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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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Pyd is required for tracheal dorsal branch fusion and cell fate specification but not for formation or maintenance of adherens junctions. (A, B) Many dorsal branches (DBs) in zygotic pydB12 mutants fail to fuse (join to contralateral branch, asterisks in B), and often incorrectly fuse to neighboring branches (arrow in B). Anti-Dysfusion (Dys; green) labels fusion cells. (C, D) Labeling for the terminal cell marker DSRF reveals that some DBs in pydB12 embryos have more than one terminal cell (arrow in D), whereas control w1118 embryos have only one terminal cell per DB (C). (E) Quantification of DB defects in zygotic and maternal/zygotic (M/Z) pydB12 mutant embryos. DB fusion phenotypes are categorized into four classes as in Jung et al. (2006): wild type (complete dorsal branch fusion), weak (only one affected metamere), intermediate (two to four affected metameres), and severe (five or more affected metameres). (F–I) Dorsal trunks (DTs, arrow in F) of zygotic and M/Z pydB12 mutants are contiguous, indicating normal adherens junction function and fusion of dorsal trunk segments. (H–I) DTs of pydMZ embryos appear tortuous but are the same length as w1118 control embryos (p = 0.20, Student's t test). DT length is normalized to embryo length. (J, K) pydMZ mutants have normal localization of the septate junction marker Coracle (Cor) and normal lumenal accumulation of the matrix marker Verm. Scale bars, A–D, 5 μm; F–H, 20 μm; J, K, 10 μm.
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Figure 7: Pyd is required for tracheal dorsal branch fusion and cell fate specification but not for formation or maintenance of adherens junctions. (A, B) Many dorsal branches (DBs) in zygotic pydB12 mutants fail to fuse (join to contralateral branch, asterisks in B), and often incorrectly fuse to neighboring branches (arrow in B). Anti-Dysfusion (Dys; green) labels fusion cells. (C, D) Labeling for the terminal cell marker DSRF reveals that some DBs in pydB12 embryos have more than one terminal cell (arrow in D), whereas control w1118 embryos have only one terminal cell per DB (C). (E) Quantification of DB defects in zygotic and maternal/zygotic (M/Z) pydB12 mutant embryos. DB fusion phenotypes are categorized into four classes as in Jung et al. (2006): wild type (complete dorsal branch fusion), weak (only one affected metamere), intermediate (two to four affected metameres), and severe (five or more affected metameres). (F–I) Dorsal trunks (DTs, arrow in F) of zygotic and M/Z pydB12 mutants are contiguous, indicating normal adherens junction function and fusion of dorsal trunk segments. (H–I) DTs of pydMZ embryos appear tortuous but are the same length as w1118 control embryos (p = 0.20, Student's t test). DT length is normalized to embryo length. (J, K) pydMZ mutants have normal localization of the septate junction marker Coracle (Cor) and normal lumenal accumulation of the matrix marker Verm. Scale bars, A–D, 5 μm; F–H, 20 μm; J, K, 10 μm.

Mentions: Previous work showed that zygotic pydC5 and pydBG02748 mutations perturb morphogenesis of the embryonic tracheal system (Jung et al., 2006). The tracheal system is a network of epithelial tubes that function as a combined pulmonary and vascular system to deliver oxygen to target tissues (reviewed in Ghabrial et al., 2003; Affolter and Caussinus, 2008; Schottenfeld et al., 2010). During embryogenesis, the tracheal system undergoes extensive cell junction rearrangement and shape changes as epithelial cells transform from planar placodes to a ramifying tubular network. Earlier characterization of pyd revealed that existing mutations had little effect on the larger tracheal branches but had partially penetrant defects in the smaller dorsal branches (DBs) (Jung et al., 2006). To determine whether the comparatively mild phenotypes resulted from the non nature of the tested pyd alleles, we examined trachea in pydB12 mutant embryos. Zygotic pydB12 embryos exhibited defects similar to those of zygotic pydC5 and pydBG02748 embryos, with DB fusion failures (Figure 7B, asterisks) and ectopic fusions between neighboring DBs (Figure 7B, arrow). Labeling for the terminal cell marker DSRF (Drosophila serum response factor) revealed that some DBs in pydB12 embryos had more than one terminal cell (Figure 7D, arrow), whereas control w1118 embryos have only one terminal cell per DB, suggesting that Pyd helps regulate cell fate determination in the DB. Consistent with pydB12 being a stronger allele than pydC5 and pydBG02748, 60% of zygotic pydB12 mutants have severe fusion defects (five or more DBs that fail to fuse; Figure 7E), whereas previous studies reported that <40% of zygotic pydC5 and pydBG02748 embryos have severe fusion defects (Jung et al., 2006). Elimination of maternal Pyd further increased the frequency and severity of dorsal branch defects, with 100% of pydMZ mutant embryos having severe DB defects (Figure 7E). Thus Pyd has an even more important role in DB morphogenesis than was previously revealed in embryos zygotically homozygous for non alleles (Jung et al., 2006).


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)

Pyd is required for tracheal dorsal branch fusion and cell fate specification but not for formation or maintenance of adherens junctions. (A, B) Many dorsal branches (DBs) in zygotic pydB12 mutants fail to fuse (join to contralateral branch, asterisks in B), and often incorrectly fuse to neighboring branches (arrow in B). Anti-Dysfusion (Dys; green) labels fusion cells. (C, D) Labeling for the terminal cell marker DSRF reveals that some DBs in pydB12 embryos have more than one terminal cell (arrow in D), whereas control w1118 embryos have only one terminal cell per DB (C). (E) Quantification of DB defects in zygotic and maternal/zygotic (M/Z) pydB12 mutant embryos. DB fusion phenotypes are categorized into four classes as in Jung et al. (2006): wild type (complete dorsal branch fusion), weak (only one affected metamere), intermediate (two to four affected metameres), and severe (five or more affected metameres). (F–I) Dorsal trunks (DTs, arrow in F) of zygotic and M/Z pydB12 mutants are contiguous, indicating normal adherens junction function and fusion of dorsal trunk segments. (H–I) DTs of pydMZ embryos appear tortuous but are the same length as w1118 control embryos (p = 0.20, Student's t test). DT length is normalized to embryo length. (J, K) pydMZ mutants have normal localization of the septate junction marker Coracle (Cor) and normal lumenal accumulation of the matrix marker Verm. Scale bars, A–D, 5 μm; F–H, 20 μm; J, K, 10 μm.
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Figure 7: Pyd is required for tracheal dorsal branch fusion and cell fate specification but not for formation or maintenance of adherens junctions. (A, B) Many dorsal branches (DBs) in zygotic pydB12 mutants fail to fuse (join to contralateral branch, asterisks in B), and often incorrectly fuse to neighboring branches (arrow in B). Anti-Dysfusion (Dys; green) labels fusion cells. (C, D) Labeling for the terminal cell marker DSRF reveals that some DBs in pydB12 embryos have more than one terminal cell (arrow in D), whereas control w1118 embryos have only one terminal cell per DB (C). (E) Quantification of DB defects in zygotic and maternal/zygotic (M/Z) pydB12 mutant embryos. DB fusion phenotypes are categorized into four classes as in Jung et al. (2006): wild type (complete dorsal branch fusion), weak (only one affected metamere), intermediate (two to four affected metameres), and severe (five or more affected metameres). (F–I) Dorsal trunks (DTs, arrow in F) of zygotic and M/Z pydB12 mutants are contiguous, indicating normal adherens junction function and fusion of dorsal trunk segments. (H–I) DTs of pydMZ embryos appear tortuous but are the same length as w1118 control embryos (p = 0.20, Student's t test). DT length is normalized to embryo length. (J, K) pydMZ mutants have normal localization of the septate junction marker Coracle (Cor) and normal lumenal accumulation of the matrix marker Verm. Scale bars, A–D, 5 μm; F–H, 20 μm; J, K, 10 μm.
Mentions: Previous work showed that zygotic pydC5 and pydBG02748 mutations perturb morphogenesis of the embryonic tracheal system (Jung et al., 2006). The tracheal system is a network of epithelial tubes that function as a combined pulmonary and vascular system to deliver oxygen to target tissues (reviewed in Ghabrial et al., 2003; Affolter and Caussinus, 2008; Schottenfeld et al., 2010). During embryogenesis, the tracheal system undergoes extensive cell junction rearrangement and shape changes as epithelial cells transform from planar placodes to a ramifying tubular network. Earlier characterization of pyd revealed that existing mutations had little effect on the larger tracheal branches but had partially penetrant defects in the smaller dorsal branches (DBs) (Jung et al., 2006). To determine whether the comparatively mild phenotypes resulted from the non nature of the tested pyd alleles, we examined trachea in pydB12 mutant embryos. Zygotic pydB12 embryos exhibited defects similar to those of zygotic pydC5 and pydBG02748 embryos, with DB fusion failures (Figure 7B, asterisks) and ectopic fusions between neighboring DBs (Figure 7B, arrow). Labeling for the terminal cell marker DSRF (Drosophila serum response factor) revealed that some DBs in pydB12 embryos had more than one terminal cell (Figure 7D, arrow), whereas control w1118 embryos have only one terminal cell per DB, suggesting that Pyd helps regulate cell fate determination in the DB. Consistent with pydB12 being a stronger allele than pydC5 and pydBG02748, 60% of zygotic pydB12 mutants have severe fusion defects (five or more DBs that fail to fuse; Figure 7E), whereas previous studies reported that <40% of zygotic pydC5 and pydBG02748 embryos have severe fusion defects (Jung et al., 2006). Elimination of maternal Pyd further increased the frequency and severity of dorsal branch defects, with 100% of pydMZ mutant embryos having severe DB defects (Figure 7E). Thus Pyd has an even more important role in DB morphogenesis than was previously revealed in embryos zygotically homozygous for non alleles (Jung et al., 2006).

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