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Gen Comp Endocrinol
2017 Jul 01;248:79-86. doi: 10.1016/j.ygcen.2017.02.007.
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In-vivo regulation of Krüppel-like factor 9 by corticosteroids and their receptors across tissues in tadpoles of Xenopus tropicalis.
Shewade LH
,
Schneider KA
,
Brown AC
,
Buchholz DR
.
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Corticosteroids are critical for normal development and for mediating effects of stress during development in all vertebrates. Even though gene knockout studies in mouse and zebrafish have identified a number of developmental roles of corticosteroids and their receptors, the numerous pleiotropic actions of these hormones affecting various aspects of development are understudied. For the most part, neither the endogenous hormone(s) nor their receptor(s) regulating developmental processes during natural development have been determined. Here, we address this issue by elucidating the endogenous regulation of the transcription factor Krüppel-like factor 9 (klf9) across tissues during development by corticosteroid hormones (aldosterone and corticosterone) and their nuclear receptors (type-I and type-II receptors). First, we measured the developmental expression profiles of klf9 and type-I and type-II corticosteroid receptors in key target tissues, brain, lungs, and tail, during larval and metamorphic stages in Xenopus tropicalis. We also studied the corticosteroid regulation of klf9 in these tissues in-vivo using exogenous hormone treatments and receptor antagonists. Klf9 and the corticosteroid receptors were expressed in each tissue and significantly increased in expression reaching a peak at metamorphic climax, except for the type-II receptor in brain and tail whose expression did not change significantly across stages. Both corticosteroid hormones induced klf9 in each tissue, although aldosterone required a five times higher dose than corticosterone to cause a significant induction. The upregulation of klf9 by both corticosteroids was completely blocked by the use of the type-II receptor antagonist RU486 and not the type-I receptor antagonist spironolactone. These results are consistent with previous in-vitro studies and indicate for the first time in-vivo that corticosteroid regulation of klf9 occurs exclusively via corticosterone and type-II receptor interaction across tissues.
Fig. 1. Developmental profile of klf9 expression in the brain, lungs, and tail during Xenopus metamorphosis. The expression of klf9 increased significantly during metamorphosis in each tissue. Total RNA was collected from tadpole tissues at the indicated developmental stages to measure klf9 mRNA expression by quantitative PCR. Bars show the mean mRNA levels relative to the reference gene rpL8. Error bars represent SEM. The letters above the error bars indicate significance groups among stages based on Tukey’s honest significant difference test (p < 0.05, n = 5 for brain and tails and n = 3 for lungs per stage).
Fig. 2. Developmental profiles of type-I and type-II corticosteroid receptor expression in the brain, lungs, and tail during Xenopus metamorphosis. Expression of both receptors was detectable in each tissue at each stage and increased significantly during metamorphic climax in most cases. Total RNA was extracted from tadpole tissues at the indicated developmental stages to measure receptor mRNA expression by quantitative PCR. Bars show the mean mRNA levels relative to the reference gene rpL8. Error bars indicate SEM. The letters above the error bars indicate significance groups among stages based on Tukey’s honest significant difference test (p < 0.05, n = 5 for brain and tail and n = 3 for lungs per stage). No letters above bars indicates a lack of significant difference among stages within the tissue.
Fig. 3. Dose response of klf9 expression after CORT and ALDO treatment in tadpole tails. Both CORT and ALDO induced klf9 expression in the tail but with different dose response curves. Total RNA was extracted from tails harvested from premetmorphic tadpoles (NF54) treated with 100 and 500 nM of CORT and ALDO or vehicle controls for 24 h to measure Klf9 mRNA expression by quantitative PCR. Bars show mean mRNA levels relative to the reference gene rpL8. Error bars indicate SEM. The letters above the error bars indicate significance groups among treatments based on Tukey’s honest significant difference test (p < 0.05, n = 5 for brain and tails and n = 3 for lungs per treatment group).
Fig. 4. Time course profile of klf9 induction by CORT for brain, lungs, and tail. The expression of klf9 peaked at 4 h in brain and lungs and at 24 h in the tail. Total RNA was extracted from tissues harvested from premetmorphic tadpoles (NF54) treated with 100 CORT or vehicle controls at the indicated time points to measure Klf9 mRNA expression by quantitative PCR. Bars show mean mRNA levels relative to the reference gene rpL8. Error bars indicate SEM. The letters above the error bars indicate significance groups among treatments based on Tukey’s honest significant difference test (p < 0.05, n = 5 tails per treatment group).
Fig. 5. Effect of receptor antagonists on CORT-induced klf9 expression in brain, lungs, and tail. CORT-induced klf9 expression was completely suppressed by type-II receptor antagonist but not by type-I receptor antagonist in each tissue. Tadpoles were raised to NF54 and treated with 100 nM CORT, 150 nM RU486, 150 nM spironolactone (SL), and EtOH control as indicated before tissue harvest at 4 h (brain and lungs) and 24 h (tails). Total RNA was isolated from brain, lungs, and tail, and klf9 mRNA levels were measured using quantitative PCR and normalized to the reference gene rpL8. Error bars indicate SEM. The letters above the error bars indicate significance groups among treatments based on Tukey’s honest significant difference test (p < 0.05, n = 5 for brain and tails and n = 3 for lungs per treatment group).
Fig. 6. Effect of receptor antagonists on ALDO-induced klf9 expression in tails. As for CORT, ALDO-induced klf9 expression was completely suppressed by type-II receptor antagonist but not by type-I receptor antagonist. Tadpoles were raised to NF54 and treated with 100 nM ALDO, 150 nM RU486, 150 nM spironolactone (SL), and EtOH control as indicated before tail harvest at 24 h. Total RNA was isolated from tails, and klf9 mRNA levels were measured using quantitative PCR and normalized to the reference gene rpL8. Error bars indicate SEM. The letters above the error bars indicate significance groups among treatments based on Tukey’s honest significant difference test (p < 0.05, n = 5 tails per treatment group).
