Regulation of cell movements by chemokine apelin during zebrafish development

Proper embryonic development requires precise cell movements, which are coordinated by multiple signaling pathways. Formation of specific organs is initiated during gastrulation when organ precursors acquire their initial cell fates. Additionally, precursors move to specific locations where they engage in additional inductive interactions to continue their differentiation, and form the specialized tissues required for organ functions. Impaired cell movements can cause severe embryonic malformation, developmental arrest and also many related diseases. In this work, I used the zebrafish, Danio rerio, a well-established vertebrate model system to study the involvement of G-protein coupled receptor (GPCR) signaling in cell movements.

Apelin and its GPCR receptor Agtrl1 regulate adult physiology, in particular cardiovascular functions, and blood vessel development. Here we show that the zebrafish Apelin and Agtrl1b homologs control heart field formation during gastrulation. Cardiac precursors, specified in the lateral plate mesoderm territories, converge toward the embryonic midline during gastrulation and extend rostrally to form bilateral heart fields. We found that agtrl1b is expressed in the forming mesendoderm before gastrulation, and in the lateral plate mesoderm later, while apelin expression is confined to the midline. Suppressing the function of Agtrl1b or its ligand Apelin using morpholino antisense oligonucleotides resulted in a deficiency of cardiac precursors and a subsequent absence or reduction of heart. Embryos injected with apelin RNA formed no heart. Our cell tracing experiments demonstrated that in embryos with excess Apelin, cardiac precursors failed to move to the correct location and to express heart markers. Time-lapse analyses of Apelin overexpressing gastrulae revealed reduced migration and defective morphology of mesodermal cells including cardiac precursors. Moreover, in Apelin deficient gastrulae, the cardiac precursors moved less efficiently to the correct location, and showed broadened and ectopic distribution. Our results demonstrate an essential developmental role for the Apelin-Agtrl1b GPCR signaling system in mesodermal cell movements and migration of cardiac precursors to form the heart field during vertebrate gastrulation.

During our investigation, we found that Apelin also regulates the migration of zebrafish primordial germ cells (PGCs), a process previously shown to be regulated by Sdf1a/Cxcr4b GPCR signaling. During gastrulation and somitogenesis, apelin mRNA is expressed in the dorsal midline, while its receptor agtrl1b gene is broadly expressed in the mesendoderm, where PGCs are localized. Manipulating Apelin function by misexpression throughout the embryo or by overexpression specifically in primordial germ cells impaired movements of PGCs towards their target tissues. Suppressing Apelin function by injections of antisense morpholino oligonucleotides also resulted in a phenotype of mis-localized PGCs. The abnormal PGC movements in these loss and gain of function scenarios are not a consequence of altered sdf1a expression. Using transplantation experiments, we showed that the cells expressing Apelin in ectopic locations attracted PGCs. However, in these experiments the PGCs stopped short of Apelin overexpressing cells. Interaction between both Apelin and Sdf1a signaling was also investigated. In odysseus (ody) (-/-) mutants, which harbor a null mutation in cxcr4b gene, the majority of ectopic PGCs aggregate in the dorsal midline where apelin is expressed. Interference with both signaling pathways, by injecting apelin MO into ody (-/-) mutant embryos, significantly reduced the dorsal aggregation of PGCs. Based on the preliminary data, I hypothesize that Apelin provides an attractive cue for PGCs migration during gastrulation and segmentation stages, in addition to the previously discovered Sdf1a/Cxcr4b signaling pathway.