Principal function of mineralocorticoid signaling suggested by constitutive knockout of the mineralocorticoid receptor in medaka fish

Abstract

As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na⁺ reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper- and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se. We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling.

Figure 1. Predominant MR Expression in Brain and Eyes.

(a) Expression of MR in tissues of adult (200 days post-fertilization) medaka determined by qPCR. Data are shown as mean ± SEM (n = 5–8 in each group). Each fish was analyzed in triplicate. (b) Photomicrographs and schematics of frontal sections of the medaka telencephalon (b1), diencephalon (b2) and hindbrain (b3), showing relatively dense (stipples) regions of MRir neuronal cell bodies. (b2’) In situ hybridization of the consecutive section using mr mRNA probe, showing co-localization of the signal in MRir neurons. Abbreviations: CCe, corpus cerebelli; D, dorsal telencephalic area; Dl, lateral zone of D; Dm, medial zone of D; TeO, tectum opticum; TL, torus longitudinalis; V, ventral telencephalic area. Scale bar: 30 μm. (c) Frontal section of medaka larvae showing MRir regions (red) in the brain and retina with nuclear staining (blue, DAPI). The larvae at 7 dpf was analyzed, since MR transcripts continuously increased 4-fold between 3 and 7 dpf, and remained at this level at 9 dpf (immediately after hatch) during early development (Supplementary Fig. 1). Strong MRir signals were observed in the nuclear layer and ganglion cell layer of the retina, as well as the choroid. ONL, outer nuclear layer (photoreceptor layer); OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, retinal ganglion cell layer. Scale bar: 50 μm.

Figure 2. TALEN design and results of indel mutations in medaka mr.

Alignment of WT mr sequence with mutated PCR amplicons. A pair of TALEN was designed near the start ATG codon. The target sequences of the TAL effector DNA-binding domains are highlighted in red. Δ2, Δ11 and Δ13 alleles with 2–11- and 13-nucleotide deletions, respectively.

Figure 3. Muscle water content in MR-KO adult medaka in fresh water and after seawater transfer.

MR-KO was confirmed by fin biopsy genotyping. Data are shown as mean + SEM (n = 7). There was no significant difference for each time period (P > 0.05).

Figure 4. Schematic of the experimental set-up for the behavioral test.

Stimuli were presented within an area of 615 × 72 pixels (170 × 20 mm²) located in the center of a 24-inch liquid-crystal display with a refresh rate of 60 Hz and resolution of 1,920 × 1080 pixels, and were controlled by computer software. The tip of the head of the fish and the dot was tracked using computer software. The distance to the dot and acceleration of the fish were analyzed.

Figure 5. Abnormal responses to visual motion stimuli in MR-KO medaka.

Naive fish were used in the behavioral test (n = 6). (a) Distance to the stimulus dot from the head and trajectory of a representative WT fish during stimulus presentation. (b) Distance to the dot from the head and trajectory of a representative MR-KO medaka during stimulus presentation. (c) Averaged distance from the head to the stimulus dot. (d) Acceleration during observation. The first 30 s at baseline (left) and the first 15 s and last 2.25 min of the stimulus presentation period (right) are not included. The WT fish (upper) swam at approximately constant speed, whereas the MR-KO fish (lower) swam in spurts during stimulus presentation. (e) Acceleration averaged during periods at baseline (before) and in stimulus presentation (after). The MR-KO fish swam significantly more than the WT fish during stimulus presentation, but the acceleration before stimulus presentation is similar in the two groups. Error bars show ± SEM.