Mike Boxem (website)
Polarization of epithelial cells into apical and basolateral domains is essential for the functioning of epithelia as selectively permeable barriers. The basolateral Scribble group proteins (Scrib, Lgl, Dlg) were originally identified as tumor suppressors in D. melanogaster and were later shown to play key roles in the establishment and maintenance of polarity in a broad range of cell types. In addition to promoting basolateral identity, Scribble group proteins regulate diverse processes including tissue growth, differentiation and directed cell migration, and therefore are major regulators of tissue development and homeostasis. In C. elegans, LET-413/Scrib and DLG-1/Dlg are essential for embryonic development, and their loss results in defects in epithelial polarization and junction formation. However, their importance in larval epithelia is less well understood.
Here, we study the role of LET-413 in the maintenance of tissue integrity in the C. elegans seam cell epithelium, a polarized epithelium that undergoes a series of asymmetric, stem cell-like divisions during larval development.
Summary of Results
To be able to investigate the role of LET-413 specifically in the seam cells, we made use of the auxin-inducible protein degradation (AID) system . We used CRISPR/Cas9 genome engineering to endogenously tag LET-413 with the degron sequence, combined with the green fluorescent protein (GFP) to visualize the localization of LET-413 and monitor the efficiency of degradation. We examined the effects of inactivating LET-413 ubiquitously and in specific epithelial tissues. We degraded LET-413 from the start of larval development by adding auxin immediately following hatching. Levels of GFP::AID::LET-413 became undetectable within 2 hours of the addition of auxin. Ubiquitous depletion of LET-413, and depletion of LET-413 in the seam and hyp7 epidermal cells resulted in a dramatic developmental arrest and lethality, while depletion in the intestine or excretory canal had little effect on development (Figure 1).
We next investigated the effects of LET-413 depletion on the development and division pattern of seam cells. We observed no defects up to the first round of division (5-7 h into development). Following the first division, the anterior seam cell daughters fuse with the surrounding hypodermis, leaving gaps between the seam cells. In control animals not exposed to auxin, the remaining seam cells extend laterally and form attachments to their neighbors. In LET-413 depleted cells however, seam cells failed to extend laterally, and remained isolated (Figure 2A). We also examined the effect of depleting LET-413 prior to L2 divisions, after the first round of divisions had completed and the seam cells reattached. We again observed a failure of cells to reattach to their neighbors, as well as additional defects. These include a deviation from the normal anterior-posterior axis of division, as well as the appearance of what appear to be cells or cell fragments that lack a nucleus (Figure 2B).
Finally, we examined the effect of LET-413 degradation on seam cell junctions, using an endogenous HMR-1::GFP fusion (generated as part of PolarNet) to label the cadherin-catenin complex, and an endogenous DLG-1::GFP fusion to label the DLG-1/AJM-1 complex. Both junctional complexes were strongly affected by depletion of LET-413 (Figure 3). Together, these results define a novel requirement for LET-413/Scribble in a stem cell-like epithelium in C. elegans.
- Zhang, L., Ward, J.D., Cheng, Z., and Dernburg, A.F. (2015). The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Dev. Camb. Engl. 142, 4374–4384.
Riga, A., Castiglioni, V.G., and Boxem, M. (2019). New insights into apical-basal polarization in epithelia. Current opinion in cell biology. In Press