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Corticosteroids are critical for development and for mediating stress responses across diverse vertebrate taxa. Study of frog metamorphosis has made significant breakthroughs in our understanding of corticosteroid signaling during development in non-mammalian vertebrate species. However, lack of adequate corticosterone (CORT) response genes in tadpoles make identification and quantification of CORT responses challenging. Here, we characterized a CORT-response gene frzb (frizzled related protein) previously identified in Xenopus tropicalis tadpoletailskin by an RNA-seq study. We validated the RNA-seq results that CORT and not thyroid hormone induces frzb in the tails using quantitative PCR. Further, maximum frzb expression was achieved by 100-250 nM CORT within 12-24 hours. frzb is not significantly induced in the liver and brain in response to 100 nM CORT. We also found no change in frzb expression across natural metamorphosis when endogenous CORT levels peak. Surprisingly, frzb is only induced by CORT in X. tropicalis tails and not in Xenopus laevis tails. The exact downstream function of increased frzb expression in tails in response to CORT is not known, but the specificity of hormone response and its high mRNA expression levels in the tail render frzb a useful marker of exogenous CORT-response independent of thyroid hormone for exogenous hormone treatments and in-vivo endocrine disruption studies.
FIGURE 1 Evaluation of CORT response in selected genes. Premetamorphic tadpoles at Nieuwkoop and Faber stage 54 (NF54) were treated with vehicle control or 100 nM CORT (corticosterone) for 24 hours followed by tail RNA extraction and quantitative PCR to quantify mRNA expression using gene specific primers for (A) calmodulin binding transcription activator 1 (camta1), (B) frizzled related protein (frzb), (C) GrpE like 1, mitochondrial (grpel1) (D) muscle associated receptor tyrosine kinase (musk), (E) serine dehydratase (sds), (F) sulfotransferase family 6B member 1 (sult6b1), and (G) ubiquitin specific peptidase 2 (usp2). Bars represent mean mRNA levels relative to the housekeeping gene rpl8 and normalized to a vehicle control sample. n = 10 tail samples per treatment. Error bars represent SE. Letters indicate significant groups, p < 0.05.
FIGURE 2 Hormone induction specificity of frzb in tadpole tails. Premetamorphic tadpoles (NF 54) were treated with vehicle, 100 nM CORT (corticosterone), 10 nM T3 (triiodothyronine), and CORT+T3 for 24 h. Tails were dissected, followed by RNA extraction and quantitative PCR for frzb and the housekeeping gene rpl8. Bars represent mean mRNA levels relative to rpl8 and normalized to a vehicle control sample. n = 10 tail samples per treatment. Error bars represent SE. Letters indicate significant groups, p < 0.05.
FIGURE 3 Tissue expression profile of frzb in tadpoles. Premetamorphic tadpoles (NF 54) were treated with vehicle and 100 nM CORT (corticosterone) for 24 hours. Tails (A), livers (B), and brains (C) were dissected, followed by RNA extraction and quantitative PCR to measure mRNA expression of frzb and the housekeeping gene rpl8. Bars represent mean mRNA levels relative to rpl8 and normalized to a vehicle control sample. n = 10 tail samples per treatment. Error bars represent SE. Letters indicate significant groups, p < 0.05.
FIGURE 4 Developmental expression profile of klf9, frzb, and ush1g in tadpole tails throughout metamorphosis. Tails from tadpoles at the indicated NF stages were harvested, followed by tail RNA extraction and quantitative PCR to measure mRNA expression levels of klf9 (A), frzb (B), ush1g (C) and the housekeeping gene rpl8. Bars represent mean mRNA levels relative to rpl8 and normalized to a vehicle control sample. n = 10 tail samples per treatment. Error bars represent SE. Letters indicate significant groups, p < 0.05.
FIGURE 5 Impaired frzb induction in glucocorticoid receptor knockout (GRKO) tails. Premetamorphic wild-type and GRKO tadpoles (NF 54) were treated with vehicle control or 100 nM CORT (corticosterone) for 24 hours followed by tail RNA extraction and quantitative PCR to quantify mRNA expression of frzb and the housekeeping gene rpl8. Bars represent mean mRNA levels relative to rpl8 and normalized to a vehicle control sample. n = 10 tail samples per treatment. Error bars represent SE. Letters indicate significant groups, p < 0.05.
FIGURE 6 Dose response and time course for CORT induction of frzb, ush1g, and klf9 in tadpole tails. (A-C) Premetamorphic tadpoles (NF 54) were treated with vehicle and 50, 100, 250, and 500 nM of CORT (corticosterone) for 24 hours. (D-F) Premetamorphic tadpoles (NF 54) were treated with 100 nM CORT for 0, 3, 6, 12, 24, and 48 hours. Tails were dissected from tadpoles followed by RNA extraction and quantitative PCR to measure mRNA expression of frzb, ush1g, and klf9 and the housekeeping gene rpl8. Bars represent mean mRNA levels relative to rpl8 and normalized to a vehicle control sample. n = 10 tail samples per treatment. Error bars represent SE. Letters indicate significant groups, p < 0.05.
FIGURE 7 Induction of klf9, frzb and ush1g in X. laevis. Premetamorphic (NF 54) X. laevis tadpoles were treated with vehicle control or 100 nM CORT (corticosterone) for 24 hours followed by tail RNA extraction and quantitative PCR to quantify mRNA expression of klf9 (A) , frzb (B), and ush1g (C). Bars represent mean mRNA levels relative to the housekeeping gene rpl8 and normalized to a vehicle control sample. n = 10 tail samples per treatment. Error bars represent SE. Letters indicate significant groups, p < 0.05.
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