The water-absorption region of
ventral skin of several semiterrestrial and aquatic amphibians
identified by aquaporins
Yuji Ogushi, Azumi Tsuzuki, Megumi
Sato, Hiroshi Mochida, Reiko Okada,
Masakazu Suzuki, Stanley D. Hillyard
and Shigeyasu Tanaka
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
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
Study Species
Hyla japonica
(tree frog)
Rana japonica
(semi-aquatic)
Bufo marinus
(terrestrial toad)
Xenopus laevis
(aquatic)
Rana nigromaculata
(semi-aquatic)
Rana catesbaiana
aka bullfrog
(semi-aquatic)
Table 1. Phylogenetics of aquaporins in
ventral pelvic skins of anuran species living
in different habitats
Habitat
Species
Arboreal
Hyla japonica
Terrestrial
Bufo japonica
Semi-aquatic
Rana catesbeiana
Semi-aquatic
Rana nigromaculata
Semi-aquatic
Rana japonica
Aquatic
Xenopus laevis
Pelvic Skin
Bladder
AQP-h2-like
AQP-h3-Like
Protein
cDNA
( Bladder- (Ventral PelvicType)
Type)
AQP-h2-Like
Protein
(Bladder-Type)
+
+
+
+
+
+
-
+
+
+
+
-
+
+ (but not
expressed)
+
+
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
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
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
Materials and Methods:
Western Blot Analysis
III
II
I
• 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
Protein
Molecular
Weight
Values
I II III
Materials and Methods:
RT-PCR of Xenopus Ventral Skin AQP-x3
• RNA extracted from ventral skin and reverse
transcribed
• Gel electrophoresis
• DNA Sequenced
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
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 SteelDwass’s test using software
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 japonica
Rana nigromaculata
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
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
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
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 japonica
Rana nigromaculata
Results: Water permeability and movement
of AQPs after stimulation with AVT
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
hindlimb
pelvic
pectoral
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
• 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
For Bufo marinus
Hindlimb
Skin-type
AQP-h3
Bladdertype
AQP-h2
Pelvic
Pectoral
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)
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 AQPh2-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
• HgCl2 inhibited water flux across hindlimb skin under AVTstimulation.
– AQP proteins are mercury sensitive, so this proves waterflux
was mediated by AQPs
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
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
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 h2and 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
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
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 posttranslational 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
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 AQPh3-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