miRNA miR-430 regulates chemokine signaling and provides robustness to germ cell migration

microRNAs (miRNAs) are small RNA molecules that are widely expressed throughout development and in adult organisms. Through binding to partially complementary sequences in the 3'UTRs of mRNA transcripts, miRNAs function post-transcriptionally to modulate gene output. Based on this regulatory role, miRNAs have been proposed as a mechanism for reducing the consequences of genetic variability, thereby providing genetic robustness. However, few studies have provided evidence for this role of miRNAs. In this work, I investigate the ability of a particular miRNA, miR-430, to buffer against perturbations in chemokine signaling. This signaling pathway directs cell migration in both developmental and homestatic processes. In zebrafish, chemokine signaling guides the primordial germ cells (PGCs) from random locations in the early embryo to their ultimate position where the somatic gonad forms. Because this process requires precise control of gene expression at both the temporal and spatial level, I have examined the role of miR-430 in regulating chemokine signaling during zebrafish PGC migration.;I have demonstrated that in the absence of miR-430, PGC migration is disrupted and expression of the chemokine signal, sdf1a, is dramatically enhanced. I used GFP reporters to show that both sdf1a and its antagonistic receptor, cxcr7b, are targets of miR-430. Because miR-430 is predicted to regulate many transcripts, it is difficult to determine which misregulated genes are responsible for the miRNA loss of function phenotype. To dissect the role of chemokine signaling genes regulation, I employed Target Protectors. These antisense oligonucleotides bind to the miRNA binding site in the transcript and prevent binding by the miRNA. Upon protection of sdf1a or cxcr7b, there is an increase in mismigration of PGCs. In situ hybridization revealed that in the absence of miR-430, sdf1a expression is both enhance and extended outside of the appropriate domain. Finally, I have shown that regulation of sdf1a and cxcr7b allows the embryo to compensate for changes in gene dosage from either overexpression or heterozygous mutations.;In summary, I have identified a novel regulatory layer of the chemokine signaling pathway that ensures correct cell migration during development. I have shown that miR-430 (i) facilitates the dynamic expression pattern of sdf1a by clearing the ligand RNA from previous expression domains, (ii) modulates the expression levels of the decoy receptor Cxcr7b to avoid excessive depletion of Sdf1a and (iii) buffers against variation in gene dosage for components of the chemokine signaling pathway to ensure accurate germ cell migration. This work supports the role of miRNAs in both shaping gene expression as well as generally dampening the level of expression. These results indicate that losing miRNA-mediated regulation can expose otherwise buffered genetic lesions leading to developmental defects.