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Tissue-specific requirements for specific domains in the FERM protein Moe/Epb4.1l5 during early zebrafish development.

Christensen AK, Jensen AM - BMC Dev. Biol. (2008)

Bottom Line: In zebrafish, loss-of-function mutations in moe result in defects in brain ventricle formation, retinal pigmented epithelium and neural retinal development, pericardial edema, and tail curvature.We found that injection of the Epb4.1l5short chimera (Epb4.1l5short+long_PBD), containing the PBD from Epb4.1l5long, could rescue retinal and RPE defects in moe- mutants, but not brain ventricle formation.Additionally, our data provide further evidence that Moe is a negative regulator of rod outer segment size.

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Affiliation: Department of Biology and the Molecular and Cellular Biology Program, University of Massachusetts, Amherst, MA, 01003, USA. arnec@bio.umass.edu

ABSTRACT

Background: The FERM domain containing protein Mosaic Eyes (Moe) interacts with Crumbs proteins, which are important regulators of apical identity and size. In zebrafish, loss-of-function mutations in moe result in defects in brain ventricle formation, retinal pigmented epithelium and neural retinal development, pericardial edema, and tail curvature. In humans and mice, there are two major alternately spliced isoforms of the Moe orthologue, Erythrocyte Protein Band 4.1-Like 5 (Epb4.1l5), which we have named Epb4.1l5long and Epb4.1l5short, that differ after the FERM domain. Interestingly, Moe and both Epb4.1l5 isoforms have a putative C' terminal Type-I PDZ-Binding Domain (PBD). We previously showed that the N' terminal FERM domain in Moe directly mediates interactions with Crumbs proteins and Nagie oko (Nok) in zebrafish, but the function of the C'terminal half of Moe/Epb4.1l5 has not yet been examined.

Results: To define functionally important domains in zebrafish Moe and murine Epb4.1l5, we tested whether injection of mRNAs encoding these proteins could rescue defects in zebrafish moe- embryos. Injection of either moe or epb4.1l5long mRNA, but not epb4.1l5short mRNA, could rescue moe- embryonic defects. We also tested whether mRNA encoding C' terminal truncations of Epb4.1l5long or chimeric constructs with reciprocal swaps of the isoform-specific PBDs could rescue moe- defects. We found that injection of the Epb4.1l5short chimera (Epb4.1l5short+long_PBD), containing the PBD from Epb4.1l5long, could rescue retinal and RPE defects in moe- mutants, but not brain ventricle formation. Injection of the Epb4.1l5long chimera (Epb4.1l5long+short_PBD), containing the PBD from Epb4.1l5short, rescued retinal defects, and to a large extent rescued RPE integrity. The only construct that caused a dominant phenotype in wild-type embryos, was Epb4.1l5long+short_PBD, which caused brain ventricle defects and edema that were similar to those observed in moe- mutants. Lastly, the morphology of rod photoreceptors in moe- mutants where embryonic defects were rescued by moe or epb4.1l5long mRNA injection is abnormal and their outer segments are larger than normal.

Conclusion: Taken together, the data reveal tissue specificity for the function of the PBD in Epb4.1l5long, and suggest that additional C' terminal sequences are important for zebrafish retinal development. Additionally, our data provide further evidence that Moe is a negative regulator of rod outer segment size.

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Both the PDZ-binding domain and unique sequences in Epb4.1l5long required for rescue of moe- mutant defects. (A-C) At 30 hpf in wild-type embryos, Moe localizes cortically in brain and retinal neuroepithelial cells and is concentrated at the apical surface (A) and ZO-1 (green) and panCrb (red) localize to the apical surface of the retina (B) and brain (C). (D-F) At 30 hpf in moe- embryos, there is no Moe labeling (D) and ZO-1 (green) and panCrb (red) fail to localize to the apical surface of the retina (E) and brain (F). (G) At 30 hpf in moe- embryos injected with epb4.1l5long mRNA, Epb4.1l5long immunoreactivity is cortically localized in most retinal and brain neuroepithelial cells and ZO-1 (green) and panCrb (red) localize to the apical surface in the retina (H) and brain (I). Upper inset (G), magnified section of anti-Epb4.1l5long labeling and lower inset, uninjected moe- embryos shows no labeling with anti-Epb4.1l5long. (J) At 30 hpf in moe- mutants injected with epb4.1l5short mRNA, anti-Epb4.1l5short is cytoplasmically localized and ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (K) and brain (L). Upper inset (G), magnified section of anti-Epb4.1l5short labeling and lower inset, uninjected moe- embryos shows no labeling with anti-Epb4.1l5short. (M, N) At 30 hpf in moe- mutants injected with myc-epb4.1l5FERM mRNA, ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (M) and brain (N). (O, P) At 30 hpf in moe- mutants injected with myc-epb4.1l5long_ΔPBD mRNA, ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (O) and brain (P). (Q, R) At 30 hpf in moe- mutants injected with myc-epb4.1l5short+long_PBD mRNA, ZO-1 (green) and panCrb (red) localize to the apical surface in moe- retina (Q) and brain (R). (S, T) At 30 hpf in moe- mutants injected with myc-epb4.1l5long+short_PBD mRNA, ZO-1 (green) and panCrb (red) localize to the apical surface in moe- retina (S) and brain (T). (U) Western analysis of zebrafish embryos injected with epb4.1l5long and epb4.1l5short mRNA were tested for expression of protein product with isoform-specific antibodies at time points from 6 hpf to 96 hpf. (V) Western analysis of zebrafish embryos injected with epb4.1l5FERM (anti-Myc), epb4.1l5long_ΔPBD (anti-Epb4.1l5long), epb4.1l5short+long_PBD (anti-Myc), epb4.1l5long+short_PBD (anti-Epb4.1l5long). Blots stripped and reprobed with Anti-α-Tubulin as a loading control. Scale bars, 10 μm (A, D, G, J), 50 μm (lower insets in G, J), 10 μm (remaining panels). (A-T), single confocal z-sections.
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Figure 3: Both the PDZ-binding domain and unique sequences in Epb4.1l5long required for rescue of moe- mutant defects. (A-C) At 30 hpf in wild-type embryos, Moe localizes cortically in brain and retinal neuroepithelial cells and is concentrated at the apical surface (A) and ZO-1 (green) and panCrb (red) localize to the apical surface of the retina (B) and brain (C). (D-F) At 30 hpf in moe- embryos, there is no Moe labeling (D) and ZO-1 (green) and panCrb (red) fail to localize to the apical surface of the retina (E) and brain (F). (G) At 30 hpf in moe- embryos injected with epb4.1l5long mRNA, Epb4.1l5long immunoreactivity is cortically localized in most retinal and brain neuroepithelial cells and ZO-1 (green) and panCrb (red) localize to the apical surface in the retina (H) and brain (I). Upper inset (G), magnified section of anti-Epb4.1l5long labeling and lower inset, uninjected moe- embryos shows no labeling with anti-Epb4.1l5long. (J) At 30 hpf in moe- mutants injected with epb4.1l5short mRNA, anti-Epb4.1l5short is cytoplasmically localized and ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (K) and brain (L). Upper inset (G), magnified section of anti-Epb4.1l5short labeling and lower inset, uninjected moe- embryos shows no labeling with anti-Epb4.1l5short. (M, N) At 30 hpf in moe- mutants injected with myc-epb4.1l5FERM mRNA, ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (M) and brain (N). (O, P) At 30 hpf in moe- mutants injected with myc-epb4.1l5long_ΔPBD mRNA, ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (O) and brain (P). (Q, R) At 30 hpf in moe- mutants injected with myc-epb4.1l5short+long_PBD mRNA, ZO-1 (green) and panCrb (red) localize to the apical surface in moe- retina (Q) and brain (R). (S, T) At 30 hpf in moe- mutants injected with myc-epb4.1l5long+short_PBD mRNA, ZO-1 (green) and panCrb (red) localize to the apical surface in moe- retina (S) and brain (T). (U) Western analysis of zebrafish embryos injected with epb4.1l5long and epb4.1l5short mRNA were tested for expression of protein product with isoform-specific antibodies at time points from 6 hpf to 96 hpf. (V) Western analysis of zebrafish embryos injected with epb4.1l5FERM (anti-Myc), epb4.1l5long_ΔPBD (anti-Epb4.1l5long), epb4.1l5short+long_PBD (anti-Myc), epb4.1l5long+short_PBD (anti-Epb4.1l5long). Blots stripped and reprobed with Anti-α-Tubulin as a loading control. Scale bars, 10 μm (A, D, G, J), 50 μm (lower insets in G, J), 10 μm (remaining panels). (A-T), single confocal z-sections.

Mentions: To identify functionally important sequences in the Moe and orthologous protein Epb41.l5, we tested whether injection of mouse epb4.1l5long mRNA could substitute for moe and rescue moe- mutant defects. We injected epb4.1l5long mRNA into 1–4 cell moe- embryos and found that it rescued brain ventricle formation, retinal pigmented epithelial integrity, and retinal lamination and straightened the tail like injection of moe mRNA (Table 1 and data not shown). Injection of epb4.1l5long also rescued apical localization of ZO-1 and anti-panCrb labeling in the retina and brain (Figure 3H, I). Using antibodies we raised against the unique sequence in Epb4.1l5long, we found that Epb4.1l5long protein in mRNA injected moe- mutants localized cortically like endogenous Moe in wild-type embryos (Figure 3A, G). Because all the proteins shown to interact with Moe do so through its FERM domain [21], we tested whether injection of epb4.1l5 mRNA encoding a myc-tagged FERM domain (amino acids 1–346, Epb4.1l5FERM) could rescue moe- mutant defects like full length moe and epb4.1l5long mRNA injection. Injection of epb4.1l5FERM mRNA at the 1–4 cell stage failed to rescue the defects in moe- mutants (data not shown) and did not lead to apical relocalization of ZO-1 and anti-panCrb labeling (Figure 3M, N). We also ruled out the possibility that the N' terminal myc-tag interfered with protein function by showing that Epb4.1l5long myc-tagged at its N' terminus was still able to rescue moe- mutant defects as well as untagged Epb4.1l5long (data not shown)


Tissue-specific requirements for specific domains in the FERM protein Moe/Epb4.1l5 during early zebrafish development.

Christensen AK, Jensen AM - BMC Dev. Biol. (2008)

Both the PDZ-binding domain and unique sequences in Epb4.1l5long required for rescue of moe- mutant defects. (A-C) At 30 hpf in wild-type embryos, Moe localizes cortically in brain and retinal neuroepithelial cells and is concentrated at the apical surface (A) and ZO-1 (green) and panCrb (red) localize to the apical surface of the retina (B) and brain (C). (D-F) At 30 hpf in moe- embryos, there is no Moe labeling (D) and ZO-1 (green) and panCrb (red) fail to localize to the apical surface of the retina (E) and brain (F). (G) At 30 hpf in moe- embryos injected with epb4.1l5long mRNA, Epb4.1l5long immunoreactivity is cortically localized in most retinal and brain neuroepithelial cells and ZO-1 (green) and panCrb (red) localize to the apical surface in the retina (H) and brain (I). Upper inset (G), magnified section of anti-Epb4.1l5long labeling and lower inset, uninjected moe- embryos shows no labeling with anti-Epb4.1l5long. (J) At 30 hpf in moe- mutants injected with epb4.1l5short mRNA, anti-Epb4.1l5short is cytoplasmically localized and ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (K) and brain (L). Upper inset (G), magnified section of anti-Epb4.1l5short labeling and lower inset, uninjected moe- embryos shows no labeling with anti-Epb4.1l5short. (M, N) At 30 hpf in moe- mutants injected with myc-epb4.1l5FERM mRNA, ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (M) and brain (N). (O, P) At 30 hpf in moe- mutants injected with myc-epb4.1l5long_ΔPBD mRNA, ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (O) and brain (P). (Q, R) At 30 hpf in moe- mutants injected with myc-epb4.1l5short+long_PBD mRNA, ZO-1 (green) and panCrb (red) localize to the apical surface in moe- retina (Q) and brain (R). (S, T) At 30 hpf in moe- mutants injected with myc-epb4.1l5long+short_PBD mRNA, ZO-1 (green) and panCrb (red) localize to the apical surface in moe- retina (S) and brain (T). (U) Western analysis of zebrafish embryos injected with epb4.1l5long and epb4.1l5short mRNA were tested for expression of protein product with isoform-specific antibodies at time points from 6 hpf to 96 hpf. (V) Western analysis of zebrafish embryos injected with epb4.1l5FERM (anti-Myc), epb4.1l5long_ΔPBD (anti-Epb4.1l5long), epb4.1l5short+long_PBD (anti-Myc), epb4.1l5long+short_PBD (anti-Epb4.1l5long). Blots stripped and reprobed with Anti-α-Tubulin as a loading control. Scale bars, 10 μm (A, D, G, J), 50 μm (lower insets in G, J), 10 μm (remaining panels). (A-T), single confocal z-sections.
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Figure 3: Both the PDZ-binding domain and unique sequences in Epb4.1l5long required for rescue of moe- mutant defects. (A-C) At 30 hpf in wild-type embryos, Moe localizes cortically in brain and retinal neuroepithelial cells and is concentrated at the apical surface (A) and ZO-1 (green) and panCrb (red) localize to the apical surface of the retina (B) and brain (C). (D-F) At 30 hpf in moe- embryos, there is no Moe labeling (D) and ZO-1 (green) and panCrb (red) fail to localize to the apical surface of the retina (E) and brain (F). (G) At 30 hpf in moe- embryos injected with epb4.1l5long mRNA, Epb4.1l5long immunoreactivity is cortically localized in most retinal and brain neuroepithelial cells and ZO-1 (green) and panCrb (red) localize to the apical surface in the retina (H) and brain (I). Upper inset (G), magnified section of anti-Epb4.1l5long labeling and lower inset, uninjected moe- embryos shows no labeling with anti-Epb4.1l5long. (J) At 30 hpf in moe- mutants injected with epb4.1l5short mRNA, anti-Epb4.1l5short is cytoplasmically localized and ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (K) and brain (L). Upper inset (G), magnified section of anti-Epb4.1l5short labeling and lower inset, uninjected moe- embryos shows no labeling with anti-Epb4.1l5short. (M, N) At 30 hpf in moe- mutants injected with myc-epb4.1l5FERM mRNA, ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (M) and brain (N). (O, P) At 30 hpf in moe- mutants injected with myc-epb4.1l5long_ΔPBD mRNA, ZO-1 (green) and panCrb (red) do not localize to the apical surface in moe- retina (O) and brain (P). (Q, R) At 30 hpf in moe- mutants injected with myc-epb4.1l5short+long_PBD mRNA, ZO-1 (green) and panCrb (red) localize to the apical surface in moe- retina (Q) and brain (R). (S, T) At 30 hpf in moe- mutants injected with myc-epb4.1l5long+short_PBD mRNA, ZO-1 (green) and panCrb (red) localize to the apical surface in moe- retina (S) and brain (T). (U) Western analysis of zebrafish embryos injected with epb4.1l5long and epb4.1l5short mRNA were tested for expression of protein product with isoform-specific antibodies at time points from 6 hpf to 96 hpf. (V) Western analysis of zebrafish embryos injected with epb4.1l5FERM (anti-Myc), epb4.1l5long_ΔPBD (anti-Epb4.1l5long), epb4.1l5short+long_PBD (anti-Myc), epb4.1l5long+short_PBD (anti-Epb4.1l5long). Blots stripped and reprobed with Anti-α-Tubulin as a loading control. Scale bars, 10 μm (A, D, G, J), 50 μm (lower insets in G, J), 10 μm (remaining panels). (A-T), single confocal z-sections.
Mentions: To identify functionally important sequences in the Moe and orthologous protein Epb41.l5, we tested whether injection of mouse epb4.1l5long mRNA could substitute for moe and rescue moe- mutant defects. We injected epb4.1l5long mRNA into 1–4 cell moe- embryos and found that it rescued brain ventricle formation, retinal pigmented epithelial integrity, and retinal lamination and straightened the tail like injection of moe mRNA (Table 1 and data not shown). Injection of epb4.1l5long also rescued apical localization of ZO-1 and anti-panCrb labeling in the retina and brain (Figure 3H, I). Using antibodies we raised against the unique sequence in Epb4.1l5long, we found that Epb4.1l5long protein in mRNA injected moe- mutants localized cortically like endogenous Moe in wild-type embryos (Figure 3A, G). Because all the proteins shown to interact with Moe do so through its FERM domain [21], we tested whether injection of epb4.1l5 mRNA encoding a myc-tagged FERM domain (amino acids 1–346, Epb4.1l5FERM) could rescue moe- mutant defects like full length moe and epb4.1l5long mRNA injection. Injection of epb4.1l5FERM mRNA at the 1–4 cell stage failed to rescue the defects in moe- mutants (data not shown) and did not lead to apical relocalization of ZO-1 and anti-panCrb labeling (Figure 3M, N). We also ruled out the possibility that the N' terminal myc-tag interfered with protein function by showing that Epb4.1l5long myc-tagged at its N' terminus was still able to rescue moe- mutant defects as well as untagged Epb4.1l5long (data not shown)

Bottom Line: In zebrafish, loss-of-function mutations in moe result in defects in brain ventricle formation, retinal pigmented epithelium and neural retinal development, pericardial edema, and tail curvature.We found that injection of the Epb4.1l5short chimera (Epb4.1l5short+long_PBD), containing the PBD from Epb4.1l5long, could rescue retinal and RPE defects in moe- mutants, but not brain ventricle formation.Additionally, our data provide further evidence that Moe is a negative regulator of rod outer segment size.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and the Molecular and Cellular Biology Program, University of Massachusetts, Amherst, MA, 01003, USA. arnec@bio.umass.edu

ABSTRACT

Background: The FERM domain containing protein Mosaic Eyes (Moe) interacts with Crumbs proteins, which are important regulators of apical identity and size. In zebrafish, loss-of-function mutations in moe result in defects in brain ventricle formation, retinal pigmented epithelium and neural retinal development, pericardial edema, and tail curvature. In humans and mice, there are two major alternately spliced isoforms of the Moe orthologue, Erythrocyte Protein Band 4.1-Like 5 (Epb4.1l5), which we have named Epb4.1l5long and Epb4.1l5short, that differ after the FERM domain. Interestingly, Moe and both Epb4.1l5 isoforms have a putative C' terminal Type-I PDZ-Binding Domain (PBD). We previously showed that the N' terminal FERM domain in Moe directly mediates interactions with Crumbs proteins and Nagie oko (Nok) in zebrafish, but the function of the C'terminal half of Moe/Epb4.1l5 has not yet been examined.

Results: To define functionally important domains in zebrafish Moe and murine Epb4.1l5, we tested whether injection of mRNAs encoding these proteins could rescue defects in zebrafish moe- embryos. Injection of either moe or epb4.1l5long mRNA, but not epb4.1l5short mRNA, could rescue moe- embryonic defects. We also tested whether mRNA encoding C' terminal truncations of Epb4.1l5long or chimeric constructs with reciprocal swaps of the isoform-specific PBDs could rescue moe- defects. We found that injection of the Epb4.1l5short chimera (Epb4.1l5short+long_PBD), containing the PBD from Epb4.1l5long, could rescue retinal and RPE defects in moe- mutants, but not brain ventricle formation. Injection of the Epb4.1l5long chimera (Epb4.1l5long+short_PBD), containing the PBD from Epb4.1l5short, rescued retinal defects, and to a large extent rescued RPE integrity. The only construct that caused a dominant phenotype in wild-type embryos, was Epb4.1l5long+short_PBD, which caused brain ventricle defects and edema that were similar to those observed in moe- mutants. Lastly, the morphology of rod photoreceptors in moe- mutants where embryonic defects were rescued by moe or epb4.1l5long mRNA injection is abnormal and their outer segments are larger than normal.

Conclusion: Taken together, the data reveal tissue specificity for the function of the PBD in Epb4.1l5long, and suggest that additional C' terminal sequences are important for zebrafish retinal development. Additionally, our data provide further evidence that Moe is a negative regulator of rod outer segment size.

Show MeSH
Related in: MedlinePlus