Shortly after fertilization, cells of the vertebrate embryo undergo substantial movement and rearrangement. This dramatic restructuring of embryo morphology involves epiboly, a process by which the embryonic cell mass spreads over the surface of the yolk. Epiboly is followed by gastrulation, a large-scale rearrangement of tissues leading to formation of germ layers that ultimately give rise to all the organs and tissues of the body. An important focus of developmental biology research is to understand the molecular mechanisms controlling epiboly and gastrulation. Using molecular genetic approaches, Dr Garrity's and Dr Horne's laboratories recently identified the calcium channel beta subunit as essential for normal epiboly in zebrafish. Classically, the beta subunit is thought to function as a critical auxiliary subunit of voltage-gated calcium channels (VGCCs), multi-protein complexes that mediate calcium entry into the cell. The auxiliary beta subunits (beta1- beta4) help transport the pore-forming alpha1 subunit to the cell membrane and modulate the electrophysiological properties of the calcium channel. However, recent research suggests that beta subunits may have additional functions in the cell independent of their roles in VGCCs. Mutations in the beta subunit lead to uncoordinated, lethargic behavior and seizures in mice, and have been associated with epilepsy in humans. Ongoing research in Dr Garrity's and Dr Horne's laboratories will use a combination of molecular genetic and biochemical approaches to test potential roles for the beta subunit in interacting with cytoskeletal components or in cell division or differentiation. The proposed research in zebrafish is expected to define new cellular roles for the beta subunit family, which could extend to other cell types in different physiological contexts, and may lead to a better understanding of disease phenotypes in mice and humans. The research will also contribute to our basic understanding of the molecular mechanisms driving epiboly. This collaboration will provide training and research opportunities for graduate and undergraduate students on two campuses, utilizing video conferencing. It will also provide summer internship opportunities for minority undergraduate students enrolled at Colorado State University-Pueblo, an affiliated Hispanic Serving Institution.
The project investigated mechanisms underlying the earliest cell movements in zebrafish embryos. ‘Gastrulation’ is a process in early embryonic development of animals in which cells generated by initial cleavages become extensively rearranged. Understanding how cells move during gastrulation is critical to science, since these cell rearrangements will: 1) establish the germ layers (ectoderm, mesoderm and endoderm) that give rise to subsequent body organs, and 2) determine body axes of the embryo (anterior/posterior, dorsal/ventral, left/right). In this project, we explored the function of a gene called β4 during ‘epiboly’, the earliest movement of zebrafish gastrulation. During epiboly, about 1000 embryonic cells in a pile atop the large yolk cell will spread out, rearranging into thin layers, until they eventually completely surround the yolk cell. A main goal of our project was to understand how β4 protein function contributes to movements of epiboly. The calcium channel beta [CACNB4 or β4] gene encodes a protein that serves as an auxiliary subunit in voltage-gated calcium channels, binding to a large pore-forming subunit called CACNA. Recent work suggests that genes in the β family may also have non-calcium channel related functions in the cell. Therefore, additional goals of our project were to discover what these functions were, and where they occurred in the cell. We first cloned two zebrafish β4 genes (termed β4.1 and β4.2). Both genes were active during epiboly, and were translated into mRNA in both embryonic cells and in extra-embryonic tissue. In zebrafish, an important extra-embryonic tissue is the yolk cell, which contains multiple nuclei in a thin layer of cytoplasm spread on top of the large yolk mass. The layer of cytoplasm is called the ‘yolk syncytial layer’ or YSL. To discover the normal function of β4 in the embryo, we analyzed embryos that lacked this protein. Using genetic techniques (morpholinos), we specifically depleted β4.1 or β4.2 protein throughout the embryo. As a consequence, epiboly was halted or delayed, and embryos died, telling us that embryos absolutely required β4.1 and β4.2 to complete epiboly and survive. If we removed β4.1 or β4.2 protein only within the YSL, epiboly was still delayed and embryos died, indicating that β4 protein function in the YSL in particular was important. To discover whether the β4 proteins were acting in the YSL as auxiliary subunits for calcium channels, we created a mutant version of human β4, in which three amino acids were changed, making the mutant protein unable to bind to the calcium channel. In control experiments, when wildtype human β4 mRNA was added back to β4-depleted zebrafish embryos, they now survived. If mutant human β4 mRNA was added back to β4-depleted zebrafish embryos, these also survived, telling us that the function of β4 in the context of epiboly did not involve its "classical" role as a calcium channel subunit – instead β4 functioned in a channel-independent fashion. We next tested which portions of the β4 protein were the most critical for its function during epiboly. We concluded that the first 199 amino acids of β4 were critical, while the remainder of the protein could be removed without serious consequences. Within the 199 amino acids, we identified a small group of amino acids (PVVLV) which were shared among β4 proteins in many species. If these amino acids were changed individually or deleted, the β4 protein became non-functional in epiboly. Work by others suggested that the PVVLV amino acids formed a protein domain that interacted with another family of proteins called chromobox proteins. The chromobox proteins operate within the nucleus, where they help regulated gene expression. Our next question was whether β4 protein similarly localized to the cell nucleus. We therefore tagged the β4 protein with a green fluorescent protein, so that we could track where it accumulated within the cell. We expected (and found) its presence near the plasma membrane, where calcium channels reside. In addition, tagged β4 protein was clearly present in the nucleus! Our current model therefore is that β4 proteins act in the nuclei of the YSL, where interactions with partner proteins possibly including the chromobox proteins, leads to regulation of downstream genes needed for cell movements during epiboly. The project was extended to study two additional β family members: β2.1 and β2.2, both expressed in the embryonic heart. Genetic studies indicated that embryos required β2.1 function in the heart for normal development. Specifically, β2.1 function helped to regulate the number of cells present in the forming heart, and helped establish normal tight connections among cardiac cells that hold the heart tube together as the heartbeats begin. Preliminary data suggests β2.1 protein does operate as part of the calcium channel in the context of cardiac development. Broader impacts include training of 1 sabbatical faculty on the zebrafish system, 1 postdoc, 5 graduate students, several undergraduate students and 6 REU summer students.
