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 arginine vasotocin (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 “urinary bladder-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 and Rana species

4. Study Species Hyla japonica (tree frog) Bufo marinus (terrestrial toad) Xenopus laevis (aquatic) Rana japonica (semi-aquatic) Rana nigromaculata (semi-aquatic) Rana catesbaiana aka bullfrog (semi-aquatic)

5. Table 1. Phylogenetics of aquaporins in ventral pelvic skins of anuran species living in different habitats Pelvic SkinBladder HabitatSpecies AQP-h2-like Protein ( Bladder- Type) AQP-h3-Like cDNA (Ventral Pelvic- Type) AQP-h2-Like Protein (Bladder-Type) ArborealHyla japonica +++ TerrestrialBufo japonica +++ Semi-aquaticRana catesbeiana -++ Semi-aquaticRana nigromaculata -++ Semi-aquaticRana japonica -++ AquaticXenopus laevis -+ (but not expressed) +

6. Introduction AQP-x3 mRNA homologous to AQP-h3 expressed in pelvic skin of aquatic species, Xenopus laevis – but not translated to protein 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 Hyla japonica

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-  m 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 I II III 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 kDa I II III Protein Molecular Weight Values

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 hydrins 1 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: Aquaporins in 3 skin regions Rana japonica and Rana nigromaculata: AQP-h3 (skin-type) in hindlimb region only Rana japonica: in basolateral, apical, and cytoplasm of FRC Rana nigromaculata: basolateral plasma membrane Rana japonicaRana nigromaculata

14. Results: Aquaporins in 3 skin regions Rana catesbeiana: Greatest AQP-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 Hindlimb Pelvic Pectoral

15. Results: Aquaporins in 3 skin regions Skin-type AQP-h3 Hindlimb Bladder-type AQP-h2 Pectoral Pelvis 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

16. Results: aquaporins in 3 skin regions Xenopus laevis: Detected AQP-x3 mRNA expression in skin from pectoral, pelvic, and hindlimb regions but not dorsal skin X. laevis skin not stimulated by AVT

17. Results: Water permeability and movement of AQPs after stimulation with AVT Rana japonica and Rana nigromaculata: Stimulation at hindlimb AQP-h3 in apical plasma membrane in FRC layer Rana japonicaRana nigromaculata

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

19. 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 Translocation of AQP-h3 and AQP-h2 to apical plasma membrane of cells in FRC layer of hindlimb, pelvic, and pectoral regions Results: Water permeability and movement of AQPs after stimulation with AVT

20. Skin-type AQP-h3 Bladder- type AQP-h2 Hindlimb PelvicPectoral For Bufo marinus

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 Rana japonica – 15 X in Rana nigromaculata – 8–12 X in Rana catesbeina – 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 Rana Catesbeiana: AQP-h3-like AQP observed in all skin regions Rana japonica and Rana nigromaculata: AQP-h3-like AQP observed only in hindlimb Greater response of Toad vs. Rana species in vivo could result from relative area of skin that contains AQPs rather than an area-specific response HgCl 2 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 toad skin 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 Rana Catesbeiana, while only present in hindlimbs of Rana japonica and Rana nigromaculata “New World” Rana genus recently reclassified as Lithobates, including Rana Catesbeiana Rana japonica and Rana nigromaculata remain 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 Xenopus tropicalis, 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 Xenopus laevis : secretes hydrin 1 and AVT but shows no hydro-osmotic response to either in skin Xenopus laevis : AVT and hydrin 1 stimulate water reabsorption from bladder – May be involved in water balance during aestivation

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 toad and tree-frog species express AQP- h3-like AQPs and AQP-h2-like AQPs in skin Semi-aquatic Ranids express only AQP-h3 in skin, primarily in ventral surface of hindlimbs Aquatic Xenopus laevis transcribes 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 phylogenetic relationships associated with water balance adaptations