Temporal control over the initiation of cell motility by a regulator of G-protein signaling

rgs14a RNA Is a Previously Unidentified Component of the Germ Plasm Strongly Expressed in Premigratory PGCs.

Whereas the molecular and cellular mechanisms controlling PGC migration and their response to the guidance cues were extensively studied (12⇓–14), the mechanisms dictating the timing of cell motility acquisition are not known.

To identify molecules controlling early PGC behavior, we conducted a microarray-based screen for genes whose RNA expression is elevated in PGCs before the onset of their migration, i.e., 4 hpf. Based on the microarray results (Table S1), we identified the regulator of G-protein signaling 14a (Rgs14a; Fig. 1A) as one such gene that could be important for PGC development at this early stage. Specifically, relative to somatic cells at the same stage, the RNA encoding for Rgs14a is enriched more than 18-fold in PGCs before the initiation of migration (Fig. 1B and Table S1), whereas its relative expression level progressively drops at later stages (Fig. 1B, note that 4.7, 5.3, and 10 hpf embryos were overstained to detect the reduced rgs14a expression at those stages and Fig. S1A).

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Fig. 1.

Zebrafish regulator of G-protein signaling 14a (rgs14a) RNA expression and Rgs14a protein localization. (A) A schematic representation of the Rgs14a protein structure showing the N-terminal RGS domain, the two Raf-like Ras-binding domains (RBD), and the C-terminal GoLoco domain. (B) Whole-mount in situ RNA hybridization using rgs14a (Left) and nanos (Right) antisense RNA probes at the indicated embryonic stages; 5.3- and 10-hpf embryos were overstained in the case of rgs14a in situ hybridization to detect the weak expression in the PGCs. (C) GFP-Rgs14a fusion protein expressed in PGCs is localized to the cytoplasm and the plasma membrane, with enrichment in perinuclear granules (in 91% of the 46 PGCs analyzed; Top) or in the nucleus (in 9% of the 46 PGCs analyzed; Middle). The perinuclear granules where the GFP-Rgs14a protein is localized harbor Vasa-DsRed protein (Bottom), defining them as germ cell granules. (Scale bars, 10 µm.)

Similar to several PGC markers in zebrafish (e.g., nanos1, tdrd7, and vasa) (15⇓–17), the maternally provided mRNA of rgs14a is localized to the cleavage-planes of four-cell embryos, the position where the germ plasm resides (Fig. 1B, Upper) (15, 18). rgs14a mRNA is then incorporated into the PGCs while its level progressively declines (Fig. 1B and Fig. S1A) compared with nanos1 mRNA, whose expression is maintained until later stages (Fig. 1B). Zygotic expression of rgs14a could be detected around the time zygotic transcription is initiated. This expression that might reflect low basal transcription of the gene in somatic cells, or very low expression in PGCs, has apparently only little effect on the gradual depletion of the mRNA (Fig. S1 A and B).

To determine the subcellular localization of Rgs14a, we expressed a GFP-tagged version of the protein in PGCs. Interestingly, in addition to a diffuse cytosolic and membranal subcellular distribution of the protein (Fig. 1C), we observed an enrichment of the GFP-Rgs14a protein in germ cell perinuclear granules, where the Vasa-dsRed protein also accumulates (Fig. 1C, Lower).

Unlike some other germ cell-enriched transcripts whose 3′UTR can direct expression of proteins to the germ line (16, 19), the 3′UTR of rgs14a did not appear to do so, as seen on injection of gfp-rgs14a-rgs14a3′UTR RNA into one-cell stage embryos (Fig. S1C). The localization of rgs14a transcripts to the germ plasm and later to germ cells is thus likely to depend on earlier events that the injected RNA can no longer take part in.

Coordination of the Onset of PGC Migration with the Presentation of Guidance Cues.

For accurate arrival at the target, the onset of cell migration should be coordinated with the presentation of the guidance cues; those are provided by the graded distribution of the chemokine Cxcl12a in the case of zebrafish PGCs (12). Specifically, whereas the germ line is established at 3 hpf, it is only at 5.3–6 hpf (between 50% epiboly to shield stages) that spatially restricted cxcl12a transcription, which can provide directional information, is observed at the embryonic blastoderm margin (Fig. 2A and Fig. S2A), the position where PGCs normally reside at this developmental stage (12). These findings were confirmed using a more sensitive in situ hybridization procedure using the RNAscope probes (20), revealing weak uniform early cxcl12a expression followed by spatially restricted expression that can direct PGC migration at 6 hpf (Fig. S2B). cxcl12b, a gene encoding for a second Cxcl12 protein, shows only little activity in the context of PGC migration (21), and its expression starts at an even later stage (7.5 hpf based on RT-PCR analysis, Fig. S2C; with no signal detected by in situ hybridization, Fig. S2A).

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Fig. 2.

The role of Rgs14a in the temporal control of migration initiation. (A) Whole-mount in situ hybridization of embryos with a cxcl12a antisense RNA probe showing the initiation of transcription of the chemokine between 5.3 and 6.0 hpf. (B) Premigratory PGCs develop polarized actin brushes. (C) Morpholino-mediated knockdown of rgs14a leads to an increase in the average number of ectopic PGCs at 11 hpf (number of ectopic PGCs divided by total PGC number; Fig. S3). (D and E) PGC groups at 4.6 hpf (dotted circles) in Rgs14a knockdown embryos cover 53% larger areas than those in control embryos. (F and G) Early overexpression of Rgs14a, using the Xenopus globin3′UTR, leads to significantly smaller PGC group areas at 6.5 hpf and an increase in the average number of ectopic PGCs at 11 hpf; target defined analogous to Fig. S3 (H). PGC groups in D–G were labeled with a nanos1 antisense RNA probe. Control embryos in F–H expressed a pa-(photoactivatable)-gfp-globin3′UTR mRNA. For E and G, PGC group sizes were averaged for each embryo, and 32–45 embryos were included per experimental point. n = number of embryos analyzed. (C and H) *P < 0.05, **P < 0.01, and ***P < 0.001 using Student t test. (E and G) *P < 0.05 using Mann–Whitney u test. Error bars indicate SEM.

Interestingly, even before the onset of migration and the establishment of spatially restricted directional cues, the PGCs already show polarized actin accumulation at the cell front (Fig. 2B). Thus, whereas shortly after their formation the PGCs can polarize and form structures that normally function in promoting their migration (22), they are initially immotile and express high levels of E-cadherin (9). The gradual decrease in rgs14a RNA expression level observed in the course of the transition from stationary to motile behavior is consistent with the idea that the Rgs14a protein plays a role in regulating the process.

Rgs14a Controls the Onset of PGC Migration.

To probe the function of maternally provided rgs14a mRNA in PGC migration, we inhibited its function using two different translation-blocking morpholino oligonucleotides (MOs). Indeed, as assayed at the three-somites stage (11 hpf), the time by which the majority of zebrafish PGCs already arrived at their target site (Fig. S3, dashed brackets), rgs14a knockdown reduced the efficiency of PGC arrival at that site (Fig. S3) with a significantly higher number of PGCs located at ectopic positions (Fig. 2C). This result suggests that premature (compared with the normal decay in rgs14a RNA level) inhibition of rgs14a function bears phenotypic consequences for PGC migration.

To determine the basis for this phenotype, we examined the behavior of the cells at the time of motility acquisition (4.6 hpf) (9). We first determined the area of circles occupied by PGC groups at the four locations where they originated to assess the degree of cell motility at those early stages (Fig. 2D). Interestingly, PGC groups in embryos depleted for Rgs14a occupied a significantly larger area than in control embryos. The two different nonoverlapping antisense oligonucleotides used in this experiment yielded the same results (Fig. 2 D and E). Conversely, early overexpression of rgs14a led to a reduction in the area PGC groups occupy relative to control at 6.5 hpf (Fig. 2 F and G) and to an increase in ectopic PGCs (Fig. 2H). These findings could stem from a premature onset of motility in embryos knocked down for Rgs14a and therefore prompted us to follow closely the behavior of PGCs at those stages.

As the first step in this analysis, we monitored the dynamics of PGC migration onset by live imaging from 3.5 to 6.5 hpf in transgenic embryos whose PGCs express GFP at these stages. To label the environment within which the PGCs migrate, we expressed mCherry-H2B protein in all cells (Fig. 3A and Movie S1, PGC migration onset events are marked with arrows while the movie is momentarily paused). Significantly, we observed that the migration onset of PGCs depleted for Rgs14a occurred earlier than in control PGCs. Conversely, mild overexpression of Rgs14a (100 pg rgs14a-globin3′UTR RNA) resulted in a delayed onset of PGC migration. Combining rgs14a overexpression and knockdown of the endogenous mRNA restored the migration onset to WT timing [shown in minutes postfertilization (mpf) in Fig. 3B and Fig. S4A), demonstrating the specificity of the treatment. Overexpression of Rgs14a at higher amounts (400 pg rgs14a-globin3′UTR RNA) resulted in a stronger effect on PGC motility acquisition (Fig. S4B and Movie S2). Specifically, whereas at 6.5 hpf, only 26.3 ± 4.9% of the rgs14a-globin3′UTR expressing PGCs acquired motility, 78.5 ± 4.8% of the PGCs expressing control-globin3′UTR were motile (Fig. S4B).

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Fig. 3.

The acquisition of cell motility is inhibited by Rgs14a. (A) Snapshots from time-lapse movies showing PGC migration-onset events (mpf = minutes post fertilization). Rgs14a morphants (MO1) exhibit earlier PGC migration onset events (white arrows) compared with control morphants (Movie S1). (B) PGC migration onset events for Rgs14a and control morphants, as well as Rgs14a overexpression. Knockdown of Rgs14a leads to premature migration onset, whereas overexpression of 100 pg rgs14a-globin3′UTR delays PGC migration onset. Combination of both treatments restored WT motility acquisition timing. Time point zero is defined as the PGC specification event at 180 mpf (9). pa(photoactivatable)-gfp-globin3′UTR was used as a control mRNA. n = number of cells analyzed. (B) ***P < 0.001 using Mann–Whitney u test. Error bars indicate SEM.

Thus, as previously shown (9), shortly after PGC formation, the cells can generate cellular protrusions and can even polarize, as manifested by polar formation of actin structures at the cell front (Fig. 2B). Nevertheless, the cells are rendered immotile until the time chemotactic cues in the environment are established. We propose that the maternally provided rgs14a RNA is translated and functions in inhibiting cell motility. Gradual degradation of the RNA relieves this inhibition as the guidance cues are being established, thereby coordinating cell motility with the emergence of directional cues (Fig. 2A and Fig. S2 A and B). The importance of this coordination is signified by the fact that embryos in which Rgs14a is knocked down exhibited a higher proportion of ectopic cells. We assume that the premature, nondirectional migration of those cells positioned them in locations distant from the expression domains of the attractive cues, from which they failed to reach the correct target. Although we could induce a migration phenotype in PGCs by inhibiting mRNA translation, it could be that in addition to the maternally provided RNA, maternally deposited Rgs14a protein masks the complete loss-of-function phenotype.

Rgs14a Controls PGC Motility by Regulating Polar Protrusion Formation and E-Cadherin Expression.

The results presented above are consistent with the idea that Rgs14a suspends the onset of migration, synchronizing cell motility with the establishment of directional cues. To further substantiate this notion and gain insight into the mechanisms underlying Rgs14a function, we expressed the protein in PGCs at later stages of their development using the nanos3′UTR (16) and determined the effect on the behavior of the cells. Strikingly, expression of Rgs14a in the PGCs beyond early stages of their development resulted in a strong germ cell migration phenotype manifested by the presence of a large number of ectopic cells in 24 hpf embryos (Fig. 4A, yellow arrowheads). Specifically, an average 32% of PGCs expressing Rgs14a failed to reach the gonad compared with 10% in the control embryos (Fig. 4C). The inefficient arrival at the target was correlated with a significant reduction in motility of the experimental cells, as demonstrated by the shorter migration tracks of the experimental cells during gastrulation stages (6–8 hpf; Fig. 4B). The inhibition of cell motility under such conditions resulted from the combined effect on several motility parameters, namely a reduction in cell displacement, migration speed, and track straightness (Fig. 4D and Movies S3 and S4). Along the same lines, PGCs expressing Rgs14a exhibited a reduction in the duration of run phases, the phases of active motility relative to surrounding cells, and a concomitant increase in the duration of the apolar, immotile tumbling phases (13) (Fig. 4E). Consistently, high-magnification time-lapse movies (Movies S5 and S6) revealed that the manipulated cells were less polar and produced protrusions in all directions at a high frequency (Fig. 4 F and G).

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Fig. 4.

Expression of Rgs14a during migration phases impairs PGC motility. (A and C) Forced expression of rgs14a-nanos3′UTR in PGCs during migratory stages results in inefficient colonization of the gonad region at 24 hpf. Ectopic cells exemplarily labeled with arrowheads in A, n = number of embryos analyzed. (B) PGCs expressing Rgs14a exhibit shorter migration tracks, stemming from impaired motility parameters (D) and longer immotile phases (E). The results presented in B, D, and E were derived from time-lapse movies spanning 70 min (Movies S3 and S4) captured in three independent experiments, with 21 control and 35 rgs14a expressing PGCs analyzed. (F and G) PGCs expressing Rgs14a display an apolar cell shape and extensive protrusion formation (protrusions indicated by white arrowheads) (Movies S5 and S6), n = number of cells analyzed. (H) The RGS domain of Rgs14a is essential for the induction of ectopic PGC migration when expressed in PGCs under the control of the nanos 3′ UTR. n = number of embryos analyzed. A, C, F, G, and H were controlled by injection of cd14-nanos3′UTR RNA. B, D, and E were controlled by injection of gfp-nanos3′UTR RNA. *P < 0.05 and **P < 0.01 using Student t test. Error bars indicate SEM.

To gain insight into the molecular mechanisms of Rgs14a function and define the protein domains required for its role in interfering with PGC migration, we next expressed either the full-length protein (Fig. 4H, protein version 1) or mutated versions of it (Fig. 4H, proteins 2–7). The function of the different proteins was then assayed by determining the number of ectopic PGCs at 24 hpf. Significantly, this analysis highlighted the importance of the regulator of G-protein signaling (RGS) domain in induction of the phenotype, because its deletion abrogated the function of the protein without affecting protein translation or localization (protein version 6 in Fig. 4H and Fig. S5A).

Importantly, consistent with its RNA expression pattern, Rgs14a appears to exert its function within the PGCs, as determined by comparing the migration tracks of Rgs14a-expressing PGCs within an untreated environment with those of untreated PGCs migrating in an environment expressing the protein (Fig. S6 and Movie S7). Significantly, alterations of Rgs14a levels had no effect on gastrulation speed (Fig. S7 A and B), nor on the group cell migration of the posterior lateral line primordium (Fig. S7 C and D), underlining the specificity of the Rgs14a effect on PGC migration. Nevertheless, a small proportion of embryos overexpressing the protein (but not embryos knocked down for it) did show developmental malformations when assayed at 24 hpf (Fig. S7 E and F).

It has been previously shown that acquisition of PGC motility correlates with a reduction in the level of the cell–cell adhesion molecule E-cadherin (9, 11). We therefore sought to examine the possibility that Rgs14a regulates E-cadherin levels at this stage. To this end, we determined the level of E-cadherin on PGC membranes relative to that on membranes of neighboring somatic cells in control PGCs in which rgs14a level naturally declined and in PGCs engineered to express the RNA at later stages (Fig. 5 A and B). As described before (9), at the time motility is initiated, PGCs exhibit reduced E-cadherin levels compared with somatic cells around them. No such reduction was observed in PGCs expressing Rgs14a during migratory stages (8 hpf), as evidenced by an average ratio of 1 between the levels in PGCs and somatic cells (Fig. 5C). Conversely, premature E-cadherin level decrease during stages of PGC motility acquisition (4.6 hpf) was observed when rgs14a was knocked down (Fig. S8C). Upon Rgs14a expression (Fig. 5D), the signal level of E-cadherin-GFP on the PGC membrane was increased relative to the cytoplasmic signal, presumably reflecting a change in intracellular trafficking of the molecule. The suggestion that Rgs14a functions by regulating E-cadherin level is supported by the finding that a protein lacking the RGS domain (version 6 in Fig. 4H) and has no effect PGC migration does not influence the level of E-cadherin (Fig. 5C). In addition, overexpression of E-cadherin in embryos depleted of rgs14a restored the normal migration onset time of PGCs (Fig. 5E), further highlighting the relevance of E-cadherin for this event. Together, our results are consistent with the idea that Rgs14a controls PGC motility initiation, at least in part, by regulating cell–cell adhesion.

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Fig. 5.

Rgs14a elevates E-cadherin levels on PGC membranes thereby affecting PGC motility. (A) The experimental setup and the scheme for evaluating E-cadherin level. (A and B) Embryos expressing F-EGFP on their membranes were injected with rgs14a-nanos3′UTR and control mRNA (cd14-nanos3′UTR), fixed, and stained for E-cadherin at 8 hpf. E-cadherin signal on the membrane of PGCs (light blue dashed lines) was normalized by dividing it by the corresponding signal on the membranes of neighboring somatic cells (yellow dashed lines). (C) PGCs expressing the full-length Rgs14a (protein 1) during migratory stages exhibit more E-cadherin on their membrane compared with PGCs expressing control RNA or RNA encoding for versions 3 and 6 of the Rgs14a protein. n = number of cell pairs analyzed. (D) Expression of Rgs14a in PGCs enhances the membrane localization of E-cadherin-GFP relative to the cytoplasm. (E) Forced E-cadherin expression abrogates the premature motility acquisition of MO1-treated PGCs. (F) Summary of the findings. The initiation of spatially restricted cxcl12a expression (red) coincides with a rapid decrease in rgs14a mRNA levels in PGCs (orange), suggesting a role in PGC migration onset (vertical dotted line). The onset of motile activity of PGCs occurs prematurely in germ cells knocked down (kd) for rgs14a, whereas the overexpression (oex) of Rgs14a led to motility defects, correlated with enhanced E-cadherin levels in the cells, whereas rgs14a knockdown prematurely reduced E-cadherin levels. n = number of cells analyzed. (C and E) ***P < 0.001 using Mann–Whitney u test. (D) *P < 0.05 using Student t test. Error bars indicate SEM.

Importantly, overexpression of E-cadherin in PGCs resulted in a PGC migration phenotype and increased protrusion formation frequency (Fig. S8 A and B) (9). Both findings are comparable to the phenotype induced by Rgs14a expression at later stages of PGC development (Fig. 4 C and G). The increase in protrusion frequency in cells overexpressing E-cadherin could stem from the regulation of Rho-kinase dependent myosin II activity at the cell cortex (23, 24) or from uneven adhesion around the cell perimeter under these conditions.

Taken together, we assigned a role for Rgs14a, whose RNA constitutes a previously unidentified germ plasm component, in regulating the onset of PGC migration (Fig. 5F). We provide evidence suggesting that Rgs14a exerts its function, at least in part, by regulating the level of E-cadherin on the cell membrane, thereby controlling the acquisition of cell motility. The link between Rgs14a and E-cadherin expression in this case could be relevant in other contexts such as the repression of synaptic plasticity in hippocampal neurons by Rgs14 (25), a phenomenon that is highly dependent on cell–cell adhesion as well (26). In the same direction, the role Rgs14 plays in determining the degree of interaction of migratory cells with cells in the environment where they are specified could be relevant in the case of dendritic cells and B lymphocytes. Here, Rgs14 is down-regulated on activation of the two cell populations (27, 28).

The precise mechanisms by which Rgs14 regulates migration and adhesion levels are currently unknown, but our results point to the importance of the RGS domain, presumably acting in regulating a G_α-protein signaling pathway. A conceivable scenario could include an interaction between a G-protein and Rgs14a acting as a GTPase activating (GAP) protein that promotes GTP hydrolysis. Such an interaction could lead to termination of a G-protein signaling cascade. In support of this option, G_iα signaling has been shown to be critical for proper PGC migration, as demonstrated by expression of pertussis toxin in PGCs (29). In addition, as G13_α is known to control E-cadherin and Rho-mediated contractility (30⇓–32) and because Rgs14 was shown to regulate G_iα- and G13_α-mediated signaling pathways (33, 34), a plausible option is that the protein exerts its function by interacting with those proteins. Whereas we could not provide evidence supporting the idea that the RBD domain of Rgs14a plays a role in PGC motility, interactions with other effector proteins such as H-Ras/Raf and MAP kinase (35) should be investigated in other contexts as well.

Conclusions.

The identification of rgs14a as a previously unidentified germ plasm component regulating early germ cell behavior opens new research directions in the germ-line biology and the cell migration fields. It would be interesting to determine whether this component is present in the germ plasm of other organisms and to examine its function in germ cell behavior. Similarly, it would be important to determine how motility acquisition is coordinated in other cell types and to examine if Rgs14, regulation over E-cadherin function, or G-protein signaling is involved.