Shape Change and Epithelial Morphogenesis: “The Force Is Strong in You”
Epithelial cells form sheets and tubes. Their ability to form such structures often depends on cell shape changes that usually involve cadherins and the actin cytoskeleton. The extracellular domains of cadherins bind groups of cells together, while the intracellular domains of the cadherins alter the actin cytoskeleton. The proteins mediating this cadherin-dependent remodeling of the cytoskeleton are usually (1) non-muscle myosin, which provides the energy for actin contraction, and (2) the Rho family of GTPases, which convert soluble actin into fibrous actin cables that anchor at the cadherins. These Rho GTPases are generally divided into three groups that have different but overlapping functions. RhoA primarily organizes stress fibers, such as those that transiently anchor cells during cell migration. Rac1 is involved in producing lamellipodia (the broad, membranous sheet extending from migrating cells). Cdc42 is used primarily in constructing filopodia (the thin extensions of cytoplasm used to sense the cells’ environment).
Two examples of cadherin-dependent remodeling of the cytoskeleton are the formation of the neural tube in vertebrates and the internalization of the mesoderm in Drosophila. In both cases, the cells (neural ectoderm in vertebrates, mesoderm in Drosophila) are on the outside of the embryo, and it is critical that they migrate to the inside.
Involution of the frog neural tube
In the early frog embryo, each cell membrane can contain several types of cadherins. Each cell of the gastrula is covered with C-cadherin. However, the presumptive neural tube ectoderm cell membranes also contain N-cadherin concentrated in their apical (upper) regions; the presumptive epidermal cells of the ectoderm express E-cadherin on their lateral and basal (lower) surfaces. The actin organized in the apical region of the neural cells causes them to change shape and enter the internal region of the embryo as a neural tube. The actin organized on the lateral sides of the epidermal cells enables the migratory movements of the epidermal (skin) cells over the surface of the embryo. If N-cadherin is experimentally removed from a frog gastrula, the cells still adhere (thanks to the C-cadherin that is still present), but the actin (and the activated myosin that binds to it) fail to assemble apically and there is no neurulation: the presumptive neural cells do not enter the embryo, and no neural tube forms (Figure 1; Nandadasa et al. 2009).
Figure 1 Importance of cadherin in cell adhesion and morphogenetic movements. (A) Frog gastrula injected with a nonfunctioning N-cadherin gene on one side. The uninjected side (right) develops normally; on the injected side (left), the epidermis and neural tissue fail to separate. (B) Cross section of a Xenopus neurula stained for F-actin (green). The red-staining cells of the neural plate (uppermost cells) and notochord (the mesodermal rod beneath them) have in them a morpholino oligonucleotide that prevents N-cadherin mRNA from being translated. The neural plate cells fail to invaginate into the embryo or to form a neural tube because of the loss of the N-cadherin-based actin assembly in their apical cytoplasm. (A from Kintner et al. 1992, courtesy of C. Kintner; B from Nandadasa et al. 2009, courtesy of C. Wylie.)
Drosophila mesoderm formation
In Drosophila, the mesoderm is formed from epithelial cells on the ventral side of the embryo. These cells form a furrow and then migrate inside the embryo (Figure 2). To create this furrow, the cube-shaped cells become wedge-shaped, constricting at their apical surfaces. This transition creates a force that pushes the ventral cells inside the embryo. What creates this force? The apical constriction is brought about by the rearrangement of actin microfilaments and myosin II (a “non-skeletal myosin”) to the apical end of the cell (Figure 3). Actin microfilaments are part of the cytoskeleton and are often found on the periphery of the cell. (Indeed, they are critical for producing the cleavage furrows of cell division.) The instructions for this apical constriction appear to emanate from the Twist gene, which is only expressed in the nuclei of the ventralmost cells (Kölsch et al. 2007). The Twist protein activates other genes, whose protein products cause the actin cytoskeleton to build up on the apical side of the cell. This build-up is accomplished by the binding of a Rho GTPase and β-catenin to E-cadherin on the apical portion of the membrane in the most ventral cells. Once stabilized, the actin-myosin complex in the apical cortex constricts like the drawstring of a purse, causing the cells to change shape, buckle inward, and enter the embryo to form the mesoderm (Dawes-Hoang et al. 2005).
Figure 2 Ventral furrow formation during Drosophila gastrulation internalizes the cells that will become the mesoderm. (A) Ventral furrow in the Drosophila embryo seen by scanning electron microscopy. (B) Cross section through the center of such an embryo, demonstrating cell shape changes and the redistribution of protein products along the dorsal-ventral axis. Apical constrictions can be observed, mediated by G proteins in the furrow. (A courtesy of F. R. Turner; B courtesy of V. Kölsch and M. Leptin.)
Figure 3 Getting mesodermal cells inside the embryo during Drosophila gastrulation by regulation of the cytoskeleton. (A) Schematic representation of ventral furrow formation shown as cross sections through the Drosophila embryo, progressing in time from left to right. The ventral cells are defined by the expression of transcription factors Twist and Snail. These cells accumulate myosin II at their apical surfaces. When myosin II interacts with actin already present, the cells begin to constrict apically and thus invaginate. (B) Close-ups of the ventral domain. Before the initiation of ventral furrow formation, Rho GTPase (green) and β-catenin (orange) both reside along the basal surface (facing the interior of the embryo) of the ventral cells. β-catenin is also found in a subapical region in all cells. Formation of the ventral furrow begins with the relocalization of Rho and β-catenin, which move from the basal surface to accumulate apically, at the opposite end of the cell. (From Kölsch et al. 2007.)
External signals: Insect trachea
In the above cases, the instructions for folding come from inside the cell. Instructions for cell shape change can also arise outside the cell. For instance, the tracheal (respiratory) system in Drosophila embryos develops from epithelial sacs. The approximately 80 cells in each of these sacs become reorganized into primary, secondary, and tertiary branches without any cell division or cell death (Ghabrial and Krasnow 2006). This reorganization is initiated when nearby cells secrete a protein called Branchless, which acts as a chemoattractant.* Branchless binds to a receptor on the cell membranes of the epithelial cells. The cells receiving the most Branchless protein lead the rest, while the followers (connected to each other by cadherins) receive a signal from the leading cells to form the tracheal tube (Figure 4). It is the lead cell that will change its shape (by rearranging its actin-myosin cytoskeleton via a Rho GTPase-mediated process, just like the mesodermal cells) to migrate and to form the secondary branches. During this migration, cadherin proteins are regulated such that the epithelial cells can migrate over one another to form a tube while keeping their integrity as an epithelium (Cela and Llimagas 2006).
Figure 4 Tracheal development in Drosophila. (A) Diagram of dorsal tracheal branch budding from tracheal epithelium. Nearby cells secrete Branchless protein (Bnl; blue dots), which activates Breathless protein (Btl) on tracheal cells. The activated Btl induces migration of the leader cells and tube formation; the dorsal branch cells are numbered 1–6. Branchless also induces unicellular secondary branches (stage 15). (B) Larval Drosophila tracheal system visualized with a fluorescent red antibody. Note the intercalated branching pattern. (A after Ghabrial and Krasnow 2006; B from Casanova 2007.)
But another external force is also at work. The dorsalmost secondary branches of the sacs move along a groove that forms between the developing muscles. These tertiary cell migrations cause the trachea to become segmented around the musculature (Franch-Marro and Casanova 2000). In this way, the respiratory tubes are placed close to the larval musculature.
*Chemoattractants are usually diffusible molecules that attract a cell to migrate along an increasing concentration gradient toward the cells secreting the factor. There are also chemorepulsive factors that send the migrating cells in an opposite direction. Generally speaking, chemotactic factors—soluble factors that cause cells to move in a particular direction—are assumed to be chemoattractive unless otherwise described.
Literature Cited
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