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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

Expressions of ectodermal patterning factors appear normal in arl6ip1-MO1-injected embryos.Dorsal views of wild-type (A–E) and arl6ip1 morphant (F–J) embryos at the 3-somite stage. Anterior to the top. The neural ectodermal patterning factors of wnt8 (A, F), bmp4 (B, G), msxb (C, H), dlx3b (D, I), and pax3 (E, J). Normal expressions of these genes reveal that ectodermal patterning factors are not affected in arl6ip1 morphants, suggesting that Arl6ip1 does not have a role in ectodermal patterning. Scale bar: 100 µm.
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pone-0032899-g004: Expressions of ectodermal patterning factors appear normal in arl6ip1-MO1-injected embryos.Dorsal views of wild-type (A–E) and arl6ip1 morphant (F–J) embryos at the 3-somite stage. Anterior to the top. The neural ectodermal patterning factors of wnt8 (A, F), bmp4 (B, G), msxb (C, H), dlx3b (D, I), and pax3 (E, J). Normal expressions of these genes reveal that ectodermal patterning factors are not affected in arl6ip1 morphants, suggesting that Arl6ip1 does not have a role in ectodermal patterning. Scale bar: 100 µm.

Mentions: To study the function of arl6ip1 during NC development, we first detected the expressions of the ectodermal patterning factors wnt8, bmp4, pax3, and msxb. The ectodermal patterning factors were all normally expressed in the arl6ip1-MO1- injected embryos (Figs. 4A and F; 4B and G; 4C and H; 4D and I). In addition, the expression of the pre-placodal marker dlx3b was also normal in the arl6ip1-MO1- injected embryos (Figs. 4E and J, n = 64). Furthermore, since Trainor and Krumlauf (2000) [28] reported that segmentation and patterning of the hindbrain are necessary for cranial NC development; we also examined the expressions of two hindbrain patterning markers, fgf3 and fgf8. Results showed that fgf3 and fgf8 were also expressed normally in the hindbrain of arl6ip1 morphants (Figs. S2A–D, n = 87) and that 83.2% of morphants exhibited a normal hindbrain segmentation at 24 hpf (Figs. S2E–F, n = 101). These findings suggested that the induction of both early NC progenitors and placodal region cells results in normal development of arl6ip1 morphants. The early patterning and later segmentation of the hindbrain in the arl6ip1-MO1-injected embryos were also similar to those of wild-type. Thus, even though arl6ip1 may not function in hindbrain development, it is expressed ubiquitously until 24 hpf, and knockdown of Arl6ip1 did cause small head morphology. This line of evidence suggests that neural crest defects in arl6ip1-MO1 embryos did not result from abnormal head development. On the other hand, when we observed the expressions of pre-migratory NC markers in the progenitors of the neural plate border, such as tfap2α, foxd3, snai1b, and sox10, we found that the expressions of foxd3, snai1b and sox10 were greatly reduced in the arl6ip1 morphants at 3-somite (Figs. 5B and F; 5C and G; 5D and H) through 8-somite stages (Figs. 5I and L; 5J and M; 5K and N). Meanwhile, tfap2a expression in NC progenitors was present in both wild-type and arl6ip1-MO1 embryos (Figs. 5A and E). Double-staining of sox2 (labeling neural plate) and dlx3b (labeling the pre-placodal region) exhibited a patch of unlabeled cells between these two expression domains which was presumed to be the NC. Thus, since the induction of presumptive NC was normal in arl6ip1-MO1 embryos (Fig. S3), we concluded that NC induction occurred normally in arl6ip1 morphants. The occurrence rates of down-regulation of foxd3, snai1b and sox10 in the arl6ip1-MO1-injected embryos were 75.6% (31/41), 82.2% (37/45) and 66.7% (30/45), respectively. Again, since down-regulation of these genes could have resulted from early cell death by the off-targeting effect of MO, we injected 6 ng p53-MO together with 4 ng arl6ip1-MO1 to inhibit apoptosis and nonspecific effects. We found that the level of cell death was dramatically reduced in the embryos injected with p53-MO. In addition, Cole and Ross (2001) [29] have detected the temporal and spatial distribution of apoptotic cells during normal development of the zebrafish embryo from 12 to 96 hpf. According to their data, only a few apoptotic cells could be seen in the optic vesicle at 12hpf, and no apoptosis was observed in the pre-migratory neural crest cell region. Robu et al. (2007) [26] also demonstrated that about 15–20% of MO used in zebrafish show off-targeting effects, as represented by a signature neural death peaking at the end of segmentation (1 dpf). They also showed that the neural cell death induced by MO was observed as early as 14 hpf. Moreover, Ekker and Larson (2001) [30] indicated that the onset of cell death induced by MO technology was 14 hpf. In our data, we detected pre-migratory neural crest markers at the 3-somite stage (about 10.33 hpf), even earlier than 12 hpf; consequently, we did not observe off-targeting effects at the 3-somite stage. On the other hand, when p53 was inhibited, cell survival could not rescue expressions of foxd3, snai1b and sox10 at the 8-somite stage (Figs. 5L–N). Therefore, we concluded that the down-regulation of these gene markers in existing NC cells was caused by defects in NC derivate specification, not cell death. Furthermore, such defects subsequently resulted in the reduction of the cell population, especially in pharyngeal arches of cranial NC sublineages. Finally, we found that the reduced expression of foxd3 induced by arl6ip1-MO1 could be restored by injection of wobble arl6ip1 mRNA in arl6ip1- MO1-injected embryos (Figs. S4A–C). These findings implicated that Arl6ip1 plays a key function in specification of NC sublineages, but not induction.


Zebrafish arl6ip1 is required for neural crest development during embryogenesis.

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

Expressions of ectodermal patterning factors appear normal in arl6ip1-MO1-injected embryos.Dorsal views of wild-type (A–E) and arl6ip1 morphant (F–J) embryos at the 3-somite stage. Anterior to the top. The neural ectodermal patterning factors of wnt8 (A, F), bmp4 (B, G), msxb (C, H), dlx3b (D, I), and pax3 (E, J). Normal expressions of these genes reveal that ectodermal patterning factors are not affected in arl6ip1 morphants, suggesting that Arl6ip1 does not have a role in ectodermal patterning. Scale bar: 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3298456&req=5

pone-0032899-g004: Expressions of ectodermal patterning factors appear normal in arl6ip1-MO1-injected embryos.Dorsal views of wild-type (A–E) and arl6ip1 morphant (F–J) embryos at the 3-somite stage. Anterior to the top. The neural ectodermal patterning factors of wnt8 (A, F), bmp4 (B, G), msxb (C, H), dlx3b (D, I), and pax3 (E, J). Normal expressions of these genes reveal that ectodermal patterning factors are not affected in arl6ip1 morphants, suggesting that Arl6ip1 does not have a role in ectodermal patterning. Scale bar: 100 µm.
Mentions: To study the function of arl6ip1 during NC development, we first detected the expressions of the ectodermal patterning factors wnt8, bmp4, pax3, and msxb. The ectodermal patterning factors were all normally expressed in the arl6ip1-MO1- injected embryos (Figs. 4A and F; 4B and G; 4C and H; 4D and I). In addition, the expression of the pre-placodal marker dlx3b was also normal in the arl6ip1-MO1- injected embryos (Figs. 4E and J, n = 64). Furthermore, since Trainor and Krumlauf (2000) [28] reported that segmentation and patterning of the hindbrain are necessary for cranial NC development; we also examined the expressions of two hindbrain patterning markers, fgf3 and fgf8. Results showed that fgf3 and fgf8 were also expressed normally in the hindbrain of arl6ip1 morphants (Figs. S2A–D, n = 87) and that 83.2% of morphants exhibited a normal hindbrain segmentation at 24 hpf (Figs. S2E–F, n = 101). These findings suggested that the induction of both early NC progenitors and placodal region cells results in normal development of arl6ip1 morphants. The early patterning and later segmentation of the hindbrain in the arl6ip1-MO1-injected embryos were also similar to those of wild-type. Thus, even though arl6ip1 may not function in hindbrain development, it is expressed ubiquitously until 24 hpf, and knockdown of Arl6ip1 did cause small head morphology. This line of evidence suggests that neural crest defects in arl6ip1-MO1 embryos did not result from abnormal head development. On the other hand, when we observed the expressions of pre-migratory NC markers in the progenitors of the neural plate border, such as tfap2α, foxd3, snai1b, and sox10, we found that the expressions of foxd3, snai1b and sox10 were greatly reduced in the arl6ip1 morphants at 3-somite (Figs. 5B and F; 5C and G; 5D and H) through 8-somite stages (Figs. 5I and L; 5J and M; 5K and N). Meanwhile, tfap2a expression in NC progenitors was present in both wild-type and arl6ip1-MO1 embryos (Figs. 5A and E). Double-staining of sox2 (labeling neural plate) and dlx3b (labeling the pre-placodal region) exhibited a patch of unlabeled cells between these two expression domains which was presumed to be the NC. Thus, since the induction of presumptive NC was normal in arl6ip1-MO1 embryos (Fig. S3), we concluded that NC induction occurred normally in arl6ip1 morphants. The occurrence rates of down-regulation of foxd3, snai1b and sox10 in the arl6ip1-MO1-injected embryos were 75.6% (31/41), 82.2% (37/45) and 66.7% (30/45), respectively. Again, since down-regulation of these genes could have resulted from early cell death by the off-targeting effect of MO, we injected 6 ng p53-MO together with 4 ng arl6ip1-MO1 to inhibit apoptosis and nonspecific effects. We found that the level of cell death was dramatically reduced in the embryos injected with p53-MO. In addition, Cole and Ross (2001) [29] have detected the temporal and spatial distribution of apoptotic cells during normal development of the zebrafish embryo from 12 to 96 hpf. According to their data, only a few apoptotic cells could be seen in the optic vesicle at 12hpf, and no apoptosis was observed in the pre-migratory neural crest cell region. Robu et al. (2007) [26] also demonstrated that about 15–20% of MO used in zebrafish show off-targeting effects, as represented by a signature neural death peaking at the end of segmentation (1 dpf). They also showed that the neural cell death induced by MO was observed as early as 14 hpf. Moreover, Ekker and Larson (2001) [30] indicated that the onset of cell death induced by MO technology was 14 hpf. In our data, we detected pre-migratory neural crest markers at the 3-somite stage (about 10.33 hpf), even earlier than 12 hpf; consequently, we did not observe off-targeting effects at the 3-somite stage. On the other hand, when p53 was inhibited, cell survival could not rescue expressions of foxd3, snai1b and sox10 at the 8-somite stage (Figs. 5L–N). Therefore, we concluded that the down-regulation of these gene markers in existing NC cells was caused by defects in NC derivate specification, not cell death. Furthermore, such defects subsequently resulted in the reduction of the cell population, especially in pharyngeal arches of cranial NC sublineages. Finally, we found that the reduced expression of foxd3 induced by arl6ip1-MO1 could be restored by injection of wobble arl6ip1 mRNA in arl6ip1- MO1-injected embryos (Figs. S4A–C). These findings implicated that Arl6ip1 plays a key function in specification of NC sublineages, but not induction.

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