RhoA GTPase inhibition organizes contraction during epithelial morphogenesis.

During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding.

Stable Force Balance between Epithelial Cells Arises from F-Actin Turnover.

The propagation of force in epithelial tissues requires that the contractile cytoskeletal machinery be stably connected between cells through E-cadherin-containing adherens junctions. In many epithelial tissues, the cells' contractile network is positioned at a distance from the junction. However, the mechanism or mechanisms that connect the contractile networks to the adherens junctions, and thus mechanically connect neighboring cells, are poorly understood. Here, we identified the role for F-actin turnover in regulating the contractile cytoskeletal network's attachment to adherens junctions. Perturbing F-actin turnover via gene depletion or acute drug treatments that slow F-actin turnover destabilized the attachment between the contractile actomyosin network and adherens junctions. Our work identifies a critical role for F-actin turnover in connecting actomyosin to intercellular junctions, defining a dynamic process required for the stability of force balance across intercellular contacts in tissues.

Apical domain polarization localizes actin-myosin activity to drive ratchet-like apical constriction.

Apical constriction promotes epithelia folding, which changes tissue architecture. During Drosophila gastrulation, mesoderm cells exhibit repeated contractile pulses that are stabilized such that cells apically constrict like a ratchet. The transcription factor Twist is required to stabilize cell shape. However, it is unknown how Twist spatially coordinates downstream signals to prevent cell relaxation. We find that during constriction, Rho-associated kinase (Rok) is polarized to the middle of the apical domain (medioapical cortex), separate from adherens junctions. Rok recruits or stabilizes medioapical myosin II (Myo-II), which contracts dynamic medioapical actin cables. The formin Diaphanous mediates apical actin assembly to suppress medioapical E-cadherin localization and form stable connections between the medioapical contractile network and adherens junctions. Twist is not required for apical Rok recruitment, but instead polarizes Rok medioapically. Therefore, Twist establishes radial cell polarity of Rok/Myo-II and E-cadherin and promotes medioapical actin assembly in mesoderm cells to stabilize cell shape fluctuations.

Genome wide analysis of transcript levels after perturbation of the EGFR pathway in the Drosophila ovary.

Defects in the epidermal growth factor receptor (EGFR) pathway can lead to aggressive tumor formation. Activation of this pathway during normal development produces multiple outcomes at the cellular level, leading to cellular differentiation and cell cycle activation. To elucidate the downstream events induced by this pathway, we used genome-wide cDNA microarray technology to identify potential EGFR targets in Drosophila oogenesis. We focused on genes for which the transcriptional responses due to EGFR pathway activation and inactivation were in opposite directions, as this is expected for genes that are directly regulated by the pathway in this tissue type. We perturbed the EGFR pathway in epithelial follicle cells using seven different genetic backgrounds. To activate the pathway, we overexpressed an activated form of the EGFR (UAS-caEGFR), and an activated form of the signal transducer Raf (UAS-caRaf); we also over- or ectopically expressed the downstream homeobox transcription factor Mirror (UAS-mirr) and the ligand-activating serine protease Rhomboid (UAS-rho). To reduce pathway activity we used loss-of-function mutations in the ligand (gurken) and receptor (torpedo). From microarrays containing 6,255 genes, we found 454 genes that responded in an opposite manner in gain-of-function and loss-of-function conditions among which are many Wingless signaling pathway components. Further analysis of two such components, sugarless and pangolin, revealed a function for these genes in late follicle cell patterning. Of interest, components of other signaling pathways were also enriched in the EGFR target group, suggesting that one reason for the pleiotropic effects seen with EGFR activity in cancer progression and development may be its ability to regulate many other signaling pathways.