One of the earliest decisions a developing embryo must make is what fraction of its cells to commit to each of three distinct "germ" lineages known as the ectoderm, mesoderm and endoderm. The Nissen lab seeks to better understand the fundamental mechanisms governing these extremely early cell fate decisions. While ectoderm cells give rise to skin and brain, mesoderm and endoderm cells give rise to most other tissues including heart, liver and muscle. The "Nodal signaling pathway" plays a critical role in determining the number of cells devoted to mesoderm and endoderm and does so at the expense of ectoderm cells. Hence, precise control over the amount of Nodal signaling pathway activity is important to maintaining the proper balance between many adult tissues. The Nodal signaling pathway is composed of a series of genes whose sequential functions are to receive, process, transmit and execute the command regarding how many cells to commit towards mesoderm and endoderm fates. Although a significant amount is understood about the Nodal signaling pathway, several genes involved in processing and transmitting the command signal are not well understood and some genes likely remain unidentified. The focus of this project is to explore the roles that two recently identified genes, wdr68 and dyrk1b, play in the Nodal signaling pathway. The proposed research uses a combination of genetic and biochemical approaches in the zebrafish model organism. Since fertilization is external and because fish lay their eggs, the very early stages of embryonic development are easily observed in zebrafish. The Broader Impacts on integrating research and education are that these studies are being performed at a federally designated Title III and Hispanic Serving Institution. These predominantly female and minority students are directly engaged in the research project, thereby enhancing their science education and our knowledge about embryonic development. In addition, because of the nature of the work, the research has implications for the development of regenerative medical therapies.
One of the earliest commitments a developing embryo must make is the proper allocation of cells to particular developmental fates. For example, if too many cells are devoted to one particular fate, such as brain, and not enough to another fate, such as gut, the result will be a malformed animal. The earliest decision of this type that an embryo must make is the establishment of the three germ layers, ectoderm, mesoderm and endoderm, from which all the different types of cells in the adult ultimately arise. One very important role of the Nodal signaling pathway is to allocate cells to particular fates early in embryonic development. Specifically, high levels of Nodal activity specify cells towards endoderm fates while lower levels of Nodal activity specify cells towards mesoderm fates. In the complete absence of Nodal signaling, cells take on ectoderm fates. In this manner, the Nodal signaling pathway is like a dimmer switch controlling cell fates. There are several steps involved in turning the Nodal signaling pathway toward the "on" position. Each step is composed of a protein molecule or complex of protein molecules that transmits a piece of information to the next step in the pathway much like runners in a relay race. The Nodal ligand is the initiating step in the pathway that transmits a piece of information turning the pathway toward the "on" position. The amount of Nodal ligand present in a particular location of the embryo is directly proportional to the "brightness" of Nodal signaling in that local cellular environment and hence whether the cells within the immediate region will become ectoderm, mesoderm, or endoderm. While the Nodal ligand acts positively to turn the pathway toward the "on" position, another set of proteins, the Leftys, act negatively to turn the pathway toward the "off" position. Acting together, it is therefore the relative ratio of Nodal ligand to Lefty protein that determines the overall "brightness" of Nodal signaling in a particular embryonic region. Complicating the situation further, Nodal ligand triggers the production of more Nodal ligand as well as production of the Lefty proteins. In this manner, the Nodal ligand drives a feedback loop with both positive and negative impacts on the overall level of Nodal signaling. The work of this project focused on two proteins called Wdr68 and Dyrk1b. These two proteins form a physical complex that we suspect represents a new step in the series of events known as the Nodal signaling pathway. When we initially set out upon our investigations, our data showed us that Dyrk1b and Wdr68 are both important for expression of the Leftys. Since the Leftys antagonize Nodal signaling, we made the simple prediction that the Wdr68-Dyrk1b complex is important for turning Nodal signaling down, rather than up. One important prediction of this model is that in the absence of Dyrk1b, we would expect to see more cells headed towards mesoderm and endoderm fates. Upon further examination, supported by this NSF grant, we found that while there is a modest expansion of mesoderm in embryos lacking Dyrk1b, there is a dramatic reduction in endoderm. In other words, we found that Dyrk1b was needed for turning Nodal signaling up, rather than down! Notably, in the zebrafish, there are two Nodal ligands, Ndr1 and Ndr2, that function in the embryo. It is also well known that Ndr2 plays the more direct role in the expression of the Lefty antagonists. Our further analyses revealed that both Dyrk1b and Wdr68 are important for robust expression of Ndr2 but not Ndr1. So, even though the Lefty antagonists are not robustly expressed, neither is the Nodal ligand that they antagonize. Since high levels of Ndr2 are needed for both expression of the Lefty antagonists as well as for specifying endoderm fates, embryos lacking Dyrk1b fail to produce sufficient numbers of endoderm cells while the remaining low levels of Nodal signaling are sufficient for inducing mesoderm cells. Thus, our experiments are intellectually meritorious because they have contributed to the discipline the fundamental knowledge that Dyrk1b works at a step in the Nodal signaling pathway between Ndr1 and Ndr2. As broader impacts, this information is important because it refines our model of Nodal signaling and how the early cell fate decisions in a developing embryo are made. For example, these findings might be useful for other studies aimed at the directed differentiation of cells, such as stem cells, into cell types desired for therapeutic uses. Future experiments will explore how Wdr68 and Dyrk1b act at the molecular level.
