Analysis of Vertebrate Developmental Temperature Tolerance

Animals, including vertebrates like fish or humans, start their existence as a single cell that divides until the embryo consist of several hundred similar cells. These cells need to know their position within the embryo and communicate among each other to decide which cell will contribute to which tissue in the developing body. First, cells partition themselves into three broad categories called the germ layers: ectoderm, mesoderm, and endoderm. Each germ layer will eventually produce different tissue types: ectodermal cells will form the nervous system and the skin, the mesoderm forms tissues like blood, bone, and muscles, and the endoderm will give rise to the digestive tract and its associated organs. Cells communicate by releasing signaling molecules that move through the extracellular space within the embryo and are read out by target cells that adapt their behavior accordingly. In the case of germ layer patterning an important signaling molecule is called Nodal. High levels of Nodal causes cells to become endoderm, medium levels specify mesoderm, and low levels lead to ectoderm. A second molecule that also moves through the extracellular space, Lefty, affects how cells sense Nodal. Both molecules are produced by the same cells, and the faster Lefty can bind to the slower Nodal and inactivate it. Therefore, the distance of receiving cells from this source decides the future fate of these cells. The body temperature of fish embryos depends on the ambient temperature. This affects both the speed of developmentfor example, cell division and cell movementand the speed of signaling molecules, like Nodal and Lefty. However, for every increase of 10 C the developmental speed roughly doubles, while molecule movement increases by only about 4%. This discrepancy should cause cells to receive the wrong amount of signaling molecules or receive these amounts at the wrong time, negatively affecting embryo development. Interestingly, embryos from some species can tolerate a wide range of temperatures, but how they do it is not understood. Since climate change increases water temperatures around the globe, it is important to understand why some species are more sensitive to temperature than others. In this project I will investigate the mechanisms that make development, robust to temperature fluctuations, using mathematical modeling, microscopy and molecular experiments with the Japanese medaka fish (Oryzias latipes) as a model system. Medaka embryos can develop happily between 15 C and 40 C and even pause their development at temperatures as low as 4 C. I will compare embryo development in this temperature- tolerant species to the development of zebrafish embryos (Danio rerio), a temperature- sensitive species. This comparison will help us to understand why some embryos can tolerate a wide range of temperatures while others can only develop close to their optimum.

Cells in a developing embryo communicate with each other by molecular signaling pathways. Only a handful of such pathways are required to complete the first periods of development in which the full body plan, with head and tail, and progenitors of organs and the nervous system, is established from a population of very similar cells. Like musicians in an orchestra, different signaling pathways fulfil different roles. The initial division of cells into three distinct populations, known as germ layers, for example is regulated by pathways called Nodal and FGF, while patterning along the future back to belly axis is controlled by BMP. Therefore, loss of function in a specific pathway manifests in a specific way, known as phenotype. Since developmental processes are complex, it can be difficult to correctly identify specific defects. For example, perturbations in the Nodal pathway can lead to cyclopia - embryos with only one eye in the center of the head. The same defect can occur if another pathway called Sonic Hedgehog is disrupted, although the mechanisms are different. Developmental processes are to some extend resilient against chemical, genetical and environmental perturbations, but the extend of this robustness varies dramatically between species and the stage of development. Embryos of the japanese ricefish medaka for example can develop over a large temperature range of more than 20 C and can even transiently pause their development when it gets colder, while tropical zebrafish embryos require a relatively stable temperature. In this project we developed an artificial intelligence application we dubbed EmbryoNet that can automatically and reliably identify defects in signaling pathways in fish embryos based on microscopy images. We used imaging data of embryos with or without defects in seven important signaling pathways and manually annotated phenotypes as training data. The resulting network is not only faster than humans but also outcompeted biology students and experienced researchers in terms of classification accuracy. By modifying the annotation in these training data, we could also create a second application that can identify defects up to 4 hours before they are visible to human researchers. We also built smaller AI networks to detect defects in Nodal signaling in medaka and the three-spine sticklebacks. Finally, we tested EmbryoNet on around 1000 FDA-approved drugs and bioactive molecules with high-throughput microscopy. The AI faithfully identified molecules of known function, but also returned unexpected results. An especially interesting finding was that cholesterol-lowering drugs known as Statins, have the capacity to dampen the FGF signaling pathway and thus cause harmful phenotypes in fish embryos. The paper was published open access in Nature Methods and we also provide the source code and the imaging data as a resource for the research community.