Correspondence to Lilianna Solnica-Krezel: lilianna.solnica-krezel{at}Vanderbilt.edu; or Heidi Hamm: heidi.hamm{at}vanderbilt.edu
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Introduction |
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Noncanonical Wnt signaling, an equivalent of the planar cell polarity signaling in Drosophila melanogaster (Wnt-PCP), is a major regulator of the mediolateral cell polarization required for cell intercalation in frog and fish, and fast dorsal migration in fish gastrulae (Keller, 2002; Myers et al., 2002b; Wallingford et al., 2002). Mutants of several genes involved in this pathway, such as trilobite (strabismus; Jessen et al., 2002), knypek (glypican4/6; Topczewski et al., 2001), silberblick (wnt11; Heisenberg et al., 2000) display shortened axis and defective mesodermal cell polarization. Recent evidence indicates that Heterotrimeric G proteins may participate in the Wnt/Ca2+ branch of the noncanonical pathway, which involves intracellular Ca2+ release and activation of PKC (Sheldahl et al., 1999; Malbon et al., 2001). The Wnt signaling pathway is activated by the binding of Wnt ligands to the Frizzled receptors, which have seven transmembrane domains, a structural characteristic of G proteincoupled receptors (GPCRs). There is evidence that like GPCRs, Frizzled receptors may activate G proteins to mediate their signal transduction. In cultured cells, coupling of the Frizzled receptor to Go, G
q, and G
t has been reported (Liu et al., 1999; Liu et al., 2001; Ahumada et al., 2002). In addition, it has been shown that G proteins are involved in Wnt signaling pathways that mediate gastrulation. Expression of pertussis toxin (which ADP-rybosylates G
i and G
o and uncouples them from their cognate receptors) disrupts tissue separation during Xenopus laevis gastrulation, an effect also seen with Xfz7 depletion. Moreover, PKC can rescue the defect in tissue separation in both Xfz7-depleted and PTX-injected embryos, suggesting that PTX-sensitive G proteins and PKC are involved in Xenopus gastrulation movements (Winklbauer et al., 2001). In addition, PKC
and PKC
are activated by Frizzled receptors, possibly through G proteins and Dishevelled to regulate C&E movements in Xenopus (Kuhl et al., 2001; Kinoshita et al., 2003). Furthermore, G
o is required for both the canonical Wnt and PCP signaling in Drosophila (Katanaev et al., 2005). Recently, it has been reported that Gß
subunits may also play important roles in C&E movements. In Xenopus gastrulae, inhibition of Gß
signaling by overexpression of G
i and G
t (which sequester free Gß
) rescued C&E defects that resulted from activation of Wnt11/Xfz7 (Penzo-Mendez et al., 2003). In addition, inhibition of Gß
signaling in the Xenopus dorsal marginal zone resulted in gastrulation arrest. However, exactly which G
-proteins are involved in Wnt-PCPmediated gastrulation remains unknown.
G proteins consist of four classes: Gs, G
i, G
q, and G
12/13 (Simon et al., 1991). G
12 /13 subunits are the most divergent G protein family and have been implicated in numerous cellular processes such as Rho-mediated cytoskeletal rearrangements, thereby affecting cell shape and migration (Buhl et al., 1995; Gohla et al., 1999; Sugimoto et al., 2003). Studies in Drosophila indicate that G
12 /13 signaling plays a role in gastrulation, as inactivation of the Drosophila G
12 homologue, concertina, impairs cell shape changes underlying mesoderm internalization during gastrulation (Parks and Wieschaus, 1991). In mice, disruption of G
13 gene led to embryonic death at midgestation, due to the failure of endothelial cells to form an organized vascular system (Offermanns et al., 1997). In addition, G
12 /13 have been shown to induce primitive endoderm formation in mouse F9 cells (Lee et al., 2004). However, the role of G
12/13 in vertebrate gastrulation has not been analyzed.
Here, we used zebrafish as a model to investigate the role of G12/13 in early vertebrate embryogenesis. Using dominant negative receptor blocking peptides and antisense morpholino oligonucleotides (MOs), we demonstrate that G
12 and G
13 have overlapping and essential roles in C&E. Cell movement analyses show that G
12 /13 signaling regulates slow dorsal migration of lateral mesoderm cells independent of noncanonical Wnt signaling. In the notochord, G
12 /13 are required for mediolateral cell intercalation, acting cell-autonomously, and likely in parallel to noncanonical Wnt signaling. Our studies for the first time suggest a central role for G
12/13 signaling in generating the diversity of gastrulation cell behaviors in vertebrate embryos.
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Results |
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In cultured cells, mammalian G12/13 induce stress fiber formation via a RhoA/Rho kinase (Rok)-dependent pathway (Buhl et al., 1995; Gohla et al., 1998). To evaluate if zebrafish G
12/13 have similar activities, wild-type (WT) and constitutively active G
12/13 mutant proteins were transiently expressed in human embryonic kidney (HEK) cells. Cells overexpressing either WT human or zebrafish G
12 or G
13 (Fig. 1, C, E, and F) or constitutively active human or zebrafish G
13 (Fig. 1, D and G) displayed stress fibers even in the absence of agonist stimulation. Formation of stress fibers was blocked by pretreatment with 10 µM of Rok inhibitor, Y-27623 (Fig. 1 H; Uehata et al., 1997). These results indicate that like their human counterparts, zebrafish G
12/13 can promote actin rearrangements in cultured cells, through a RhoA/Rok-dependent pathway.
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Interference with G12/13 function does not alter cell fate specification during gastrulation
Gastrulation defects might be a consequence of altered embryonic patterning and consequent changes in cell movements, or might be due to defects in cell movements alone (Myers et al., 2002b). Therefore, we tested whether dorsoventral patterning is affected in G12/13-CT or MO injected embryos by analyzing expression of dorsoventral patterning genes, bmp4 and chordin (Hammerschmidt and Mullins, 2002). Our results revealed that bmp4 expression was not altered in early and late gastrulae injected with three MOs (Fig. 4 M, n =34, and not depicted). Likewise, expression of chordin gene encoding a Bmp antagonist was confined to its normal dorsal expression domain during early gastrulation in embryos injected with the combination of 3MO (Fig. 4 O, n = 32), or with RNAs encoding G
12/13-CT peptides (not depicted). Moreover, embryos injected with CT peptide RNA or 3MO displayed normal expression of several cell type specific markers at late gastrulation, consistent with normal cell fate specification (Fig. 3, IL; Fig. 4 F). Finally, cell tracing experiments revealed that the labeled lateral mesodermal cells acquired somitic fates in G
12/13-depleted embryos (not depicted), consistent with their positions in the early gastrula (Sepich et al., 2000). Based on these results, we conclude that morphogenetic defects observed in G
12/13 depleted embryos are likely not associated with significant patterning or cell fate changes during gastrulation.
G12/13 are required for efficient directed cell migration during early dorsal convergence movements
Shortened anteroposterior and enlarged mediolateral dimensions of the embryonic axes in G12/13-depleted gastrulae could be a consequence of defective C&E movements (Sepich et al., 2000). Recent studies reveal that convergence movements in zebrafish mesoderm are accomplished by a stereotyped sequence of cell behaviors, including slow and fast directed cell migration (Jessen et al., 2002). To investigate if any of the convergence cell behaviors were altered in G
12/13-depleted embryos, we performed Nomarski time-lapse analyses in WT (6 embryos, 144 cells) and 3MO-injected (6 embryos, 134 cells) embryos at midgastrulation. Analysis of total cell speed, accounting for movement in all directions, revealed that in G
12/13-depleted gastrulae, cells moved at a reduced speed (70% of WT total speed; P = 9.8 x 1027; Fig. 5 B). Interestingly, the net dorsal speed of G
12/13-depleted cells was especially strongly compromised, accounting only for 28% of the WT net dorsal speed (P = 2.1 x 1013, Fig. 5 B). Further analysis of cell migration paths revealed that, similar to the WT cells, G
12/13-depleted cells migrated predominantly dorsally (Fig. 6 A). However, compared with WT, these cells more frequently changed their movement direction (Fig. 6, A and B), at the expense of movement in the dorsal direction (Fig. 6 C). To determine how efficiently WT and 3MO cells corrected their path direction when they were off-course, we examined cells moving toward dorsal, animal, ventral or vegetal direction (± 15°) and assayed the direction of their next step (Fig. 6, DG). We found that in WT embryos, cells moving dorsally largely maintained this direction in the next step. Moreover, WT cells that had been moving in the animal or vegetal direction turned toward dorsal in the next movement step, very few cells from these populations moved away from dorsal. By contrast, equivalent cell populations in embryos injected with 3MO were less persistent in dorsal movement (Fig. 6 D). Moreover, when these cells moved in animal or vegetal direction, they less frequently corrected their movement toward dorsal compared with WT cells (Fig. 6, E and F). Cells moving ventrally made large turns, and no clear difference is apparent between WT and 3MO cells (Fig. 6 G). Consequently, cells in G
12/13-depleted gastrulae migrated less efficiently toward dorsal. In support of the notion that this defect is specific to the depletion of G
12/13 proteins, cells in embryos injected with an equivalent dose of gna13a-MO (12 ng) migrated dorsally at comparable speed to WT cells (unpublished data). Together, these studies revealed that G
12/13 function is required for directed dorsal migration underlying the early, slow convergence movements of lateral mesodermal cells.
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Interference with G12/13 function disrupts mediolateral cell intercalation
Mediolaterally oriented intercalation of cells at the dorsal midline drives robust axial extension in zebrafish (Crawford et al., 2003; Glickman et al., 2003) and Xenopus gastrulae (Shih and Keller, 1992). During gastrulation, notochord precursor cells elongate, align mediolaterally and intercalate between one another to lengthen the notochord anteroposteriorly and narrow its mediolateral dimension, decreasing from the initial width of 45 to 12 cells (Glickman et al., 2003). To investigate whether G12/13 signaling is required for mediolateral intercalation of midline cells, we analyzed shape (LWR) and orientation of notochord cells at the 4 and 6 somite stage. We found that at the 4 somite stage, the WT notochord was one to two cells wide, and cells were aligned mediolaterally (at an average angle of 6 ± 5° relative to a line perpendicular to the embryonic axis as represented by the notochord) and were well elongated with LWR of 3.24 ± 1.19 (166 cells, 6 embryos; Fig. 7, AC). At the 6 somite stage, notochord cells were further elongated with LWR of 4.84 ± 1.92 and aligned mediolaterally with an angle of 4 ± 3° (241 cells, 8 embryos; Fig. 7, A and B). In contrast, in G
12/13-depleted embryos at the 4 somite stage, the notochord was two or three cells wide revealing an intercalation defect, and these cells were rounder, exhibiting an average LWR of 2.25 ± 0.79 (254 cells, 9 embryos, P = 3.3 x 1018; Fig. 7, A and D), however, these cells still aligned mediolaterally but at a slightly greater angle of 11 ± 12° (P = 4.2x 109; Fig. 7, B and D). At the 6 somite stage, notochord cells in G
12/13-depleted embryos continued to show impaired elongation and orientation defects with LWR of 2.95 ± 1.2 and angle of 8 ± 8° relative to the mediolateral axis (202 cells, 6 embryos, P = 5.1x 1031; Fig. 7, A and B). These results indicate G
12/13 signaling is also required for elongation and intercalation of axial mesodermal cells, and consequently for C&E of embryonic axis.
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The relationship between G12/13 and the noncanonical Wnt signaling during zebrafish gastrulation
The noncanonical Wnt signaling pathway mediates mediolateral cell polarization underlying normal C&E movements (Keller, 2002; Myers et al., 2002b; Wallingford et al., 2002). The morphological changes in embryos with altered G12/13 signaling are strikingly similar to those reported for mutants of slb (wnt11) and knypek (glypican4/6) that resulted from the disruption of the noncanonical Wnt signaling (Heisenberg et al., 2000; Topczewski et al., 2001). Moreover, G
12/13-depleted mesodermal cells also exhibited impaired cell elongation during late gastrulation (Fig. 5), similar to embryos overexpressing dominant negative Rok2 and trilobite and knypek mutants (Topczewski et al., 2001; Jessen et al., 2002; Marlow et al., 2002). However, our studies showed that G
12/13 are required for two types of directed cell migration for which noncanonical signaling does not appear to be required: early slow convergence and epiboly (Fig. 5; unpublished data), suggesting that G
12/13 mediate these movements independent of Wnt signaling. To elucidate the functional relationship between G
12/13 signaling and noncanonical Wnt signaling during gastrulation, we analyzed the effect of modulation of G
12/13 signaling on noncanonical Wnt signaling mutant phenotypes.
The small GTPase Rho, is the main effector of G12 and G
13 (Buhl et al., 1995) and is also implicated in noncanonical Wnt signaling (Habas et al., 2001). Accordingly, Rho downstream mediator Rok2 can partially suppress the slb (wnt11) gastrulation defects (Marlow et al., 2002). To address whether zebrafish G
12/13 can modulate Wnt11 signaling during gastrulation, we injected RNAs encoding G
13 to enhance, or G
13-CT to inhibit the function of G
13, in homozygous slb (wnt11)tz216/tz216 embryos (Heisenberg et al., 2000). However, neither various levels of excess nor deficit of G
13 signaling could suppress the gastrulation defects in slb (wnt11)/ embryos. Rather, both perturbations of G
13 signaling exacerbated the slb (wnt11)/ phenotype (unpublished data). We also performed molecular epitasis experiments by injecting embryos obtained from knym119 heterozygotes, carrying a null mutation in the glypican 4/6 gene (Topczewski et al., 2001), with a small dose of synthetic RNAs encoding G
13 or MOs against gna13 or three MOs against both gna13 and gna12. Neither overexpression of G
13 nor down-regulation of G
13 signaling suppressed kny(/) C&E defects. Instead, depletion of G
12/13 signaling by injection with three MOs enhanced the kny(/) defects of neuroectoderm convergence and of anterior prechordal mesendoderm migration (Fig. 7, GJ). Finally, the expression pattern of wnt11, kny, or tri was unchanged in embryos with excess or deficit of G
12/13 signaling (unpublished data). These results are consistent with the notion that G
12/13 and noncanonical Wnt signaling functionally interact during zebrafish gastrulation, likely acting in parallel pathways.
To address the cellular basis of the exacerbated C&E defects in kny mutants with compromised G12/13 signaling, we focused on cell intercalation that drives notochord convergence and extension (see above; Glickman et al., 2003). Our previous studies showed that axial mesoderm C&E is impaired in kny mutants (Topczewski et al., 2001), although the underlying cell defects have not been investigated. Compared with WT embryos, notochord cells in kny mutants showed impaired elongation and orientation at the 4 somite stage with LWR of 2.37 ± 0.8 and angle of 12 ± 11° (269 cells, 8 embryos) and at 6 somite stage with LWR of 3.16 ± 1.2 and angle of 11 ± 9° (256 cells, 7 embryos; Fig. 7, A, B, and E). These results suggest that kny not only mediates lateral cell elongation, it also contributes to midline cell alignment. To test whether G
12/13 signaling functions in addition to kny in midline cells, embryos obtained from kny heterozygous parents were injected with 3MOs and notochord cells from kny homozygous mutant embryos were analyzed. Notably, deficit of G
13 signaling in kny homozygous mutant embryos exacerbated defects in notochord cells relative to kny mutant embryos. At 4 somite stage, the notochord cells were much rounder with LWR of 1.48 ± 0.35 (220 cells, 7 embryos, P = 1.7x 1044 compared with kny embryos) and lacked proper mediolateral alignment with an angle of 30 ± 25° (P = 9.3x 1020; Fig. 7, A, B, and F). Similar results were found in embryos at 6 somite stage (Fig. 7, A and B). Moreover, we also performed molecular epistasis experiments by injecting embryos obtained from kny heterozygotes with a small dose of gna13a RNA or MOs against gna13 or 3MOs against both gna13 and gna12. Overexpression of G
13 did not suppress kny C&E defects (not depicted), whereas depletion of G
12/13 signaling by injection with 3MO enhanced these defects (Fig. 7, GJ).
Collectively, these results suggest that G12/13 do not simply act as the downstream effectors of slb or kny to mediate C&E movements. However, G
12/13 appears to functionally interact with noncanonical Wnt signaling to influence cell movements, likely acting through a parallel pathway.
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Discussion |
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In vivo time-lapse analyses revealed that G12/13 signaling is required for several distinct gastrulation cell behaviors, including dorsalward migration and intercalation during C&E movements. However, G
12/13 signaling does not appear to act as a general motility factor. Indeed, mesendoderm internalization occurred without any obvious defects in G
12/13-depleted gastrulae (unpublished data). Therefore, we conclude that G
12/13 signaling affects only a subset of morphogenetic events in zebrafish gastrula. We cannot, however exclude the possibility that there is a residual activity in embryos injected with MOs and or CT-peptides, permitting other morphogenetic processes that require lower levels of G
12/13 signaling.
We demonstrated that in G12/13-depleted embryos, mediolateral cell elongation of slowly migrating cells at midgastrulation is impaired, whereas this cell behavior is normal in the tri mutant (Jessen et al., 2002). Moreover, our preliminary analyses of lateral mesodermal cells in embryos overexpressing G
13 revealed normal cell elongation (unpublished data). This suggests that the role of G
12/13 signaling in the regulation of cell elongation is distinct from that of noncanonical Wnt signaling, where either increased or decreased pathway activity impairs elongation of mesodermal cells (Wallingford et al., 2000). Consistent with this hypothesis, our mosaic analyses indicate that mediolateral cell elongation requires only cell-autonomous G
12/13 activities, whereas noncanonical Wnt signaling regulates cell elongation both cell-autonomously and nonautonomously (Jessen et al., 2002; Marlow et al., 2002). In addition, ectopic G
12/13 activity cannot suppress the kny/ and slb/ phenotypes (Fig. 5 and not depicted) and Fz2/7 morphant phenotypes (not depicted). Collectively, these results strongly argue that G
12/13 do not act as components of a linear noncanonical Wnt signaling pathway to mediate cell polarization. Identification of the ligands and receptors that regulate gastrulation behaviors acting upstream of G
12/13 will be our next main focus.
Recent studies indicate that a number of distinct cell behaviors contribute to vertebrate gastrulation (Elul and Keller, 2000; Jessen et al., 2002; Myers et al., 2002b; Montero et al., 2003). How is this diversity of cell behaviors generated? In some gastrula regions, cells are engaged in more than one cell behavior, suggesting that cells are competent to respond to several cues. Our work implicates G12/13 as key mediators of many different gastrulation cell behaviors: slow and fast dorsal convergence in lateral regions and cell intercalation in dorsal regions (Figs. 5 and 7; Jessen et al., 2002; Myers et al., 2002b; Glickman et al., 2003). Given that G
12/13 can be activated by a variety of GPCR/ligands, it is tempting to hypothesize that these proteins may underlie interaction with signals in different regions of fish gastrulae to generate the distinct gastrulation cell behaviors. In the lateral region, cells become influenced by a dorsally provided attracting system that initiates convergence movements. Evidence suggests that ß-catenin activates the STAT3 pathway in the dorsal gastrula organizer to produce a long range dorsal attractant, which could interact with G
12/13 signaling to mediate slow dorsal convergence movements (Yamashita et al., 2002). In the dorsolateral region, G
12/13 may also interact with the noncanonical Wnt signaling to generate high mediolateral elongation underlying fast dorsal migration. Finally in the dorsal region, G
12/13 and noncanonical Wnt signaling could interact with yet to be identified regulators to mediate intercalation behavior. In conclusion, we establish a central role for G
12 and G
13 proteins in mediating several distinct cell behaviors that drive vertebrate gastrulation. Identification of extracellular cues that are integrated by G
12/13 to mediate individual gastrulation cell behaviors is an important future goal for this research into the molecular mechanisms of morphogenesis.
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Materials and methods |
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Cloning zebrafish gna12/13 and generation of G12/13 COOH-terminal peptide constructs
Zebrafish gna12 and gna13 cDNAs were cloned by RT-PCR and then subcloned into the pCS2 expression vector. The conserved glutamine at residue 226 of G13a was changed to leucine to generate a constitutively active form of G
protein (Katoh et al., 1998) using the QuikChange mutagenesis kit (Stratagene). To generate constructs encoding the last 11 COOH-terminal aa of G
S (QRMHLRQYELL), G
12 (LQENLKDIMLQ), and G
13 (LHDNLKQLMLQ), two synthetic short complimentary oligonucleotides encoding the peptide sequences were obtained for each gene. The forward and reverse oligonucleotides were annealed, and subcloned into pCS2 vector. These constructs were designated as G
s-CT, G
12-CT, and G
13-CT (one peptide was used to block function of both G
13a and G
13b because they have identical COOH-terminal sequences). Longer forms of CT peptides encoding the last 50 aa of COOH termini of G
13 and G
12, which included a HA-tag at the NH2-terminus, were constructed by PCR. All constructs were verified by DNA sequencing.
Microscopy
Live embryos for still photography were mounted in 1.52% methylcellulose at 28°C, whereas embryos processed for whole-mount in situ hybridization were mounted in 75% glycerol/PBT. Embryos were photographed using an Axiophot2 microscope (Carl Zeiss MicroImaging, Inc.) and an Axiocam digital camera (Carl Zeiss MicroImaging, Inc.). For confocal imaging, embryos were mounted in 75% glycerol/PBT, and a laser scanning inverted microscope (model LSM 510; Carl Zeiss MicroImaging, Inc.) with a 40x lens and 2x digital Zoom was used. All images acquired were compiled and edited using Adobe Photoshop and Illustrator software.
In situ hybridization
Sense and antisense RNA probes for gna12, gna13a, and gna13b were synthesized using the NH2-terminal EST clones as templates. Antisense RNA probes hgg1, dlx3, krox20, shh, deltaC, ntl, bmp4, and chordin were synthesized as described previously (Jessen et al., 2002). Whole-mount in situ hybridization was performed as described previously (Thisse and Thisse, 1998), except that BM purple (Roche) was used for the chromogenic reaction. Sense probes produced no signal.
Cell culture stress fiber formation assay
HEK cells were transiently transfected with GFP or with G protein constructs as indicated. To block Rok activity, ROCK inhibitor, Y-27623 was added at 10 µM to media after transfection (Uehata et al., 1997). Stress fiber formation assay was performed as described previously (Gilchrist et al., 2001). Anti-G12 or G
13 antibodies (1:100) generated against the last 11 AAs of human G
12 or G
13 (Hallak et al., 1994) were used to identify the G proteinexpressing cells. Cells were mounted in Vectashield mounting medium (Vector Laboratories) and confocal images were acquired as described in Microscopy.
Whole-mount immunostaining
Zebrafish embryos were fixed in 4%PFA/PBS/4% sucrose at shield and whole mount immunohistochemistry was performed as described previously (Topczewska et al., 2001). Primary anti-G12 or G
13 antibodies and Cy2-conjugated pAb (1:100) were used. No signal was detected when the primary antibodies were preincubated with the peptides encoding the COOH-terminal 11 residues of the G
12 or G
13, respectively, or when only the secondary antibody was used. Confocal images were acquired.
RNA and antisense MO injections
Capped sense RNAs encoding the G13, G
12, G
S CT-peptides, mGFP (Wallingford et al., 2000) or a full-length zebrafish G
13a and human G
13 were synthesized using mMessage Machine system (Ambion). RNAs were injected into embryos at 12 cell stage.
Antisense MOs (Gene-Tools) targeted against the zebrafish gna12, gna13a, and gna13b transcripts were designed according to the manufacturer's suggestions and injected into embryos at 1 cell stage. Two distinct MOs were designed to target either the sequences overlapping the ATG initiation codon (MO1) or the 5' untranslated sequences (MO2) of gna13a transcript. For gna13b and gna12, one MO against sequence overlapping the translation start site was designed for each transcript.
Time-lapse and cell shape analysis
Nomarski time-lapse images of lateral gastrula mesodermal cells at midgastrulation (80% epiboly) were collected as described previously (Myers et al., 2002a). Dechorionated zebrafish embryos were mounted in 0.81% low melting point agarose in 30% Danieau's buffer (100% Danieau's buffer: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM CaCl2, 5 mM Hepes, pH 7.6), to view the lateral mesoderm (90° from the dorsal midline). The microscope room was maintained at 28°C during recordings. Single focal plane time-lapse recordings were collected at 30-s intervals using DIC optics and a 20x objective (0.5 NA Plan Neofluor) on an Axiophot2 microscope (Carl Zeiss MicroImaging, Inc.) and an Axiocam digital camera (Carl Zeiss MicroImaging, Inc.). Images were analyzed using Object-Image software (Norbert Vischer, http://simon.bio.uva.nl/object-image.html). Data was exported to Excel (Microsoft) where cell migration speed, path, direction, turning angle, and LWRs were determined according to Topczewski et al. (2001). The direction of movements of lateral mesodermal cells was determined at 30-s intervals and change in movement direction was calculated. A turn was defined as a change in direction of >60°. We also determined the actual direction of lateral mesendodermal cells at every 60-s interval.
To investigate shape of notochord cells, WT embryos or embryos obtained from knym119 heterozygous parents were injected with mgfp RNA (Wallingford et al., 2000) alone or with 3MOs, and were then fixed in 4% PFA. Images of notochord were collected on a confocal microscope and LWR of notochord cells and the angle of the long axis relative to a line perpendicular to the embryonic axis as represented by the notochord were analyzed using Object-Image software.
Transplantation experiments
For cell autonomy analyses, cells from embryos injected with dextran-Rhodamine or together with 3MO were transplanted into host embryos injected with mgfp RNA and 3MO or mgfp RNA alone at 1K-dome stage (mgfp RNA was injected to visualize cell shape), as described previously (Jessen et al., 2002). Host embryos were then fixed in 4% PFA at 4s stage and stained with anti-Rhodamine, then LWR and the angle of the long axis of notochord cells relative to a line perpendicular to the embryonic axis were determined.
Statistical analysis
Data are presented as the mean ± 1 SD. Statistical analyses were performed using unpaired t tests unequal variance. In all analyses, the asterisk indicates P < 0.001.
Accession nos.
GenBank/EMBL/DDBJ accession nos. for the zebrafish gna12, gna13a, and gna13b are AY386359, AY386360, and AY386361, respectively.
Online supplemental material
Fig. S1 shows the sequence alignment of human and zebrafish G12/13. Two synthetic short complementary oligonucleotides encoding the peptide sequences were obtained for gna12, gna13, and gnaS. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200501104/DC1.
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Acknowledgments |
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D.S. Sepich is supported by NIH Vascular Biology Training grant (T32HL07751). The work in L. Solnica-Krezel lab is supported by NIH GM55101 grant. H. Hamm lab is supported by NIH grants EY10291 and HL60678.
Submitted: 19 January 2005
Accepted: 26 April 2005
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