Your Gut Is Right to Turn LeftReferencesDevelopmental CellPreviewsYour Gut Is Right to Turn LeftOlga Klezovitch1and Valeri Vasioukhin1,*1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA*Correspondence: [email protected]://dx.doi.org/10.1016/j.devcel.2013.08.018Transcriptional factor Pitx2 is a key regulator of left-right asymmetry in the developing gut. In this issue ofDevelopmental Cell, Welsh et al. (2013) identify the formin Daam2, an effector of Wnt signaling, as a keycellular target of Pitx2 required for morphogenetic events during asymmetric gut formation.Despite the (almost) perfect exterior sym-metry of our bodies, the internal organs(including the gut) of virtually all verte-brates are patterned asymmetrically(Burn and Hill, 2009; Levin, 2005). Theprimitive gut starts as an endoderm-derived straight midline epithelial tubesurrounded by mesenchymal cells. Itconsists of the foregut, midgut (precursorof the small intestine), and hindgut(precursor of the large intestine). As theembryo develops, the midgut continuesto grow, and this disproportionate elon-gation leads to a complex process of‘‘looping,’’ during which the gut tubeundergoes asymmetric counterclockwiserotation of a total of 270!. This rotation ismandatory for the proper positioning ofthe developing intestine inside the bodycavity. In amniotes, the ‘‘looping’’ is initi-ated by the primitive gut tilting leftward,a critical event that sets up a left-right(LR) direction for the subsequent gutcoiling. Why would a relatively symmetriccylindrical tube exhibit such an asym-metric behavior? Is there an external drivethat forces the gut to rotate? Indeed,previous studies in birds and mice haveestablished that this intestinal leftwardtilt is potentiated by LR differences in thecellular morphology of the dorsal mesen-tery (DM), a mesoderm-derived structurethat connects the gut tube to the dorsalbody wall (Davis et al., 2008; Kurpioset al., 2008).The DM of embryonic gut consists offour morphologically and functionallydistinct compartments: a left epithelium,a left mesenchyme, a right mesenchyme,and a right epithelium. At first, the DMforms as a completely symmetric struc-ture that eventually becomes asymmetri-cally organized, with dense mesenchymeand columnar epithelium on the leftside and sparse mesenchymal cells andcuboidal epithelium on the right side.These asymmetric changes in the cellularmorphology of the left side of the DM aretranslated into a shortening of the surfacearea of the left epithelium relative to itsright counterpart. This event, when takentogether with the condensation of theleft mesenchyme, induces a change inthe shape of the DM and, as a result,forces the gut tube to tilt leftward. Previ-ous studies in birds and mice unequivo-cally demonstrated that the exclusiveleft-sided expression of transcriptionfactors Pitx2 and Isl1 (downstream ofNodal) (Davis et al., 2008) and the adhe-sion molecule N-cadherin (downstreamof Pitx2/Isl1) is responsible for the LRasymmetry of the DM (Kurpios et al.,2008). However, the direct downstreamtargets and cellular mechanism ofPitx2 remain unknown. In this issue ofDevelopmental Cell, Welsh et al. (2013)provide new mechanistic insights intothe function of Pitx2 by showing that itpotentiates a noncanonical Wnt pathwayto activate the formin Daam2, which inturn interacts with and links N-cadherin-mediated adhesion complexes to theactin cytoskeleton, thus inducing polar-ized condensation in the left DM.To identify the downstream targetsof Pitx2, the authors performed lasercapture microdissection and microarrayanalyses of the left (Pitx2+) and right(Pitx2") parts of chicken DM at stageHH21, when the DM acquires its uniqueasymmetric organization and whenthe leftward gut tilting is first observed.Interestingly, positive mediators of theWnt pathway such as Frizzled (Frz)receptors 4/8, Gpc3, and the forminDaam2 were found to be expressedexclusively in the left DM, whereas theWnt inhibitors Srfp1/2 were presentexclusively in the right DM, and thenoncanonical Wnt/planar cell polaritycore member Prickle-1 was expressed ina decreasing right-to-left gradient. Theauthors corroborated these microarraydata by performing RNA in situ hybridi-zation using both chicken and mouseembryos. Of note, the noncanonical Wntligand Wnt5a was absent in the DM butwas bilaterally expressed in the ventrallyadjacent midgut mesenchyme. Based onthese data, the authors hypothesizedthat the Wnt5a secreted by the adjacentmidgut will find the left DM to be a per-missive environment for the positivenoncanonical Wnt signaling, where it willdrive the activation of the formin Daam2.In turn, activated Daam2 will functionto potentiate actin polymerization andcytoskeleton reorganization, and this setof events will result in the condensationof the left mesenchyme, thus providingan initiation clue for the leftward tilting ofthe gut.To demonstrate that Pitx2 is indeed akey upstream regulator of Daam2 ex-pression, the authors electroporatedPitx2 cDNA into the right side of chickenDM, where it is normally not expressed.This misexpression strongly inducedboth Daam2 and Gpc3 in the rightDM, making them expressed bilaterally.Moreover, in wild-type mouse embryos,Daam2 was present in the left DM, butthis expression was lost in the Pitx2"/"mutants. In addition, by performing com-parative genomics and computationalsequence analyses, the authors identifiedPitx2 binding sites at the promoters ofGpc3, Fzd4, and Daam2.Next, the authors asked whetheractivation of Daam2 is required andsufficient to drive the mesenchymalcondensation in the left DM. By over-expressing wild-type Daam2 and itsdominant-negative (DN) and constitu-tively active (CA) truncated mutant formsin either left or right chicken DM, as wellas knocking down the endogenousDevelopmental Cell 26, September 30, 2013 ª2013 Elsevier Inc. 553Daam2 with specific small hairpin RNA,the authors demonstrated that Daam2represents a key mediator of Pitx2signaling and is indispensable for LRasymmetry in the DM.It has previously been shown thatasymmetric changes in the cell architec-ture in the DM are partially dependent onthe exclusive left-side expression of thecell adhesion protein N-cadherin (Kurpioset al., 2008; Plageman et al., 2011). To un-cover the role of Daam2 in this process,the authors inhibited Daam2 activity inthe left DM, which resulted in perturbedintercellular N-cadherin-mediated adhe-sion. Conversely, the
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