Zebrafish

RESEARCH REPORT

57

Development 139, 57-62 (2012) doi:10.1242/dev.073924 © 2012. Published by The Company of Biologists Ltd

G signaling controls the polarization of zebrafish primordial germ cells by regulating Rac activity
Hui Xu1, Elena Kardash2, Songhai Chen3, Erez Raz2 and Fang Lin1,*
SUMMARY
During development, primordial germ cells (PGCs) migrate from the sites of theirspecification towards the region in which the future gonad develops. This cell migration requires polarization of PGCs and their responsiveness to external guidance cues. In zebrafish, the directed migration and polarization of PGCs are regulated independently, by the chemokine Cxcl12a and the Rho GTPase Rac1, respectively. However, the upstream signals controlling Rac activity in this context have notyet been identified. By investigating the role of G proteins in PGC migration, we found that signaling mediated by G protein subunits G is required to regulate cell polarization. PGCs that are defective for G signaling failed to polarize, and developed multiple protrusions in random locations, resembling the defects observed in PGCs with decreased Rac activity. These defects render PGCsincapable of migrating actively and responding to directional cues. FRET-based assays showed that PGCs require G signaling for polarized Rac activation and actin organization at the leading front, as well as for maintaining overall Rac levels in these cells. Conversely, overexpression of G in PGCs increases Rac activity. Our results indicate that during PGC migration in vivo, G signaling regulatesRac activity to control cell polarity, which is required for the responsiveness to chemokine signaling.
KEY WORDS: G signaling, Cell migration, Primordial germ cells, Zebrafish

INTRODUCTION Directed cell migration is involved in diverse processes during embryogenesis, immune response and disease (Friedl and Gilmour, 2009; Raz and Mahabaleshwar, 2009). Elucidating the underlying mechanisms isthus highly relevant for understanding both normal development and pathological conditions. In many species, primordial germ cells (PGCs) migrate a long distance from the sites of their specification to the region in which the future gonad develops (Richardson and Lehmann, 2010; Tarbashevich and Raz, 2010). They, thus, represent a powerful model for studying directed cell migration in the contextof the developing organism. Like other cell types, PGCs rely on both intrinsic cell motility and external guidance cues to migrate efficiently and precisely to their targets. In zebrafish, the chemokine Cxcl12a/Sdf1a guides PGC migration by activating its cognate G protein-coupled receptor Cxcr4b, which is expressed in PGCs (Doitsidou et al., 2002; Knaut et al., 2003; Boldajipour et al., 2008).However, PGCs lacking Cxcl12a or Cxcr4b are able to polarize and migrate actively, albeit non-directionally (Reichman-Fried et al., 2004; Kardash et al., 2010). Recent studies demonstrated that PGC polarization involves restricted Rac1 activation and the formation of actin-rich structures called ‘actin brushes’ at the leading edge (Kardash et al., 2010). Interestingly, the as yet unidentifiedsignals that regulate the activation of Rac1 act independently of chemokine signaling (Kardash et al., 2010).

Investigating the function of G proteins in PGC migration, we found that the G protein subunits G are crucial for the efficient polarization of PGCs and their migration. Inhibition of G signaling resulted in a loss of polarity and the ectopic distributions of PGCs. Live-cell imagingindicated that, unlike PGCs with reduced chemokine signaling, PGCs with compromised G signaling failed to become polarized and to migrate actively, suggesting that G is involved in a chemokine-independent pathway. We further show that this phenotype stems from a lack of persistent Rac activation and a consequent failure to accumulate polymerized actin at the leading edge. Our data thus provide...