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The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled.

Choi W, Jung KC, Nelson KS, Bhat MA, Beitel GJ, Peifer M, Fanning AS - Mol. Biol. Cell (2011)

Bottom Line: Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure.The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together.Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, USA.

ABSTRACT
Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of alleles. We generated alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

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cno and ena exhibit strong, dose-sensitive genetic interactions. (A–C) Cuticles of embryos, exhibiting representative phenotypes scored in the different genotypes. (A) Example of mild to severe defects in the head skeleton (arrow), indicative of defects in head involution. (B) Example of the more severe phenotypic class, exhibiting either a head hole due to complete failure of head involution (arrow; illustrated here), a large dorsal hole, indicating failure of dorsal closure (not shown), or both. (C) Example of novel phenotypic class only seen in genetic interaction experiments, in which only the ventral cuticle (identified by the presence of denticle belts; arrowhead) remained. (D) Percentages of dead embryos in each phenotypic class for different genetic interaction crosses. Column at far right indicates embryonic lethality—expected percentages assume that only embryos homozygous for cno, ena, or both die. Scale bar, 75 μm.
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Figure 11: cno and ena exhibit strong, dose-sensitive genetic interactions. (A–C) Cuticles of embryos, exhibiting representative phenotypes scored in the different genotypes. (A) Example of mild to severe defects in the head skeleton (arrow), indicative of defects in head involution. (B) Example of the more severe phenotypic class, exhibiting either a head hole due to complete failure of head involution (arrow; illustrated here), a large dorsal hole, indicating failure of dorsal closure (not shown), or both. (C) Example of novel phenotypic class only seen in genetic interaction experiments, in which only the ventral cuticle (identified by the presence of denticle belts; arrowhead) remained. (D) Percentages of dead embryos in each phenotypic class for different genetic interaction crosses. Column at far right indicates embryonic lethality—expected percentages assume that only embryos homozygous for cno, ena, or both die. Scale bar, 75 μm.

Mentions: These data suggested that Pyd and Cno might act through Ena. To further test this hypothesis, we looked for genetic interactions between ena and cno, using embryonic cuticles to assess effects on morphogenesis. ena23 zygotic mutants have little effect on the epidermis—90% of mutants have virtually wild-type cuticles, whereas 8% have head holes (Gertler et al., 1990; Gates et al., 2007; Figure 11) (ena embryonic lethality is thought to be due to defects in axon guidance). In contrast, 84% of cnoR2 zygotic mutants have defects in head involution ranging from mild to severe defects in the head skeleton hole, whereas 16% have a head or dorsal cuticle hole, indicating failure of head involution and/or dorsal closure (Figure 11) (Sawyer et al., 2009). Simply reducing maternal and zygotic Ena levels enhanced the cno zygotic phenotype (cnoR2/+;ena23/+ × cnoR2/+; mothers and some embryos thus heterozygous for ena)—now 38% of the progeny had head and/or dorsal holes, and 4% had a novel, stronger phenotype in which only ventral cuticle remained (Figure 11). Reducing maternal and zygotic Cno levels (cnoR2/+;ena23/+ × ena23/+; mothers and some embryos thus heterozygous for cno) also strongly enhanced the ena zygotic phenotype—now 50% of the progeny had head holes, consistent with all ena mutant embryos that were maternally and zygotically cno/+ having this phenotype (Figure 11). When we crossed females and males heterozygous for both cno and ena, thus generating both double mutants and single mutants with a reduced maternal dose of the other gene, we saw even more striking enhancement of the cno phenotype (lethal embryos are a 2:2:1 mix of ena mutants, cno mutants, and ena;cno double mutants), with 10% of the total lethal mutants and thus likely about half of the double mutants now having only ventral cuticle remaining (Figure 11). These results are consistent with essentially all double mutants having completely failed in dorsal closure. Thus ena and cno exhibit strong, dose-sensitive genetic interactions.


The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled.

Choi W, Jung KC, Nelson KS, Bhat MA, Beitel GJ, Peifer M, Fanning AS - Mol. Biol. Cell (2011)

cno and ena exhibit strong, dose-sensitive genetic interactions. (A–C) Cuticles of embryos, exhibiting representative phenotypes scored in the different genotypes. (A) Example of mild to severe defects in the head skeleton (arrow), indicative of defects in head involution. (B) Example of the more severe phenotypic class, exhibiting either a head hole due to complete failure of head involution (arrow; illustrated here), a large dorsal hole, indicating failure of dorsal closure (not shown), or both. (C) Example of novel phenotypic class only seen in genetic interaction experiments, in which only the ventral cuticle (identified by the presence of denticle belts; arrowhead) remained. (D) Percentages of dead embryos in each phenotypic class for different genetic interaction crosses. Column at far right indicates embryonic lethality—expected percentages assume that only embryos homozygous for cno, ena, or both die. Scale bar, 75 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 11: cno and ena exhibit strong, dose-sensitive genetic interactions. (A–C) Cuticles of embryos, exhibiting representative phenotypes scored in the different genotypes. (A) Example of mild to severe defects in the head skeleton (arrow), indicative of defects in head involution. (B) Example of the more severe phenotypic class, exhibiting either a head hole due to complete failure of head involution (arrow; illustrated here), a large dorsal hole, indicating failure of dorsal closure (not shown), or both. (C) Example of novel phenotypic class only seen in genetic interaction experiments, in which only the ventral cuticle (identified by the presence of denticle belts; arrowhead) remained. (D) Percentages of dead embryos in each phenotypic class for different genetic interaction crosses. Column at far right indicates embryonic lethality—expected percentages assume that only embryos homozygous for cno, ena, or both die. Scale bar, 75 μm.
Mentions: These data suggested that Pyd and Cno might act through Ena. To further test this hypothesis, we looked for genetic interactions between ena and cno, using embryonic cuticles to assess effects on morphogenesis. ena23 zygotic mutants have little effect on the epidermis—90% of mutants have virtually wild-type cuticles, whereas 8% have head holes (Gertler et al., 1990; Gates et al., 2007; Figure 11) (ena embryonic lethality is thought to be due to defects in axon guidance). In contrast, 84% of cnoR2 zygotic mutants have defects in head involution ranging from mild to severe defects in the head skeleton hole, whereas 16% have a head or dorsal cuticle hole, indicating failure of head involution and/or dorsal closure (Figure 11) (Sawyer et al., 2009). Simply reducing maternal and zygotic Ena levels enhanced the cno zygotic phenotype (cnoR2/+;ena23/+ × cnoR2/+; mothers and some embryos thus heterozygous for ena)—now 38% of the progeny had head and/or dorsal holes, and 4% had a novel, stronger phenotype in which only ventral cuticle remained (Figure 11). Reducing maternal and zygotic Cno levels (cnoR2/+;ena23/+ × ena23/+; mothers and some embryos thus heterozygous for cno) also strongly enhanced the ena zygotic phenotype—now 50% of the progeny had head holes, consistent with all ena mutant embryos that were maternally and zygotically cno/+ having this phenotype (Figure 11). When we crossed females and males heterozygous for both cno and ena, thus generating both double mutants and single mutants with a reduced maternal dose of the other gene, we saw even more striking enhancement of the cno phenotype (lethal embryos are a 2:2:1 mix of ena mutants, cno mutants, and ena;cno double mutants), with 10% of the total lethal mutants and thus likely about half of the double mutants now having only ventral cuticle remaining (Figure 11). These results are consistent with essentially all double mutants having completely failed in dorsal closure. Thus ena and cno exhibit strong, dose-sensitive genetic interactions.

Bottom Line: Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure.The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together.Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, USA.

ABSTRACT
Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of alleles. We generated alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

Show MeSH
Related in: MedlinePlus