How Aqp3b Influence Convergent Extension Through Noncanonical WntSignaling

Kaitlyn See, Dr. Jennifer Forecki, and Dr. Christa MerzdorfDepartment of Cell Biology and Neuroscience, Montana State University, Bozeman, MT

How Aqp3b Influence Convergent Extension Through Noncanonical WntSignaling

Kaitlyn See, Dr. Jennifer Forecki, and Dr. Christa MerzdorfDepartment of Cell Biology and Neuroscience, Montana State University, Bozeman, MT

How Aqp3b Influence Convergent Extension Through Noncanonical WntSignaling

Kaitlyn See, Dr. Jennifer Forecki, and Dr. Christa MerzdorfDepartment of Cell Biology and Neuroscience, Montana State University, Bozeman, MT

How Aqp3b Influence Convergent Extension Through Noncanonical WntSignaling

Kaitlyn See, Dr. Jennifer Forecki, and Dr. Christa MerzdorfDepartment of Cell Biology and Neuroscience, Montana State University, Bozeman, MT August, 2016

How Aqp3b Influence Convergent Extension Through Noncanonical WntSignaling

Kaitlyn See, Dr. Jennifer Forecki, and Dr. Christa MerzdorfDepartment of Cell Biology and Neuroscience, Montana State University, Bozeman, MT August, 2016

INTRODUCTION RESULTS SIGNIFICANCE6

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

INTRODUCTION RESULTS

Figure 13. Rescue withDvl1ΔDIX. We have shown thatDvl1ΔDIX is capable of rescuingnormal development afterinhibition of Aqp3b. Dvl1ΔDIX isinvolved in all noncanonical Wntpathways, indicating that Aqp3bacts through noncanonical Wntsignaling.

SIGNIFICANCETo quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.

5 6

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 13. Rescue withDvl1ΔDIX. We have shown thatDvl1ΔDIX is capable of rescuingnormal development afterinhibition of Aqp3b. Dvl1ΔDIX isinvolved in all noncanonical Wntpathways, indicating that Aqp3bacts through noncanonical Wntsignaling.

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 13. Rescue withDvl1ΔDIX. We have shown thatDvl1ΔDIX is capable of rescuingnormal development afterinhibition of Aqp3b. Dvl1ΔDIX isinvolved in all noncanonical Wntpathways, indicating that Aqp3bacts through noncanonical Wntsignaling.

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 13. Rescue withDvl1ΔDIX. We have shown thatDvl1ΔDIX is capable of rescuingnormal development afterinhibition of Aqp3b. Dvl1ΔDIX isinvolved in all noncanonical Wntpathways, indicating that Aqp3bacts through noncanonical Wntsignaling.

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 13. Rescue withDvl1ΔDIX. We have shown thatDvl1ΔDIX is capable of rescuingnormal development afterinhibition of Aqp3b. Dvl1ΔDIX isinvolved in all noncanonical Wntpathways, indicating that Aqp3bacts through noncanonical Wntsignaling.

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 6. Noncanonical Wnt signalingpathways. Secreted Wnt ligands bind to theFrizzled (Fzd) membrane receptor. In thecytoplasm, Disheveled directs Wnt signalingthrough either the calcium modulated pathway(Wnt/Ca2+), the planar cell polarity pathway(Wnt/PCP), or the Wnt/Ror2 pathway. The Ca2+

pathway involves PKC as cytoplasmic transducer,while the PCP pathway involves RhoA and Rac1.The Ror2 pathway utilizes a co-receptor andinvolves cdc42.

Figure 14. Rescue with PKC.PKC is a component of the

Wnt/Ca2+ pathway. We haveshown that rescuing with PKC

restores normal convergentextension activity. Aqp3b

influences the Wnt/Ca2+ pathway

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 6. Noncanonical Wnt signalingpathways. Secreted Wnt ligands bind to theFrizzled (Fzd) membrane receptor. In thecytoplasm, Disheveled directs Wnt signalingthrough either the calcium modulated pathway(Wnt/Ca2+), the planar cell polarity pathway(Wnt/PCP), or the Wnt/Ror2 pathway. The Ca2+

pathway involves PKC as cytoplasmic transducer,while the PCP pathway involves RhoA and Rac1.The Ror2 pathway utilizes a co-receptor andinvolves cdc42.

Figure 14. Rescue with PKC.PKC is a component of the

Wnt/Ca2+ pathway. We haveshown that rescuing with PKC

restores normal convergentextension activity. Aqp3b

influences the Wnt/Ca2+ pathway

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 6. Noncanonical Wnt signalingpathways. Secreted Wnt ligands bind to theFrizzled (Fzd) membrane receptor. In thecytoplasm, Disheveled directs Wnt signalingthrough either the calcium modulated pathway(Wnt/Ca2+), the planar cell polarity pathway(Wnt/PCP), or the Wnt/Ror2 pathway. The Ca2+

pathway involves PKC as cytoplasmic transducer,while the PCP pathway involves RhoA and Rac1.The Ror2 pathway utilizes a co-receptor andinvolves cdc42.

Figure 14. Rescue with PKC.PKC is a component of the

Wnt/Ca2+ pathway. We haveshown that rescuing with PKC

restores normal convergentextension activity. Aqp3b

influences the Wnt/Ca2+ pathway

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 6. Noncanonical Wnt signalingpathways. Secreted Wnt ligands bind to theFrizzled (Fzd) membrane receptor. In thecytoplasm, Disheveled directs Wnt signalingthrough either the calcium modulated pathway(Wnt/Ca2+), the planar cell polarity pathway(Wnt/PCP), or the Wnt/Ror2 pathway. The Ca2+

pathway involves PKC as cytoplasmic transducer,while the PCP pathway involves RhoA and Rac1.The Ror2 pathway utilizes a co-receptor andinvolves cdc42.

Figure 14. Rescue with PKC.PKC is a component of the

Wnt/Ca2+ pathway. We haveshown that rescuing with PKC

restores normal convergentextension activity. Aqp3b

influences the Wnt/Ca2+ pathway

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.Figure 15. Rescue with RhoA.

RhoA is capable of rescuingnormal development afterinhibition of Aqp3b. RhoA is partof the Wnt/PCP pathway andprimarily involved in contractileforces such as convergentextension.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 6. Noncanonical Wnt signalingpathways. Secreted Wnt ligands bind to theFrizzled (Fzd) membrane receptor. In thecytoplasm, Disheveled directs Wnt signalingthrough either the calcium modulated pathway(Wnt/Ca2+), the planar cell polarity pathway(Wnt/PCP), or the Wnt/Ror2 pathway. The Ca2+

pathway involves PKC as cytoplasmic transducer,while the PCP pathway involves RhoA and Rac1.The Ror2 pathway utilizes a co-receptor andinvolves cdc42.

Figure 5. Convergent Extension.Cells merge (intercalate) to form along column of cells that migrate intothe embryo's interior. A short widesection of cells forms a long, narrowcolumn of cells. Early in development,this process helps drive gastrulationand elongation of the body axis.

Figure 19.Anencephaly.Anencephaly is thecondition where thecerebrum of thebrain does not formdue to improperclosure of the rostralneural tube. Somereflexes may befunctional if a brainstem has developed,but infants aretypically still-born ordie within hours ordays following birth.

To quote Louis Wolpert, "it is not birth, marriage or death, butgastrulation, which is truly the most important time in yourlife". Defects in embryonic develop that occur duringgastrulation are often lethal and convergent extension is afundamental process during gastrulation. Convergentextension is important in many other developmental stages,not just during gastrulation. During neurulation, convergentextension aids in the closure of the neural tube by directingcells to migrate medially and intercalate to form a long,narrow neural tube. When convergent extension is defectiveduring this process, the neural tube does not close properly(Wallingford 2002). The resulting neural tube closure defectsinclude spina bifida and anencephaly (Figures 18 and 19).Neural tube closure defects occur in about 1 in 2000 babiesborn in the United States. Later in development, convergentextension is important, for example, in the formation of theheart. Here, we use convergent extension that occurs duringXenopus gastrulation as a paradigm for all these otherconvergent extension events during embryonic development.Figure 15. Rescue with RhoA.

RhoA is capable of rescuingnormal development afterinhibition of Aqp3b. RhoA is partof the Wnt/PCP pathway andprimarily involved in contractileforces such as convergentextension.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 5. Convergent Extension.Cells merge (intercalate) to form along column of cells that migrate intothe embryo's interior. A short widesection of cells forms a long, narrowcolumn of cells. Early in development,this process helps drive gastrulationand elongation of the body axis.

Figure 19.Anencephaly.Anencephaly is thecondition where thecerebrum of thebrain does not formdue to improperclosure of the rostralneural tube. Somereflexes may befunctional if a brainstem has developed,but infants aretypically still-born ordie within hours ordays following birth.

Figure 15. Rescue with RhoA.RhoA is capable of rescuingnormal development afterinhibition of Aqp3b. RhoA is partof the Wnt/PCP pathway andprimarily involved in contractileforces such as convergentextension.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 5. Convergent Extension.Cells merge (intercalate) to form along column of cells that migrate intothe embryo's interior. A short widesection of cells forms a long, narrowcolumn of cells. Early in development,this process helps drive gastrulationand elongation of the body axis.

Figure 19.Anencephaly.Anencephaly is thecondition where thecerebrum of thebrain does not formdue to improperclosure of the rostralneural tube. Somereflexes may befunctional if a brainstem has developed,but infants aretypically still-born ordie within hours ordays following birth.

Figure 15. Rescue with RhoA.RhoA is capable of rescuingnormal development afterinhibition of Aqp3b. RhoA is partof the Wnt/PCP pathway andprimarily involved in contractileforces such as convergentextension.

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 5. Convergent Extension.Cells merge (intercalate) to form along column of cells that migrate intothe embryo's interior. A short widesection of cells forms a long, narrowcolumn of cells. Early in development,this process helps drive gastrulationand elongation of the body axis.

Figure 18. Spina Bifida. If the neural tubefails to close at the caudal end, it may lead tospina bifida. Severe cases create a cyst-likestructure, called the myelomeningocele, on theback, as spinal nerves protrude from the spinalcanal, filling with cerebrospinal fluid.

Figure 19.Anencephaly.Anencephaly is thecondition where thecerebrum of thebrain does not formdue to improperclosure of the rostralneural tube. Somereflexes may befunctional if a brainstem has developed,but infants aretypically still-born ordie within hours ordays following birth.

Figure 7. Xenopus laevis. Xenopuslaevis frogs are a well-established

model for the study of earlyembryonic development, as they are

large and develop outside the bodyat room temperature.

Figure 15. Rescue with RhoA.RhoA is capable of rescuingnormal development afterinhibition of Aqp3b. RhoA is partof the Wnt/PCP pathway andprimarily involved in contractileforces such as convergentextension.

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 18. Spina Bifida. If the neural tubefails to close at the caudal end, it may lead tospina bifida. Severe cases create a cyst-likestructure, called the myelomeningocele, on theback, as spinal nerves protrude from the spinalcanal, filling with cerebrospinal fluid.

Figure 19.Anencephaly.Anencephaly is thecondition where thecerebrum of thebrain does not formdue to improperclosure of the rostralneural tube. Somereflexes may befunctional if a brainstem has developed,but infants aretypically still-born ordie within hours ordays following birth.

Figure 7. Xenopus laevis. Xenopuslaevis frogs are a well-established

model for the study of earlyembryonic development, as they are

large and develop outside the bodyat room temperature.

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

a

Aquaporin-3b, Aqp3b, is an aquaglyceroporin, a membranebound water channel, that is present during gastrulation andother stages of early embryonic development (Figures 1 and2). Gastrulation organizes cells into germ layers, which willlater form different body tissues. Convergent extension cellmovements are critical in driving gastrulation. Duringconvergent extension, cells fold into the embryo, then mergeto form the long body axis (Figure 5). These cell movementsare regulated by noncanonical Wnt signaling, an intercellularsignaling pathway that controls the patterning, migration,and polarity of tissues. In previous experiments, whenAqp3b is inhibited using a morpholino oligonucleotide,convergent extension does not occur properly, suggesting alink between Aqp3b and noncanonical Wnt signaling. Inorder to assay these defects, We use the Keller tissueexplanting method to observe convergent extension (Figure11). Our goal is to determine which parts of the Wntsignaling pathway are influenced by Aqp3b. We conductedrescue experiments by inhibiting Aqp3b and injecting anRNA or DNA construct of several proteins involved in Wntsignaling. We have shown that Aqp3b is involved innoncanonical Wnt signaling, as demonstrated by successfulrescue using Dvl1ΔDix and Dvl2ΔDix. Aqp3b appears to actthrough the Wnt/Ca2+ subpathway, since PKC is able torescue the defects caused by the inhibition of Aqp3b. Inconclusion, I have demonstrated that the ability of Aqp3b toinfluence convergent extension is dependent onnoncanonical Wnt signaling, specifically the Wnt/Ca2+

subpathway.

Figure 18. Spina Bifida. If the neural tubefails to close at the caudal end, it may lead tospina bifida. Severe cases create a cyst-likestructure, called the myelomeningocele, on theback, as spinal nerves protrude from the spinalcanal, filling with cerebrospinal fluid.

Figure 19.Anencephaly.Anencephaly is thecondition where thecerebrum of thebrain does not formdue to improperclosure of the rostralneural tube. Somereflexes may befunctional if a brainstem has developed,but infants aretypically still-born ordie within hours ordays following birth.

Figure 1.Aquaporins.Aquaporins aremembrane channelsthat allow for thepassage of wateracross cellmembranes.

Figure 7. Xenopus laevis. Xenopuslaevis frogs are a well-established

model for the study of earlyembryonic development, as they are

large and develop outside the bodyat room temperature.

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

Figure 18. Spina Bifida. If the neural tubefails to close at the caudal end, it may lead tospina bifida. Severe cases create a cyst-likestructure, called the myelomeningocele, on theback, as spinal nerves protrude from the spinalcanal, filling with cerebrospinal fluid.

Figure 19.Anencephaly.Anencephaly is thecondition where thecerebrum of thebrain does not formdue to improperclosure of the rostralneural tube. Somereflexes may befunctional if a brainstem has developed,but infants aretypically still-born ordie within hours ordays following birth.

Figure 1.Aquaporins.Aquaporins aremembrane channelsthat allow for thepassage of wateracross cellmembranes.

Figure 7. Xenopus laevis. Xenopuslaevis frogs are a well-established

model for the study of earlyembryonic development, as they are

large and develop outside the bodyat room temperature.

METHODS

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

Figure 19.Anencephaly.Anencephaly is thecondition where thecerebrum of thebrain does not formdue to improperclosure of the rostralneural tube. Somereflexes may befunctional if a brainstem has developed,but infants aretypically still-born ordie within hours ordays following birth.

Figure 1.Aquaporins.Aquaporins aremembrane channelsthat allow for thepassage of wateracross cellmembranes.

METHODSCONCLUSION

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

Figure 1.Aquaporins.Aquaporins aremembrane channelsthat allow for thepassage of wateracross cellmembranes.

CONCLUSION• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

b

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

Figure 8. Morpolino Oligonucleotides.The Aqp3b morpholino oligonucleotide(MO) binds to the aqp3b mRNA bycomplementary base pairing. The 5'UTRaqp3bMO binds in the 5'UTR of aqp3band blocks ribosome progression, whichprevents translation of the aqp3b mRNA..The morpholino backbone differs fromthe DNA phosphodiester backbone,protecting it from degradation.

Figure 9. Injections.The Aqp3b morpholinooligonucleotide, as wellas the RNA for therescuing protein areinjected into the twodorsal cells of 4-cellembryos. Dorsal cellsare often smaller andlighter in color.

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 16.Extension.

In explants withinhibited aqp3bexpression, We

rescued convergentextension with

various constructs,the explants were

inhibited with eitherthe aqp3b 5’UTRMO (a) or aqp3b

ATG MO (b). Thepercent elongation

is normalized touninjected,comparing

development tohealthy embryos

Figure 2. Aquaporins and Aquaglyceroporins.Aquaporins allow only the passage of water. Aquaglyceroporinshave a slightly wider pore, which allows for the passage of glycerolin addition to water. Both exclude ions. Aqp3b is anaquaglyceroporin.

Figure 8. Morpolino Oligonucleotides.The Aqp3b morpholino oligonucleotide(MO) binds to the aqp3b mRNA bycomplementary base pairing. The 5'UTRaqp3bMO binds in the 5'UTR of aqp3band blocks ribosome progression, whichprevents translation of the aqp3b mRNA..The morpholino backbone differs fromthe DNA phosphodiester backbone,protecting it from degradation.

Figure 9. Injections.The Aqp3b morpholinooligonucleotide, as wellas the RNA for therescuing protein areinjected into the twodorsal cells of 4-cellembryos. Dorsal cellsare often smaller andlighter in color.

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 2. Aquaporins and Aquaglyceroporins.Aquaporins allow only the passage of water. Aquaglyceroporinshave a slightly wider pore, which allows for the passage of glycerolin addition to water. Both exclude ions. Aqp3b is anaquaglyceroporin.

Figure 8. Morpolino Oligonucleotides.The Aqp3b morpholino oligonucleotide(MO) binds to the aqp3b mRNA bycomplementary base pairing. The 5'UTRaqp3bMO binds in the 5'UTR of aqp3band blocks ribosome progression, whichprevents translation of the aqp3b mRNA..The morpholino backbone differs fromthe DNA phosphodiester backbone,protecting it from degradation.

Figure 9. Injections.The Aqp3b morpholinooligonucleotide, as wellas the RNA for therescuing protein areinjected into the twodorsal cells of 4-cellembryos. Dorsal cellsare often smaller andlighter in color.

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 2. Aquaporins and Aquaglyceroporins.Aquaporins allow only the passage of water. Aquaglyceroporinshave a slightly wider pore, which allows for the passage of glycerolin addition to water. Both exclude ions. Aqp3b is anaquaglyceroporin.

Figure 8. Morpolino Oligonucleotides.The Aqp3b morpholino oligonucleotide(MO) binds to the aqp3b mRNA bycomplementary base pairing. The 5'UTRaqp3bMO binds in the 5'UTR of aqp3band blocks ribosome progression, whichprevents translation of the aqp3b mRNA..The morpholino backbone differs fromthe DNA phosphodiester backbone,protecting it from degradation.

Figure 9. Injections.The Aqp3b morpholinooligonucleotide, as wellas the RNA for therescuing protein areinjected into the twodorsal cells of 4-cellembryos. Dorsal cellsare often smaller andlighter in color.

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.

Figure 8. Morpolino Oligonucleotides.The Aqp3b morpholino oligonucleotide(MO) binds to the aqp3b mRNA bycomplementary base pairing. The 5'UTRaqp3bMO binds in the 5'UTR of aqp3band blocks ribosome progression, whichprevents translation of the aqp3b mRNA..The morpholino backbone differs fromthe DNA phosphodiester backbone,protecting it from degradation.

Figure 10. Dorsal Lip of theBlastopore. The dorsal lip of the

blastopore forms as a dark crescent onthe bottom of the embryo, marking the

beginning of gastrulation.

Figure 9. Injections.The Aqp3b morpholinooligonucleotide, as wellas the RNA for therescuing protein areinjected into the twodorsal cells of 4-cellembryos. Dorsal cellsare often smaller andlighter in color.

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 3. Gastrulation.During gastrulation cells fold intothe interior of the embryo to form

the three germ layers(endoderm, mesoderm, and

ectoderm) which lays thefoundation for tissue

differentiation during laterstages.

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.

a

Figure 10. Dorsal Lip of theBlastopore. The dorsal lip of the

blastopore forms as a dark crescent onthe bottom of the embryo, marking the

beginning of gastrulation.

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 3. Gastrulation.During gastrulation cells fold intothe interior of the embryo to form

the three germ layers(endoderm, mesoderm, and

ectoderm) which lays thefoundation for tissue

differentiation during laterstages.

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.

Figure 10. Dorsal Lip of theBlastopore. The dorsal lip of the

blastopore forms as a dark crescent onthe bottom of the embryo, marking the

beginning of gastrulation.

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 3. Gastrulation.During gastrulation cells fold intothe interior of the embryo to form

the three germ layers(endoderm, mesoderm, and

ectoderm) which lays thefoundation for tissue

differentiation during laterstages.

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.

FUTURE WORKFigure 11. Keller Explants.The dorsal mesendoderm islocate just above the dorsal lipof the blastopore. It is removedfrom the embryo by makingcuts on either side of the dorsallip of the blastopore. Theresulting explant is thencultivated under a coverslipand scored the following day.

• We have shown that when Aqp3b is inhibited with amorpholino oligonucleotide, convergent extension ingastrulation is adversely affected. Thus, Aqp3b is requiredfor convergent extension movements of cells.

• Dvl1ΔDIX restores normal convergent extension afterinhibition of Aqp3b, which indicates that Aqp3b acts throughnoncanonical Wnt signaling.

• PKC rescues normal development after inhibition of Aqp3b,demonstrating that Aqp3b influences the Wnt/Ca2+ pathway

•RhoA is capable of rescuing convergent extension defects,showing that Aqp3b may play a part in Wnt/PCP

Our results suggest that aquaporins engage in specificsignaling during convergent extension. This is a significantdeparture from the general picture of aquaporins, which havemainly been considered passive pores that allow bidirectionalpassage of water through cell membranes, depending on theosmotic gradient. Thus, this continued research mayultimately have a very significant impact on understanding therole of aquaporins in cellular processes, such as convergentextension and cell migration.

Figure 3. Gastrulation.During gastrulation cells fold intothe interior of the embryo to form

the three germ layers(endoderm, mesoderm, and

ectoderm) which lays thefoundation for tissue

differentiation during laterstages.

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.

FUTURE WORKFigure 11. Keller Explants.The dorsal mesendoderm islocate just above the dorsal lipof the blastopore. It is removedfrom the embryo by makingcuts on either side of the dorsallip of the blastopore. Theresulting explant is thencultivated under a coverslipand scored the following day.

•We will continue Rescue experiments involving Rac1(Another component within the Wnt/PCP pathway) andcdc42 (Wnt/Ror2). As well as working with the ATGMorpholino Oligonucleotide

Figure 4.Noncanonical Wntsignaling. TheWnt/PCP pathway,involves both RhoAand Rac1. Theseproteins directactivity differently,where RhoAprimarily controls

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.

Figure 11. Keller Explants.The dorsal mesendoderm islocate just above the dorsal lipof the blastopore. It is removedfrom the embryo by makingcuts on either side of the dorsallip of the blastopore. Theresulting explant is thencultivated under a coverslipand scored the following day.

•We will continue Rescue experiments involving Rac1(Another component within the Wnt/PCP pathway) andcdc42 (Wnt/Ror2). As well as working with the ATGMorpholino Oligonucleotide

Figure 4.Noncanonical Wntsignaling. TheWnt/PCP pathway,involves both RhoAand Rac1. Theseproteins directactivity differently,where RhoAprimarily controls

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.

b

ACKNOWLEDGMENTS

Figure 11. Keller Explants.The dorsal mesendoderm islocate just above the dorsal lipof the blastopore. It is removedfrom the embryo by makingcuts on either side of the dorsallip of the blastopore. Theresulting explant is thencultivated under a coverslipand scored the following day.

•We will continue Rescue experiments involving Rac1(Another component within the Wnt/PCP pathway) andcdc42 (Wnt/Ror2). As well as working with the ATGMorpholino Oligonucleotide

Figure 4.Noncanonical Wntsignaling. TheWnt/PCP pathway,involves both RhoAand Rac1. Theseproteins directactivity differently,where RhoAprimarily controls

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.

ACKNOWLEDGMENTS

Figure 11. Keller Explants.The dorsal mesendoderm islocate just above the dorsal lipof the blastopore. It is removedfrom the embryo by makingcuts on either side of the dorsallip of the blastopore. Theresulting explant is thencultivated under a coverslipand scored the following day.

Figure 12. ExplantScoring. The explantsform long narrowprotrusions by convergentextension cell movements.When convergentextension is inhibited, the

I would like to acknowledge The Merzdorf Lab, Montana INBRE, USP,and MAS for funding this project.

Research reported in this publication was supported by the NationalInstitute of General Medical Sciences of the National Institutes ofHealth under Award Number P20GM103474. The content is solely theresponsibility of the authors and does not necessarily represent theofficial views of the National Institutes of Health.

Figure 4.Noncanonical Wntsignaling. TheWnt/PCP pathway,involves both RhoAand Rac1. Theseproteins directactivity differently,where RhoAprimarily controls

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.Figure 12. Explant

Scoring. The explantsform long narrowprotrusions by convergentextension cell movements.When convergentextension is inhibited, the

I would like to acknowledge The Merzdorf Lab, Montana INBRE, USP,and MAS for funding this project.

Research reported in this publication was supported by the NationalInstitute of General Medical Sciences of the National Institutes ofHealth under Award Number P20GM103474. The content is solely theresponsibility of the authors and does not necessarily represent theofficial views of the National Institutes of Health.

Figure 4.Noncanonical Wntsignaling. TheWnt/PCP pathway,involves both RhoAand Rac1. Theseproteins directactivity differently,where RhoAprimarily controls PCP Wnt/Ca2+ Ror2contractile forces and Rac1 is involved in protrusive forces. The Wnt/Ca2+

pathway influences gene expression to facilitate cell migration and tissuestructure.

Figure 17.Convergence. Inexplants withinhibited aqp3bexpression, Werescuedconvergentextension withvariousconstructs, theexplants wereinhibited witheither the aqp3b5’UTR MO (a) oraqp3b ATG MO(b) Out of thosethat extendedproperly, thepercent of thosethat convergedproperly is shown.Figure 12. Explant

Scoring. The explantsform long narrowprotrusions by convergentextension cell movements.When convergentextension is inhibited, the

I would like to acknowledge The Merzdorf Lab, Montana INBRE, USP,and MAS for funding this project.

Research reported in this publication was supported by the NationalInstitute of General Medical Sciences of the National Institutes ofHealth under Award Number P20GM103474. The content is solely theresponsibility of the authors and does not necessarily represent theofficial views of the National Institutes of Health.

contractile forces and Rac1 is involved in protrusive forces. The Wnt/Ca2+

pathway influences gene expression to facilitate cell migration and tissuestructure.

Figure 12. ExplantScoring. The explantsform long narrowprotrusions by convergentextension cell movements.When convergentextension is inhibited, the

I would like to acknowledge The Merzdorf Lab, Montana INBRE, USP,and MAS for funding this project.

Research reported in this publication was supported by the NationalInstitute of General Medical Sciences of the National Institutes ofHealth under Award Number P20GM103474. The content is solely theresponsibility of the authors and does not necessarily represent theofficial views of the National Institutes of Health.protrusion does not form (0 or 1). When only convergence is inhibited,

the protrusion is wide (3).

contractile forces and Rac1 is involved in protrusive forces. The Wnt/Ca2+

pathway influences gene expression to facilitate cell migration and tissuestructure.

protrusion does not form (0 or 1). When only convergence is inhibited,the protrusion is wide (3).