The water-absorption region of ventral skin of several semi-terrestrial and aquatic amphibians - PowerPoint Presentation

Slide1

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

Slide2

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

Slide3

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

F

ound

in urinary

bladder of all study species

F

ound 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

Slide4

Study Species

H

yla

japonica

(tree

frog)

Bufo

marinus (terrestrial toad)

X

enopus laevis(aquatic)

Rana japonica(semi-aquatic)

Rana

nigromaculata(semi-aquatic)

Rana

catesbaiana

a

ka bullfrog

(semi-aquatic)

Slide5

Table 1. Phylogenetics of aquaporins

in ventral pelvic skins of anuran species living in different habitats

Pelvic Skin

Bladder

Habitat

Species

AQP-h2-like

Protein

( Bladder-Type)AQP-h3-Like cDNA(Ventral Pelvic-Type)AQP-h2-Like Protein

(Bladder-Type)Arboreal

Hyla japonica+++

TerrestrialBufo japonica

+++Semi-aquaticRana catesbeiana-++Semi-aquaticRana nigromaculata-++Semi-aquatic

Rana japonica

-++AquaticXenopus laevis-+ (but not expressed)

+

Slide6

Introduction

AQP-x3 mRNA homologous to AQP-h3 expressed in pelvic skin of aquatic species,

Xenopus

laevis

but

not translated

to

proteinHydrins: intermediate peptides derived from a provasotocin-neurophysin precursorStimulate osmotic water movement across skin and bladderOnly present in anurans Have stimulatory effects on water permeability across pelvic skin in Hyla japonica

Slide7

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 speciesExtend observations and compare them with response of Ranid and toad species to AVT

Slide8

Materials and Methods: Immunohistochemistry

4-

m

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

Slide9

Materials and Methods: Western Blot Analysis

I

II

III

S

kin 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 WeightValues

Slide10

Materials and Methods: RT-PCR of Xenopus

Ventral Skin

AQP-x3

RNA

extracted from ventral skin

and reverse transcribed

G

el

electrophoresisDNA Sequenced

Slide11

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) solutionMucosal (outer side) chamber filled with water Water movement from mucosal to serosal side recorded over 30 min with Ringer solution in mucosal chamberFollowed by 30 min of Ringer solution with AVTSkins examined by immuno-fluorescence microscopy to evaluate incorporation of AQPs into apical membrane of First Reacting Cell (FRC) layerFRC layer: continuous barrier between outside and inside of body

Slide12

Materials and Methods: Water Permeability

Effect of AVT on

hindlimb

skin permeability

compared

with

hydrins

1 and 2Skins pretreated with AVT to increase number of AQPs inserted in apical plasma membraneSkins treated with HgCl2Water movement with continued AVT treatment measured for additional 30 minResults from 5 or 6 individuals expressed as meansStatistical Analysis: data compared by Steel-Dwass’s test using software

Slide13

Results: A

quaporins

in

3

skin regions

Rana

japonica and Rana nigromaculata: AQP-h3 (skin-type) in hindlimb region onlyRana japonica: in basolateral, apical, and cytoplasm of FRCRana nigromaculata: basolateral plasma membraneRana japonica

Rana

nigromaculata

Slide14

Results: Aquaporins in 3 skin regions

Rana

catesbeiana

:

G

reatest

AQP-h3 in hindlimb Present in small number pelvic skin cells In hindlimb and pelvic skin, localized in basolateral plasma membrane in FRC layerIn pectoral region, dot spot only in cytoplasm of few cells in FRC layer Intensity of labeling decreased from hindlimb to pectoral skinHindlimb

Pelvic

Pectoral

Slide15

Results: Aquaporins in 3 skin regions

Skin-type

AQP-h3

Hindlimb

Bladder-type

AQP-h2PectoralPelvis

B.

marinus

:

AQP-h3 and AQP-h2 in all regionsPredominantly in cytoplasm just beneath apical membraneNumber of cells varied among toads (less in pectoral skin of some)Western Blot: Intensity of bands decreased from hindlimb to pectoral skin

Slide16

Results: aquaporins in 3

skin regions

Xenopus

laevis

:

Detected

AQP-x3 mRNA expression in skin from pectoral, pelvic, and

hindlimb regions but not dorsal skinX. laevis skin not stimulated by AVT

Slide17

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 layerRana japonica Rana nigromaculata

Slide18

Bullfrog: S

timulation

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 regionResults: Water permeability and movement of AQPs after stimulation with AVThindlimb pelvicpectoral

Slide19

B. marinus:

S

timulation

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 regionsResults: Water permeability and movement of AQPs after stimulation with AVT

Slide20

Results: Water permeability and movement of AQPs after stimulation with AVT

Skin-type

AQP-h3

Bladder-

type

AQP-h2

Hindlimb

Pelvic

Pectoral

For

Bufo marinus

Slide21

Results: Water permeability and dynamic movement of AQPs after stimulation of AVT

and

h

ydrins

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 japonica15 X in Rana nigromaculata8–12 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)

Slide22

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 regionsRana japonica and Rana nigromaculata: AQP-h3-like AQP observed only in hindlimbGreater response of Toad vs. Rana species in vivo could result from relative area of skin that contains AQPs rather than an area-specific responseHgCl2 inhibited water flux across hindlimb skin under AVT-stimulation.AQP proteins are mercury sensitive, so this proves waterflux

was mediated by AQPs

Slide23

Discussion: Physiological and behavioral variables that affect water absorption

V

ariable area-specific

water flux across toad

skin could result from greater dependence on vascular perfusion relative to thinner frog

skin

Behavioral

water absorption responseSkin pressed to moist surfaceLarge increase in blood flow to absorbing area of seat patchInsertion of AQPs into apical membranes of FRC skin layer

Slide24

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 patchesApomorphic (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

Slide25

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 lineageFor contemporary anurans h2-like AQPa2 occurs in bladder, while h3-like AQPa2 is expressed in ventral skinIn 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 bladderMight give terrestrial species an advantage: cutaneous water absorption / adaptiaton to drier environments

Slide26

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

Slide27

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 receptorBoth peptides generated from down-regulation in post-translational processingXenopus laevis : secretes hydrin 1 and AVT but shows no hydro-osmotic response to either in skinXenopus laevis : AVT and hydrin 1 stimulate water reabsorption from bladder May be involved in water balance during aestivation

Slide28

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 skinSemi-aquatic Ranids express only AQP-h3 in skin, primarily in ventral surface of hindlimbsAquatic Xenopus laevis transcribes mRNA for homologs of both isoforms but a C-terminal sequence prevents translationFuture studies needed to examine species differences in expression of AQP-h2 and AQP-h3 to examine phylogenetic relationships associated with water balance adaptations