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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: 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.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.

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.

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The outer limiting membrane (OLM) is not restored in moe- mutants by injection of epb4.1l5long mRNA. (A) In wild-type larvae at 6 dpf, Crumbs proteins localize just apical to the OLM, which is labeled by anti-ZO-1. (B) In moe- mutants, very little Crumbs protein is visible and ZO-1 labeling is disorganized. (C) In epb4.1l5long mRNA injected moe- mutants, streaks of panCrb labeling are visible, but an organized OLM is absent. Ultrastructural transmission electron microscopic analysis at 6 dpf in wild-type (D), moe- mutants (E) and epb4.1l5long mRNA injected moe- mutants (F) retinas. Electron dense outer segments are seen in all individuals. Insets, higher magnifications of rod outer segments showing regular disc stacking is present in all individuals (100,000X). RPE, retinal pigmented epithelium; OS, outer segments; IS, inner Segments; CB, cell body. (A-C) are confocal z-projections, scale bar 10 μm (D-F). Scale bars, 5 μm (D-F), 100 nm (insets).
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Figure 6: The outer limiting membrane (OLM) is not restored in moe- mutants by injection of epb4.1l5long mRNA. (A) In wild-type larvae at 6 dpf, Crumbs proteins localize just apical to the OLM, which is labeled by anti-ZO-1. (B) In moe- mutants, very little Crumbs protein is visible and ZO-1 labeling is disorganized. (C) In epb4.1l5long mRNA injected moe- mutants, streaks of panCrb labeling are visible, but an organized OLM is absent. Ultrastructural transmission electron microscopic analysis at 6 dpf in wild-type (D), moe- mutants (E) and epb4.1l5long mRNA injected moe- mutants (F) retinas. Electron dense outer segments are seen in all individuals. Insets, higher magnifications of rod outer segments showing regular disc stacking is present in all individuals (100,000X). RPE, retinal pigmented epithelium; OS, outer segments; IS, inner Segments; CB, cell body. (A-C) are confocal z-projections, scale bar 10 μm (D-F). Scale bars, 5 μm (D-F), 100 nm (insets).

Mentions: Whereas Müller glial morphology and retinal lamination are rescued in moe- mutants by injection of either moe or epb4.15long mRNA, the morphology of photoreceptors (rods and double cones) is not; instead of standing perpendicular to the normal RPE (Fig. 5A, D, G and Fig. 6A), those in moe- mutants injected with either moe or epb4.15long mRNA, lie collapsed in a twisted heap adjacent to the RPE (Fig. 5C, F, I and Fig. 6C, and data not shown). The failure to rescue photoreceptor morphology is likely because Moe or Epb4.15long protein from injected mRNA is lost by the time photoreceptors undergo morphogenesis.


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)

The outer limiting membrane (OLM) is not restored in moe- mutants by injection of epb4.1l5long mRNA. (A) In wild-type larvae at 6 dpf, Crumbs proteins localize just apical to the OLM, which is labeled by anti-ZO-1. (B) In moe- mutants, very little Crumbs protein is visible and ZO-1 labeling is disorganized. (C) In epb4.1l5long mRNA injected moe- mutants, streaks of panCrb labeling are visible, but an organized OLM is absent. Ultrastructural transmission electron microscopic analysis at 6 dpf in wild-type (D), moe- mutants (E) and epb4.1l5long mRNA injected moe- mutants (F) retinas. Electron dense outer segments are seen in all individuals. Insets, higher magnifications of rod outer segments showing regular disc stacking is present in all individuals (100,000X). RPE, retinal pigmented epithelium; OS, outer segments; IS, inner Segments; CB, cell body. (A-C) are confocal z-projections, scale bar 10 μm (D-F). Scale bars, 5 μm (D-F), 100 nm (insets).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266719&req=5

Figure 6: The outer limiting membrane (OLM) is not restored in moe- mutants by injection of epb4.1l5long mRNA. (A) In wild-type larvae at 6 dpf, Crumbs proteins localize just apical to the OLM, which is labeled by anti-ZO-1. (B) In moe- mutants, very little Crumbs protein is visible and ZO-1 labeling is disorganized. (C) In epb4.1l5long mRNA injected moe- mutants, streaks of panCrb labeling are visible, but an organized OLM is absent. Ultrastructural transmission electron microscopic analysis at 6 dpf in wild-type (D), moe- mutants (E) and epb4.1l5long mRNA injected moe- mutants (F) retinas. Electron dense outer segments are seen in all individuals. Insets, higher magnifications of rod outer segments showing regular disc stacking is present in all individuals (100,000X). RPE, retinal pigmented epithelium; OS, outer segments; IS, inner Segments; CB, cell body. (A-C) are confocal z-projections, scale bar 10 μm (D-F). Scale bars, 5 μm (D-F), 100 nm (insets).
Mentions: Whereas Müller glial morphology and retinal lamination are rescued in moe- mutants by injection of either moe or epb4.15long mRNA, the morphology of photoreceptors (rods and double cones) is not; instead of standing perpendicular to the normal RPE (Fig. 5A, D, G and Fig. 6A), those in moe- mutants injected with either moe or epb4.15long mRNA, lie collapsed in a twisted heap adjacent to the RPE (Fig. 5C, F, I and Fig. 6C, and data not shown). The failure to rescue photoreceptor morphology is likely because Moe or Epb4.15long protein from injected mRNA is lost by the time photoreceptors undergo morphogenesis.

Bottom Line: 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.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.

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