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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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Loss of Pyd or reduction of Cno disrupts Ena localization to LE dots and mimics Ena loss of function. (A–C) ena23 maternal/zygotic mutants. Note the wavy leading edge (A, arrow), very deep segmental grooves (B, arrowheads), and uneven cell shapes of LE cells, with hyperconstricted (C, white arrows) and splayed open (C, blue arrows) leading edges. (D–P) pydMZ and control wild-type embryos stained and imaged in parallel to visualize DE-cad and Ena. (H, I) Close-ups of D, E. (J, K) Close-ups of F, G. (N, O) Close-ups of L, M. (D, E, H, I) Early stage 14. Blue arrows indicate LE dots. Red arrows in D’ and E’ indicate amnioserosal cell junctions. Red arrows in H and I indicate Ena enrichment in segmental groove cells. White arrows indicate Ena in tricellular junctions. (F, G, J–Q) Late stage 14. Arrows indicate LE dots. In pydMZ mutants, the amount of Ena in LE dots is reduced (F, G, J, K, P, Q) or almost eliminated (L–O). (R–U) cnoR2 mutants and heterozygous wild-type siblings visualized identically. In cnoR2 mutants, Ena in LE dots is strongly reduced (S and U, arrows). Scale bars, 10 μm.
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Figure 10: Loss of Pyd or reduction of Cno disrupts Ena localization to LE dots and mimics Ena loss of function. (A–C) ena23 maternal/zygotic mutants. Note the wavy leading edge (A, arrow), very deep segmental grooves (B, arrowheads), and uneven cell shapes of LE cells, with hyperconstricted (C, white arrows) and splayed open (C, blue arrows) leading edges. (D–P) pydMZ and control wild-type embryos stained and imaged in parallel to visualize DE-cad and Ena. (H, I) Close-ups of D, E. (J, K) Close-ups of F, G. (N, O) Close-ups of L, M. (D, E, H, I) Early stage 14. Blue arrows indicate LE dots. Red arrows in D’ and E’ indicate amnioserosal cell junctions. Red arrows in H and I indicate Ena enrichment in segmental groove cells. White arrows indicate Ena in tricellular junctions. (F, G, J–Q) Late stage 14. Arrows indicate LE dots. In pydMZ mutants, the amount of Ena in LE dots is reduced (F, G, J, K, P, Q) or almost eliminated (L–O). (R–U) cnoR2 mutants and heterozygous wild-type siblings visualized identically. In cnoR2 mutants, Ena in LE dots is strongly reduced (S and U, arrows). Scale bars, 10 μm.

Mentions: Given that both loss of Pyd and reduction of Cno lead to similar defects in cell shape change and morphogenesis, we further explored the mechanism by which they act. The suite of defects shared by pydMZ and cno mutants was reminiscent of that we previously observed in embryos in which the actin regulator Ena was inactivated (Figure 10, A–C) (Gates et al., 2007). ena23 maternal/zygotic mutant embryos share with pydMZ and cno mutants a wavy leading edge (Figure 10, A and C, red arrows), very similar defects in LE cell shape (Figure 10C, arrows), a reduced rate of zippering of the two sheets together (Gates et al., 2007), and, strikingly, deep and persistent segmental grooves (Figure 10B, arrowheads). Furthermore, segmental groove cells normally have elevated and planar polarized Ena (Gates et al., 2007). We thus considered the hypothesis that Pyd and Cno might act via Ena.


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)

Loss of Pyd or reduction of Cno disrupts Ena localization to LE dots and mimics Ena loss of function. (A–C) ena23 maternal/zygotic mutants. Note the wavy leading edge (A, arrow), very deep segmental grooves (B, arrowheads), and uneven cell shapes of LE cells, with hyperconstricted (C, white arrows) and splayed open (C, blue arrows) leading edges. (D–P) pydMZ and control wild-type embryos stained and imaged in parallel to visualize DE-cad and Ena. (H, I) Close-ups of D, E. (J, K) Close-ups of F, G. (N, O) Close-ups of L, M. (D, E, H, I) Early stage 14. Blue arrows indicate LE dots. Red arrows in D’ and E’ indicate amnioserosal cell junctions. Red arrows in H and I indicate Ena enrichment in segmental groove cells. White arrows indicate Ena in tricellular junctions. (F, G, J–Q) Late stage 14. Arrows indicate LE dots. In pydMZ mutants, the amount of Ena in LE dots is reduced (F, G, J, K, P, Q) or almost eliminated (L–O). (R–U) cnoR2 mutants and heterozygous wild-type siblings visualized identically. In cnoR2 mutants, Ena in LE dots is strongly reduced (S and U, arrows). Scale bars, 10 μm.
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Figure 10: Loss of Pyd or reduction of Cno disrupts Ena localization to LE dots and mimics Ena loss of function. (A–C) ena23 maternal/zygotic mutants. Note the wavy leading edge (A, arrow), very deep segmental grooves (B, arrowheads), and uneven cell shapes of LE cells, with hyperconstricted (C, white arrows) and splayed open (C, blue arrows) leading edges. (D–P) pydMZ and control wild-type embryos stained and imaged in parallel to visualize DE-cad and Ena. (H, I) Close-ups of D, E. (J, K) Close-ups of F, G. (N, O) Close-ups of L, M. (D, E, H, I) Early stage 14. Blue arrows indicate LE dots. Red arrows in D’ and E’ indicate amnioserosal cell junctions. Red arrows in H and I indicate Ena enrichment in segmental groove cells. White arrows indicate Ena in tricellular junctions. (F, G, J–Q) Late stage 14. Arrows indicate LE dots. In pydMZ mutants, the amount of Ena in LE dots is reduced (F, G, J, K, P, Q) or almost eliminated (L–O). (R–U) cnoR2 mutants and heterozygous wild-type siblings visualized identically. In cnoR2 mutants, Ena in LE dots is strongly reduced (S and U, arrows). Scale bars, 10 μm.
Mentions: Given that both loss of Pyd and reduction of Cno lead to similar defects in cell shape change and morphogenesis, we further explored the mechanism by which they act. The suite of defects shared by pydMZ and cno mutants was reminiscent of that we previously observed in embryos in which the actin regulator Ena was inactivated (Figure 10, A–C) (Gates et al., 2007). ena23 maternal/zygotic mutant embryos share with pydMZ and cno mutants a wavy leading edge (Figure 10, A and C, red arrows), very similar defects in LE cell shape (Figure 10C, arrows), a reduced rate of zippering of the two sheets together (Gates et al., 2007), and, strikingly, deep and persistent segmental grooves (Figure 10B, arrowheads). Furthermore, segmental groove cells normally have elevated and planar polarized Ena (Gates et al., 2007). We thus considered the hypothesis that Pyd and Cno might act via Ena.

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