Medaka fish, a vertebrate genetic model system

We use medaka (Oryzias latipes) as a vertebrate genetic model system to study population genetics and developmental processes.
 

Fig. 1: Wild male medaka. Medaka is a freshwater teleost from East Asia and well established model system (Aida, 1921). Small body size, short generation time, high fecundity, compact genome and high tolerance to inbreeding render medaka well suited for many studies.

A population genetic resource

Individuals of wild populations are genetically polymorphic. Thus, even though they share the same set of genes, their genomes contain many sequence polymorphisms that affect phenotypic traits, for example morphology, physiology and behaviour. Often several polymorphisms, mostly SNPs, act on a given trait and lead to subtle quantitative phenotypic variations. A classical example is craniofacial variation of human individuals (Fig.1). The repertoire of genetic variations in a population and their effects on a given trait are studied by genome wide association studies (GWAS) that use statistical analysis to elucidate phenotype-genotype association.
 

  
Fig. 2: Individual facial variation in humans and wild Kiyosu medaka (6 billion others, Auer)

Medaka has a high tolerance to inbreeding from the wild (Hyodo-Taguchi and Egami, 1985). Furthermore medaka wild catches can easily be obtained from their natural habitat in Japan (Spivakov et al., 2014). In collaboration with Jochen Wittbrodt (COS, HD Germany), Ewan Birney (EBI, UK), and Kiyoshi Naruse (NBRP, Japan) we have established a medaka population genetic resource to be used as a vertebrate animal model for GWAS. Importantly in addition to association studies, the inbred panel can be used to test associated SNPs by in vivo functional assays.
 

wild polymorphic population inbreeding by single  brother-sister crosses  for 9 generations   panel of inbred strains

Fig. 3: Starting with random crosses of individuals from the wild population a panel of inbred strains was established by inbreeding for several generations. Each inbred line harbors a different set of genetic variants.

By successive single brother-sister crosses over 9 generations we have generated a panel of more than 100 near-isogenic inbred strains starting from a wild population of medaka (Kiyosu). Whole genome sequence analysis showed that more than 75% of the strains are more than 80% homozygous, a value which is comparable to the Drosophila genetic reference panel. The entire panel captures more than 25 million SNPs of the wild population (Tom Fitzgerald, Ewan Birney; EBI). 

We use quantitative assays to search for phenotypc variation across the inbred panel. In collaboration with Tilo Baumbach, IPS/KIT we have developed an X-ray micro-CT imaging pipeline including automated segmentation for quantitative morphometric analysis of embryonic and adult medaka fish. This detailed morphometric analysis aims to identify genetic variants regulating morphometric variation.
 

Fig. 4: Pipeline for quantitative 3D morphometric analysis. (a) fish husbandry; (b) fixation, staining with contrast agent and embedding; (c) X-ray micro-CT at the synchrotron radiation facility; (d) 3D reconstruction and manual egmentation of the reference atlas; (e) automatic segmentations and morphometric analysis. (Weinhardt et al., unpublished).

To complement this morphological analysis we are using organ function assays, such as resting heart rate analysis. Recent studies in humans reveal an association of heart rate genetic variants and all cause mortality, such that 5bpm increased heart rate results in a decreased life span of about 2.5 years, underscoring the importance of resting heart rate as a key risk factor.  (Eppinga et al., 2016).
 

Fig. 5: Quantitative assay to determine resting heart rate. Native resting heart rate of embryos is extracted from short videos by automated image analysis. Right panel shows variance of normalized heart rate in some strains of the inbred panel (Gierten et al., unpublished). 

Our assay enables a quantitative measurement of native embryonic heart rate in a 96 well format. The Kiyosu panel allows to model this key trait and to functionally test associated genetic variants. In addition the inbred strains will be used to test human SNPs for functional relevance, which is not possible in humans.

Comparison of data sets from different phenotyping assays across the panel will allow to search for pleiotropic effects of genetic variants. Cross species comparison by comparative genomics will enhance the association power of GWAS data. Associated genetic variants will be tested by genome editing in specific strains of the medaka inbred panel. We are currently developing further assays to phenotype physiological and behavioural traits with the aim to provide a more complete phenotypic description of the individual strains and to establish a data base of functionally relevant genetic variants of the medaka Kiyosu population.
 

Cell polarity and proliferation

Cells within epithelial sheets are highly polarized with distinct apical and basal domains. This cellular organization is critical to both epithelial morphology and function, for example the uptake of nutrients in the gut, the secretion in epithelial glands or light reception in the neural retina of the eye. Our research aims to identify genes that function in epithelial cell polarity of the vertebrate nervous system and to analyse their specific function.
Prior to organogenesis, the progenitor cells of organs require information concerning apico-basal polarity for their proper development. Mutations that affect apico-basal polarity often perturb the epithelial arrangement of cells and by that also the morphology of the affected tissue (Figure 6). 
 

Fig. 6: Wildtype (A) and med mutant (B) retinae of medaka fish hatchlings. The mutation affects apico-basal polarity resulting in an abnormal shape and cellular architecture of the neural retina (Herder et al., 2013).

Epithelial polarity is tightly linked to proliferation. Mutations in epithelial polarity genes often result in ectopic proliferation, leading to an increased size of the affected tissue (Fig. 7). Thus, epithelial polarity controls two aspects that play a critical role in tumour formation, namely epithelial-mesenchymal transition and cell proliferation. In fact, many genes that function in apico-basal polarity have initially been isolated as tumour supressor genes.
 

Fig. 7: Proliferation in wild type (A) and med mutant (B) retinae. The anti-phospho-Histone 3 antibody labels M-phase nuclei (red). A high number of ectopic M-phase nuclei are visible in the mutant retina.

The development of the transparent medaka embryos can be examined by non invasive microscopy. Thus, their development is not affected by the analysis. To visualize apico-basal polarity in vivo we use GFP-tagged proteins that are specifically localized along the apico-basal axis. The simple architecture of neuroepithelia during early fish development allows to visualize the subcellular localization in the living embryo using a fluorescence dissection microscope (Fig. 8).
 

Fig. 8: Transversal section showing apical localization of aPKC in the developing CNS by antibody staining  (A, green). B: dorsal view of a live embryo that expresses apically localized Par3-GFP (green).

References:

Aida, T. (1921). On the Inheritance of Color in a Fresh-Water Fish, APLOCHEILUS LATIPES Temmick and Schlegel, with Special Reference to Sex-Linked Inheritance. Genetics 6, 554–573.

Eppinga, R. N., Hagemeijer, Y., Burgess, S., Hinds, D. A., Stefansson, K., Gudbjartsson, D. F., van Veldhuisen, D. J., Munroe, P. B., Verweij, N. and van der Harst, P. (2016). Identification of genomic loci associated with resting heart rate and shared genetic predictors with all-cause mortality. Nat Genet 48, 1557–1563.

Herder, C., Swiercz, J. M., Müller, C., Peravali, R., Quiring, R., Offermanns, S., Wittbrodt, J. and Loosli, F. (2013). ArhGEF18 regulates RhoA-Rock2 signaling to maintain neuro-epithelial apico-basal polarity and proliferation. 140, 2787–2797.

Hyodo-Taguchi, Y. and Egami, N. (1985). Establishment of Inbred Strains of the Medaka Oryzias latipes and the Usefulness of the Strains for Biomedical Research. Zool. Sci. 2, 305–316.

Spivakov, M., Auer, T. O., Peravali, R., Dunham, I., Dolle, D., Fujiyama, A., Toyoda, A., Aizu, T., Minakuchi, Y., Loosli, F., et al. (2014). Genomic and phenotypic characterization of a wild medaka population: towards the establishment of an isogenic population genetic resource in fish. G3 (Bethesda) 4, 433–445.