Novel zebrafish behavioral assay to identify modifiers of the rapid, nongenomic stress response

Abstract

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response. Rapid changes in heart rate and blood glucose levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic‐pituitary‐adrenal axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent hypothalamic‐pituitary‐adrenal axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires hypothalamic‐pituitary‐interrenal (HPI) axis activation. In teleost fish, interrenal cells are functionally homologous to the adrenocortical layer. We derived eight frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), five in exon 2 or 5 of nr3c1 (glucocorticoid receptor [GR]) and two in exon 2 of nr3c2 (mineralocorticoid receptor [MR]). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show that this locomotor response requires a functioning HPI axis via the action of mc2r and the canonical GR encoded by nr3c1 gene, but not MR ( nr3c2 ). Our rapid behavioral assay paradigm based on HPI axis biology can be used to screen for genetic and environmental modifiers of the hypothalamic‐pituitary‐adrenal axis and to investigate the effects of corticosteroids and their cognate receptor interactions on behavior.

Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity

“…1E). We conclude that the previously described nr3c1 exon 2 frameshift mutations [27][28][29] strongly abrogate but do not abolish the potential to translate a GR with transcriptional function, whereas GR 369eliminates potential for translating a transcriptionally active GR.…”

“…31 ). One potential in-frame initiation codon upstream of the DNA binding domain lies downstream of all previously reported frameshift mutations in exon 2 [27][28][29] (Fig. 1 C,D), suggesting that nr3c1 mRNA transcribed in those mutants might still allow translation of an N-terminally truncated GR isoform with an intact DNA binding domain.…”

“…Thus, the zebrafish homolog of the HPA axis is the Hypothalamus-Pituitary-Interrenal (HPI) axis. Eight different zebrafish GR mutants have been described in the literature to date [26][27][28][29] . The first of these, gr s357 , is a missense mutation that substitutes a cysteine for an arginine in the DNA binding domain, abolishing GR DNA binding activity 26 .…”

“…The first of these, gr s357 , is a missense mutation that substitutes a cysteine for an arginine in the DNA binding domain, abolishing GR DNA binding activity 26 . The other mutations, all produced by targeted mutagenesis using CRISPR-Cas9 or TALEN technology, consist of frameshift indels in exons 2 and 5 [27][28][29] , which respectively encode domains N-and C-terminal to the DNA binding domain (see Fig. 1).…”

“…1D). The N310 mRNA corresponds to the shortest translation initiation variant with a DNA binding domain potentially available to the previously described exon 2 mutants [27][28][29] , whereas the N369 corresponds to what is available in GR 369mutants. The embryos were collected at 6 hours post fertilization (hpf) and quantitative reverse transcription and polymerase chain reaction (qRT-PCR) was used to measure the expression of the GR-target gene fkbp5.…”