YOUR GUT IS RIGHT TO TURN LEFT

Abstract

Transcriptional factor Pitx2 is a key regulator of left-right asymmetry in the developing gut. In this issue of Developmental Cell, Welsh et al. (2013) identify the formin Daam2, an effector of Wnt signaling, as a key cellular target of Pitx2 required for morphogenetic events during asymmetric gut formation.

Despite the (almost) perfect exterior symmetry of our bodies, the internal organs (including the gut) of virtually all vertebrates are patterned asymmetrically (Burn and Hill, 2009; Levin, 2005). The primitive gut starts as an endoderm-derived straight midline epithelial tube surrounded by mesenchymal cells. It consists of the foregut, midgut (precursor of the small intestine) and hindgut (precursor of the large intestine). As the embryo develops, the midgut continues to grow, and this disproportionate elongation leads to a complex process of “looping” during which the gut tube undergoes asymmetric counterclockwise rotation of a total of 270°. This rotation is mandatory for the proper positioning of the developing intestine inside the body cavity. In amniotes, the “looping” is initiated by the primitive gut tilting leftwards, a critical event that sets up a left-right (LR) direction for the subsequent gut coiling. Why would a relatively symmetric cylindrical tube exhibit such an asymmetric behavior? Is there an external drive that forces the gut to rotate? Indeed, previous studies in birds and mice have established that this intestinal leftward tilt is potentiated by LR differences in the cellular morphology of the dorsal mesentery (DM), a mesoderm-derived structure that connects the gut tube to the dorsal body wall (Davis et al., 2008; Kurpios et al., 2008).

The DM of embryonic gut consists of four morphologically and functionally distinct compartments: a left epithelium, left mesenchyme, right mesenchyme, and a right epithelium. At first, the DM forms as a completely symmetric structure, which eventually becomes asymmetrically organized, with dense mesenchyme and columnar epithelium on the left side and sparse mesenchymal cells and cuboidal epithelium on the right side. These asymmetric changes in the cellular morphology of the left side of the DM are translated into a shortening of the surface area of the left epithelium relative to its right counterpart. This event, when taken together with the condensation of the left mesenchyme, induces a change in the shape of the DM and, as a result, forces the gut tube to tilt leftwards. Previous studies in birds and mice unequivocally demonstrated that the exclusive left-sided expression of transcription factors Pitx2 and Isl1 (downstream of Nodal) (Davis et al., 2008) and the adhesion molecule N-cadherin (downstream of Pitx2/Isl1) is responsible for the LR asymmetry of the DM (Kurpios et al., 2008). However, the direct downstream targets and cellular mechanism of Pitx2 remain unknown. In this issue of Developmental Cell, Welsh et al. (2013) provide new mechanistic insights into the function of Pitx2 by showing that it potentiates a non-canonical Wnt pathway to activate the formin Daam2, which in turn interacts with and links N-cadherin-mediated adhesion complexes to the actin cytoskeleton, thus inducing polarized condensation in the left DM.

To identify the downstream targets of Pitx2, the authors performed laser capture microdissection and microarray analyses of the left (Pitx2+) and right (Pitx2−) parts of chicken DM at stage HH21, when the DM acquires its unique asymmetric organization and when the leftward gut tilting is first observed. Interestingly, positive mediators of the Wnt pathway such as Frizzled (Frz) receptors 4/8, Gpc3, and the formin Daam2 were found to be expressed exclusively in the left DM, whereas the Wnt inhibitors Srfp1/2 were present exclusively in the right DM, and the non-canonical Wnt/planar cell polarity core member Prickle-1 was expressed in a decreasing R-to-L gradient. The authors corroborated these microarray data by performing RNA in situ hybridization using both chicken and mouse embryos. Of note, the non-canonical Wnt ligand Wnt5a was absent in the DM but was bilaterally expressed in the ventrally adjacent midgut mesenchyme. Based on these data, the authors hypothesized that the Wnt5a secreted by the adjacent midgut will find the left DM to be a permissive environment for the positive non-canonical Wnt signaling, where it will drive the activation of the formin Daam2. In turn, activated Daam2 will function to potentiate actin polymerization and the cytoskeleton reorganization, and this set of events will result in the condensation of the left mesenchyme, thus providing an initiation clue for the leftward tilting of the gut.

To demonstrate that Pitx2 is indeed a key upstream regulator of Daam2 expression, the authors electroporated Pitx2 cDNA into the right side of chicken DM, where it is normally not expressed. This misexpression strongly induced both Daam2 and Gpc3 in the right DM, making them expressed bilaterally. Moreover, in wild-type mouse embryos, Daam2 was present in the left DM, but this expression was lost in the Pitx2−/− mutants. In addition, by performing comparative genomics and computational sequence analyses the authors identified Pitx2 binding sites at the promoters of Gpc3, Fzd4, and Daam2.

Next, the authors asked whether activation of Daam2 is required and sufficient to drive the mesenchymal condensation in the left DM. By overexpressing WT Daam2 and its dominant-negative (DN) and constitutively active (CA) truncated mutant forms in either left or right chicken DM, as well as knocking down the endogenous Daam2 with specific shRNA, the authors demonstrated that Daam2 represents a key mediator of Pitx2 signaling and is indispensable for LR asymmetry in the DM.

It has previously been shown that asymmetric changes in the cell architecture in the DM are partially dependent on the exclusive left-side expression of the cell adhesion protein N-cadherin (Kurpios et al., 2008; Plageman et al., 2011). To uncover the role of Daam2 in this process, the authors inhibited Daam2 activity in the left DM, which resulted in perturbed intercellular N-cadherin-mediated adhesion. Conversely, the introduction of CA-Daam2 into the right DM produced an accumulation of both N-cadherin and α-catenin, as well as lengthening of the cell-cell junctions. This suggests the intriguing possibility that Daam2 may play a role in stabilizing N-cadherin based junctions. Indeed, the authors demonstrated not only that Daam2 partially co-localizes with α-catenin at cell borders, but also that it forms a protein complex with α-catenin and N-cadherin. Although it still remains to be determined whether this interaction is direct or requires additional components, this finding is nevertheless very important as it provides new insights into the mechanism of Daam2 action.

Thus, the work by Welsh et al. (2013) uncovers a connection between two major conserved signaling pathways, Pitx2 and non-canonical Wnt, in the context of LR asymmetry establishment in the developing embryonic gut. The findings presented in this study will help to clarify the molecular mechanisms of midgut malrotations that usually occur in early embryonic development and lead to devastating gut disorders. Future studies will surely focus on whether this connection between Pitx2 and non-canonical Wnt signaling represents a general mechanism that governs polarization and LR asymmetry in other internal organs.

This is a commentary on article Welsh IC, Thomsen M, Gludish DW, Alfonso-Parra C, Bai Y, Martin JF, Kurpios NA. Integration of left-right Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2. Dev Cell. 2013;26(6):629-44.