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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 tailtissue, 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.
Figure 1.
Genomic analysis of cyp21a2 targeting by CRISPR. A, The CRISPR target site of cyp21a2 is in the eighth exon. The region targeted has been expanded, showing the single-guide RNA binding site (shaded), the exact cut site (black triangle), and the forward and reverse polymerase chain reaction (PCR) primer binding sites (underlined) used to amplify the targeted region. Amino acids are listed below the messenger RNA sequence. E, exon. Black bars at the beginning and end of exon 1 and exon 10 represent the 5′ and 3′ untranslated regions, respectively. B, Sequence analysis of cyp21a2 mutation shows an 11-bp deletion. The wild-type sequence is shown at the top of the panel with the highlighted portion representing the CRISPR target site, while cyp21a2 mutant tadpoles demonstrate an 11-bp deletion at the target site. Amino acid translation is shown below each sequence, and the cyp21a2 mutant sequence predicts a frameshift and early stop codon. The first 373 amino acids are in common followed by amino acids that were changed due to the frameshift mutation. C, Heteroduplex mobility assay (HMA). PCR amplification of wild-type (lane 1), cyp21a2 knockout (lane 2), and cyp21a2 heterozygous (lane 3) tadpole DNA followed by boiling and reannealing of the PCR products results in diagnostic banding patterns on an ethidium bromide-stained polyacrylamide gel electrophoresis gel. The wild-type (180 bp) and knockout (169 bp) bands run at the predicted PCR product sizes, and the addition of heteroduplex bands running more slowly through the gel were expected in the heterozygote sample.
Figure 2.
Delayed growth and development in cyp21a2 mutant tadpoles. A, At 21 days post fertilization (dpf), 56 tadpoles from a cyp21a2 F2 clutch were genotyped and staged according to Nieuwkoop and Faber (NF); there were 12 wild-type (WT, white bars), 30 heterozygous (HET, gray bars), and 14 cyp21a2 mutant (KO, black bars) tadpoles. Sibling tadpoles of each genotype (WT, HET, KO) were reared individually starting at 21 dpf, n = 10 per genotype. B, The average number of days taken to reach NF 58 after fertilization; C, the average number of days taken to reach NF 66 starting from NF 58; and D, the average snout-vent-length (SVL) at NF 58 were recorded. Letters indicate statistically significant differences between bars determined for each genotype based on non-parametric Kruskal-Wallis test followed by pairwise comparisons using Wilcoxon rank sum exact test (P < 0.05). Error bars represent SE. KO, cyp21a2 mutants.
Figure 3.
Reduced corticosterone and aldosterone quantities in cyp21a2 mutants. Tails (∼50 mg each) from genotyped Nieuwkoop and Faber (NF) stage 62 (metamorphic climax) tadpoles were harvested to measure A, corticosterone (CORT) and B, aldosterone (ALDO) from a cyp21a2 clutch and C, CORT and D, ALDO from a pomc clutch via liquid-chromatography tandem mass-spectrometry. White bars, wild-type (WT); black bars, knockout (KO). n = 8-10 tails/genotype. Error bars represent SE. Letters indicate statistically significant groups based on A and C, unpaired t test and B and D, nonparametric Wilcoxon rank sum test; P less than .05.
Figure 4.
Impaired corticosterone (CORT) signaling in cyp21a2 mutants. Total RNA was collected from tails of wild-type (WT, white bars), heterozygous (HET, gray bars), and cyp21a2 mutant (KO, black bars) tadpoles at Nieuwkoop and Faber (NF) 54, 58, and 62 and from brains of tadpoles at NF 62 to analyze messenger RNA (mRNA) expression of A, the CORT-only response gene Usher syndrome 1G (ush1g) in tails; B, CORT and TH response gene Krüppel-like factor 9 (klf9) in tails; and C, klf9 in brains at NF 62. Bars represent mean mRNA levels relative to the housekeeping gene rpl8. Error bars represent SE. Letters indicate statistically significant differences between bars determined for each genotype by nonparametric Kruskal-Wallis test followed by pairwise comparisons using Wilcoxon rank sum exact test for. P less than .05. n = 8-10 tails or brains per genotype per stage.
Figure 5.
cyp21a2 mutants have a hyperactive hypothalamus-pituitary-interrenal (HPI) axis. Total RNA was collected from brain and kidney of wild-type (WT, white bars), heterozygous (HET, gray bars), and cyp21a2 mutant (KO, black bars) tadpoles at Nieuwkoop and Faber (NF) 62 to analyze messenger RNA (mRNA) expression of A, corticotropin-releasing hormone (crh); B, proopiomelanocortin (pomc); C, steroidogenic acute regulatory protein (star); and D, aldosterone synthase (cyp11b2). Bars represent mean mRNA levels relative to the housekeeping gene rpl8. n = 8-10 brain and kidney samples per genotype. Error bars represent SE. Letters indicate statistically significant differences between bars determined for each genotype based on nonparametric Kruskal-Wallis test followed by pairwise comparisons using Wilcoxon rank sum exact test. P less than .05.
Figure 6.
Quantification of progesterone and cortisol in cyp21a2 mutant and pomc mutant tadpoles. Tails (∼50 mg each) from genotyped Nieuwkoop and Faber (NF) stage 62 (metamorphic climax) tadpoles were harvested to measure A, progesterone and B, cortisol from a cyp21a2 clutch and C, progesterone and D, cortisol from a pomc clutch via liquid-chromatography tandem mass-spectrometry. White bars, wild-type (WT); black bars, knockout (KO). n = 8-10 tails/genotype. Error bars represent SE. Letters indicate statistically significant groups based on B and D, unpaired t test and A and C, nonparametric Wilcoxon rank sum text; P less than .05.
Figure 7.
Transactivation of corticosterone (CORT) response gene by progesterone through glucocorticoid receptor (GR). GR knockout (GRKO) or wild-type (WT) Nieuwkoop and Faber (NF) 54 tadpole tails were treated in vitro with vehicle, 100 nM CORT, or 500 nM progesterone (PROG) for 24 hours, and tails were processed for RNA extraction and quantitative polymerase chain reaction. Messenger RNA (mRNA) expression levels of klf9 were measured relative to the normalizing gene rpl8. n = 6 per genotype per treatment. Error bars represent SE. Expression levels were analyzed across genotypes and treatment groups. Letters above bars represent statistically significant groups based on nonparametric Kruskal-Wallis test followed by pairwise comparisons using Wilcoxon rank sum exact test; P less than .05.
