FORMING TUBES BY DE NOVO EPITHELIALIZATION

Figure 3: Three stages of pharynx tube formation: rotation of pharyngeal cells (A to B), epithelialization of arcade cells (C) and contraction (D). Right: yellow dotted line, pharynx primordium; red, cell boundaries; green, adherens junctions.

 

Figure 4: zen-4 mutant (left) fails to produce the arcade cell epithelium (orange arrowheads) compared to a wildtype embryo (right). Red, nuclei; green, adherens junctions.

Many organs, including the C. elegans pharynx, are systems of epithelial tubes that provide an essential function by transporting gases or liquids. All epithelia are composed of polarized cells, with apical and basolateral domains at the cell surface, separated by intercellular junctions. However, generation of epithelial tubes differs from that of epithelial sheets because tubes are three-dimensional structures, with their apical domains facing the lumen of the tube and intercellular junctions aligned longitudinally. Epithelial tubes can arise by remodeling of pre-existing epithelia, (e.g. branching morphogenesis of the lung). Epithelial tubes are also constructed from groups of cells induced to form epithelia de novo, (e.g. nephron formation in the kidney). While significant progress has been made towards understanding the mechanisms that govern branching morphogenesis, very little is known about tubulogenesis by de novo epithelialization. The C. elegans pharynx offers a powerful genetic system to investigate tube formation by this latter mechanism.

Three stages of pharynx tubulogenesis. We began our studies with an in-depth analysis of the cellular behaviors that accompany pharyngeal tube formation. Initially, the pharynx primordium consists of an epithelial ball located deep within the embryo. In the first stage, cells located at the anterior of the pharyngeal primordium reorganize their polarity to convert the ball into a short tube. This behavior requires remodelling of adherens junctions to reorient the cells' apical and basolateral domains relative to the axis of the embryo body. In the second stage, nine mesenchymal cells (the arcade cells) located anterior to the pharynx primordium are converted into an epithelium that links the nascent pharynx tube to the external epidermis. In the final stage, the apical surfaces of the pharynx and arcade cells constrict along the AP axis while expanding circumferentially to form the lumen.

We have combined experimental embryology with forward and reverse genetics to examine the molecular mechanisms that govern remodelling of intercellular junctions (Stage 1) and epithelium formation (Stage 2). The first gene identified by forward genetics was zen-4, which encodes a mitotic kinesin-like protein (MKLP1, CHO1, pavarotti). Previously, zen-4 and its partner the RhoGAP cyk-4 (MgcRacGAP), had been implicated in cytokinesis. Our studies revealed a new, post-mitotic role for zen-4 and cyk-4 to construct the apical domain and adherens junctions during epithelium assemblyI (e.g. Figure 4). Our data suggest that these proteins organize the actin or microtubule cytoskeletons in response to an external cue. Conversely, the RhoGEF Pebble, which also has a role during cytokinesis and interacts with ZEN-4 and CYK-4 homologs in Drosophila, has recently been implicated in migration of Drosophila mesodermal cells. The phenotypes of Pebble, zen-4 and cyk-4 suggest that these cytokinesis factors have been co-opted for a second role, to control the mesenchymal-epithelial transition of cells. ZEN-4/CYK-4 promote epithelium formation whereas Pebble is required for conversion of epithelia to mesenchyme. Outstanding questions for pharynx tubulogenesis are the nature of the signal that initiates polarization and the events downstream of ZEN-4 and CYK-4 that promote formation of adherens junctions and apical domains.

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