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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.

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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Genomic structure of the mouse epb4.1l5 locus and expression of its two major splice isoforms. (A) Diagram of the inton/exon structure of the Mus musculus epb4.1l5 locus. Exons that are common to both isoforms are black, the exons unique to epb4.1l5long are indicated in red and the unique exon in epb4.1l5short is indicated in green. Bars represent 1 kb and 10 kb scales for exon and intron lengths, respectively. (B) ClustalX alignment of mouse Epb4.1l5long and zebrafish Moe. Ymo1long and Moe share a high degree of homology within the FERM domain (black). Ymo1long and Ymo1short are identical up to Lysine 444 (blue arrow) and then alternately spliced into the long (red) and short (green) isoforms. Moe and Epb4.1l5long, and Epb4.1l5short have predicted C'terminal PDZ-binding domains (Pink [TTEL]) and (light green [MTEI]). (*) identical, (:) highly conserved, (.) moderately conserved. (C) Western analysis of mouse tissues with antibodies raised against the unique C'terminal sequences of Epb4.1l5long and Epb4.1l5short. Two bands are immunoreactive with the anti-Epb4.1l5long antibody, one migrates at the expected molecular weight of Epb4.1l5long (100 kDa) and is present in the eye, brain, heart, lung, kidney, and testis. An additional band migrates at 75 kDa, which is probably non-specific. Two bands are recognized by the affinity purified Epb4.1l5short antibody. A lower band migrates at the predicted molecular weight of 56 kDa and is present in brain, liver, lung, kidney, pancreas, and gut. A second band with a broad expression pattern migrates at approximately 75 kDa. Anti-α-Tubulin was used as a loading control.
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Figure 2: Genomic structure of the mouse epb4.1l5 locus and expression of its two major splice isoforms. (A) Diagram of the inton/exon structure of the Mus musculus epb4.1l5 locus. Exons that are common to both isoforms are black, the exons unique to epb4.1l5long are indicated in red and the unique exon in epb4.1l5short is indicated in green. Bars represent 1 kb and 10 kb scales for exon and intron lengths, respectively. (B) ClustalX alignment of mouse Epb4.1l5long and zebrafish Moe. Ymo1long and Moe share a high degree of homology within the FERM domain (black). Ymo1long and Ymo1short are identical up to Lysine 444 (blue arrow) and then alternately spliced into the long (red) and short (green) isoforms. Moe and Epb4.1l5long, and Epb4.1l5short have predicted C'terminal PDZ-binding domains (Pink [TTEL]) and (light green [MTEI]). (*) identical, (:) highly conserved, (.) moderately conserved. (C) Western analysis of mouse tissues with antibodies raised against the unique C'terminal sequences of Epb4.1l5long and Epb4.1l5short. Two bands are immunoreactive with the anti-Epb4.1l5long antibody, one migrates at the expected molecular weight of Epb4.1l5long (100 kDa) and is present in the eye, brain, heart, lung, kidney, and testis. An additional band migrates at 75 kDa, which is probably non-specific. Two bands are recognized by the affinity purified Epb4.1l5short antibody. A lower band migrates at the predicted molecular weight of 56 kDa and is present in brain, liver, lung, kidney, pancreas, and gut. A second band with a broad expression pattern migrates at approximately 75 kDa. Anti-α-Tubulin was used as a loading control.

Mentions: To help identify functionally important domains in the Moe and Epb4.1l5 proteins, we first compared their sequences (Figure 2). The mammalian epb4.1l5 locus encodes two major splice isoforms that are represented by ESTs in both the human and mouse databases, which we term Epb4.1l5short and Epb4.1l5long. We provide the exon/intron structure of the mouse epb4.1l5 locus that has 25 exons: Epb4.1l5short is encoded by exons 1–16 and Epb4.1l5long by exons 1–15 and 17–25 (Figure 2A). We have not found a zebrafish transcript that encodes a protein similar to Epb4.1l5short.


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)

Genomic structure of the mouse epb4.1l5 locus and expression of its two major splice isoforms. (A) Diagram of the inton/exon structure of the Mus musculus epb4.1l5 locus. Exons that are common to both isoforms are black, the exons unique to epb4.1l5long are indicated in red and the unique exon in epb4.1l5short is indicated in green. Bars represent 1 kb and 10 kb scales for exon and intron lengths, respectively. (B) ClustalX alignment of mouse Epb4.1l5long and zebrafish Moe. Ymo1long and Moe share a high degree of homology within the FERM domain (black). Ymo1long and Ymo1short are identical up to Lysine 444 (blue arrow) and then alternately spliced into the long (red) and short (green) isoforms. Moe and Epb4.1l5long, and Epb4.1l5short have predicted C'terminal PDZ-binding domains (Pink [TTEL]) and (light green [MTEI]). (*) identical, (:) highly conserved, (.) moderately conserved. (C) Western analysis of mouse tissues with antibodies raised against the unique C'terminal sequences of Epb4.1l5long and Epb4.1l5short. Two bands are immunoreactive with the anti-Epb4.1l5long antibody, one migrates at the expected molecular weight of Epb4.1l5long (100 kDa) and is present in the eye, brain, heart, lung, kidney, and testis. An additional band migrates at 75 kDa, which is probably non-specific. Two bands are recognized by the affinity purified Epb4.1l5short antibody. A lower band migrates at the predicted molecular weight of 56 kDa and is present in brain, liver, lung, kidney, pancreas, and gut. A second band with a broad expression pattern migrates at approximately 75 kDa. Anti-α-Tubulin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Genomic structure of the mouse epb4.1l5 locus and expression of its two major splice isoforms. (A) Diagram of the inton/exon structure of the Mus musculus epb4.1l5 locus. Exons that are common to both isoforms are black, the exons unique to epb4.1l5long are indicated in red and the unique exon in epb4.1l5short is indicated in green. Bars represent 1 kb and 10 kb scales for exon and intron lengths, respectively. (B) ClustalX alignment of mouse Epb4.1l5long and zebrafish Moe. Ymo1long and Moe share a high degree of homology within the FERM domain (black). Ymo1long and Ymo1short are identical up to Lysine 444 (blue arrow) and then alternately spliced into the long (red) and short (green) isoforms. Moe and Epb4.1l5long, and Epb4.1l5short have predicted C'terminal PDZ-binding domains (Pink [TTEL]) and (light green [MTEI]). (*) identical, (:) highly conserved, (.) moderately conserved. (C) Western analysis of mouse tissues with antibodies raised against the unique C'terminal sequences of Epb4.1l5long and Epb4.1l5short. Two bands are immunoreactive with the anti-Epb4.1l5long antibody, one migrates at the expected molecular weight of Epb4.1l5long (100 kDa) and is present in the eye, brain, heart, lung, kidney, and testis. An additional band migrates at 75 kDa, which is probably non-specific. Two bands are recognized by the affinity purified Epb4.1l5short antibody. A lower band migrates at the predicted molecular weight of 56 kDa and is present in brain, liver, lung, kidney, pancreas, and gut. A second band with a broad expression pattern migrates at approximately 75 kDa. Anti-α-Tubulin was used as a loading control.
Mentions: To help identify functionally important domains in the Moe and Epb4.1l5 proteins, we first compared their sequences (Figure 2). The mammalian epb4.1l5 locus encodes two major splice isoforms that are represented by ESTs in both the human and mouse databases, which we term Epb4.1l5short and Epb4.1l5long. We provide the exon/intron structure of the mouse epb4.1l5 locus that has 25 exons: Epb4.1l5short is encoded by exons 1–16 and Epb4.1l5long by exons 1–15 and 17–25 (Figure 2A). We have not found a zebrafish transcript that encodes a protein similar to Epb4.1l5short.

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