Introduction

1. The water-absorption region of ventral skin of several semi-terrestrial and aquatic amphibians identified by aquaporins Yuji Ogushi, Azumi Tsuzuki, Megumi Sato, Hiroshi Mochida, Reiko Okada, Masakazu Suzuki, Stanley D. Hillyard and Shigeyasu Tanaka

2. Introduction Semi-terrestrial water balance strategy • Used by many tree frog and toad species • Use ventral pelvic patch to absorb water cutaneously • Capillaries contact basement membrane beneath epithelium • Store dilute urine in bladder for re-absorption while foraging far from water • Aquaporins (AQPs): plasma membrane proteins forming water channels into cells (present in almost all organisms) • Control water permeability across membranes • Stimulated by argininevasotocin(AVT): causes fusion of vesicles containing AQPs with apical membrane of epithelial water absorption/reabsorption tissues

3. Introduction • Researchers used Real Time Polymerase Chain Reaction (RT-PCR) to identify 2 forms of AQP in epithelial tissues • AQP-h2 (isoform) • Termed “urinarybladder-type” AQP • Found in urinary bladder of all study species • Found in pelvic skin region of toad and tree frog • AQP-h3 (isoform) • Termed “ventral skin-type” AQP • Found in skin but not bladder of tree frogs, toads andRanaspecies

4. Study Species Hyla japonica (treefrog) Bufomarinus (terrestrial toad) Xenopuslaevis (aquatic) Ranacatesbaiana aka bullfrog (semi-aquatic) Rana japonica (semi-aquatic) • Rananigromaculata • (semi-aquatic)

5. Table 1. Phylogenetics of aquaporins in ventral pelvic skins of anuran species living in different habitats

6. Introduction • AQP-x3 mRNA homologous to AQP-h3 expressed in pelvic skin of aquatic species, Xenopuslaevis • but not translated toprotein • Hydrins: intermediate peptides derived from a provasotocin-neurophysin precursor • Stimulate osmotic water movement across skin and bladder • Only present in anurans • Have stimulatory effects on water permeability across pelvic skin in Hylajaponica

7. Objectives • Examine relationship between AQP distribution in apical membranes and ATV stimulation of water permeability in hindlimb, pelvic and pectoral zones of ventral skin • Examine expression of AQP-x3 mRNA in skin of hindlimb, pelvic, pectoral, dorsal regions • Different patterns of regional specialization present in terrestrial, arboreal, and semiaquatic species • Extend observations and compare them with response of Ranid and toad species to AVT

8. Materials and Methods: Immunohistochemistry • 4-mm sections of ventral skin mounted on slides • Reacted with fluorescent labeled anti-bodies • Nuclei stained with DAPI (appear blue) • Pelvic skin type AQP proteins (AQP-h3) stained using Alexa Fluor 488 (appears green) • Urinary bladder-type AQP proteins (AQP-h2) stained using Cy3 (appears red) • Specimens examined with microscope equipped with fluorescence attachment

9. Materials and Methods: Western Blot Analysis • Skin from hind-limb (I), pelvic (II) and pectoral (III) regions removed and homogenized • Proteins separated via gel electrophoresis, transferred to membrane, and probed (detected) using antibodies III II kDa I II III Protein Molecular Weight Values I

10. Materials and Methods: RT-PCR of Xenopus Ventral Skin AQP-x3 • RNA extracted from ventral skin and reverse transcribed • Gel electrophoresis • DNA Sequenced

11. Materials and Methods: Water Permeability • Skin from pectoral, pelvic, and hindlimb regions mounted between two chambers connected by a small opening • Chamber on serosal (inner)side of skin filled with Ringer (salt) solution • Mucosal (outer side) chamber filled with water • Water movement from mucosal to serosal side recorded over 30 min with Ringer solution in mucosal chamber • Followed by 30 min of Ringer solution with AVT • Skins examined by immuno-fluorescence microscopy to evaluate incorporation of AQPs into apical membrane of First Reacting Cell (FRC) layer • FRC layer: continuous barrier between outside and inside of body

12. Materials and Methods: Water Permeability • Effect of AVT on hindlimb skin permeability compared with hydrins1 and 2 • Skins pretreated with AVT to increase number of AQPs inserted in apical plasma membrane • Skins treated with HgCl2 • Water movement with continued AVT treatment measured for additional 30 min • Results from 5 or 6 individuals expressed as means • Statistical Analysis: data compared by Steel-Dwass’s test using software

13. Results:Aquaporinsin 3 skin regions Ranajaponica and Rananigromaculata: • AQP-h3 (skin-type) in hindlimb region only • Ranajaponica: inbasolateral, apical, and cytoplasm of FRC • Rananigromaculata: basolateral plasma membrane Ranajaponica Rananigromaculata

14. Results:Aquaporins in 3 skin regions Ranacatesbeiana: • GreatestAQP-h3 in hindlimb • Present in small number pelvic skin cells • In hindlimb and pelvic skin, localized in basolateral plasma membrane in FRC layer • In pectoral region, dot spot only in cytoplasm of few cells in FRC layer • Intensity of labeling decreased from hindlimb to pectoral skin Pelvic Pectoral Hindlimb

15. Results:Aquaporins in 3 skin regions B. marinus: • AQP-h3 and AQP-h2 in all regions • Predominantly in cytoplasm just beneath apical membrane • Number of cells varied among toads (less in pectoral skin of some) • Western Blot: Intensity of bands decreased from hindlimb to pectoral skin Pectoral Hindlimb Pelvis Skin-type AQP-h3 Bladder-type AQP-h2

16. Results: aquaporinsin 3 skin regions Xenopuslaevis: • Detected AQP-x3 mRNA expression in skin from pectoral, pelvic, and hindlimb regions but not dorsal skin • X. laevisskin not stimulated by AVT

17. Results: Water permeability and movement of AQPs after stimulation with AVT Ranajaponica and Rananigromaculata: • Stimulation at hindlimb • AQP-h3 in apical plasma membrane in FRC layer Ranajaponica Rananigromaculata

18. Results: Water permeability and movement of AQPs after stimulation with AVT Bullfrog: • Stimulation increased in order of pectoral, pelvic, hindlimbregions • Translocation of AQP-h3 protein to apical plasma membrane of FRC layer greater in hindlimb region and decreased in pelvic and pectoral region hindlimb pectoral pelvic

19. Results: Water permeability and movement of AQPs after stimulation with AVT B. marinus: • Stimulation variable depending on individuals and regions of skin but above controls • ½ of toads: response greatest in hindlimb, declined in pelvic and pectoral skin • Other ½: response greatest in pelvic skin • Translocationof AQP-h3 and AQP-h2 to apical plasma membrane of cells in FRC layer of hindlimb, pelvic, and pectoral regions

20. Results: Water permeability and movement of AQPs after stimulation with AVT For Bufo marinus Hindlimb Pelvic Pectoral Skin-type AQP-h3 Bladder- type AQP-h2

21. Results: Water permeability and dynamic movement of AQPs after stimulation of AVT and hydrins • AVT and hydrin 1 and 2 increased water permeability of hindlimb skin in R. japonica > R. nigromaculata > R. catesbeina > B. marinus • No differences among hormone response within species • Increased water flux rates (relative to controls): • 30–38 X in Ranajaponica • 15 X in Rananigromaculata • 8–12 X in Ranacatesbeina • 3 or 4 X in Bufo marinus • When hindlimb skin from each species stimulated with AVT following HgCl2 treatment, ratio of water flux decreased (compared with AVT stimulation groups)

22. Discussion: Importance of AQP-rich hindlimbs for water absorption • Area-specific rate of AVT-stimulated water flow across hindlimb skin similar for moist and dry-adapted species • Toad: AVT-stimulated water flow correlated with presence of AQP-h2-like water channel in all skin regions • RanaCatesbeiana: AQP-h3-like AQP observed in all skin regions • Ranajaponica and Rananigromaculata: AQP-h3-like AQP observed only in hindlimb • Greater response of Toadvs. Ranaspecies in vivo could result from relative area of skin that contains AQPs rather than an area-specific response • HgCl2 inhibited water flux across hindlimb skin under AVT-stimulation. • AQP proteins are mercury sensitive, so this proves waterflux was mediated by AQPs

23. Discussion: Physiological and behavioral variables that affect water absorption • Variable area-specific water flux across toadskin could result from greater dependence on vascular perfusion relative to thinner frog skin • Behavioral water absorption response • Skin pressed to moist surface • Large increase in blood flow to absorbing area of seat patch • Insertion of AQPs into apical membranes of FRC skin layer

24. Discussion: Phylogenetic significance of AQPs in ventral pelvic skin • Largest superfamilies of anurans are Hyloidea (includes modern tree frog and toad species) and Ranoidea (includes Ranids (typical frogs) • AQP-h2-like proteins not only in bladder, but in skin of tree frog and toad species, which also have more pelvic patches • Apomorphic (only these lineages have this character) • AQP-h3 found in toad, tree frog, and Ranid species • Pleisiomorphic (likley shared with common ancestors) • Present in all ventral skin regions of RanaCatesbeiana, while only present in hindlimbs of Ranajaponica andRananigromaculata • “New World” Rana genus recently reclassified as Lithobates, including RanaCatesbeiana • Ranajaponica andRananigromaculataremain in “Old World” Rana genus

25. Discussion: Expression of 2 AVT-stimulated AQPs in skin of toad and tree frog species • AQP-h2 homolog detected in bladder of all species examined but in skin of only toad and tree frog species • mRNA encoding AQP-h3 homolog identified in skin but not bladder of all species examined • Based on genetic analyses of Xenopustropicalis, likely that h2- and h3-like AQPa2 genes were generated by local gene duplication of AQP2 in anuran lineage • For contemporary anurans h2-like AQPa2 occurs in bladder, while h3-like AQPa2 is expressed in ventral skin • In toad and tree frog species, h2-like AQPa2 gene may have undergone a change to express this gene in the ventral skin, not just the bladder • Might give terrestrial species an advantage: cutaneous water absorption / adaptiaton to drier environments

26. Discussion: A unique AQP in aquatic Xenopus • No hydro-osmotic response to AVT • Identified mRNA for AQP-x3 in pelvic skin homologous to that for AQP-h3, but contains extra C-terminal tail preventing translation • AQP-x3 present in all 3 skin regions • Data lacking on possibility of expression during dry periods

27. Discussion: Regulation of AQP expression by AVT and related peptides • Hydrin 1 and 2 stimulated water permeability of hindlimb skin of toad and tree frog species at level equivalent to AVT • Km values for cAMP production by tree frog V2-type AVT receptor suggests hydrin 1 and 2 share a common receptor • Both peptides generated from down-regulation in post-translational processing • Xenopuslaevis: secretes hydrin 1 and AVT but shows no hydro-osmotic response to either in skin • Xenopuslaevis: AVT and hydrin 1 stimulate water reabsorption from bladder • May be involved in water balance duringaestivation

28. Perspectives and Significance • Anurans have 2 AQP isoforms stimulated by AVT to increase water absorption across ventral skin and re-absorption from bladder • All species examined express AQP-h2-like AQPs in bladder • Only semi-terrestrial toadand tree-frog species express AQP-h3-like AQPs and AQP-h2-like AQPs in skin • Semi-aquatic Ranids express onlyAQP-h3 in skin, primarily in ventral surface of hindlimbs • Aquatic Xenopuslaevistranscribes mRNA for homologs of both isoforms but a C-terminal sequence prevents translation • Future studies needed to examine species differences in expression of AQP-h2 and AQP-h3 to examine phylogeneticrelationships associated with water balance adaptations