-??Acriticalaspectofgutrotationisinitiationofaleftwardtiltdirectedbytheconservedleft-??right (LR) Pitx2 transcription factor. Failure to establish proper gut chirality leads to gut malrotation and catastrophic volvulus in pediatric patients. Whereas the direction of rotation has long been assumed to be intrinsic to the tube itself we demonstrated that rotation is instead driven by asymmetric cellular behavior withinthedorsalmesentery(DM)thatsuspendstheguttubeandwhosecellulararchitectureisdownstreamof Pitx2 expressed strictly on the DM left side. In contrast, the mechanisms governing the right side of the DM remainentirelyunknownandarethemajorgoalofthisproposal.WerecentlyshowedthattheECMexpansion uniquetotherightsideprecedesthecellularasymmetriestakingplaceontheleft,makingECMexpansionthe firstsymmetry-??breakingeventandpointingtoanovelpathwayduringgutrotationinitiatedbytherightside of the embryo. Hyaluronan (HA), a unique and highly conserved glycosaminoglycan, predominates in the ECMoftherightDMandisastrongcandidatefordrivingECMexpansion.InhibitionofHAsynthesis,or,loss oftheinflammatoryenzymetumornecrosisfactorstimulatedgene6(Tsg6),theonlyenzymethatcovalently modifies HA, prevents DM expansion and results in randomized gut looping. Tsg6 transfers heavy chains (HCs) to HA and transforms the normally inert HA matrix into a unique HC-??HA complex implicated in a variety of inflammatory diseases. The only known developmental function of HC-??HA is to ensure female fertility. Blood vessels in the gut mesentery are the cause of tissue death during midgut volvulus, but how these vessels become positioned inside the DM isn?t known. We now show that gut arteries develop only on the left side because they are progressively excluded from the right side, dependent on HA and Tsg6. Thus, HAmaybeacommonlinkintheglobalphenotypeobservedontherightsideoftheDM.Inthisproposal,we hypothesizethatECMchangesontherightinitiategutrotation.UsingchickenandmouseDM,ourgoalisto identify the mechanisms regulating HA function in the DM, the relationship between Tsg6 and HA production, and the changes in tissue architecture that establish gut and vascular chirality.
In Aim 1, we propose to define the role of Tsg6 during ECM expansion and vascular exclusion.
In Aim 2, we characterize themechanismsbywhichHAinhibitsvasculardevelopmentontheright.WehypothesizethatHAregulates endothelialmigrationbyinhibitingtheCxcl12/Cxcr4axisintherightDM.
In Aim3, weproposeamechanism responsible for the opposing pro-??angiogenic and anti-??angiogenic phenotypes within the LR DM. Our studies willidentifymechanismsthatgovernLRorganandvascularmorphogenesisdownstreamofHA-??matricesand may shed light on the origin of gut and vascular anomalies. Vasculogenesis -?? induction of new vessels and destruction of exiting ones -?? is among the most important processes in human biology. Our DM system is perfectlysuitedtostudythesedynamicandclinicallyrelevanteventsintheintestine.

Public Health Relevance

Gut malrotation is a birth defect of abnormal intestinal rotation that occurs once in 500 live births. While malrotationpredisposesaffectedbabiestovolvulus,acatastrophicstrangulationoftheintestineanditsblood supply, the origin of this anomaly remains unknown. We have developed the genetic tools to answer the fundamentalquestionssurroundingpropergutrotationmakinguseoftheaccessibilityofthechickeneggand geneticmousemodelstoultimatelyimprovediagnosisinneonates.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Intercellular Interactions Study Section (ICI)
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Greenwel, Patricia
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Cornell University
Other Basic Sciences
Schools of Veterinary Medicine
United States
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Sivakumar, Aravind; Mahadevan, Aparna; Lauer, Mark E et al. (2018) Midgut Laterality Is Driven by Hyaluronan on the Right. Dev Cell 46:533-551.e5
Sivakumar, Aravind; Kurpios, Natasza A (2018) Transcriptional regulation of cell shape during organ morphogenesis. J Cell Biol 217:2987-3005
Welsh, Ian C; Kwak, Hojoong; Chen, Frances L et al. (2015) Chromatin Architecture of the Pitx2 Locus Requires CTCF- and Pitx2-Dependent Asymmetry that Mirrors Embryonic Gut Laterality. Cell Rep 13:337-49
Mahadevan, Aparna; Welsh, Ian C; Sivakumar, Aravind et al. (2014) The left-right Pitx2 pathway drives organ-specific arterial and lymphatic development in the intestine. Dev Cell 31:690-706
Welsh, Ian C; Thomsen, Michael; Gludish, David W et al. (2013) Integration of left-right Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2. Dev Cell 26:629-44