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Figure 1. Alignment of Ccl19 and Ccl21 and expression patterns of ccl19 and ccl21. (a and b) Alignment of Xenopus laevis L, Xenopus laevis S, Xenopus tropicalis, human, mouse, and zebrafish Ccl19 (a) and Ccl21 (b). Asterisks (*) indicate conserved amino acids. The conserved cysteine residues are shaded gray. The positively charged 41 amino acids at the C-terminus of human Ccl21 are shaded black. (c and d) Expression patterns of ccl19 and ccl21. odc1 was used as an internal control. (c) Temporal expression patterns. U indicates unfertilized eggs and the numbers indicate developmental stages. (d) Spatial expression patterns. Indicated genes under odc1 were used as positive control markers for each germ layer and tissue. Dissections at several developmental stages were performed as shown in the bottom panels. D, dorsal; Ec, ectoderm; En, endoderm; H, head; Me, mesoderm; V, ventral.
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Figure 2. Phenotypes of dorsally ccl19.L and ccl21.L mRNA-injected embryos and Keller sandwich explants. (a) Phenotypes of embryos when injected with ccl19.L and ccl21.L mRNA into two dorsal blastomeres at the four-cell stage. The developmental stages are indicated in upper panels. Vegetal view (st. 12), dorsal view (st. 18), lateral view (st. 28). In the stage 28 images of mRNA-injected embryos, which had very short and bending anterior–posterior axes, the left panels show a representative example of the weak phenotypes with head structures, and the right panels show a representative example of the severe phenotype without head structures. Bars, 0.5 mm. (b and c) The appearance rates of phenotypes in Figure 2a. Normal phenotype (light gray), weak phenotype (dark gray), and severe phenotype (black) are shown. The quantity of injected mRNA is indicated at the bottom of the graph. (b) ccl19.L mRNA-injected embryos. Lane 1: n = 58; normal (41.4%), weak (53.4%), and severe (5.2%). Lane 2: n = 58; normal (0%), weak (46.6%), and severe (53.4%). Lane 3: n = 60; normal (0%), weak (10.0%), and severe (90.0%). (c) ccl21.L mRNA-injected embryos. Lane 1: n = 58; normal (31.0%), weak (56.9%), and severe (12.1%). Lane 2: n = 58; normal (0%), weak (34.5%), and severe (65.5%). Lane 3: n = 57; normal (0%), weak (12.1%), and severe (87.9%). (d) Keller sandwich explants of embryos injected dorsally with ccl19.L or ccl21.L mRNA. Upper panels indicate the stages of time-control embryos. Right images indicate fluorescent images of the explants. Overexpression of ccl19.L and ccl21.L mRNA inhibited the convergent extension cell movements. Bars, 0.5 mm. (e) Length-to-width ratio of Keller sandwich explants (st. 25). The length of the bending parts of the explants was measured along the axial elongation. The longest axis of each explant was divided by its widest perpendicular aspect except for the epidermal ectoderm region. Eight explants were measured in each category.
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Figure 3. Analysis of chemotactic cell migration. (a) Reverse transcription-PCR (RT-PCR) analysis of spatial expression patterns of ccl19.L and ccl21.L in dorsal halves of gastrulae (st. 10). Dissections were performed as shown on the right. L, left; C, center; R, right. chrd.1 or myod1 was used as an axial or a paraxial marker gene, respectively. odc1 was used as an internal control. (b) Schematics of the protocol of the cell migration assay. ccl19.L (1000 pg/blastomere) or ccl21.L (500 pg/blastomere) mRNA was co-injected into two ventral blastomeres of four-cell embryos with green fluorescent tracer. Control-MO (20 ng/blastomere) or ccr7.S-MO (20 ng/blastomere) was co-injected into two lateral blastomeres of four-cell embryos with red fluorescent tracer. The ventral mesodermal sectors were dissected from stage 10 embryos. Epithelium was removed to use as the ventral mesodermal explants. The lateral mesodermal sectors were dissected from stage 10 embryos and dissociated in a calcium- and magnesium-free solution. The dissociated lateral mesodermal cells were placed approximately 0.5–1.0 mm from the ventral mesodermal explants on a fibronectin-coated cover glass. (c) Migration of scattered lateral mesodermal cells. The ventral mesodermal explants were placed on the left side. Injected reagents into the ventral and lateral mesodermal sectors are indicated on the left. V, ventral mesoderm; L, lateral mesoderm. Each combination of ventral mesodermal explant and lateral mesodermal cells is numbered. The elapsed time is indicated at the bottom. Bars, 0.5 mm. (d) Schematics of the experiments are shown on the left. The graph of the ratio (Distancetime/Distanceinitial) of lateral mesodermal cell migration (n = 10) is shown on the right. The ratio of the distance between ventral mesodermal explants and lateral mesodermal cells was measured hourly. In the numbers in Figure 3c, the average ratio is shown by the black line for number 1, the red line for number 2, the green line for number 3, and the blue line for number 4.
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Figure 4. Analysis of haptotactic cell migration. Schematics of the method to make the combined ventral mesodermal explants. ccl19.L mRNA (1000 pg/blastomere) or ccl21.L mRNA (500 pg/blastomere) was co-injected into two ventral blastomeres of four-cell embryos with a green fluorescent tracer. Control-MO (20 ng/blastomere) or ccr7.S-MO (20 ng/blastomere) was co-injected into two ventral blastomeres of four-cell embryos with a red fluorescent tracer. The different fluorescent labeled ventral mesodermal sectors were dissected from stage 10 embryos and their epithelia were removed. Each ventral mesoderm sector was combined for use as combined ventral mesodermal explants. The combined ventral mesodermal explants were placed on a cover glass coated with bovine serum albumin. (b) Observation of the combined ventral mesodermal explants. Reagents injected into each ventral mesodermal sector are indicated on the left. Each combination of ventral mesodermal sectors is numbered. The elapsed time is indicated at the bottom. The two-color versions are indicated in the upper panels. The grayscale versions of red fluorescent labeled ventral mesoderm are indicated in the bottom panels because their contrasts are better than those of the two-color versions. For the number 1, 3, and 5 combinations, the combined ventral mesoderm explants remained in the same locations and the red labeled ventral mesodermal cells did not deeply migrate into the green labeled ventral mesodermal sectors. For the number 2 combination, the red labeled ventral mesodermal cells migrated around the green labeled ventral mesoderm. Several control ventral mesodermal cells invaded into neighboring green labeled ventral mesoderm. For the number 4 combination, the red labeled ventral mesodermal cells deeply invaded into the neighboring green labeled ventral mesodermal sector. Bars, 0.5 mm. (c) Schematics of movement of the combined ventral mesodermal explant. (d) Highly magnified images of the red labeled ventral mesodermal sector of Figure 4b-2 are indicated. Arrows indicate migrating cells around the green labeled ventral mesoderm and arrowheads indicate non-polar shaped cells. Bar, 0.25 mm. (e) Highly magnified images of the red labeled ventral mesodermal sector of Figure 4b-4 are indicated in the upper panels. Overlapping cells with green labeled ventral mesodermal sector 2 h previously were pseudo-colored and are displayed in bottom panels. Arrows indicate monopolar shaped cells, and arrowheads indicate non-polar shaped cells. Bars, 0.25 mm.
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Figure 5. Effects of ccl19.L and ccl21.L on differentiation. (a) Phenotypes after injection of ccl19.L (1000 pg/blastomere) and ccl21.L (500 pg/blastomere) mRNA into two ventral blastomeres at the four-cell stage (st. 28). All injected embryos had ventral gastrulation defects and induced small secondary axis-like structures without head structures (n = 60, ccl19.L injection; n = 60, ccl21.L injection). Arrowheads indicate partial secondary axis-like structures. Bars, 1.0 mm. (b) RT-PCR analysis of the dorsal or ventral sectors of the ccl19.L and ccl21.L mRNA-injected embryos (st. 10). (c) RT-PCR analysis of the animal caps of the ccl19.L (500 pg/blastomere) and ccl21.L (250 pg/blastomere) mRNA-injected embryos (st. 9.5). (d) RT-PCR analysis of the animal caps after co-injection of ccl19.L and ccl21.L mRNA with ccr7.s mRNA (st. 9.5). The injected amounts are indicated. (e) The relative intensity of each gel electrophoresis band in Figure 5d. Signal intensity was measured by ImageJ, and the chrd.1/odc1 and bmp4/odc1 ratios were calculated. Values were normalized to the value for uninjected embryos (lane 1). (f) RT-PCR analysis of the animal caps after co-injection of ccl19.L (500 pg/blastomere) and ccl21.L (250 pg/blastomere) mRNA with Control-MO (20 ng/blastomere) or ccr7.s-MO (20 ng/blastomere) (st. 9.5).
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Figure 6. Knockdown effects of Ccl19.L and Ccl21.L on morphogenesis and differentiation. (a and b) Results of western blot analysis. Indicated reagents were injected. hbg1-MYC mRNA was co-injected as the loading control. Specificity of ccl19.L-MO (a) and ccl21.L-MO (b). (c) Phenotypes of embryos after injection with ccl19.L- and ccl21.L-MO into two dorsal blastomeres at the four-cell stage. The developmental stages are indicated in the upper panels. Vegetal view (st. 12), dorsal view (st. 18), lateral view (st. 28). In the stage 18 images of morpholino oligonucleotide (MO)-injected embryos, which were affected in neural fold closure, the left panels show a representative example of the weak phenotypes with delaying neural fold closure and the right panels show a representative example of the severe phenotype of embryos whose neural folds were wide open. In the stage 28 images of MO-injected embryos, which had no head structure, the left panels show a representative example of the weak phenotypes with normal trunk axes and the right panels show a representative example of the severe phenotype with short and bending axes. Bars, 0.5 mm. (d and e) The appearance rates of phenotypes (st. 28) in Figure 6c. Normal phenotype (light gray), weak phenotype (dark gray), and severe phenotype (black) are shown. Injected MO amounts are indicated at the bottom of the graph. (d) ccl19.L-MO-injected embryos. Lane 1: n = 57, normal (94.7%), weak (5.3%), and severe (0%). Lane 2: n = 54; normal (3.7%), weak (88.9%), and severe (7.4%). Lane 3: n = 55; normal (0%), weak (54.5%), and severe (45.5%). (e) ccl21.L-MO-injected embryos. Lane 1: n = 57; normal (94.7%), weak (5.3%), and severe (0%). Lane 2: n = 57; normal (0%), weak (77.2%), and severe (22.8%). Lane 3: n = 58; normal (0%), weak (12.1%), and severe (87.9%). (f) Keller sandwich explants of embryos injected dorsally with ccl19.L-, ccl21.L-, or ccr7.S-MO (30, 15, or 20 ng/blastomere, respectively). Upper panels indicate the stages of time-control embryos. Right images are fluorescence images of the explants. Injection of ccl19.L- and ccr7.S-MO only marginally affected convergent extension cell movements. In contrast, injection of ccl21.L-MO severely inhibited convergent extension cell movements. Bars, 0.5 mm. (g) Length-to-width ratio of Keller sandwich explants (st. 25). The length of the bending parts of the explants was measured along the axial elongation. The longest axis of each explant was divided by its widest perpendicular aspect except for the epidermal ectoderm region. Eight explants were measured in each category. (h) Phenotypes of the ventrally ccl19.L- and ccl21.L-MO-injected embryos (st. 28). All injected embryos had ventral gastrulation defects (n = 45, ccl19.L-MO injection, and n = 44, ccl21.L-MO injection). Bars, 1.0 mm. (i and j) Data of rescue experiments by co-injection with each mRNA (5-mis-ccl19.L mRNA, 1000 pg/blastomere, or 5-mis-ccl21.L mRNA, 500 pg/blastomere) and each MO (ccl19.L-MO, 30 ng/blastomere, or ccl21.L-MO, 15 ng/blastomere). (i) Phenotypes of the dorsally and ventrally injected embryos (st. 28). Arrowheads indicate small secondary axis-like structures. All dorsally injected embryos showed severe gastrulation defects (n = 43, 5-mis-ccl19.L mRNA + ccl19.L-MO-injection, and n = 42, 5-mis-ccl21.L mRNA + ccl21.L-MO-injection). All ventrally injected embryos showed ventral gastrulation defects with very small axis-like structures (n = 44, 5-mis-ccl19.L mRNA + ccl19.L-MO-injection, and n = 45, 5-mis-ccl21.L mRNA + ccl21.L-MO-injection). Bars, 1.0 mm. (j) RT-PCR analysis of the dorsal or ventral sectors of each mRNA- and MO-co-injected embryo (st. 10).
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Figure 7. Effects of human ccl19 and ccl21 on early Xenopus development. (a) Phenotypes of embryos injected with human ccl19 and ccl21 mRNA (500 pg/blastomere) (st. 28). All dorsally injected embryos showed gastrulation defects (n = 43, human ccl19 injection, and n = 44, human ccl21.L injection). All ventrally injected embryos showed ventral gastrulation defects with small secondary axis-like structures (n = 44, human ccl19 injection, and n = 43, human ccl21.L injection). Arrowheads indicate small secondary axis-like structures. Bars, 1.0 mm. (b and c) RT-PCR analysis of the dorsal or ventral sectors (st. 10) (b) and the animal caps (st. 9.5) (c) of embryos injected with human ccl19 and ccl21 mRNA.
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FIGURE S1: Analysis of haptotactic cell migration.
Highly magnified images of the red labeled ventral mesodermal sector of Figure 4b-1, b-3, b-5 are indicated in the top, center, and bottom panels, respectively. The elapsed time is indicated at the top. Bars, 0.5 mm.
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