Transthyretin expression evolved more recently in liver than in brain

Comp. Biochem. Physiol. Vol. 105B, No. 2, pp. 317-325, 1993 0305-0491/93 $6.00 + 0.00 Printed in Great Britain © 1993 Pergamon Press Lid

TRANSTHYRETIN EXPRESSION EVOLVED MORE RECENTLY IN LIVER THAN IN BRAIN

GERHARD SCHREIBER,* TOM M. PETTERSSON, BRIDGET R. SOUTHWELL, ANGELA R. ALDRED, PAUL J. HARMS, SAMANTHA J. RICHARDSON, RICHARD E. H. WETTENHALL, WEI DUAN and

STEWART C. N1COL~" The Russell Grimwade School of Biochemistry, University of Melbourne, ParkviUe, 3052, Australia (Tel: 61-3-344 7955; Fax: 61-3-3477730); and tDepartment of Physiology, University of Tasmania,

Sandy Bay, Hobart, 7001, Australia

(Received 21 October 1992; accepted 27 November 1992)

Abstract--1. Transthyretin was found to be synthesized and secreted by choroid plexus from rats, echidnas, and lizards, but not toads.

2. Transthyretin was observed in blood from placental mammals, birds, and marsupials, but not reptiles and monotremes.

3. The obtained data suggest that transthyretin synthesis by the liver evolved independently in the lineage leading to the placental mammals and marsupials and in that leading to the birds.

4. It is proposed that transthyretin gene expression in mammalian liver appeared about 200 million years later than its first occurrence in the choroid plexus of the stem reptiles.

INTRODUCTION

Thyroid hormones reach their targets within cells from the surrounding extracellular fluid. In this fluid, they are transported either as free or protein-bound hormones. The two plasma proteins which bind thyroxine with high affinity (Ka > 10SM -1) are thyroxine- binding globulin and transthyretin. Thyroxine- binding globulin is not found in the blood of birds and smaller mammals (Larsson et al., 1985). Albumin, present already in the fishes (Byrnes and Gannon, 1990), binds thyroxine with low affinity (Tabachnick, 1967).

For humans and rats, it is well documented that thyroid hormone-binding plasma proteins are synthesized and secreted by the liver (for review see Schreiber, 1987). However, much higher concentrations of transthyretin mRNA were found in choroid plexus than in liver from rats (Schreiber et al., 1990) and chickens (Duan et al., 1991). The epithelial cells of the choroid plexus constitute the blood-cerebrospinal fluid barrier, which is part of the blood-brain barrier (Davson et al., 1987). The choroid plexus produces most of the cerebrospinal fluid surrounding the brain (Cserr, 1971). The transthyretin synthesized by the choroid plexus epithelial cells is secreted exclusively towards the brain (Schreiber et al., 1990), and is proposed to be involved in the transport of thyroid hormones from the bloodstream to the brain (Dickson et al., 1987; Schreiber et al., 1990). A protein, similar to that

*To whom correspondence should be addressed.

identified for rats (Dickson and Schreiber, 1986) as transthyretin, was also found to be synthesized and secreted by in vitro incubated choroid plexus from cattle, pigs, rabbits, guinea pigs, mice, rats, lizards and chickens (Harms et al., 1991). It is composed of subunits of a molecular weight of about 15,000, and its rate of synthesis and secretion by the choroid plexus greatly exceeds that of all other secreted proteins. The wide distribution among species and the strict conservation of the domains of transthyretin involved in thyroxine binding (Duan et al., 1991) suggest an important and early role during evolution for the transthyretin-mediated transport of thyroxine in the brain. Here, it was investigated whether the choroid plexus from echidnas and Eastern grey kangaroos synthesizes and secretes transthyretin or contains the mRNA coding for this protein.

The proteins in blood plasma are synthesized and secreted by the liver (for review see Schreiber, 1987). Therefore, transthyretin levels in blood plasma reflect transthyretin synthesis in liver. Transthyretin was found in the blood of 12 species of placental mammals (Larsson et al., 1985), and in chickens (Duan et al., 1991). In order to determine the time of the first appearance of transthyretin gene expression in the liver during evolution, the distribution of transthyretin in blood plasma was studied for representa- tive species for all four extant Orders of Reptilia, two monotreme, one marsupial, and two placental mammal species. The results of this study, and previously obtained data, were then related to the fossil record in the construction of a phylogenetic tree for the expression of the transthyretin gene in the brain and in the liver.

317

318 GERHARD SCHREIBER etal.

MATERIALS AND METHODS

Animals and tissues

The animal species studied were rat (Rattus norvegicus), Buffalo strain; sheep (Ovis aries), Merino crossbred; domestic chicken (Gallus domesticus), White leghorn/Black Australorp crossbred; Eastern grey kangaroo (Macropus giganteus); echidna ( Tachyglossus aculeatus ); platypus ( Ornithorhynchus anatinus ); salt-water crocodile ( Crocodylus porosus ); shingle-back or stumpy-tailed lizard (Tiliqua rugosa); Krefft's tortoise (Emydura krefftii); tuatara ( Sphenodon punctatus ) and cane toad ( Bufo marinus ). Only adult animals were used.

Rats were from the Biochemistry Department, University of Melbourne. They were killed by CO 2 inhalation. Sheep plasma, chicken plasma and tissues were obtained from local abattoirs. Stumpy-tailed lizards were collected from the wild in Victoria under permits Y20992, and RP-92-059 from the Victorian Department of Conservation and Environment. Lizards were anaesthetized by intraperitoneal injec- tion of sodium phenobarbitone, 75 mg/kg body weight, and killed by inducing a pneumothorax. Cane toads were collected in Queensland, Australia, and imported under permit 91-C-1. Toads were anaesthetized by transcutaneous absorption of benzocaine from a 0.02% solution in HzO, and then killed by decapitation. Echidna plasma and choroid plexus and liver were obtained under permit from the Tasmanian Department of Parks, Wildlife and Heritage. Platy- pus plasma was provided by Dr K. Handasyde, Zoology Department, University of Melbourne. Kangaroo plasma, liver and choroid plexus were obtained from the Royal Zoological Gardens, Melbourne. Crocodile plasma and liver were obtained from a commercial crocodile farm (C. Webb P. L.), Darwin, Northern Territory, Australia and imported to Victoria under permit RP-91-021 from the Victorian Department of Conservation and Environment. Plasma from the Krefft's tortoise was provided by Dr R. Booth, Healesville Sanctuary, Victoria. Tuatara plasma and liver were from Drs A. Cree, C. Kray and C. Daugherty, Victoria University of Wellington, New Zealand, and imported under permit 16844 from the Australian National Parks and Wildlife Service. Tissues were removed within 15 min after death. For RNA preparation, tissues were frozen in liquid nitrogen immediately after dissection and stored at -70°C.

Materials

Sodium phenobarbitone was purchased from Bomac Laboratory, New South Wales, [ct-32p]dATP (3 Ci//~mol) and L-[UJ4C]leucine (350 Ci/mol) were from Amersham, Sepharose 4B from Pharmacia and GeneScreen Plus membrane from Dupont NEN Research Products. The sources of other chemicals and enzymes were as described previously (Larsson et al., 1985; Harms et al., 1991; Duan et al., 1991).

Two-dimensional gel electrophoresis for the analysis of proteins in plasma from various species

Transthyretin from 0.5 mi plasma from rats, East- ern grey kangaroos, echidnas, platypus, domestic chickens, salt-water crocodiles, stumpy-tailed lizards and tuataras was enriched by precipitating other plasma proteins with phenol, essentially as described by De Nayer et al. (1966). Briefly, a phenol solution (4%) was added dropwise to an equal volume of plasma containing NaC1 (200 g/I) at 0°C with vigor- ous mixing. This resulted in the precipitation of most of the serum albumin. Samples of 30/d of the resulting supernatant were dialysed, concentrated and then separated by electrophoresis in two dimen- sions. The electrophoresis was carried out under non-denaturing conditions (10% polyacrylamide gel, pH 8.6) in the first dimension, and under denaturing conditions (0.1% SDS, gradient of 10-15% polyacrylamide gel) in the second dimension with a discontinuous buffer system, according to Laemmli (1970). After electrophoresis in the first dimension, gel slices corresponding to whole lanes were cut out from the gel, incubated in 4% SDS, 0.1 M Tris-HCl, pH 8.6, for 10 min at room temperature, or boiled for 10 min in the same solution. The proteins were then separated in the second gel under denaturing conditions. Gels were finally fixed in 10% trichloroacetic acid and stained with Coomassie Brilliant Blue R-250.

Isolation of proteins with electrophoretic mobility similar to transthyretin

For the isolation of proteins, which migrated with a mobility similar to transthyretin, from plasma or from the supernatant after phenol extraction, ali- quots of 100 # 1 of plasma or phenol-extracted protein solution were analysed by electrophoresis at pH 8.6 in a non-denaturing polyacrylamide gel with a gradient of 10-15% acrylamide. The regions of the gels expected to contain transthyretin (anodal to albumin) were cut out and proteins in the gel slices were electroeluted in an ISCO electroelution apparatus.

Isolation of human retinol-binding protein and affinity chromatography of transthyretin

Retinol-binding protein was isolated from human plasma by the method described by Fex and Hanson (1979). The retinol-binding protein was coupled to CNBr-activated Sepharose 4B according to the man- ufacturer's (Pharmacia) instructions. The obtained retinol-binding protein-Sepharose was then used for affinity chromatography of transthyretin, as described previously (Larsson et al., 1985).

Analysis of protein synthesis and secretion by choroid plexus

Choroid plexus, freshly removed from the third and fourth ventricles of lizards, echidnas, rats and cane toads, were incubated in leucine-free Ham's

Evolution of transthyretin expression

F-12 medium with L-[U-14C]leucine, 10 #Ci/ml incubation medium, for up to 8 hr with shaking. Choroid plexus from rats and echidnas were incubated at 37°C, while choroid plexus from lizards and cane toads were incubated at 25°C. Radioactive proteins in the incubation medium (10,000 cpm in protein; 10 to 50#1 volume) were analysed by electrophoresis in 14% SDS-polyacrylamide gels, using 4.5% polyacrylamide stacking gels and the discontinuous buffering system of Laemmli (1970). Following electrophoresis, gels were fixed with 40% methanol, 12% acetic acid, stained with Coomassie Brilliant Blue, impregnated with scintillant (Amplify, Amersham), dried and then fluorographed at -70°C for 7 days using Kodak X-AR film.

Analysis o f the N-terminal amino acid sequence of proteins

Proteins migrating faster than albumin in electrophoresis were purified by reversed phase HPLC, using a Vydac wide pore C4 column (0.21 cm x 10cm), developed with a continuous gradient of acetonitrile in aqueous trifluoroacetic acid (1:1000, v:v), as described previously (Achen et al., 1992), and their N-termini were sequenced. Automated Edman degradation of the protein immobilized in a Bioprene (Applied Biosystems) matrix was carried out on an Applied Biosystems model 477A gas-liquid-phase sequenator (Hewick et al., 1981) and followed by on-line quantitative HPLC analysis of phenylthio- hydantoin derivatives (Zimmerman et aL, 1977), using an Applied Biosystems model 120 HPLC analyser.

Binding of thyroxine to plasma proteins

Plasma (10 #1) was incubated with 2.5 nCi L-[3',5'- 125I]thyroxine (0.58fmol, purified as described by Mendel et al., 1989), at room temperature for 30 min before being subjected to non-denaturing 10% polyacrylamide gel electrophoresis in 0.05 M Tris-glycine, pH 8.6, 4°C. Electrophoresis of samples was carried out in duplicate, for autoradiography and staining by Coomassie Blue R-250.

319

RESULTS

Synthesis and secretion of proteins by echidna choroid plexus

Choroid plexus from rat, echidna, stumpy-tailed lizard and cane toad were incubated in vitro with medium containing [~4C]leucine. Samples of incubation medium were analysed, as described in Materials and Methods, by SDS--gel electrophoresis. One major radioactive band was present in samples of medium containing protein secreted by choroid plexus from rat, echidna and lizard (Fig. 1). The polypeptide corresponding to this band was much more abundantly synthesized than all other secreted polypeptides. This polypeptide has been identified previously as the subunit of transthyretin, in rats, sheep and chickens (Dickson et al., 1987; Schreiber et al., 1990; Duan et al., 1991; Harms et al., 1991). The protein with a molecular weight of about 20,000 secreted by cane toad choroid plexus has been shown by amino acid and nucleotide sequencing not to be related to transthyretin (Achen et al., 1992).

M W x 10 "3

Purification of RNA and Northern analysis

RNA was isolated from the livers from two echidnas, two kangaroos and one sheep, and from choroid plexus from two kangaroos, as described previously (Tu et al., 1990). RNA samples were separated in 1.5% agarose gel containing formaldehyde, transferred to GeneScreen Plus membrane, then hybridized with 32p-labelled sheep transthyretin cDNA (Tu et al., 1989) at 35°C. The membrane was washed twice for 5 min at 25°C in 0.3M NaCI, 0.3M trisodium citrate containing 1% SDS, then twice for 30min at 35°C in 0.3 M NaCI, 0.3 M trisodium citrate, 1% SDS. The filter was exposed to Kodak X-AR film at - 70°C for 14 days.

Fig. 1. Electrophoretic analysis of proteins synthesized and secreted by choroid plexus. Fluorograph of proteins synthesized and secreted by in vitro incubated choroid plexus from rat, echidna, stumpy-tailed lizard and cane toad. Choroid plexus pieces were incubated for 6 hr with medium containing [t4C]leucine, as described in Materials and Methods. Samples of incubation medium were analysed by electrophoresis in polyacrylamide gels containing SDS, followed by fluorography. The position of the transthyretin subunit is labelled with an arrow and "TTR". The protein with an apparent molecular weight of about 20,000, secreted by cane toad choroid plexus, has been shown by amino acid and nucleotide sequencing of its eDNA not to be related to transthyretin (Aehen et al., 1992). Molecular weight markers were phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,000), and ~t-lactalbumin

(14,000).

320 GERHARD SCHREIBER et al.

Presence of transthyretin in the plasma of vertebrate species

The supernatants obtained from 0.5ml blood plasma, after removing albumin by phenol precipitation, from a placental mammal (rat), a marsupial (Eastern grey kangaroo), two monotreme species (echidna and platypus), an avian species (domestic chicken) and three reptilian species (salt-water crocodile, stumpy-tailed lizard and tuatara) were analysed by two-dimensional polyacrylamide gel electrophoresis. The identification of transthyretin using this electrophoretic separation has been described previously in detail for human transthyretin (Pettersson et al., 1987). Proteins with electrophoretic mobility similar to the transthyretin monomer were found in plasma from all species (Fig. 2). The protein labelled "TTR" in rat and chicken plasma was assumed to be transthyretin based on the following characteristics: migration anodal to albumin and dissociation pattern of the tetramer into dimers and monomers depending on temperature and presence of detergent (Pettersson et al., 1987); binding to thyroxine (data not shown), and binding to retinol-binding protein (coupled to Sepharose 4B).

In plasma from the Eastern grey kangaroo, a multimeric protein migrated to the same positions as the monomers, dimers and the tetramers of transthyretin. It was present in similar amounts as

transthyretin in plasma from placental mammals and birds. The area of the gel containing the protein was excised. The protein was eluted and identified as transthyretin by determination of the amino acid sequence from the N-terminus. This was val-his-his- glu-ser-glu-his-ser-lys-_-pro-leu-met -val-lys-val-leu- leu-ala, i.e. similar to that of other mammalian transthyretins, from the eighth amino acid onwards (Fig. 3).

Only very small amounts of a protein with an electrophoretic mobility and subunit size similar to that of transthyretin could be demonstrated in echidna plasma after enrichment by phenol extraction. Under non-denaturing conditions, this protein was present as a tetramer. Upon denaturation, it dissociated into monomers, similarly to known transthyretins. It was also found to bind to human retinol-binding protein (coupled to Sepharose), a feature characteristic of transthyretins. For plasma from the platypus and the reptiles (Fig. 2), faint spots were present in the area of the gel where transthyretin was expected to be found. The areas of the gel containing each of these spots were excised and analysed further to determine the identity of proteins, The proteins in the spot labelled "d" in lizard plasma were recovered from the gel, purified by reversed- phase HPLC and analysed by N-terminal amino acid sequencing. Five polypeptides were obtained by HPLC. The N-terminal amino acid sequences of two

m . ~ v

It- .

Fig. 2. Analysis of proteins in plasma from various species. Analysis of plasma proteins from a placental mammal (rat), a marsupial (Eastern grey kangaroo), two monotreme species (echidna, and platypus), a bird (chicken), and three reptilian species (salt-water crocodile, stumpy-tailed lizard, and tuatara). Phenol-extracted proteins from 0.5 ml blood plasma were analysed by two-dimensional polyacrylamide gel electrophoresis. First dimension: from left to right, non-denaturing electrophoresis (10% polyacrylamide); second dimension: from top to bottom, SDS-polyacrylamide gel electrophoresis (10-15% polyacrylamide). Gel slices from the non-denaturing gel were boiled for 10 min with 4% SDS before transfer to the denaturing gel. Positions of transthyretin tetramers, a; dimers, b; and monomers, c, are indicated. For samples from platypus and the reptiles, the faint spots, present in the area where transthyretin is expected to be found, are not transthyretin-related: protein, d, recovered from the gel and purified by reversed-phase HPLC was analysed by N-terminal sequencing; e, protein was shown by electrophoresis to be composed of a single polypeptide chain, in contrast to the tetrameric structure of

transthyretin (for details see text).

P

R e s i d u e N o .

Evolution of transthyretin expression

1 2 3 4 5 6 7 8 9 1011 12 131415161718 19

321

H u m a n G P T G T G E S K C P L M V K V L D A

S h e e p S * A * A . . . . . . . . . . . . . *

B o v i n e * S B * A * * P * * * * * * * * * * *

R a b b i t * * V * * * D * * * * * * * * * * * *

R a t * * G * A * * * * * * * * * * * * * *

M o u s e * * A * A * * * * * * * * * * * * * *

K a n g a r o o V H H E S E H * * - * * * * * * * L *

C h i c k e n A * L V S H * S V D * * * * * * * * * * * *

Fig. 3. Comparison of the N-terminal amino acid sequence of kangaroo transthyretin with those of other vertebrates. The N-terminal amino acid sequence of kangaroo transthyretin was determined by automated Edman degradation as described in Materials and Methods. The amino acid residues are numbered 1-19. The amino acid sequences of rabbit transthyretin (Sundelin et al., 1985), and those deduced from the cDNA sequences for sheep (Tu et al., 1989), rat (Duan et al., 1989), mouse (Wakasugi et al., 1985), chicken (Duan et al., 1991), and the amino acid sequence of kangaroo transthyretin are aligned with the deduced amino acid sequence of human transthyretin (Mita et al., 1984). Those residues in sheep, bovine, rabbit, rat, mouse, chicken and kangaroo transthyretins, which are identical to those in human transthyretin, are

represented by asterisks.

were homologous to that of complement C3a. Two other polypeptides had the sequence glu-ala-glu-pro- ala-gln-ala-gln-asp-phe-glu-ile-leu-gln. Thus these four proteins were not related to transthyretin. A discernable sequence could not be obtained from the fifth polypeptide. The proteins giving rise to the spots labelled "e", (for platypus, crocodile, lizard and tuatara) were analysed in separate experiments by electrophoresis. In each case, after electrophoresis under denaturing conditions without prior boiling, the protein migrated into the same position as protein treated by previous boiling. In contrast, boiling in detergent solution was required to dissociate transthyretin from rats, humans, sheep, chickens, kangaroos, echidnas, and lizards into their four subunits. A transthyretin-like protein was also not detectable in the plasma of the Krefft's tortoise (data not shown in a figure).

Northern analysis o f transthyretin m R N A f r o m liver and choroid plexus

Polyadenylated RNA was isolated from the livers of two echidnas, and total RNA was isolated from the livers of two Eastern grey kangaroos and one sheep. Total RNA was also isolated from the choroid plexus from two Eastern grey kangaroos. RNAs were separated by electrophoresis, transferred to GeneScreen Plus membrane and analysed by Northern blot analysis, using radioactive sheep transthyretin cDNA as a probe. A strong signal for transthyretin mRNA was present in RNA isolated from both liver and choroid plexus from the kangaroo (Fig. 4). The proportion of transthyretin mRNA in RNA from kangaroo choroid plexus was much greater than in RNA from the kangaroo liver. The ratio of the transthyretin mRNA level in RNA from liver to that from choroid plexus of the kangaroo was similar to the ratios observed previously for other

placental mammals (Harms et al., 1991) and birds (Duan et al., 1991; Southweil et al., 1991). In contrast, no transthyretin mRNA was detected in total RNA from echidna liver, (data not shown). A weak signal was obtained for preparations of polyadenylated RNA from echidna liver (Fig. 4).

DISCUSSION

The fossil record suggests that a common ancestor (Cotylosauria=stem reptiles) for mammals and birds existed about 350 million years ago (see Young, 1981). The expression in choroid plexus and the very strong conservation of the structure of transthyretin in mammalian species and chickens (Duan et al., 1991) suggest that transthyretin was already synthesized in the choroid plexus of this common ancestor of birds and mammals. The amino acids involved in the binding of thyroxine by human transthyretin are unchanged in all mammalian and avian transthyretins studied (Duan et al., 1991).

Transthyretin is by far the most abundant protein synthesized and secreted by choroid plexus of mammals and birds. The data presented here show that the choroid plexus of a monotreme species, the echidna, also synthesized and secreted one protein much more abundantly than other proteins, and that this protein migrated similarly to transthyretin from rat and lizard (Fig. 1). Synthesis and secretion of transthyretin by lizard choroid plexus has also been recently demonstrated (Harms et al., 1991). However, in an amphibian, Bufo marinus, the most abundantly synthesized and secreted protein was found to be a lipocalin, and not transthyretin (Achen et al., 1992). Two radioactive proteins of apparent molecular weights of 20,000 and 50,000 were observed in proteins secreted by cane toad choroid plexus (Fig. 1). Thus, transthyretin synthesis in the choroid plexus

322 GERHarD SCHKEIBER et al.

I*g RNA

origin --~

~N)~ total RNA

I ! 20 20 2 2

28,$

18S

"FrR

Fig. 4. Northern analysis of transthyretin mRNA from liver and choroid plexus from echidna and kangaroo. Polyadeny- lated RNA, "poly(A) + RNA", or total RNA were separated in 1.5% agarose gel containing formaldehyde, transferred to GeneScreen Plus membrane, the hybridized with 32P-labelled sheep transthyretin eDNA at 35°C. RNA was from two echidnas, two kangaroos and one sheep. The positions of 18S and 28S rRNA and of transthyretin mRNA

" T T R " , are shown in the left margin.

seems to have evolved for the transport of thyroxine to the brain between or around the transition from amphibians to reptiles.

The reptilian brain shows the first traces of a cortex (see Kent, 1987). The development of the very large and complex system of synaptic connections between the dendrites of neurons, beginning at the reptilian stage, has been proposed to be related to the evolution of consciousness (Eccles, 1992).

Before the discovery of transthyretin gene expression in choroid plexus (Dickson et al., 1985a,b; Herbert et al., 1986; Mita et al., 1986), in the yolk sac (Soprano et al., 1986; Fung et al., 1988; Thomas et al., 1990), and in very small amounts in the pancreas (Jacobsson, 1989; Jacobsson e t al. , 1989) and in the retina (Martone e t al. , 1988; Cavallaro et al., 1990; Dwork et al., 1990), the liver was believed

to be the only site of transthyretin synthesis in the body. A quantitative analysis of transthyretin pools, turnover, plasma concentrations and rate of secretion by l~rfused rat liver showed that the liver produced virtually all of the transthyretin found in the bloodstream (Dickson et al., 1982). There seems to be only one copy of the transthyretin gene per haploid genome in the rat (Fung et al., 1988). In the human, the transthyretin gene is located on chromosome 18 (Wallace et al., 1985). Therefore, the question arose of whether the evolution of the thyroxine-binding features of transthyretin and the expression of its gene first occurred in the brain or in the liver. Previously, no transthyretin could be detected by electrophoresis and [125I]thyroxine binding in the sera from cane toads, and from lizards, (Harms et al., 1991). Similarly, the results of the analysis by two- dimensional gel electrophoresis, presented in Fig. 2, show that representatives of other orders of the class reptilia, namely the salt-water crocodile, the stumpy- tailed lizard and the tuatara, do not possess transthyretin in their blood. Together with the observation of a lack of transthyretin in serum of a tortoise (results not shown), and the absence of transthyretin from the blood of the lizard published earlier (Harms et al., 1991), and confirmed here (Fig. 2), the available information suggests that the transthyretin gene is not yet expressed in the liver at the reptilian stage. Surprisingly, two primitive mammalian species, the echidna and the platypus, both monotremes, also exhibit no, or only traces of transthyretin in their sera (Fig. 2), and only traces of transthyretin mRNA are found in echidna liver. A "more advanced" mammalian species, the Eastern grey kangaroo, however, displays abundant transthyretin in the serum (Fig. 2) and transthyretin mRNA in liver (Fig. 3), as do the birds (Fig. 2, Southwell et al., 1991).

For a more comprehensive understanding of the evolution of the structure and the expression of the transthyretin gene, the data described here and previously obtained results were related to a fossil record of vertebrate evolution (Fig. 5). The common ancestors of placental mammals and marsupials lived about 150 million years ago (Air et al., 1971), those of mammals, including the monotremes, perhaps 200-220 million years ago (Young, 1981). It is unlikely that the ancestors common to all mammals expressed transthyretin in their livers, since transthyretin gene expression is absent from monotreme livers. Similarly, it is unlikely that the common ancestors of the birds and the reptiles expressed the transthyretin gene in their livers, since crocodiles, lizards, tuataras and tortoises, representing the four extant orders of the reptilia, do not express transthyretin in their livers. One might conclude then, that transthyretin gene expression in the liver is a rela- tively late evolutionary event which occurred independently i n the lineages leading to the placental mammals and marsupials, and to the birds. All mammalian, avian and reptilian species studied,

Evolution of transthyretin expression 323

MILLION YEARS B E F O R E PRESENT

O R D E R

species (ref)

TRANSTHYRETIN EXPRESSION

Choroid Plexus Liver

I 400

O

I 200

I 0

PLACENTAL MAMMALS human (4) dog (5) cat (6) rat (Figs. 1 & 2A,

5,7,8)

+ +

+ +

+ +

+ +

mouse (5) + + guinea pig (5) + + rabbit (5) + + pig (5) + + cow (5) + + sheep (5,7,9) + +

MARSUPIALS kangaroo (Fig. 2) wallaby (6)

MONOTREMES echidna (Figs. 1 & 2) platypus (Fig. 2A)

+ +

n.d. +

+ t race

n.d.

BmaS chicken (Fig. 2A, 5, + +

]o,1]) duck ( I ] ) + + pigeon (11) + + quail ( I ] ) + +

REPTILES

crocodile (Fig. 2A) n.d.

lizard (5, Fig. 1 & 2) +

tuatara (Fig. 2A) n.d.

tortoise (6) n.d.

AMPHIBIANS toad (Fig. 1, 5,12)

If tog (12) n.d.

400 200 0 I J I

Fig. 5. Evolution of transthyretin gene expression in choroid plexus and liver. L~ft half of figure, phylog~netic tree based on fossil records adapted from Young (1981); right half of figure, transthyretin gene expression detcx~tcd by analysis of transthyretin mRNA in tissues, analysis of transthyretin synthesized and socrvted by incubated choroid plexus, or analysis of transthyretin in blood plasma. (D Appearance of transthyrvtin gene expression in the choroid plexus of the stem reptiles, about 350 million years ago; (~) appearance of transthyretin gene expression in the livers of the ancestors of placental mammals and marsupials; (~) appearance of transthyretin gene expression in the liver of birds, after the separation of the lineages leading to birds and modern reptiles; + , presence of transthyretin gene expression; - - , absence of transthyretin gene expression; n.d., transthyretin gene expression was not determined; trace, less than 2% of value for transthyretin gene expression found for rat serum. References: (4) Dickson and Schreiber, 1986; Herbert et aL, 1986; Mita et aL, 1986; (5) Harms et aL, 1991; (6) unpublished observation by authors; (7) Schreiber et aL, 1990; (8) Dickson et al., 1986; Kato et al., 1986; Fung el ai., 1988; (9) Tu et al., 1990; (10) Duan et al., 1991; (11) Southwell et al., 1991; (12) Achen

et al., 1992.

324 GERHARD SCHREIBER et al.

synthesize transthyretin in the choroid plexus, but transthyretin was not synthesized in amphibian choroid plexus. Thus, these data suggest that transthyretin originally evolved as a thyroxine- binding protein in the brain, and that a later change in the tissue distribution of gene expression resulted in it becoming a plasma protein.

A major function of thyroid hormone-binding proteins is to ensure the appropriate distribution of thyroxine in the body. By binding thyroxine with high affinity, thyroxine-binding serum proteins prevent the very lipophilic thyroxine from disappearing from the bloodstream by partitioning into the lipid phase of cells (Mendel et al., 1987). Lipid contents, total volumes of mitochondrial membranes, phospholipid concentrations, and the relative sizes of membrane- rich organs, such as liver and kidney, are larger in endothermic than ectothermic animals of similar size (Hulbert and Else, 1989). One might speculate, that the increase of the total volume of the lipid phase available to thyroxine during the evolution of the endotherms from the ectotherms provided a selection pressure leading to the synthesis and secretion of the thyroxine-binding protein transthyretin in the liver. Synthesis of this protein had already evolved much earlier in the brain.

Acknowledgements--We thank Professor A. Hulbert for critical discussions, J. Guest, M. Moritz and J. Morley for help in preparation of the manuscript, Drs Cree, Kray, Daugherty, Handasyde and the Melbourne Zoo for donation of samples. Some of the data in this paper were presented in preliminary format at the meeting of the Royal Zoological Society of New South Wales, Sydney, July 7 I0, 1991, and appeared in the proceedings of this meeting which were published as a monograph: Platypus and Echidnas (Edited by Augee M. L.), The Royal Zoological Society of New South Wales, Sydney.

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Transthyretin expression evolved more recently in liver than in brain

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