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Zebrafish arl6ip1 is required for neural crest development during embryogenesis.

Tu CT, Yang TC, Huang HY, Tsai HJ - PLoS ONE (2012)

Bottom Line: Expressions of the ectodermal patterning factors msxb, dlx3b, and pax3 were normal, but the expressions of the neural crest specifier genes foxd3, snai1b, and sox10 were greatly reduced.We found that this migration defect was induced by dampened Shh signaling, which may have resulted from defective cilia.Therefore, we concluded that arl6ip1 is required for neural crest migration and sublineage specification.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Although the embryonic expression pattern of ADP ribosylation factor-like 6 interacting protein 1 (Arl6ip1) has been reported, its function in neural crest development is unclear.

Methods/principal findings: We found that knockdown of Arl6ip1 caused defective embryonic neural crest derivatives that were particularly severe in craniofacial cartilages. Expressions of the ectodermal patterning factors msxb, dlx3b, and pax3 were normal, but the expressions of the neural crest specifier genes foxd3, snai1b, and sox10 were greatly reduced. These findings suggest that arl6ip1 is essential for specification of neural crest derivatives, but not neural crest induction. Furthermore, we revealed that the streams of crestin- and sox10-expressing neural crest cells, which migrate ventrally from neural tube into trunk, were disrupted in arl6ip1 morphants. This migration defect was not only in the trunk neural crest, but also in the enteric tract where the vagal-derived neural crest cells failed to populate the enteric nervous system. We found that this migration defect was induced by dampened Shh signaling, which may have resulted from defective cilia. These data further suggested that arl6ip1 is required for neural crest migration. Finally, by double-staining of TUNEL and crestin, we confirmed that the loss of neural crest cells could not be attributed to apoptosis.

Conclusions/significance: Therefore, we concluded that arl6ip1 is required for neural crest migration and sublineage specification.

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Related in: MedlinePlus

Cell death does not cause the loss of neural crest cells.Lateral views of TUNEL-labeled wild-type (WT) embryos (A–C) or arl6ip1-MO1-injected embryos (D–F) at 3-somite stage (A, D), 8-somite stage (B, E), and 15-somite stage (C, F), respectively. (A, D) There were no TUNEL-positive cells in WT and arl6ip1-MO1 embryos. (B, C, E, F) Compared to WT embryos, arl6ip-deficient embryos displayed a significant increase of TUNEL-positive cells in the dorsal region of embryos at the 8- and 15-somite stages (indicated by arrowheads) (B and C vs. E and F). (G, H, I) Double-labeling analysis with crestin (dark blue) and TUNEL (red fluorescent) showed that only limited cell death occurred at the expression region of crestin. (G′, H′, I′) The views of the embryos shown in panels G′, H′ and I′ were at higher magnification.
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pone-0032899-g008: Cell death does not cause the loss of neural crest cells.Lateral views of TUNEL-labeled wild-type (WT) embryos (A–C) or arl6ip1-MO1-injected embryos (D–F) at 3-somite stage (A, D), 8-somite stage (B, E), and 15-somite stage (C, F), respectively. (A, D) There were no TUNEL-positive cells in WT and arl6ip1-MO1 embryos. (B, C, E, F) Compared to WT embryos, arl6ip-deficient embryos displayed a significant increase of TUNEL-positive cells in the dorsal region of embryos at the 8- and 15-somite stages (indicated by arrowheads) (B and C vs. E and F). (G, H, I) Double-labeling analysis with crestin (dark blue) and TUNEL (red fluorescent) showed that only limited cell death occurred at the expression region of crestin. (G′, H′, I′) The views of the embryos shown in panels G′, H′ and I′ were at higher magnification.

Mentions: In arl6ip1-deficient embryos, pharyngeal arches, dorsal root ganglia and enteric neurons at the later stages of embryonic development were severely reduced or absent. The loss of these cells during development might result from cell death. To investigate the role of Arl6ip1 in NC survival, TUNEL assay was performed to examine cell death. At the 3-somite stage, there were no significant TUNEL signal differences in cell death between wild-type and arl6ip1-MO1-injected embryos (Figs. 8A and D). However, we detected a great increase of TUNEL-positive cells around the dorsal ectoderm in arl6ip1-MO1-injected embryos from the 3- to 15-somite stages, compared to wild-type embryos (Figs. 8B and E; 8C and F). The expression of pre-migratory NC markers was also severely reduced in the arl6ip1-MO1 embryos between the 3- and 8-somite stages (Fig. 5). We detected a great increase of TUNEL-positive cells around the dorsal ectoderm in arl6ip1-MO embryos from the 3- to 15-somite stages, and this increase ended at 36 hpf. To confirm whether these dying cells were overlapped with crestin-positive NC cells, we performed double- labeling with crestin and fluorescent TUNEL (Figs. 8G–I; G′–I′). The significant expression of crestin in wild-type embryos indicated that cranial NC appeared at the anterior region of trunk somites at this stage (Figs. 8G and G′, indicated by arrows). When we compared dying cells with crestin-positive NC cells using double-labeling assay in arl6ip1-MO1-injected embryos, apoptotic cells tended to localize at the more anterior and superficial region, while crestin-positive NC cells were inclined to localize at the posterior region (Figs. 8H and H′). Since cell death occurring in arl6ip1-MO1-injected embryos was inhibited by p53-MO, cell death was observed to be correspondingly decreased in the arl6ip1-MO1/p53-MO-injected embryos (Fig. 8I). Compared with wild-type embryos, we also found the number of crestin-positive cells in arl6ip1-MO1-injected embryos and arl6ip1-MO1/p53-MO-injected embryos to be reduced (Figs. 8G′ vs. 8H′; 8G′ vs. 8I′, indicated by brackets). In addition, most crestin-positive cells failed to show cell death signaling in arl6ip1-MO1-injected embryos (Figs. 8H and H′). To account for the overlapping signals of crestin- and TUNEL-positive cells under fluorescent stereomicroscopy, we found an average of 9.6, 6.7 and 1.9 out of 10 cells to be positive to both TUNEL and crestin signals in arl6ip-MO1-injected embryos, wild-type embryos, and arl6ip1-MO1/p53-MO- injected embryos, respectively. However, the appearance of TUNEL-positive cells did not correlate with most crestin-labeled cells at this stage, which could be attributed to other defects in the arl6ip1 morphants. Thus, we concluded that cell death does not play a role in the NC survival of arl6ip1-MO1-injected embryos.


Zebrafish arl6ip1 is required for neural crest development during embryogenesis.

Tu CT, Yang TC, Huang HY, Tsai HJ - PLoS ONE (2012)

Cell death does not cause the loss of neural crest cells.Lateral views of TUNEL-labeled wild-type (WT) embryos (A–C) or arl6ip1-MO1-injected embryos (D–F) at 3-somite stage (A, D), 8-somite stage (B, E), and 15-somite stage (C, F), respectively. (A, D) There were no TUNEL-positive cells in WT and arl6ip1-MO1 embryos. (B, C, E, F) Compared to WT embryos, arl6ip-deficient embryos displayed a significant increase of TUNEL-positive cells in the dorsal region of embryos at the 8- and 15-somite stages (indicated by arrowheads) (B and C vs. E and F). (G, H, I) Double-labeling analysis with crestin (dark blue) and TUNEL (red fluorescent) showed that only limited cell death occurred at the expression region of crestin. (G′, H′, I′) The views of the embryos shown in panels G′, H′ and I′ were at higher magnification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3298456&req=5

pone-0032899-g008: Cell death does not cause the loss of neural crest cells.Lateral views of TUNEL-labeled wild-type (WT) embryos (A–C) or arl6ip1-MO1-injected embryos (D–F) at 3-somite stage (A, D), 8-somite stage (B, E), and 15-somite stage (C, F), respectively. (A, D) There were no TUNEL-positive cells in WT and arl6ip1-MO1 embryos. (B, C, E, F) Compared to WT embryos, arl6ip-deficient embryos displayed a significant increase of TUNEL-positive cells in the dorsal region of embryos at the 8- and 15-somite stages (indicated by arrowheads) (B and C vs. E and F). (G, H, I) Double-labeling analysis with crestin (dark blue) and TUNEL (red fluorescent) showed that only limited cell death occurred at the expression region of crestin. (G′, H′, I′) The views of the embryos shown in panels G′, H′ and I′ were at higher magnification.
Mentions: In arl6ip1-deficient embryos, pharyngeal arches, dorsal root ganglia and enteric neurons at the later stages of embryonic development were severely reduced or absent. The loss of these cells during development might result from cell death. To investigate the role of Arl6ip1 in NC survival, TUNEL assay was performed to examine cell death. At the 3-somite stage, there were no significant TUNEL signal differences in cell death between wild-type and arl6ip1-MO1-injected embryos (Figs. 8A and D). However, we detected a great increase of TUNEL-positive cells around the dorsal ectoderm in arl6ip1-MO1-injected embryos from the 3- to 15-somite stages, compared to wild-type embryos (Figs. 8B and E; 8C and F). The expression of pre-migratory NC markers was also severely reduced in the arl6ip1-MO1 embryos between the 3- and 8-somite stages (Fig. 5). We detected a great increase of TUNEL-positive cells around the dorsal ectoderm in arl6ip1-MO embryos from the 3- to 15-somite stages, and this increase ended at 36 hpf. To confirm whether these dying cells were overlapped with crestin-positive NC cells, we performed double- labeling with crestin and fluorescent TUNEL (Figs. 8G–I; G′–I′). The significant expression of crestin in wild-type embryos indicated that cranial NC appeared at the anterior region of trunk somites at this stage (Figs. 8G and G′, indicated by arrows). When we compared dying cells with crestin-positive NC cells using double-labeling assay in arl6ip1-MO1-injected embryos, apoptotic cells tended to localize at the more anterior and superficial region, while crestin-positive NC cells were inclined to localize at the posterior region (Figs. 8H and H′). Since cell death occurring in arl6ip1-MO1-injected embryos was inhibited by p53-MO, cell death was observed to be correspondingly decreased in the arl6ip1-MO1/p53-MO-injected embryos (Fig. 8I). Compared with wild-type embryos, we also found the number of crestin-positive cells in arl6ip1-MO1-injected embryos and arl6ip1-MO1/p53-MO-injected embryos to be reduced (Figs. 8G′ vs. 8H′; 8G′ vs. 8I′, indicated by brackets). In addition, most crestin-positive cells failed to show cell death signaling in arl6ip1-MO1-injected embryos (Figs. 8H and H′). To account for the overlapping signals of crestin- and TUNEL-positive cells under fluorescent stereomicroscopy, we found an average of 9.6, 6.7 and 1.9 out of 10 cells to be positive to both TUNEL and crestin signals in arl6ip-MO1-injected embryos, wild-type embryos, and arl6ip1-MO1/p53-MO- injected embryos, respectively. However, the appearance of TUNEL-positive cells did not correlate with most crestin-labeled cells at this stage, which could be attributed to other defects in the arl6ip1 morphants. Thus, we concluded that cell death does not play a role in the NC survival of arl6ip1-MO1-injected embryos.

Bottom Line: Expressions of the ectodermal patterning factors msxb, dlx3b, and pax3 were normal, but the expressions of the neural crest specifier genes foxd3, snai1b, and sox10 were greatly reduced.We found that this migration defect was induced by dampened Shh signaling, which may have resulted from defective cilia.Therefore, we concluded that arl6ip1 is required for neural crest migration and sublineage specification.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Although the embryonic expression pattern of ADP ribosylation factor-like 6 interacting protein 1 (Arl6ip1) has been reported, its function in neural crest development is unclear.

Methods/principal findings: We found that knockdown of Arl6ip1 caused defective embryonic neural crest derivatives that were particularly severe in craniofacial cartilages. Expressions of the ectodermal patterning factors msxb, dlx3b, and pax3 were normal, but the expressions of the neural crest specifier genes foxd3, snai1b, and sox10 were greatly reduced. These findings suggest that arl6ip1 is essential for specification of neural crest derivatives, but not neural crest induction. Furthermore, we revealed that the streams of crestin- and sox10-expressing neural crest cells, which migrate ventrally from neural tube into trunk, were disrupted in arl6ip1 morphants. This migration defect was not only in the trunk neural crest, but also in the enteric tract where the vagal-derived neural crest cells failed to populate the enteric nervous system. We found that this migration defect was induced by dampened Shh signaling, which may have resulted from defective cilia. These data further suggested that arl6ip1 is required for neural crest migration. Finally, by double-staining of TUNEL and crestin, we confirmed that the loss of neural crest cells could not be attributed to apoptosis.

Conclusions/significance: Therefore, we concluded that arl6ip1 is required for neural crest migration and sublineage specification.

Show MeSH
Related in: MedlinePlus