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Gen Comp Endocrinol
2020 May 15;291:113419. doi: 10.1016/j.ygcen.2020.113419.
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Glucocorticoid receptor is required for survival through metamorphosis in the frog Xenopus tropicalis.
Sterner ZR
,
Shewade LH
,
Mertz KM
,
Sturgeon SM
,
Buchholz DR
.
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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.
Fig. 1. Genomic analysis of GR targeting by CRISPR A) The CRISPR target site of GR is in the first exon of 8 exons. Black bars at the start and end of the transcript represent 3′ and 5′ untranslated regions (UTRs). The region targeted has been expanded, showing the gRNA binding site (highlighted), the exact cut site (black triangle), and the forward and reverse PCR primer binding sites (underlined) used to amplify the targeted region. Amino acids are listed below the mRNA sequence. E = exon. DBD = DNA binding domain. LBD = ligand binding domain. B) Sequence analysis of GR mutation shows a 10-bp deletion. Computer generated translation is shown below each sequence. The wild-type sequence is shown at the top of the panel with the highlighted portion representing the CRISPR target site, while GRKO tadpoles demonstrate a 10-bp deletion within the target site. C) Computer translation of the wild-type and mutant GR sequences predicts a truncation mutation eliminating half of the N-terminal region and all of the the DNA-binding and the ligand-binding domains. The first 168 amino acids are in common followed by amino acids that were changed due to the frameshift mutation. NTD = N-terminal Domain. DBD = DNA-binding domain. H = Hinge Region. LBD = Ligand-binding domain. GRKO = Glucocorticoid receptor knockout.
Fig. 2. Heteroduplex mobility assay (HMA). PCR amplification of wild-type (lane 1), GRKO (lane 2), and heterozygous (lane 3) tadpole DNA followed by boiling and reannealing of the PCR products results in diagnostic banding patterns on an ethidium bromide-stained gel. The lower bands run at the predicted PCR product sizes, and heteroduplex bands running more slowly through the gel were expected in the heterozygous sample. The identity of the upper bands in the wild-type and GRKO lanes are unknown.
Fig. 3. GR expression during natural metamorphosis. Tails were harvested from wild-type, heterozygous, and GRKO individuals reared to NF 54, NF 58, and NF 61 for gene expression analysis of GR. GR expression levels were normalized to rpl8 and analyzed utilizing ANOVA. Letters above each bar represent significance groups based on Tukey’s honest significant difference test (p < 0.05). n = 5 per genotype. Error bars represent standard error. WT = Wild-type. Het = Heterozygous. GRKO = Glucocorticoid receptor knockout.
Fig. 4. Impaired CORT-response gene induction by exogenous CORT. Wild-type, heterozygous, and GRKO individuals at NF 54 were treated with vehicle, 100 nM CORT, or 500 nM CORT for 24 h, and tails were harvested for RNA extraction and quantitative PCR. mRNA expression levels relative to the normalizing gene rpl8 of the CORT-response genes (A) klf9 and (B) ush1g show lack of induction for both genes in both heterozygotes and GRKO animals. n = 5 per genotype per treatment. Expression levels were analyzed across genotypes and treatment groups. Letters above bars represent significance groups based on Tukey’s honest significant difference test (p < 0.05). Error bars represent standard error. WT = Wild-type. Het = Heterozygous. GRKO = Glucocorticoid receptor knockout.
Fig. 5. Lethality of GR mutation. Twenty GRKO individuals were genotyped at NF 50-53 and individually reared through larval development. The histogram of NF stage at death of GRKO individuals reveals that mutant tadpoles all died, predominantly at metamorphic climax (NF 62-63) when TH and CORT levels are highest in wild-type individuals and gill resorption is occurring.
Fig. 6. Growth and development anomalies in GRKO tadpoles. Sibling tadpoles of each genotype, (wild-type, heterozygotes, GRKO) were reared individually starting at NF 54 (just before metamorphosis begins), and (A) developmental stage and (B) tadpole snout-vent length (SVL) were recorded every 5 days. Also, (C) day of forelimb emergence (NF 58) and (D) start of gill resorption (NF 61) was recorded for each tadpole. (A, B) Circled shapes represent significance groups based on Tukey’s honest significant difference test (p < 0.05) assessed for each Day. (C, D) Letters above bars represent significance groups based on Tukey’s honest significant difference test (p < 0.05). n = 10 per genotype. Error bars represent standard error. WT = Wild-type. Het = Heterozygous. GRKO = Glucocorticoid receptor knockout.
Fig. 7. Impaired TH-response gene expression during natural metamorphosis. Sibling wild-type, heterozygous, and GFKO tadpoles were reared to NF 54, NF 58, or NF 61, and their tails were harvested for RNA extraction and quantitative PCR. mRNA expression levels relative to the normalizing gene rpl8 of (A) thrb, a TH-response gene (B) klf9, a gene induced by TH and/or CORT, and (C) ush1g, a CORT-response gene shows reduced TH-response gene expression at NF 61. For NF 54, n = 5 per genotype. For NF 58 and NF 61, n = 10 per genotype. Letters above each bar represent significance groups based on Tukey’s honest significant difference test (p < 0.05). Error bars represent standard error. WT = Wild-type. Het = Heterozygous. GRKO = Glucocorticoid receptor knockout.
