Developmental Cell Previews Your Gut Is Right to Turn Left Olga Klezovitch1 and Valeri Vasioukhin1 1Division of Human Biology Fred Hutchinson Cancer Research Center Seattle WA 98109 USA Correspondence vvasiouk fhcrc org http dx doi org 10 1016 j devcel 2013 08 018 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 endodermderived 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 leftward 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 a left mesenchyme a right mesenchyme and a right epithelium At first the DM forms as a completely symmetric structure that 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 leftward 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 noncanonical Wnt pathway to activate the formin Daam2 which in turn interacts with and links N cadherinmediated 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 noncanonical Wnt planar cell polarity core member Prickle 1 was expressed in a decreasing right to left gradient The authors corroborated these microarray data by performing RNA in situ hybridization using both chicken and mouse embryos Of note the noncanonical 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 noncanonical Wnt signaling where it will drive the activation of the formin Daam2 In turn activated Daam2 will function to potentiate actin polymerization and 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 wild type 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 Developmental Cell 26 September 30 2013 2013 Elsevier Inc 553 Developmental Cell Previews Daam2 with specific small hairpin RNA 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 a
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