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Show posters on the hill Internal Asymmetry in Vertebrates: Polaris and Polycystin-2 in Kupffer's Vesicle and the left-Right Axis in Zebrafish Anoush Emrazian, Brent W. Bisgrove, Brian S. Snarr, and H. Joseph Yost Huntsman Cancer Institute Center for Children, Department of Oncological Sciences H, Lumans and all vertebrates are bilaterally symmetric externally, but internally they have an important asymmetry across the Left-Right (L-R) axis in the arrangement of internal organs such as the heart and gut. The question of how the vertebrate L-R axis is determined has been at least partially answered for several organisms. During early mouse development it has been determined that monocilia associated with the node beat, creating a leftward flow of fluid. This directional fluid flow acts to initiate a left-sided signaling cascade that helps establish the L-R axis. Two proteins important for L-R patterning in mice are Polaris and Polycystin-2. Polaris is involved in assembly of cilia and Polycystin-2 is thought to be a Ca+ permeable cation channel important for the signaling function of node monocilia. To ask whether the L-R patterning process is conserved in other vertebrates, we cloned the zebrafish homologs of polaris and polycystin-2. It is thought that a region of the developing zebrafish embryo called Kupffer's vesicle (Kv) plays the same role in zebrafish as the node does in mice in establishing asymmet- Internal Asymmetry in Vertebrates: Polaris and Polycystin-2 in Kupffer's Vesicle and the Left-Right Axis in Zebrafish Anoush Emrazian, Brent Bisgrove, Brian Snarr, H. Joseph Yost Huntsman Cancer Institute Center for Children Department of Oncological Sciences normal loop reversed loop Rotation of node monocilia creates fluid flow toward the left side of the mouse node. Fluid flow is involved in establishing the L-R axis Polaris and Polycystin-2 (Pkd2) play essential but distinct roles in renal and node cilia function Morpholinos injected into the yolk at late cleavage stages are specifically taken up by dfe's and target Kupffer's vesicle - lnlraflagellar Transport Protein - function: ciliary assembly Pkd-2 - Calcium Ion Channel - mouse KO lia»"iioriiiiil"mc and L-K defects polaris and pki/2 Morpholinos randomize pression of southpaw, a mu/<i/-related gene polaris morpholinos cause a loss and/or shortening of Kv cilia when targeted to all cells by injection al the 1-2 cell stage, or nhen targeted specifically to Kupffer's vesicle by injection into the yolk at late cleavage stages immiec.ted 1-2 tell untniscted late cluvag« ric organ arrangement. Both Polaris and Polycystin-2 are expressed in dorsal forerunner cells (dfe's), which gives rise to Kv. When Polaris and Polycystin-2 function is knocked down, expression of genes normally expressed only on the left side (such as southpaw, lefty 1 and lefty2) is randomized and heart and gut looping is often reversed. When Kv cilia are stained after Polaris and Polycystin-2 knockdown, those embryos lacking Polaris expression have missing or misshapen cilia. This data supports the hypothesis that Kv function is similar to that of the node in mice and the functions of Polaris and Polycystin-2 in L-R patterning are conserved in zebraf- ish. A similar mechanism might be expected to function in establishing the L-R axis of other vertebrates, including humans. |