Dynamics of cell polarity in tissue morphogenesis: A comparative view from Drosophila and Ciona [version 1; referees: 2 approved]

Citation: Veeman, M. T., & McDonald, J. A. (2016). Dynamics of cell polarity in tissue morphogenesis: A comparative view from Drosophila and Ciona [version 1; referees: 2 approved]. F1000Research, 5. doi:10.12688/F1000RESEARCH.8011.1Tissues in developing embryos exhibit complex and dynamic rearrangements that shape forming organs, limbs, and body axes. Directed migration, mediolateral intercalation, lumen formation, and other rearrangements influence the topology and topography of developing tissues. These collective cell behaviors are distinct phenomena but all involve the fine-grained control of cell polarity. Here we review recent findings in the dynamics of polarized cell behavior in both the Drosophila ovarian border cells and the Ciona notochord. These studies reveal the remarkable reorganization of cell polarity during organ formation and underscore conserved mechanisms of developmental cell polarity including the Par/atypical protein kinase C (aPKC) and planar cell polarity pathways. These two very different model systems demonstrate important commonalities but also key differences in how cell polarity is controlled in tissue morphogenesis. Together, these systems raise important, broader questions on how the developmental control of cell polarity contributes to morphogenesis of diverse tissues across the metazoa. © 2016 Veeman MT and McDonald JA

Reciprocal and dynamic polarization of planar cell polarity core components and myosin

Citation: Newman-Smith, E., Kourakis, M. J., Reeves, W., Veeman, M., & Smith, W. C. (2015). Reciprocal and dynamic polarization of planar cell polarity core components and myosin. eLife, 2015(4). doi:10.7554/eLife.05361The Ciona notochord displays PCP-dependent polarity, with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalize with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Washout of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line aimless myosin polarization in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization. © 2015, eLife. All rights reserved

Ascidian notochord boundary morphogenesis [3a. Third place]

A six minute short film about notochord morphogenesis in ascidians showing 3D confocal renderings, timelapse sequences and other images of wildtype and mutant embryos.Componente Curricular::Educação Superior::Ciências Biológicas::Biologia Gera

Ascidian notochord boundary morphogenesis [3a. Third place]

A six minute short film about notochord morphogenesis in ascidians showing 3D confocal renderings, timelapse sequences and other images of wildtype and mutant embryos.Componente Curricular::Educação Superior::Ciências Biológicas::Biologia Gera

Ascidian notochord boundary morphogenesis [3a. Third place]

A six minute short film about notochord morphogenesis in ascidians showing 3D confocal renderings, timelapse sequences and other images of wildtype and mutant embryos.Componente Curricular::Educação Superior::Ciências Biológicas::Biologia Gera

Ascidian notochord boundary morphogenesis [3a. Third place]

A six minute short film about notochord morphogenesis in ascidians showing 3D confocal renderings, timelapse sequences and other images of wildtype and mutant embryos.Componente Curricular::Educação Superior::Ciências Biológicas::Biologia Gera

Quantitative Dissection of the Proximal Ciona brachyury Enhancer

A major goal in biology is to understand the rules by which cis-regulatory sequences control spatially and temporally precise expression patterns. Here we present a systematic dissection of the proximal enhancer for the notochord-specific transcription factor brachyury in the ascidian chordate Ciona. The study uses a quantitative image-based reporter assay that incorporates a dual-reporter strategy to control for variable electroporation efficiency. We identified and mutated multiple predicted transcription factor binding sites of interest based on statistical matches to the JASPAR binding motif database. Most sites (Zic, Ets, FoxA, RBPJ) were selected based on prior knowledge of cell fate specification in both the primary and secondary notochord. We also mutated predicted Brachyury sites to investigate potential autoregulation as well as Fos/Jun (AP1) sites that had very strong matches to JASPAR. Our goal was to quantitatively define the relative importance of these different sites, to explore the importance of predicted high-affinity versus low-affinity motifs, and to attempt to design mutant enhancers that were specifically expressed in only the primary or secondary notochord lineages. We found that the mutation of all predicted high-affinity sites for Zic, FoxA or Ets led to quantifiably distinct effects. The FoxA construct caused a severe loss of reporter expression whereas the Ets construct had little effect. A strong Ets phenotype was only seen when much lower-scoring binding sites were also mutated. This supports the enhancer suboptimization hypothesis proposed by Farley and Levine but suggests that it may only apply to some but not all transcription factor families. We quantified reporter expression separately in the two notochord lineages with the expectation that Ets mutations and RBPJ mutations would have distinct effects given that primary notochord is induced by Ets-mediated FGF signaling whereas secondary notochord is induced by RBPJ/Su(H)-mediated Notch/Delta signaling. We found, however, that ETS mutations affected primary and secondary notochord expression relatively equally and that RBPJ mutations were only moderately more severe in their effect on secondary versus primary notochord. Our results point to the promise of quantitative reporter assays for understanding cis-regulatory logic but also highlight the challenge of arbitrary statistical thresholds for predicting potentially important sites.</jats:p

Table8_Quantitative Dissection of the Proximal Ciona brachyury Enhancer.XLSX

A major goal in biology is to understand the rules by which cis-regulatory sequences control spatially and temporally precise expression patterns. Here we present a systematic dissection of the proximal enhancer for the notochord-specific transcription factor brachyury in the ascidian chordate Ciona. The study uses a quantitative image-based reporter assay that incorporates a dual-reporter strategy to control for variable electroporation efficiency. We identified and mutated multiple predicted transcription factor binding sites of interest based on statistical matches to the JASPAR binding motif database. Most sites (Zic, Ets, FoxA, RBPJ) were selected based on prior knowledge of cell fate specification in both the primary and secondary notochord. We also mutated predicted Brachyury sites to investigate potential autoregulation as well as Fos/Jun (AP1) sites that had very strong matches to JASPAR. Our goal was to quantitatively define the relative importance of these different sites, to explore the importance of predicted high-affinity versus low-affinity motifs, and to attempt to design mutant enhancers that were specifically expressed in only the primary or secondary notochord lineages. We found that the mutation of all predicted high-affinity sites for Zic, FoxA or Ets led to quantifiably distinct effects. The FoxA construct caused a severe loss of reporter expression whereas the Ets construct had little effect. A strong Ets phenotype was only seen when much lower-scoring binding sites were also mutated. This supports the enhancer suboptimization hypothesis proposed by Farley and Levine but suggests that it may only apply to some but not all transcription factor families. We quantified reporter expression separately in the two notochord lineages with the expectation that Ets mutations and RBPJ mutations would have distinct effects given that primary notochord is induced by Ets-mediated FGF signaling whereas secondary notochord is induced by RBPJ/Su(H)-mediated Notch/Delta signaling. We found, however, that ETS mutations affected primary and secondary notochord expression relatively equally and that RBPJ mutations were only moderately more severe in their effect on secondary versus primary notochord. Our results point to the promise of quantitative reporter assays for understanding cis-regulatory logic but also highlight the challenge of arbitrary statistical thresholds for predicting potentially important sites.</p