Glucocorticoid receptor is required for survival through metamorphosis in the frog Xenopus tropicalis.

Stress hormones, also known as glucocorticoids, are critical for survival at birth in mammals due at least in part to their importance in lung maturation. However, because air breathing is not always required for amphibian survival and because stress hormones have no known developmental impact except to modulate the developmental actions of thyroid hormone (TH), the requirement for stress hormone signaling during metamorphosis is not well understoodi. Here, we produced a glucocorticoid receptor knockout (GRKO) Xenopus line with a frameshift mutation in the first exon of the glucocorticoid receptor. Induction by exogenous corticosterone (CORT, the frog stress hormone) of the CORT response genes, klf9 (Krüppel-like factor 9, also regulated by TH) and ush1g (Usher's syndrome 1G), was completely abrogated in GRKO tadpoles. Surprisingly, GRKO tadpoles developed faster than wild-type tadpoles until forelimb emergence and then developed more slowly until their death at the climax of metamorphosis. Growth rate was not affected in GRKO tadpoles, but they achieved a smaller maximum size. Gene expression analysis of the TH response genes, thrb (TH receptor beta) and klf9 showed reduced expression in the tail at metamorphic climax consistent with the reduced development rate. These results indicate that glucocorticoid receptor is required for survival through metamorphosis and support dual roles for GR signaling in control of developmental rate.

A novel stress hormone response gene in tadpoles of Xenopus tropicalis.

Previous work identified a transcribed locus, Str. 34945, induced by the frog stress hormone corticosterone (CORT) in Xenopus tropicalis tails. Because thyroid hormone had no influence on its expression, Str. 34945 was dubbed the first "CORT-only" gene known from tadpoles. Here, we examine the genomic annotation for this transcript, hormone specificity, time course of induction, tissue distribution, and developmental expression profile. The location of Str. 34945 on the X. tropicalis genome lies between the genes ush1g (Usher syndrome 1G) and fads6 (fatty acid desaturase 6). A blast search showed that it maps to the same region on the X. laevis genome, but no hits were found in the human genome. Using RNA-seq data and conventional reverse transcriptase PCR and sequencing, we show that Str. 34945 is part of the 3' untranslated region of ush1g. We find that CORT but not aldosterone or thyroid hormone treatment induces Str. 34945 in tadpole tails and that expression of Str. 34945 achieves maximal expression within 12-24 h of CORT treatment. Among tissues, Str. 34945 is induced to the highest degree in tail, with lesser induction in lungs, liver, and heart, and no induction in the brain or kidney. During natural metamorphosis, Str. 34945 expression in tails peaks at metamorphic climax. The role of ush1g in metamorphosis is not understood, but the specificity of its hormone response and its expression in tail make ush1g valuable as a marker of CORT-response gene induction independent of thyroid hormone.

In-vivo regulation of Krüppel-like factor 9 by corticosteroids and their receptors across tissues in tadpoles of Xenopus tropicalis.

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.

cyp21a2 Knockout Tadpoles Survive Metamorphosis Despite Low Corticosterone.

Corticosteroids are so vital for organ maturation that reduced corticosteroid signaling during postembryonic development causes death in terrestrial vertebrates. Indeed, death occurs at metamorphosis in frogs lacking proopiomelanocortin (pomc) or the glucocorticoid receptor (GR; nr3c1). Some residual corticosteroids exist in pomc mutants to activate the wild-type (WT) GR and mineralocorticoid receptor (MR), and the elevated corticosteroids in GR mutants may activate MR. Thus, we expected a more severe developmental phenotype in tadpoles with inactivation of 21-hydroxylase, which should eliminate all interrenal corticosteroid biosynthesis. Using CRISPR/Cas9 in Xenopus tropicalis, we produced an 11-base pair deletion in cyp21a2, the gene encoding 21-hydroxylase. Growth and development were delayed in cyp21a2 mutant tadpoles, but unlike the other frog models, they survived metamorphosis. Consistent with an absence of 21-hydroxylase, mutant tadpoles had a 95% reduction of aldosterone in tail tissue, but they retained some corticosterone (∼40% of WT siblings), an amount, however, too low for survival in pomc mutants. Decreased corticosteroid signaling was evidenced by reduced expression of corticosteroid-response gene, klf9, and by impaired negative feedback in the hypothalamus-pituitary-interrenal axis with higher messenger RNA expression levels of crh, pomc, star, and cyp11b2 and an approximately 30-fold increase in tail content of progesterone. In vitro tail-tip culture showed that progesterone can transactivate the frog GR. The inadequate activation of GR by corticosterone in cyp21a2 mutants was likely compensated for by sufficient corticosteroid signaling from other GR ligands to allow survival through the developmental transition from aquatic to terrestrial life.

Corticosterone Is Essential for Survival Through Frog Metamorphosis.

Thyroid hormone (TH) is required for frog metamorphosis, and corticosterone (CORT) increases TH signaling to accelerate metamorphic progression. However, a requirement for CORT in metamorphosis has been difficult to assess prior to the recent development of gene-editing technologies. We addressed this long-standing question using transcription activator-like effector nuclease (TALEN) gene disruption to knock out proopiomelanocortin (pomc) and disrupt CORT production in Xenopus tropicalis. As expected, mutant tadpoles had a reduced peak of plasma CORT at metamorphosis with correspondingly reduced expression of the CORT-response gene Usher syndrome type-1G (ush1g). Mutants had reduced rates of growth and development and exhibited lower expression levels of 2 TH response genes, Krüppel-like factor 9 (klf9) and TH receptor ß (thrb). In response to exogenous TH, mutants had reduced TH response gene induction and slower morphological change. Importantly, death invariably occurred during tail resorption, unless rescued by exogenous CORT and, remarkably, by exogenous TH. The ability of exogenous TH by itself to overcome death in pomc mutants indicates that the CORT-dependent increase in TH signaling may ensure functional organ transformation required for survival through metamorphosis and/or may shorten the nonfeeding metamorphic transition to avoid lethal inanition.

Xenopus Tadpole Tissue Harvest.

The procedures described here apply to Xenopus tadpoles from the beginning of feeding through the major changes of metamorphosis and are appropriate for downstream postoperative snap freezing for molecular analysis, fixation for histological analysis, and sterile organ culture. To the uninitiated, the most difficult aspects of tadpole tissue dissections are likely knowing the appearance and location of organs, and the difficulty manipulating and holding tadpoles in place to carry out the oftentimes fine and precise dissections. Therefore, images and stepwise instructions are given for the harvest of external organs (tail, head, eyes, tail skin, back skin, gills, thymus, hind limbs, forelimbs) and peritoneal organs (intestine, pancreas, liver, spleen, lungs, fat bodies, kidney/gonad complex), as well as brain, heart, and blood. Dissections are typically done under a dissection stereomicroscope, and two pairs of fine straight forceps, one pair of fine curved forceps, and one pair of microdissection scissors are sufficient for most tissue harvests.