars represent the mean + SEM. Asterisks represent significant differences from the control group (n = 8 pools; âp < 0.05; âââp < 0.001). Note that the scales of the y-axis vary between genes.
Fig. 5.
Diagram summarizing the findings on TH-related gene expression, THs levels, dio activity and anatomical and morphological changes during S. tropicalis and X. laevis embryonic and larval development. The x-axis represents time of development in NF stages. Hours post-fertilization (hpf) under our husbandry conditions are also presented along with the main characteristics of each of the stages. The y-axis represents relative mRNA levels in a log scale. The results of the gene expression of tr and dio in S. tropicalis are those presented in Fig. 1. Levels of T3 and T4 and dio activity in X. laevis were taken from Berg et al., 2009 ; Morvan-Dubois et al., 2006. Iodine uptake has been detected at NF 46 in X. laevis by Brown (2005). The morphological and anatomical information was taken from Nieuwkoop and Faber (1967). Thyroid gland organogenesis begins around NF 40 and the first follicles are visible around NF 49 in X. laevis.
