Fish husbandry and sampling

The short-lived turquoise killifish strain (GRZ-AD) was used. Fish were maintained as described previously (Dodzian et al. 2018). The fish room was kept on a 12/12 h light/dark cycle. Fish were fed twice each day, one and eight hours after lights on every day.

Every fish in this study was euthanized by supplying 1.5 g/L of ethyl 3-aminobenzoate methanesulfonate (MS-222, E10521, Sigma-Aldrich) to the fish tank water. When fish gill movement ceased, dissections were performed.

The animal husbandry and experiments in this study were carried out in accordance with the animal care and use protocol that is reviewed and approved by the Institutional Animal Care and Use Committee at Daegu Gyeongbuk Institute of Science and Technology, Republic of Korea (approval number: DGIST-IACUC-17103001-00).

Gross anatomy of the turquoise killifish brain

The whole fish body of young adult fish (8-10 weeks old, fourteen female and male fish) were fixed in 4% paraformaldehyde (PFA) overnight. Fish heads were carefully dissected under a stereomicroscope. Brain images were acquired in a 4% PFA solution using a stereomicroscope (M80, Leica Microsystems).

Nissl staining of killifish brain sections

For gross anatomy analysis, fixed brains were embedded in 2.5% agarose and sectioned with a Vibratome (VT1200S, Leica) at a thickness of 100 µm. Sections were transferred onto slide glasses and dried for 10 minutes. Sections were stained for five minutes in Nissl stain solution (0.1% cresyl violet (Sigma C-1971), 0.1% glacial acetic acid) and rinsed in distilled water for five minutes. Subsequently, sections were dehydrated in an ethanol series (50%→70%→95%) and destained in 100% ethanol until the sections exhibited the desired amount of staining. Stained sections were mounted with mounting medium (Fisher Chemical^™ Permount^™ Mounting Medium, SP15-100; Fisher Scientific). Section images were acquired using a stereomicroscope (M80, Leica Microsystems).

Fig. 1.

Gross anatomy of the turquoise killifish. Yellow and red arrows indicate pineal and pituitary glands, respectively.

Fig. 2.

Cross-section of the brain containing the pineal and pituitary glands: a-d) sections depict the epiphysis on the dorsal side of the brain; e-h) sections depict the hypophysis on the ventral side of the brain. Section numbers are linked with supplemental figures. Abbreviations of each figure can be found in the glossary of brain sections in Table S1.

Whole brain immunostaining

The brains of nine six-week-old, sexually mature males were carefully dissected under a microscope and then fixed in 4% PFA overnight. Whole brains were cleared using a tissue immunostaining solution (Binaree Immuno Staining^™ Kit for Brain, BINAREE), according to the manufacturer's protocol. Cleared brains were stained with DAPI (D9542, Sigma-Aldrich) and GFAP-Alexa 647 (ab194325, abcam), Vimentin-Alexa 647 (ab195878, abcam), or VIP-Alexa 647 (bs-0077R-A647, Bioss) antibodies. The killifish glial fibrillary acidic protein (GFAP), Vimentin, and vasopressin intestinal peptide (VIP) proteins showed 69%, 68%, and 46% of amino acid sequence identity, respectively, with those of human. Antibody specificity was tested using gel blot analysis (Fig. S1). Cleared and stained brains were embedded in 2% low-melting-point agarose in capillaries with an inner diameter of 2 mm. The embedded brains were tiled into 9-30 regions to cover the whole brain and imaged using a 20 × objective lens (W Plan-APOCHROMAT 20, Zeiss) on a Light Sheet microscope (Lightsheet Z.1, Zeiss). Stacked images were converted (Imaris File Converter × 64.9.2, Bitplane) and displayed using Imaris (Imaris × 64 7.6.0, Bitplane).

The gross brain anatomy of the turquoise killifish revealing weakly-attached subregions in the dorsal and ventral parts of the brain

The gross brain anatomy of the turquoise killifish brain has been reported previously (D‘Angelo 2013). The turquoise killifish brain comprises the 1) olfactory bulbs, 2) telencephalon, 3) diencephalon, 4) optic tectum, 5) cerebellum, and 6) rhombencephalon. In addition to the six main parts, we found two additional bulging structures in the ventral and dorsal regions of the turquoise killifish brain (Fig. 1). The two bulging organs, in particular the ventral one, were weakly attached to the main body of the brain and were thus easy to lose during dissection and further processing. One of the bulging organs was located in the dorsal brain nearest to the telencephalon and between the optic tectum, measuring approximately 0.4 mm in diameter. The other bulging organ was located in the ventral brain at the anterior part of the hypothalamus and caudal to the optic nerves, measuring approximately 0.7 mm in diameter. In comparison with the brain anatomy of the zebrafish and medaka, these additional bulging organs appeared to correlate with the pineal and pituitary glands. Similar to the dorsal bulging organ of the killifish, the pineal gland of the zebrafish and medaka is located between the telencephalic area and the optic tectum of the dorsal brain. The pituitary gland of the zebrafish and medaka is attached to the ventral brain near the hypothalamus, similar to the second bulging organ observed in the killifish ventral brain. Therefore, the location of the killifish brain hypophysis is similar to the position of that in the medaka.

Characterization of the neuroendocrine organs using Nissl staining

To further characterize the pineal and pituitary glands, a whole killifish brain was sectioned and stained with Cresyl violet (Nissl staining). The internal structures of the brain sections were re-identified based on previous descriptions of N. furzeri (D‘Angelo 2013), medaka (Ralph Anken 1998), and zebrafish (Wullimann et al. 1996) brain anatomies (Table S1). GRZ-AD, a strain showing the shortest lifespan (median lifespan under our laboratory conditions, 16 weeks), was used for coronal sectioning of the brain (in rostral to caudal order). Intensive staining revealed that the pineal gland was located at the end of the telencephalon and extended to the beginning of the optic tectum. Sections of the pineal gland showed that it had a rounded triangular shape at its anterior end (Fig. 2a, b) and that it became more circular (Fig. 2c) and rod-shaped toward its posterior end (Fig. 2c). At the location of the distal pineal gland, the pituitary started to emerge and was observed as a separate, flat, and rounded shape. Similar to the pineal gland, the pituitary gland was more strongly stained with Cresyl violet than other regions of the brain (Fig. 2c). The pituitary bridged the left and right parts of the anterior hypothalamic regions in the diencephalon (Fig. 2d-h). The pineal and pituitary glands of the killifish brain could be viewed in one section depending on the sectioning angle (Fig. S2). The other parts of the killifish brain, including olfactory bulbs, telencephalon, the remaining optic tectum, cerebellum, and rhombencephalon, are annotated in an illustration (Fig. S3, S4; Table S1).

Anatomy of the turquoise killifish pituitary gland

A 3D imaging method was used to further analyse the detailed structures of the two neuroendocrine glands in the killifish brain. After increasing the transparency of the whole brain tissue, it was stained with DAPI to observe its intact cell arrangements and the internal structures of the pineal and pituitary glands. As a master gland of the endocrine system, the pituitary gland resembled a flattened sphere covered with blood vessels at the ventral part of the brain (Fig. 3a, Movie S1). To compare the internal anatomy of the killifish pituitary gland with those of other teleost fish, cell densities were compared from mid-sagittal optical sections. The pituitary gland of turquoise killifish exhibited asymmetric differences in cell density, with the anterior region being more compact than the posterior region. The anterior pituitary of the killifish correlated with the RPD, whereas the posterior hypophysis correlated with the PPD, PN, and PI (Fig. 3b). The RPD occupied approximately one third of the volume of the killifish pituitary, and cells in this area were arranged compactly. The PPD was located in the ventral pituitary near to the RPD, PN, and PI. The PN was located in the dorsal pituitary and was surrounded by the RPD, PPD, and PI. Cells in the PN formed clusters surrounded by blood vessels. Additionally, the PN was connected to the hypothalamus as a “Y-shaped” structure in the turquoise killifish brain (Fig. 3c), and a small rounded cavity was observed between the PN and hypothalamus. The PI was positioned in the ventral pituitary. The surface of the pituitary was covered by blood vessels, especially the posterior part of the pituitary gland (Fig. 3a). We conducted further analysis by staining with a vasopressin intestinal peptide (VIP) antibody because VIP regulates the release of PRL and growth hormone (Sherwood et al. 2000, Kulick et al. 2005). Interestingly, the prolactin-releasing cells are known to be located in the RPD of the medaka (Aoki & Umeura 1970). VIP staining was mostly detected in the PN. Blood vessels partly covered the RPD of the hypophysis (Fig. 3d, Movie S2).

Fig. 3.

Anatomy of the turquoise killifish pituitary gland. a) Three-dimensional features of the killifish hypophysis. The transparent killifish brain was stained with DAPI, a nuclear dye. b) A mid-sagittal section and illustration defining the internal structure of the killifish hypophysis. c) A coronal section of the killifish hypophysis. Rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI), and pars nervosa (PN). d) Mid-sagittal and coronal sections of the VIP-stained hypophysis. Red arrows indicate the position of the brain; the letters A and P are abbreviations for anterior and posterior, respectively.

Anatomy of the turquoise killifish pineal gland

The other important neuroendocrine organ in the brain is the pineal gland. The killifish pineal gland protruded out from the brain and had a spherical shape, and its tail extended into the middle of the telencephalon (Fig. 4a, Movie S3). A coronal section of the pineal gland contained dozens of elliptically-shaped cell arrays (Fig. 4a). These globular cell arrays were covered by another layer of cells that were involuted inwards to surround the cell arrays, starting from about the middle of the pineal gland and forming an inverted and tailed pocket shape. This outer cell layer section of the pineal gland was located nearest to the telencephalon and the head of the pineal gland located with optic tectum and habenula (Fig. 4a).

Fig. 4.

Anatomy of the turquoise killifish epiphysis. a) Sections of the killifish epiphysis stained with DAPI; b) Sections of the killifish epiphysis stained with Vimentin; c) Sections of the killifish epiphysis stained with GFAP. Red arrows indicate position of the brain; the capital letters A, P, D, and V are abbreviations for anterior, posterior, dorsal, and ventral, respectively.

Table 1.

Comparison of anatomical characteristics of the neuroendocrine glands of turquoise killifish, medaka, and zebrafish.

The mammalian pineal gland consists mainly of pinealocytes, as well as astrocytes and microglia (Moller & Baeres 2002, Jiang-Shieh et al. 2003, Ibanez Rodriguez et al. 2016). To identify microglia and astrocytes, we used the characteristic markers vimentin and glial fibrillary acidic protein (GFAP), respectively (Graeber et al. 1988, Wohl et al. 2011, Zhang et al. 2019). Both vimentin and GFAP were expressed in the killifish pineal gland (Fig. 4b, 4c). Vimentin was detected specifically in the proximal region of the pineal gland (Fig. 4b, Movie S4), whereas GFAP localized preferentially to the outer cell layer of the pineal gland, covering half of the globular cell arrays (Fig. 4c, Movie S5).