We maintain an orange-red medaka stock culture at INIA-CSIC, housed in flow-through Plexiglas aquaria within a recirculating system. This system uses charcoal filtrated, UV-treated and dechlorinated local tap water (City of Madrid, Madrid province). Fish are reared under a 14L: 10D (light: dark) photoperiod cycle at 25 ± 3°C, with a 2-3°C decrease in temperature during the dark period to simulate nocturnal temperature variations. The light intensity at the water surface is maintained between 500-800 lux. Water quality parameters are controlled, with CaCO3 hardness maintained at 60-90 mg/L, alkalinity at CaCO3 70-80 mg/L, pH at 7.2-7.8, and dissolved oxygen at 7.0 ± 0.5 mg/L. Ammonia and nitrite levels are always below detectable limits (< 0.1 mg/L), and nitrate levels are kept below 40 mg/L by exchanging 20% of the system water monthly. Fish are fed via automatic feeders with a blend of commercial diet and bluish-green filamentous algae (spirulina) at the 3:2 ratio. This diet is supplemented manually once or twice every week with fresh brine shrimp (Artemia salina). The feeders deliver food eight times per day with a daily regime of 5-10 % body weight. Under these conditions, female medakas spawn daily during the first few hours of light.
To obtain synchronized-aged embryos for histological analysis, eggs clusters were collected directly from females 2-4 h after the start of the daylight cycle. Individual fertilized eggs were cleaned with system water and transferred to laboratory glass dishes containing the same system water for incubation at 25°C and 14 L: 10 D. Approximately 80-90% of the incubation water was renewed daily. Under these conditions embryos hatch in around 10 days or so. Resultant larvae, which were capable of independent swimming and food capture, were transferred to individual aquaria in the recirculating system. For the first seven days, larvae were fed spirulina
ad libitum, after which feeding conditions followed those described above for the colony.
Fish were collected at specific developmental time points (see Figure 1) for histological analysis. Once collected, fish were overdosed with tricaine methanesulfonate, rinsed in dechlorinated tap water, examined grossly, and some were photographed for reference purposes. The number of fish collected at each time point is detailed in Figure 1. For earlier developmental periods (i.e. 2, 5, 10, 15 and 30), more fish were surveyed to ensure proper positioning during embedding, as these stages are more delicate. For later stages (i.e. 45, 60 and 110), fewer fish were required.
Figure 1. Schematic representation showing the chronological survey with the eight time points selected and the number of fish surveyed for the histological atlas of the medaka post-hatch development. The medaka drawings were obtained from Iwamatsu, 2004 (https://doi.org/10.1016/j.mod.2004.03.012).
Whole fish were blotted dry and fixed in a fixative mixture of picric acid saturated in methanol, formol and acetic acid (75:25:5 ratio). Fish longer than 20 mm were cut at the caudal-most region to aid fixative diffusion. Specimens were kept in fixative for 24-48 hours at room temperature to prevent crystallization of the picric acid. The dehydration process involved a series of sequential incubations in increasing grades of ethanol as follows:
- 80% ethanol, 4 h, orbital shaking (4ºC)
- 96% ethanol, 2 h, orbital shaking (4ºC)
- 96% ethanol, 2 h, orbital shaking (4ºC)
- 100% ethanol, overnight, orbital shaking (4ºC)
- 100% ethanol, 1 h, orbital shaking (4ºC)
- 100% ethanol, 1 h, orbital shaking (4ºC)
Dehydration was followed by a clearing stage in “Clear-rite 3” in the following sequence:
- Clear-rite 3, 1 h, orbital shaking (4ºC)
- Clear-rite 3, 1 h, orbital shaking (4ºC)
Specimens were embedded in paraffin and maintained at 60ºC for 4-5 hours before being positioned in different orientations while cooling. Paraffin blocks were sectioned serially into parasagittal, coronal and transverse sections (7 µm thick). The section ribbons obtained from the microtome were transferred to a warm water bath where they were allowed to stretch and cooped up onto glass slides. Slides were stained in hematoxylin and eosin, mounted with Canada balsam, and cover slipped.
Microscopic images of the histological sections were captured using a Nikon Eclipse E-600 bright-field microscope coupled to a Nikon DXM1200F digital camera. Panoramic images of whole and partial fish sections were obtained by merging sequential photographs (4x or 10x depending on the specific section) using Adobe Photoshop images. Any undesirable artifact such as dust motes present in the sections was removed with Photoshop but maintaining the integrity and originality of each sample. No modifications were made to any of the photographed images except for adjusting the luminance levels to ensure uniformity. The final images uploaded to this website were selected from thousands of histological images captured from the eight developmental time points. We implemented a Virtual Microscope feature that enables users to explore each image in detail, simulating the experience of viewing tissue sections under a real microscope. This functionality was achieved using the Zoomify Design software.