How Aqp3b Influence Convergent Extension Through Noncanonical Wnt Signaling Kaitlyn See, Dr. Jennifer Forecki, and Dr. Christa Merzdorf Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT August, 2016

INTRODUCTION 5 6 RESULTS SIGNIFICANCE Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membrane To quote Louis Wolpert, "it is not birth, marriage or death, but Figure 13. Rescue with , which is truly the most important time in your bound water channel, that is present during gastrulation and Dvl1ΔDIX. We have shown that other stages of early (Figures 1 and Dvl1ΔDIX is capable of rescuing life". Defects in embryonic develop that occur during 2). Gastrulation organizes cells into germ layers, which will normal development after gastrulation are often lethal and convergent extension is a later form different body tissues. Convergent extension cell inhibition of Aqp3b. Dvl1ΔDIX is fundamental process during gastrulation. Convergent movements are critical in driving gastrulation. During involved in all noncanonical Wnt extension is important in many other developmental stages, pathways, indicating that Aqp3b convergent extension, cells fold into the , then merge not just during gastrulation. During neurulation, convergent acts through noncanonical Wnt extension aids in the closure of the neural tube by directing to form the long body axis (Figure 5). These cell movements signaling. are regulated by noncanonical Wnt signaling, an intercellular cells to migrate medially and intercalate to form a long, narrow neural tube. When convergent extension is defective signaling pathway that controls the patterning, migration, Figure 6. Noncanonical Wnt signaling Figure 14. Rescue with PKC. and polarity of tissues. In previous experiments, when pathways. Secreted Wnt ligands bind to the during this process, the neural tube does not close properly PKC is a component of the Aqp3b is inhibited using a morpholino oligonucleotide, Frizzled (Fzd) membrane receptor. In the (Wallingford 2002). The resulting neural tube closure defects Wnt/Ca2+ pathway. We have cytoplasm, Disheveled directs Wnt signaling include spina bifida and anencephaly (Figures 18 and 19). convergent extension does not occur properly, suggesting a shown that rescuing with PKC through either the calcium modulated pathway link between Aqp3b and noncanonical Wnt signaling. In restores normal convergent Neural tube closure defects occur in about 1 in 2000 babies (Wnt/Ca2+), the planar cell polarity pathway extension activity. Aqp3b born in the United States. Later in development, convergent order to assay these defects, We use the Keller tissue (Wnt/PCP), or the Wnt/Ror2 pathway. The Ca2+ influences the Wnt/Ca2+ pathway extension is important, for example, in the formation of the explanting method to observe convergent extension (Figure pathway involves PKC as cytoplasmic transducer, 11). Our goal is to determine which parts of the Wnt while the PCP pathway involves RhoA and Rac1. heart. Here, we use convergent extension that occurs during signaling pathway are influenced by Aqp3b. We conducted The Ror2 pathway utilizes a co-receptor and Xenopus gastrulation as a paradigm for all these other rescue experiments by inhibiting Aqp3b and injecting an involves cdc42. Figure 15. Rescue with RhoA. convergent extension events during embryonic development. RNA or DNA construct of several proteins involved in Wnt Figure 5. Convergent Extension. RhoA is capable of rescuing normal development after Figure 19. signaling. We have shown that Aqp3b is involved in Cells merge (intercalate) to form a long column of cells that migrate into inhibition of Aqp3b. RhoA is part Anencephaly. noncanonical Wnt signaling, as demonstrated by successful the embryo's interior. A short wide of the Wnt/PCP pathway and Anencephaly is the rescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to act section of cells forms a long, narrow primarily involved in contractile condition where the through the Wnt/Ca2+ subpathway, since PKC is able to column of cells. Early in development, forces such as convergent cerebrum of the rescue the defects caused by the inhibition of Aqp3b. In this process helps drive gastrulation extension. does not form and elongation of the body axis. due to improper conclusion, I have demonstrated that the ability of Aqp3b to closure of the rostral influence convergent extension is dependent on Figure 16. Figure 18. Spina Bifida. If the neural tube Figure 7. Xenopus laevis. Xenopus a neural tube. Some noncanonical Wnt signaling, specifically the Wnt/Ca2+ laevis frogs are a well-established Extension. fails to close at the caudal end, it may lead to reflexes may be subpathway. model for the study of early In explants with spina bifida. Severe cases create a cyst-like functional if a brain embryonic development, as they are inhibited aqp3b structure, called the myelomeningocele, on the stem has developed, Figure 1. large and develop outside the body expression, We back, as spinal nerves protrude from the spinal but infants are Aquaporins. at room temperature. rescued convergent canal, filling with cerebrospinal fluid. typically still-born or Aquaporins are extension with die within hours or membrane channels METHODS various constructs, days following birth. that allow for the the explants were passage of water inhibited with either CONCLUSION across cell the aqp3b 5’UTR membranes. MO (a) or aqp3b • We have shown that when Aqp3b is inhibited with a ATG MO (b). The morpholino oligonucleotide, convergent extension in percent elongation b gastrulation is adversely affected. Thus, Aqp3b is required is normalized to for convergent extension movements of cells. uninjected, comparing • Dvl1ΔDIX restores normal convergent extension after development to inhibition of Aqp3b, which indicates that Aqp3b acts through healthy Figure 9. Injections. noncanonical Wnt signaling. Figure 8. Morpolino Oligonucleotides. The Aqp3b morpholino The Aqp3b morpholino oligonucleotide oligonucleotide, as well • PKC rescues normal development after inhibition of Aqp3b, (MO) binds to the aqp3b mRNA by as the RNA for the demonstrating that Aqp3b influences the Wnt/Ca2+ pathway Figure 2. Aquaporins and Aquaglyceroporins. complementary base pairing. The 5'UTR rescuing protein are Aquaporins allow only the passage of water. Aquaglyceroporins aqp3bMO binds in the 5'UTR of aqp3b injected into the two have a slightly wider pore, which allows for the passage of glycerol and blocks ribosome progression, which dorsal cells of 4-cell •RhoA is capable of rescuing convergent extension defects, in addition to water. Both exclude ions. Aqp3b is an prevents translation of the aqp3b mRNA.. embryos. Dorsal cells showing that Aqp3b may play a part in Wnt/PCP aquaglyceroporin. The morpholino backbone differs from are often smaller and the DNA phosphodiester backbone, lighter in color. Figure 17. Our results suggest that aquaporins engage in specific Figure 3. Gastrulation. protecting it from degradation. a Convergence. In signaling during convergent extension. This is a significant During gastrulation cells fold into explants with departure from the general picture of aquaporins, which have Figure 10. Dorsal Lip of the inhibited aqp3b the interior of the embryo to form mainly been considered passive pores that allow bidirectional the three germ layers Blastopore. The dorsal lip of the expression, We (endoderm, mesoderm, and blastopore forms as a dark crescent on rescued passage of water through cell membranes, depending on the ectoderm) which lays the the bottom of the embryo, marking the convergent osmotic gradient. Thus, this continued research may foundation for tissue beginning of gastrulation. extension with ultimately have a very significant impact on understanding the differentiation during later various role of aquaporins in cellular processes, such as convergent stages. constructs, the extension and cell migration. Figure 11. Keller Explants. explants were The dorsal mesendoderm is inhibited with FUTURE WORK locate just above the dorsal lip either the aqp3b Figure 4. of the blastopore. It is removed 5’UTR MO (a) or •We will continue Rescue experiments involving Rac1 from the embryo by making Noncanonical Wnt aqp3b ATG MO (Another component within the Wnt/PCP pathway) and signaling. The cuts on either side of the dorsal b (b) Out of those cdc42 (Wnt/Ror2). As well as working with the ATG Wnt/PCP pathway, lip of the blastopore. The that extended involves both RhoA resulting explant is then properly, the Morpholino Oligonucleotide and Rac1. These cultivated under a coverslip percent of those ACKNOWLEDGMENTS proteins direct and scored the following day. that converged activity differently, Figure 12. Explant properly is shown. I would like to acknowledge The Merzdorf Lab, Montana INBRE, USP, where RhoA Scoring. The explants and MAS for funding this project. primarily controls PCP Wnt/Ca2+ Ror2 form long narrow contractile forces and Rac1 is involved in protrusive forces. The Wnt/Ca2+ protrusions by convergent Research reported in this publication was supported by the National pathway influences expression to facilitate cell migration and tissue extension cell movements. Institute of General Medical Sciences of the National Institutes of structure. When convergent Health under Award Number P20GM103474. The content is solely the extension is inhibited, the responsibility of the authors and does not necessarily represent the protrusion does not form (0 or 1). When only convergence is inhibited, official views of the National Institutes of Health. the protrusion is wide (3).