Histology involves the study of the microscopic anatomy of cells and tissues that cannot be observed by the naked eye. This allows an understanding of these elements for a comprehensive approach of organ structure and function. Hence, a prior thorough knowledge of normal anatomy and histology are necessary for the observer to detect pathologies or alterations produced by disease or chemical insult. Classically conceived as based on subjective diagnoses, nowadays histology and histopathological observations are supported by unbiased morphometric and stereologic techniques thanks to the development of quantitative tools with computer-based image analysis programs (Elias and Hide, 1983; Cooke and Hinton, 1999; Gibbons et al. 2009; Justulin et al., 2008; Bhanot and Hundal 2021). Moreover, the application of immunohistochemistry and cytochemistry techniques allows for the localization of specific compounds within tissues and cells, providing more detailed insights into tissue composition. The more recent development of
in situ hybridization techniques also provides a direct visualization of the spatial localization of specific DNA or RNA sequences. All these tools are giving a more ample meaning offering a multidisciplinary environment of histology and histopathology in relation to what they were originally conceived.
Among vertebrates, teleostean fishes represent the oldest, largest, and most diverse extant group, having adapted to almost all the different aquatic environments present in earth. These characteristics and their importance as main component in human nutrition have positioned fishes as ideal organisms for a conceptual framework for other vertebrate studies. Several reviews have highlighted the advantage of these organisms for laboratory use (e.g Arcand-Hoy and Benson, 1988; Masahito et al., 1988; Powers, 1989; Bolis et al., 2001; Law, 2003; D’Angelo and Girolamo, 2021). In aquatic toxicology, fishes are established models as sentinel organisms as they occupy the top trophic level in the food chain, making them the ultimate receptors of xenobiotics present in the aquatic compartment and neighboring environments. Their use in environmental toxicity studies has gained increasing attention well over the past five decades (Stanton, 1965; Hawkins et al., 1985, 1995; Winn, 2001; Jovanovic et al., 2018) and, in the majority of these, histopathology has played a pivotal role in defining extent and mechanisms of toxicity (Hinton, 1993; Noyes et al., 2011; González-Doncel et al., 2022). Despite the emphasis placed during the last two decades in toxicogenomics, no other approach can yield such information simultaneously on so many different structures of the fish anatomy at once and so rapidly, such as histopathology offers (Hinton, 1990; Wester and Canton, 1986; Salamat and Zarei, 2016).
Among the different forms of fishes, those species of small size are increasingly being used for their numerous advantages as experimental models that are not offered by larger fish species like salmon, catfish, or bass (Overstreet et al., 2000). Several fish species are suitable for experimental research, with the zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) standing out as valuable vertebrate models across various biological disciplines (Kimmel et al., 1993; Westerfield, 1995; Ishikawa et al., 1997; Ishikawa, 2000; Wittbrodt et al., 2002; MacRae & Peterson, 2003; Furutani-Seiki & Wittbrodt, 2004; Shima & Mitani, 2004; Signore et al., 2009; Sasado et al., 2010; Lin et al., 2016; Murata et al., 2019a; Chowdhury et al., 2022). Because of their small size, both models allow maintenance and experimentation of large number of individuals in a limited space while and, at the same time, increasing statistical power. Their size also allows whole-mount histological assessments with sections of larvae and adults that can be examined in a minimum of preparations.
In particular, the Japanese medaka is a small egg-laying freshwater teleost native to South East Asia that has become an excellent organism for studying developmental genetics and biology (Briggs and Egami, 1959; Yamamoto, 1975; Kinoshita et al., 2009; Naruse et al., 2011). It has a smaller genome (approx. 800 Mb) compared to the zebrafish genome (approx. 1700 Mb). Here are some remarks indicating the importance of this species in the branch of biology: It is the first fish species where the Y-linked inheritance was demonstrated (Aida 1921), and the first vertebrate in which the complete sex reversal, in both directions, was successfully achieved by administration of exogenous hormones during the larval phase (Yamamoto 1953). More recently, the male-determining gene, DMY/Dmrt1bY located in the Y chromosome, was identified as the first non-mammalian equivalent to the sex-determining gene, SRY, present in mammals (Matsuda et al., 2002; Nanda et al., 2002). It is no coincidence that this fish species has taken part in missions onboard the US space shuttles and in expeditions to the International Space Station for research and testing (Ijiri, 2003; Nihoti et al., 2004; Chatani et al., 2015; Murata et al., 2015; Chatani et al., 2019; Wagner et al., 2023). Back to earth, the medaka has shown to be particularly sensitive to the exposure of a variety of toxicants, developing neoplastic lesions even after short periods of exposure (Bauman and Okihiro, 2000; Kawasaki and Shimizu, 2021). Thanks to a thorough prior knowledge of the normal anatomy and physiology of the different organs and tissues, researchers use medaka histological analyses to detect alterations in these structures (Ding et al., 2010; Murata et al., 2015; Mihaich, et al., 2019; Wang et al., 2019). Thus, a condition required for the widespread use of an organism as model is to have available its normal histology in the form of images at different moments of its life. To assess developmental toxicity in medaka using histological tools, it is crucial to systematically identify and classify distinct normal developmental periods. Having reference criteria for normal development provides researchers with baseline data from which to compare deviations. In line with this need, a considerable work has been made with the zebrafish for which print and online histology atlases are currently available (e.g. Menke et al. 2011; Lin et al., 2022;
https://bio-atlas.psu.edu/index.php;
https://zfin.org/hh_atlas/index.html). Interestingly, fewer reports can be found showing the histological anatomy of the medaka fish. Anken and Bourrat (1998) reported a histological brain atlas of the adult medaka depicting the entire central nervous system. Suzuki and Shibata (2004) described the process of the reproductive tract development, from 10 to 90 days after hatch. Using scanning electron microscopy, Isogai and Fujita (2011) provided an anatomical atlas of the blood vascular system of the adult medaka. Gladys et al. (2015) proposed ultra-high resolution optical coherence tomography to study the morphological development of internal organs in medaka at three post-hatch development stages (i.e. 30, 60 and 90 days post-hatch). More recently, Royan et al (2021) resorted to
in situ hybridization and immunofluorescence techniques to complete the first 3D atlas of the adult medaka pituitary gland. A book has been released exhaustively describing the medaka brain morphogenesis and development (Ishikawa et al., 2022). A substantial amount of information in the form of bright-field histology images has also been accumulated regarding specific organs within the medaka architecture as potential sites for xenobiotic-induced toxicity (e.g. Boorman et al., 1997; Bunton, 1991; Cooke and Hinton, 1999; Teh and Hinton, 1998; Seki et al., 2002; Dietrich and Krieger, 2009; González-Doncel et al., 2022). However, the reported histological images have mostly been restricted to particular phases and after specific windows of exposure. To our knowledge, few studies have attempted to show the general histological morphology of the whole medaka body. As Chief of Core Organization of the National Bioresource Project of Medaka in Japan, back in 2005, Prof. Wakamatsu stressed the need for a complete histological atlas of medaka for use by researchers available in the web (Wakamatsu, 2005). Ishikawa et al. (1999) took the first step by producing and sharing histological sections that depicted the adult wild-type medaka brain structures. To date no further uploads were made for additional histological structures of the medaka leaving this ambitious project unfinished. Perhaps the most significant contributions to the development of a histological atlas of the whole medaka body have been carried out by Bauchet (2006) and, later, by Murata et al. (2019b). Although exhaustive and showing top-notch longitudinal and transverse sections, these works were restricted to only the juvenile medaka.
Building upon the current state of the art in this field, and extending beyond the embryonic phase, we have compiled comprehensive histological study of the orange-red strain of medaka. This study provides an exhaustive description of its normal internal microscopic anatomy across eight key developmental periods (refer to Figure 1). Each period has been arranged as a series of consecutive sagittal, coronal and transverse sections of the whole fish depicting histological particularities viewed from a panoramic and from organ-specific perspectives. This approach allows examining specific organs and structures within a particular developmental period and within the context of the whole organism. This contribution is aimed to provide a comprehensive reference guide for the three-dimensional organization of the internal medaka anatomy along its development offering a baseline from which to compare deviations at specific stages of the medaka life. Although the primary purpose of this atlas is to provide a tool for medaka anatomists and histopathologists, it may equally allow comparisons of organ arrangement and growth between different piscine species. Prior works lay a foundational understanding, and this atlas aims to fill the gaps by presenting a more comprehensive and continuous view of the medaka's histological development.
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).
*Numbers are approximate.
We have taken a degree of initiative in reporting, for the first time, specific structures in medaka that, to our knowledge, have not been identified or described by previous authors. Given the limited information available, we acknowledge that these identifications could be provisional and subject to revision. As such, we welcome both scientific and non-technical comments and critiques to further refine and improve this atlas. We have drawn on both the literature cited in this text (see
References) and additional works that informed our research but are not directly cited (see
Consulted Literature). These sources collectively support the compilation and interpretation of the observations presented.
In "How We Made It", you will find the way we accomplished this histological atlas of the medaka post-hatch development.