Transcriptional Control of Developmental Cell Behaviors.

Tissue-specific transcription regulators emerged as key developmental control genes, which operate in the context of complex gene regulatory networks (GRNs) to coordinate progressive cell fate specification and tissue morphogenesis. We discuss how GRNs control the individual cell behaviors underlying complex morphogenetic events. Cell behaviors classically range from mesenchymal cell motility to cell shape changes in epithelial sheets. These behaviors emerge from the tissue-specific, multiscale integration of the local activities of universal and pleiotropic effectors, which underlie modular subcellular processes including cytoskeletal dynamics, cell-cell and cell-matrix adhesion, signaling, polarity, and vesicle trafficking. Extrinsic cues and intrinsic cell competence determine the subcellular spatiotemporal patterns of effector activities. GRNs influence most subcellular activities by controlling only a fraction of the effector-coding genes, which we argue is enriched in effectors involved in reading and processing the extrinsic cues to contextualize intrinsic subcellular processes and canalize developmental cell behaviors. The properties of the transcription-cell behavior interface have profound implications for evolution and disease.

WAVE regulates Cadherin junction assembly and turnover during epithelial polarization.

Actin is an integral component of epithelial apical junctions, yet the interactions of branched actin regulators with apical junction components are still not clear. Biochemical data have shown that α-catenin inhibits Arp2/3-dependent branched actin. These results suggested that branched actin is only needed at earliest stages of apical junction development. We use live imaging in developing C. elegans embryos to test models for how WAVE-induced branched actin collaborates with other apical junction proteins during the essential process of junction formation and maturation. We uncover both early and late essential roles for WAVE in apical junction formation. Early, as the C. elegans intestinal epithelium becomes polarized, we find that WAVE components become enriched concurrently with the Cadherin components and before the DLG-1 apical accumulation. Live imaging of F-actin accumulation in polarizing intestine supports that the Cadherin complex components and branched actin regulators work together for apical actin enrichment. Later in junction development, the apical accumulation of WAVE and Cadherin components is shown to be interdependent: Cadherin complex loss alters WAVE accumulation, and WAVE complex loss increases Cadherin accumulation. To determine why Cadherin levels rise when WVE-1 is depleted, we use FRAP to analyze Cadherin dynamics and find that loss of WAVE as well as of the trafficking protein EHD-1/RME-1 increases Cadherin dynamics. EM studies in adults depleted of branched actin regulators support that WVE-1 maintains established junctions, presumably through its trafficking effect on Cadherin. Thus we propose a developmental model for junction formation where branched actin regulators are tightly interconnected with Cadherin junctions through their previously unappreciated role in Cadherin transport.

Surrounding tissues canalize motile cardiopharyngeal progenitors towards collective polarity and directed migration.

Collectively migrating cells maintain group polarity and interpret external cues to reach their destination. The cardiogenic progenitors (also known as trunk ventral cells, TVCs) of the ascidian Ciona intestinalis provide a simple chordate model with which to study collective migration. Bilateral pairs of associated TVCs undergo a stereotyped polarized migration away from the tail towards the ventral trunk, arguably constituting the simplest possible example of directed collective migration. To identify tissues contributing to TVC polarity and migration, we quantified the contact between TVCs and surrounding tissues, and blocked the secretory pathway in a tissue-specific manner. Even though TVCs normally migrate as an invariably determined leader-trailer polarized pair of adherent cells, they are capable of migrating individually, albeit a shorter distance and with altered morphology. The mesenchyme contacts newborn TVCs and contributes to robust specification of the trailer but appears to have only minor effects on directed migration. The notochord does not contact the TVCs but contributes to the onset of migration. The trunk endoderm first contacts the leader TVC, then 'encases' both migrating cells and provides the inputs maintaining leader-trailer polarity. Migrating TVCs adhere to the epidermis and need this contact for their cohesion. These phenomenological studies reveal that inherently motile cardiopharyngeal progenitors are channeled into stereotyped behaviors by interactions with surrounding tissues.

UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph polarize F-actin during embryonic morphogenesis by regulating the WAVE/SCAR actin nucleation complex.

Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the Caenorhabditis elegans embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in C. elegans. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.

Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells.

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue-scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis.

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.

Discoidin-domain receptor coordinates cell-matrix adhesion and collective polarity in migratory cardiopharyngeal progenitors.

Integrated analyses of regulated effector genes, cellular processes, and extrinsic signals are required to understand how transcriptional networks coordinate fate specification and cell behavior during embryogenesis. Ciona cardiopharyngeal progenitors, the trunk ventral cells (TVCs), polarize as leader and trailer cells that migrate between the ventral epidermis and trunk endoderm. We show that the TVC-specific collagen-binding Discoidin-domain receptor (Ddr) cooperates with Integrin-ß1 to promote cell-matrix adhesion. We find that endodermal cells secrete a collagen, Col9-a1, that is deposited in the basal epidermal matrix and promotes Ddr activation at the ventral membrane of migrating TVCs. A functional antagonism between Ddr/Intß1-mediated cell-matrix adhesion and Vegfr signaling appears to modulate the position of cardiopharyngeal progenitors between the endoderm and epidermis. We show that Ddr promotes leader-trailer-polarized BMP-Smad signaling independently of its role in cell-matrix adhesion. We propose that dual functions of Ddr integrate transcriptional inputs to coordinate subcellular processes underlying collective polarity and migration.

Arp2/3 promotes junction formation and maintenance in the Caenorhabditis elegans intestine by regulating membrane association of apical proteins.

It has been proposed that Arp2/3, which promotes nucleation of branched actin, is needed for epithelial junction initiation but is less important as junctions mature. We focus here on how Arp2/3 contributes to the Caenorhabditis elegans intestinal epithelium and find important roles for Arp2/3 in the maturation and maintenance of junctions in embryos and adults. Electron microscope studies show that embryos depleted of Arp2/3 form apical actin-rich microvilli and electron-dense apical junctions. However, whereas apical/basal polarity initiates, apical maturation is defective, including decreased apical F-actin enrichment, aberrant lumen morphology, and reduced accumulation of some apical junctional proteins, including DLG-1. Depletion of Arp2/3 in adult animals leads to similar intestinal defects. The DLG-1/AJM-1 apical junction proteins, and the ezrin-radixin-moesin homologue ERM-1, a protein that connects F-actin to membranes, are required along with Arp2/3 for apical F-actin enrichment in embryos, whereas cadherin junction proteins are not. Arp2/3 affects the subcellular distribution of DLG-1 and ERM-1. Loss of Arp2/3 shifts both ERM-1 and DLG-1 from pellet fractions to supernatant fractions, suggesting a role for Arp2/3 in the distribution of membrane-associated proteins. Thus, Arp2/3 is required as junctions mature to maintain apical proteins associated with the correct membranes.