Toddler: An Embryonic Signal That Promotes Cell Movement via Apelin Receptors

Introduction Embryogenesis is thought to be directed by a small number of signaling pathways with most if not all embryonic signals having been identified. However, the molecular control of some embryonic processes is still poorly understood. For example, it is unclear how cell migration is regulated during gastrulation, when mesodermal and endodermal germ layers form. The goal of our study was to identify and characterize previously unrecognized signals that regulate embryogenesis. Toddler promotes gastrulation movements via Apelin receptor signaling. Toddler is an essential, short, conserved embryonic signal that promotes cell migration during zebrafish gastrulation. The internalization movement highlighted by the colored cell tracks requires Toddler signaling. Toddler signals via the G-protein–coupled APJ/Apelin receptor and may be one of several uncharacterized embryonic signals. Methods To identify uncharacterized signaling molecules, we mined zebrafish genomic data sets for previously non-annotated translated open reading frames (ORFs). One such ORF encoded a putative signaling protein that we call Toddler (also known as Apela/Elabela/Ende). We analyzed expression, production, and secretion of Toddler using RNA in situ hybridization, mass spectrometry, and Toddler-GFP fusion proteins, respectively. We used transcription activator-like effector (TALE) nucleases to generate frame-shift mutations in the toddler gene. To complement loss-of-function analyses with gain-of-function studies, Toddler was misexpressed through mRNA or peptide injection. We characterized phenotypes using marker gene expression analysis and in vivo imaging, using confocal and lightsheet microscopy. Toddler mutants were rescued thorugh global or localized toddler production. The relationship between Toddler and APJ/Apelin receptors was studied through genetic interaction and receptor internalization experiments. Results We identified several hundred non-annotated candidate proteins, including more than 20 putative signaling proteins. We focused on the functional importance of the short, conserved, and secreted peptide Toddler. Loss or overproduction of Toddler reduced cell movements during zebrafish gastrulation; mesodermal and endodermal cells were slow to internalize and migrate. Both the local and ubiquitous expression of Toddler were able to rescue gastrulation movements in toddler mutants, suggesting that Toddler acts as a motogen, a signal that promotes cell migration. Toddler activates G-protein–coupled APJ/Apelin receptor signaling, as evidenced by Toddler-induced internalization of APJ/Apelin receptors and rescue of toddler mutants through expression of the known receptor agonist Apelin. Discussion These findings indicate that Toddler promotes cell movement during zebrafish gastrulation by activation of APJ/Apelin receptor signaling. Toddler does not seem to act as a chemo-attractant or -repellent, but rather as a global signal that promotes the movement of mesendodermal cells. Both loss and overproduction of Toddler reduce cell movement, revealing that Toddler levels need to be tightly regulated during gastrulation. The discovery of Toddler helps explain previous genetic studies that found a broader requirement for APJ/Apelin receptors than for Apelin. We propose that in these cases, Toddler—not Apelin—activates APJ/Apelin receptor signaling. Our genomics analysis identifying a large number of candidate proteins that function during embryogenesis suggests the existence of other previously uncharacterized embryonic signals. Applying similar genomic approaches to adult tissues might identify additional signals that regulate physiological and behavioral processes. It has been assumed that most, if not all, signals regulating early development have been identified. Contrary to this expectation, we identified 28 candidate signaling proteins expressed during zebrafish embryogenesis, including Toddler, a short, conserved, and secreted peptide. Both absence and overproduction of Toddler reduce the movement of mesendodermal cells during zebrafish gastrulation. Local and ubiquitous production of Toddler promote cell movement, suggesting that Toddler is neither an attractant nor a repellent but acts globally as a motogen. Toddler drives internalization of G protein–coupled APJ/Apelin receptors, and activation of APJ/Apelin signaling rescues toddler mutants. These results indicate that Toddler is an activator of APJ/Apelin receptor signaling, promotes gastrulation movements, and might be the first in a series of uncharacterized developmental signals. A conserved signal is identified that activates G protein–coupled receptors to promote zebrafish gastrulation. Toddler Welcome It has been assumed that most, if not all, major signals that control vertebrate embryogenesis have been identified. Using genomics, Pauli et al. (10.1126/science.1248636, published online 9 January) have now identified several new candidate signals expressed during early zebrafish development. One of these signals, Toddler, is a short, conserved, and secreted peptide that promotes the movement of cells during zebrafish gastrulation. Toddler signals through G protein–coupled receptors to drive internalization of the Apelin receptor, and activation of Apelin signaling can rescue toddler mutants.