gnb1 (a and b) and gnb4 (a and b) are expressed in the migrating pLLP and in deposited NMs. Transcripts of the indicated genes were detected by whole-mount in situ hybridization. (A-D) Embryos at 30-32 hpf and (A′-D′) High-magnification images of the boxed areas in A-D. Dots outline the pLLP. (E-H) Embryos at 72 hpf and (E′-H′) high magnification images of NMs in the boxed areas in E-H. All images are lateral views with anterior to the left. hpf: hours post fertilization.

EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Terms:
Stage Range: Prim-15 to Protruding-mouth

Gβ1, but not Gβ4, is required for migration of the pLLP. (A-E) Epifluorescence images of 48-hpf Tg(-8.0cldnb:lynEGFP) control embryos (A), embryos injected with MOs targeting gnb1a (B, C), gnb1a/b (D) or gnb4a/b (E). All images are lateral views with anterior to the left. White arrowheads, NMs. The embryo was subdivided into six equal sections (zones 1-6) from the ear to the tip of the tail. (F) Quantification of pLLP migration relative to the position of the posterior-most deposited NM (red arrows in A-E). The number of animals/injection is indicated.

Gβ1 signaling is required for the coordinated movement of cells within the pLLP. (A, B) Snapshots from 8-h epifluorescence time-lapse movies of control (A) or gnb1a/b MO1-injected (B). Tg(-8.0cldnb:lynEGFP) embryos, at 28-36 hpf, using a 5×/NA 0.15 objective (Movies 1 and 2). Lateral views with anterior to the left. Arrowheads, pLLP; *, the anterior-most region of the pronephric duct. (C, D) Snapshots from 2-h confocal time-lapse movies of control (C) or gnb1a/b MO1-injected (D). Tg(-8.0cldnb:lynEGFP) embryos, starting at 30 hpf, using a 20× NA 0.8 objective (Movies 3 and 4). The white arrows in C and D indicate the direction of pLLP migration. (C′-D′) Kymograph analysis showing the cell traces from the 2-h movies in C and D. (E) Speed of migration (in micrometers per hour) of the pLLP in control or gnb1a/b MO1-injected embryos recorded for 6 h from 28 to 34 hpf.

Gβ1 signaling regulates protrusive activity in the leading region of the pLLP. (A, B) Snapshots of the leading region of the pLLP from 30-min confocal time-lapse movies of control (A) or gnb1a/b MO1-injected (B) Tg(-8.0cldnb:lynEGFP) embryos, at 30 hpf, using a 40×/NA 1.3 objective (Movies 7 and 8). White arrows, protrusions at the leading edge of the pLLP. (C, D) Number of protrusions per cell (C) and average length of the filopodia (D) at the leading edge of the pLLP, in control or gnb1a/b MO1-injected embryos. *, p<0.01 vs. control.

Gβ1 signaling regulates actin dynamics in the leader cells of the pLLP. Confocal time-lapse movies taken on Tg(-135bpcxcr4b:lifeact-RFP)/Tg(-8.0cldnb:lynEGFP) double-transgenic embryos at 30–32 hpf, in which actin cytoskeleton dynamics were revealed by Lifeact-RFP labeling, and pLLP cell membrane by membrane-bound EGFP. (A-A′) (A) Montage images of the control pLLP leader cells from a 4.5-min confocal time-lapse movie. A few protrusion areas (arrowheads) were highlighted (red: high actin labeling, Cyan: decreased actin labeling, and white: faint or no actin labeling). Numbers follow the same area. A 19.5-min time-lapse recording is shown in Movie 9. (A′) The kymograph image was generated from 15-min movie along the line shown in the snapshot at 0 min time point, showing the relative positions of Lifeact-RFP and LynEGFP labeling. A few cycles of association and dissociation of Lifeact-RFP enrichment (arrowheads) with GFP were shown. (B, D) Snapshots of the leading region of the pLLP of gnb1a/b MO- (B, Movies 10), cxcl12a MO- (C) or cxcr4b MO- (D) injected embryos.

Gβ1 acts synergistically with Cxcr4b to regulate migration of the pLLP. (A-D) Epifluorescence images of the following 48-hpf Tg(-8.0cldnb:lynEGFP) embryos: WT uninjected control (A), cxcr4b(+/-) (B), WT injected with a low dose of gnb1a/b MO1 (1 ng, C) and cxcr4b(+/-) injected with a low dose of gnb1a/b MO1 (1 ng, D). Lateral views with anterior to the left; white arrowheads, NMs; and red arrows, posterior-most deposited NM. (F) Quantification of pLLP migration in embryos, as indicated in Fig. 2.

Morpholinos (MOs) targeting gnb1 (a and b) and gnb4 (a and b) efficiently block translation of the corresponding Gβ proteins, at stages during which the pLLP migrates. (A) Western blot analysis showing the expression of Gb in the indicated embryos at 24 hpf. α-Tubulin was used as a loading control. (B-C) Confocal images of the pLLP showing the expression of Gβ1 (B) and total Gβ (C), in 32-hpf Tg(-8.0cldnb:lynEGFP) embryos with or without injection of the indicated MOs.

Acknowledgments
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Reprinted from Developmental Biology, 385(2), Xu, H., Ye, D., Behra, M., Burgess, S., Chen, S., and Lin, F., Gbeta1 controls collective cell migration by regulating the protrusive activity of leader cells in the posterior lateral line primordium, 316-27, Copyright (2014) with permission from Elsevier. Full text @ Dev. Biol.