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Itch is required for lateral line development in zebrafish.

Angers A, Drapeau P - PLoS ONE (2014)

Bottom Line: The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium.The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/β-catenin signaling and the establishment of specific cxcr4b and cxcr7b cytokine receptor expression domains.Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of cxcr4b in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Montreal, Montreal, Quebec, Canada.

ABSTRACT
The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium. The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/β-catenin signaling and the establishment of specific cxcr4b and cxcr7b cytokine receptor expression domains. Itch has been identified as a regulator in several different signaling pathways, including Wnt and Cxcr4 signaling. We identified two homologous itch genes in zebrafish, itcha and itchb, with generalized expression patterns. By reducing itchb expression in particular upon morpholino knockdown, we demonstrated the importance of Itch in regulating lateral line development by perturbing the patterns of cxcr4b and cxcr7b expression. Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of cxcr4b in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line.

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Primordium patterning is altered in itchb knocked-down embryos.(A–F) RNA in situ hybridization of factors required for primordium patterning in control embryos (vehicle-injected siblings) (CTRL, left panels) and MOs-injected embryos (MoB, right panels) at 30 hpf. cldnb:gfp embryos were used to ensure that the primordium had migrated pass the 10th somite before fixation. (A,B) cxcr4b expression was limited to the leading half of the primordium in control embryos, but extended throughout the primordium after itchb knockdown. (C,D) cxcr7b was limited to the trailing end of control embryos, and almost completely excluded from the primordium in itchb knockdown. (E,F) lef1 was expressed in the leading edge of control embryos. Darker staining indicated higher expression in embryos injected with MOs against itchb (MoB), but leading edge expression was maintained. Scale bar: . (G) Semi-quantitative RT-PCR showing enhanced expression of lef1 in embryos injected with MOs against itchb (MoB) as compared to control embryos (WT). Amplification of the actin gene was used as an internal control. The image was inverted to facilitate quantification. (H) Densitometry measurements from the gel presented in G. This is representative of three different experiments.
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pone-0111799-g005: Primordium patterning is altered in itchb knocked-down embryos.(A–F) RNA in situ hybridization of factors required for primordium patterning in control embryos (vehicle-injected siblings) (CTRL, left panels) and MOs-injected embryos (MoB, right panels) at 30 hpf. cldnb:gfp embryos were used to ensure that the primordium had migrated pass the 10th somite before fixation. (A,B) cxcr4b expression was limited to the leading half of the primordium in control embryos, but extended throughout the primordium after itchb knockdown. (C,D) cxcr7b was limited to the trailing end of control embryos, and almost completely excluded from the primordium in itchb knockdown. (E,F) lef1 was expressed in the leading edge of control embryos. Darker staining indicated higher expression in embryos injected with MOs against itchb (MoB), but leading edge expression was maintained. Scale bar: . (G) Semi-quantitative RT-PCR showing enhanced expression of lef1 in embryos injected with MOs against itchb (MoB) as compared to control embryos (WT). Amplification of the actin gene was used as an internal control. The image was inverted to facilitate quantification. (H) Densitometry measurements from the gel presented in G. This is representative of three different experiments.

Mentions: Migration of the primordium along the myoseptum is directed by the cytokine receptor Cxcr4b, expressed in the leading edge of the primordium. Directionality is ensured by expression of another cytokine receptor, Cxcr7b, in the trailing end of the migrating primordium, whose expression is restricted by Cxcr4b and acts as a sink for the ligand Sdf-1, thereby preventing Cxcr4b signaling in the trailing end and allowing directionality of migration [31]. Given the slow migration of the pLL primordium in itchb morphants, we examined cxcr4b and cxcr7b expression in the primordium. We visually assessed in cldnb:gfp embryos that the primordium had reached somite 10 before fixation and in situ hybridization. This occurred at about 28 hpf for control embryos and 30 hpf for itchb morphants. Comparing cxcr4b expression in these stage-matched embryos, while cxcr4b expression was restricted to the leading two-third of the primordium in control embryos (Fig. 5 A), it was clear that cxcr4b was overexpressed in itchb morphants and that its distribution encompassed the entire primordium, extending to the deposited cells behind the migrating primordium (Fig. 5 A and B; representative of 11 embryos in 4 different experiments).


Itch is required for lateral line development in zebrafish.

Angers A, Drapeau P - PLoS ONE (2014)

Primordium patterning is altered in itchb knocked-down embryos.(A–F) RNA in situ hybridization of factors required for primordium patterning in control embryos (vehicle-injected siblings) (CTRL, left panels) and MOs-injected embryos (MoB, right panels) at 30 hpf. cldnb:gfp embryos were used to ensure that the primordium had migrated pass the 10th somite before fixation. (A,B) cxcr4b expression was limited to the leading half of the primordium in control embryos, but extended throughout the primordium after itchb knockdown. (C,D) cxcr7b was limited to the trailing end of control embryos, and almost completely excluded from the primordium in itchb knockdown. (E,F) lef1 was expressed in the leading edge of control embryos. Darker staining indicated higher expression in embryos injected with MOs against itchb (MoB), but leading edge expression was maintained. Scale bar: . (G) Semi-quantitative RT-PCR showing enhanced expression of lef1 in embryos injected with MOs against itchb (MoB) as compared to control embryos (WT). Amplification of the actin gene was used as an internal control. The image was inverted to facilitate quantification. (H) Densitometry measurements from the gel presented in G. This is representative of three different experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219781&req=5

pone-0111799-g005: Primordium patterning is altered in itchb knocked-down embryos.(A–F) RNA in situ hybridization of factors required for primordium patterning in control embryos (vehicle-injected siblings) (CTRL, left panels) and MOs-injected embryos (MoB, right panels) at 30 hpf. cldnb:gfp embryos were used to ensure that the primordium had migrated pass the 10th somite before fixation. (A,B) cxcr4b expression was limited to the leading half of the primordium in control embryos, but extended throughout the primordium after itchb knockdown. (C,D) cxcr7b was limited to the trailing end of control embryos, and almost completely excluded from the primordium in itchb knockdown. (E,F) lef1 was expressed in the leading edge of control embryos. Darker staining indicated higher expression in embryos injected with MOs against itchb (MoB), but leading edge expression was maintained. Scale bar: . (G) Semi-quantitative RT-PCR showing enhanced expression of lef1 in embryos injected with MOs against itchb (MoB) as compared to control embryos (WT). Amplification of the actin gene was used as an internal control. The image was inverted to facilitate quantification. (H) Densitometry measurements from the gel presented in G. This is representative of three different experiments.
Mentions: Migration of the primordium along the myoseptum is directed by the cytokine receptor Cxcr4b, expressed in the leading edge of the primordium. Directionality is ensured by expression of another cytokine receptor, Cxcr7b, in the trailing end of the migrating primordium, whose expression is restricted by Cxcr4b and acts as a sink for the ligand Sdf-1, thereby preventing Cxcr4b signaling in the trailing end and allowing directionality of migration [31]. Given the slow migration of the pLL primordium in itchb morphants, we examined cxcr4b and cxcr7b expression in the primordium. We visually assessed in cldnb:gfp embryos that the primordium had reached somite 10 before fixation and in situ hybridization. This occurred at about 28 hpf for control embryos and 30 hpf for itchb morphants. Comparing cxcr4b expression in these stage-matched embryos, while cxcr4b expression was restricted to the leading two-third of the primordium in control embryos (Fig. 5 A), it was clear that cxcr4b was overexpressed in itchb morphants and that its distribution encompassed the entire primordium, extending to the deposited cells behind the migrating primordium (Fig. 5 A and B; representative of 11 embryos in 4 different experiments).

Bottom Line: The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium.The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/β-catenin signaling and the establishment of specific cxcr4b and cxcr7b cytokine receptor expression domains.Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of cxcr4b in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Montreal, Montreal, Quebec, Canada.

ABSTRACT
The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium. The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/β-catenin signaling and the establishment of specific cxcr4b and cxcr7b cytokine receptor expression domains. Itch has been identified as a regulator in several different signaling pathways, including Wnt and Cxcr4 signaling. We identified two homologous itch genes in zebrafish, itcha and itchb, with generalized expression patterns. By reducing itchb expression in particular upon morpholino knockdown, we demonstrated the importance of Itch in regulating lateral line development by perturbing the patterns of cxcr4b and cxcr7b expression. Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of cxcr4b in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line.

Show MeSH
Related in: MedlinePlus