Gβγ signaling controls the polarization of zebrafish primordial germ cells by regulating Rac activity

Abstract

SUMMARYDuring development, primordial germ cells (PGCs) migrate from the sites of their specification 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 not yet 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 PGCs incapable 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 regulates Rac activity to control cell polarity, which is required for the responsiveness to chemokine signaling.

Gβ1 controls collective cell migration by regulating the protrusive activity of leader cells in the posterior lateral line primordium

“…We previously identified 5 Gβ isoforms (9 members, of which all except Gβ2 are duplicated) in the zebrafish genome. Among the encoding genes, those for the Gβ1 and Gβ4 isoforms ( gnb1a, gnb1b , gnb4a, gnb4b ) are maternally and ubiquitously expressed during gastrulation (Xu et al, 2012). In this study, we found that their expression continues throughout the embryo from 30–48 hours post fertilization (hpf), particularly in the anterior region of embryo (Fig.…”

“…To investigate the functions of Gβ1 and Gβ4 during pLLP migration, we inhibited their expression using previously validated antisense morpholino oligonucleotides (MOs, in this case MO1) (Xu et al, 2012). Western blotting using an antibody that specifically recognizes zebrafish Gβ1 (both the Gβ1a and Gβ1b isoforms) confirmed that injection of MOs targeting both gnb1a and gnb1b ( gnb1a/b MO1) led to 81% reduction of the Gβ1 expression in 24-hpf embryos (Fig.…”

“…For gnb1 ( gnb1a and gnb1b ): the first set of MOs inhibits translation, as previously validated (Xu et al, 2012), and the individual MOs are referred as gnb1a MO1 (2ng, 5′-GAGTTCGCT CAT TTTCTTCTGCTTC-3′) and gnb1b MO1 (2ng, 5′-CTGGTCCAGTTCACT CAT TTTCCTC-3′), respectively; the second set of MOs inhibits gnb1a translation and blocks gnb1b splicing, as previously validated (Hippe et al, 2009), and the individual MOs are referred as gnb1a MO2 (3ng, 5′-CTGGTCGAGTTCGCT CAT TTTCTTC-3′) and gnb1b MO2 (8ng, 5′-AATTAGGTGGTTACCTGTGATAGT-3′, targets the splice site at the junction between the 1 st exon and intron). For gnb4 ( gnb4a and gnb4b ), the first set of MOs block translation, as previously validated (Xu et al, 2012), and the individual MOs are referred as gnb4a MO1 (4ng, 5′-CCGCAACTGCTC CAGCTCACT CAT G-3′) and gnb4b MO1 (4ng, 5′-GACGCAACTGCTCCAACTCACT CAT -3′); the second set of MOs targets the splicing of gnb4a and gnb4b and the individual MOs are referred as gnb4a MO2 (8ng, 5′-GCATCCTGCAATGGGAACAGCAGCA-3′, targets the splice site at the junction between the 2 nd intron and the 3 rd exon) and gnb4b MO2 (8ng, 5′-TGTTACGGAGTCACCCTTACCCGGA-3′, targets the splice site at the junction between the 2 nd exon and intron). RT-PCR performed on RNA isolated from 28-hpf embryos revealed that embryos injected with gnb4a MO2 or gnb4b MO2 produced smaller amplicons than uninjected control embryos (Fig.…”

The Expanding Roles of GβγSubunits in G Protein–Coupled Receptor Signaling and Drug Action

“…Previous work has found that a defect in Gbg signaling leads to alterations in cell migration, which requires reorganization of the actin-cytoskeleton network (Peracino et al, 1998;Hwang et al, 2005;Xu et al, 2012). Upon GPCR activation by chemokines, G protein heterotrimers are believed to dissociate and modulate many signaling pathways that control cell migration.…”

A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms

“…Biosensor imaging has been applied to classic models of embryonic development less frequently than whole animal physiology, with only a handful of successes reported in zebrafish (Andrews et al, 2016; Muto et al, 2011; Xu et al, 2012; Zhao et al, 2015), Drosophila (Markova et al, 2015), and C. elegans (Kelley et al, 2015; Vuong-Brender et al, 2018). Consequently, less is known about protein activities during development outside of gene expression.…”