In the current study we used the dorsal fold system to investigate whether specific cellular mechanisms actively regulate the extent of epithelial invagination. We showed that α-Catenin is required for the restricted invagination caused by constitutive activation of Rap1 and identified Rapgap1 as a locally expressed modulator of Rap1 that is required for the extensive invagination of the posterior fold. These data suggest a model whereby Rap1 regulates dorsal fold invagination through an α-Catenin-dependent process, and establish that differential regulation of an active, specific cellular mechanism confers distinct properties to the neighboring cells to control the extent of epithelial invagination.
Our genetic analysis identifies two separate functions of Rap1 during dorsal fold formation. The early function appears to be a general role required in all cells that is important for junctional positioning. This was established via examination of embryos that lack Rap1 activity, such as embryos that are produced by the germline clones of null alleles of Rap1
, which encodes the Drosophila
homolog of PDZ-GEF, a known guanine nucleotide exchange factor that activates Rap1 or embryos that overexpress a GDP-locked, dominant negative form of Rap1, Rap1N17 (Figure S3A–S3E
, Movie S5
Part 1; Boettner and Van Aelst, 2007
; de Rooij et al., 1999; Huelsmann et al., 2006; Spahn et al., 2012
). These embryos display normal assembly of the adherens junctions, the initial basal shift of junction positioning in the initiating cells and attempt to form dorsal folds. Subsequently, however, the junctions re-localize to the apical surface in all dorsal cells, reversing these initial attempts of dorsal fold formation and eliminating all folding structures.
Rap1 appears to maintain the junctional positioning by maintaining the junctional levels of Bazooka. This notion is supported by the lower levels of junctional Bazooka in the Rap1
mutant embryos (Figure S3F and S3G
) and by suppression of the loss-of-function phenotype of Rap1
by Bazooka overexpression, which restores the apical domain in the initiating cells and the dorsal fold structures in the Rap1
mutant embryos (Figure S3H
, , Movie S5
Part 2). Because Bazooka levels are uniform across the dorsal epithelium (Wang et al., 2012
), this early function of Rap1 appears broadly required, independently of the levels of Rapgap1 expression, and operates in addition to Rap1’s later role during epithelial invagination. The effective suppression of Rap1
loss-of-function following Bazooka overexpression suggests that the two separate functions of Rap1 – the maintenance of Bazooka levels and the regulation of junction-actin connection during epithelial invagination – could be decoupled, allowing us to compare the effect of loss of Rap1 function to that of constitutively active Rap1V12.
The later, spatially regulated function of Rap1 is independent of Bazooka and is differentially modulated by the spatially restricted expression of Rapgap1. Since active Rap1 appears to act through α-Catenin to inhibit invagination, it seems plausible that distinct Rap1 activity states modulate the coupling strength between junctions and actin, thereby conferring distinct properties of junctional restructuring to the neighboring cells of the anterior and posterior folds. The geometric measurements of the neighboring cells suggest a model whereby constitutively active Rap1 inhibits junctional mobility so that the size of the apical domain remains constant in the cells surrounding the anterior fold where Rapgap1 levels are low. In contrast, Rapgap1 expression modulates Rap1 activity to promote junctional mobility in the neighboring cells of the posterior fold so that their apical domain expands. In this view, both the initiation and invagination processes require active remodeling of the junctions, but differ in their underlying cellular mechanisms (). During initiation, the junctional shift is induced by a modification of the epithelial apical-basal polarity as a result of the downregulation of Par-1 in the initiating cells (Wang et al., 2012
). During invagination, since Par-1 levels do not decrease, mechanical stress might be the dominant force that causes the junctions to move in the neighboring cells.
A model for the initiation and invagination of dorsal fold formation
How Rap1 modulates α-Catenin-dependent junction-actin coupling remains unknown. We examined the intensities, localization and turnover kinetics (as measured by fluorescent recovery after photobleaching) of the core junctional components (E-Cadherin and Armadillo), α-Catenin, two junctional proteins that interact with both α-Catenin and actin (Canoe and Vinculin; Choi et al., 2012
; Pokutta et al., 2008
; Sawyer et al., 2009
; Yonemura et al., 2010
), but did not detect a difference between the neighboring cells of the anterior and posterior folds (data not shown). Recent work in mammalian tissue culture cells showed that the FRET (fluorescent resonance energy transfer) intensities of an E-Cadherin tension sensor correlate with the actin-coupling states of adherens junctions (Borghi et al., 2012
). The use of such a sensor in the living Drosophila
embryo might help revealing the difference in junction-actin coupling states between the anterior and posterior fold neighboring cells.
Recent work suggests that α-Catenin undergoes a conformational change upon mechanical stretch at the cell junctions (le Duc et al., 2010
; Pokutta et al., 2008
; Yonemura et al., 2010
). Such conformational change could in principle relieve α-Catenin from an intramolecular inhibition on actin binding, thereby increasing its affinity to, or stabilizing its interaction with, the junctional actin (Choi et al., 2012
). Changes in α-Catenin conformation thus may determine its ability to mediate the physical coupling between junctions and actin. It is of note that expression of a mutant form of α-Catenin that lacks the domain that modulates its conformational change can support static junctional function, but fails to effectively rescue the loss-of-α-Catenin
phenotype in dynamic morphogenetic processes (Desai et al., 2013
). It is possible that mechanical forces during morphogenesis dynamically modulate the conformational states of α-Catenin, the maintenance of which may require distinct Rap1 activity states. The dynamic changes of the α-Catenin conformations and the actin-coupling states of adherens junctions that they confer might be crucial for morphogenetic processes that involve extensive restructuring of cell-cell adhesion.
If Rapgap1 dictates the spatial extent of cell invagination, one simple model would envision that elevating the levels of Rapgap1
expression in the anterior region could promote anterior fold invagination. We explored this possibility using a UAS transgene to uniformly express Rapgap1
under the control of a maternal Gal4 driver. Two classes of phenotypes were observed: either a complete loss of dorsal fold formation (Movie S6
Part 1), or a limited degree of invagination similarly in both dorsal folds (Movie S6
Part 2). The former class suggests that the level of expression may be too high to permit the normal function of Rap1, while the latter class suggests that a reversal of the expression pattern – high in the anterior fold, but low in the posterior fold – might be necessary. We have attempted to express Rapgap1
in the cells anterior to the anterior fold using a Gal4 driver localized through the 3’UTR of the bicoid
gene and have also used the enhancer of the Kr
gene to direct the expression of Rapgap1
in cells that are posterior to the anterior fold. In neither case was there an effect on anterior fold invagination (data not shown). It is possible that driving extensive invagination for the anterior fold would require that Rapgap1
be expressed only in the surrounding cells of the anterior fold in a manner that mimics the endogenous pattern of Rapgap1
expression in the region of posterior fold. Currently, we know of no cis
-regulatory element or a Gal4 driver that can drive gene expression in such a specific pattern. Thus, it remains unresolved whether ectopic expression in the anterior fold region would be sufficient to cause extensive invagination.
In summary, our data suggest an exciting conceptual framework in which regulated coupling between junctions and actin has a profound impact on the levels of tissue reorganization and on the cellular responses to mechanical stresses that arise during tissue reorganization. We define a specific molecular pathway that produces drastically different epithelial structures from a morphogenetic process whose initiation mechanism appears similar. The regulatory principles that we unveil for Rap1 and α-Catenin might be employed in other contexts of morphogenesis in which a tissue undergoes dramatic remodeling, while unperturbed tissue integrity and cell adhesion must be maintained.