Stem Reptiles Research Articles

Cranial anatomy and phylogenetic affinities of Bolosaurus major, with new information on the unique bolosaurid feeding apparatus and evolution of the impedance-matching ear.

Resolving the phylogenetic relationships of early amniotes, in particular stem reptiles, remains a difficult problem. Three-dimensional morphological analysis of well-preserved stem-reptile specimens can reveal important anatomical data and clarify regions of phylogeny. Here, we present the first thorough description of the unusual early Permian stem reptile Bolosaurus major, including the first comprehensive description of a bolosaurid braincase. We describe previously obscured details of the palate, allowing for insight into bolosaurid feeding mechanics. Aspects of the rostrum, palate, mandible, and neurocranium suggest that B. major had a particularly strong bite. We additionally found B. major has a surprisingly slender stapes, similar to that of the middle Permian stem reptile Macroleter poezicus, which may suggest enhanced hearing abilities compared to other Paleozoic amniotes (e.g., captorhinids). We incorporated our new anatomical information into a large phylogenetic matrix (150 OTUs, 590 characters) to explore the relationship of Bolosauridae among stem reptiles. Our analyses generally recovered a paraphyletic "Parareptilia," and found Bolosauridae to diverge after Captorhinidae + Araeoscelidia. We also included B. major within a smaller matrix (10 OTUs, 27 characters) designed to explore the interrelationships of Bolosauridae and found all species of Bolosaurus to be monophyletic. While reptile relationships still require further investigation, our phylogeny suggests repeated evolution of impedance-matching ears in Paleozoic stem reptiles.

The tuatara genome reveals ancient features of amniote evolution

The tuatara (Sphenodon punctatus)—the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2—is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which—at approximately 5 Gb—is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Hearing as an extreme sport: Underwater ears, infra to ultrasonic, and surface to the abyss

It has been argued that “hearing” evolved in aquatic animals. Auditory precursors exist in fossil Agnatha and cephalopod statolithic organs, but how/when/why did a dedicated acoustic receptor, the true primordial ear, first appear? Did hearing arise linearly or independently, in parallel, in and out of the water? Modern aquatic species have an extraordinary range of auditory systems, from simple pressure receptors to complex biosonar systems. What drives this breadth of “hearing”? Vertebrate ears reflect selective pressures. While vision, touch, taste, and olfaction are important, only hearing is ubiquitous. Even natural mutes, like goldfish and sea turtles, listen. Ears capture passive and active sound cues. Auditory structures, honed by habit and habitat, delimit species abilities to detect, analyze, and act on survival cues. Cochleae, from shrews to bats to wolves to whales, evolved from the essential papilla of stem reptiles, elongating, coiling, increasing in complexity that enhanced frequency discrimination, but with heads tuned to the physics of sound in their media. Air-water parallels evolved: ultrasonic echolocators and massive infrasonic specialists. The ear then is a window into the evolutionary push-pull driven by three tasks that shaped the several thousand elements packed into every auditory system: feed, breed, and survive another day.

Zebrin II Is Expressed in Sagittal Stripes in the Cerebellum of Dragon Lizards (Ctenophorus sp.)

Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). In contrast, in snakes and turtles, ZII is not expressed heterogeneously; rather all Purkinje cells are ZII+. Here, we examined the expression of ZII in the cerebellum of lizards to elucidate the evolutionary origins of ZII stripes in Sauropsida. We focused on the central netted dragon (Ctenophorus nuchalis) but also examined cerebellar ZII expression in 5 other dragon species (Ctenophorus spp.). In contrast to what has been observed in snakes and turtles, we found that in these lizards, ZII is heterogeneously expressed. In the posterior part of the cerebellum, on each side of the midline, there were 3 sagittal stripes consisting of Purkinje cells with high ZII expression (ZII+) alternating with 2 sagittal stripes with weaker ZII expression (ZIIw). More anteriorly, most of the Purkinje cells were ZII+, except laterally, where the Purkinje cells did not express ZII (ZII-). Finally, all Purkinje cells in the auricle (flocculus) were ZII-. Overall, the parasagittal heterogeneous expression of ZII in the cerebellum of lizards is similar to that in mammals and birds, and contrasts with the homogenous ZII+ expression seen in snakes and turtles. We suggest that a sagittal heterogeneous expression of ZII represents the ancestral condition in stem reptiles which was lost in snakes and turtles.

A large, multiple-tooth-rowed captorhinid reptile (Amniota: Eureptilia) from the Upper Permian of Mallorca (Balearic Islands, western Mediterranean)

SUPPLEMENTAL DATA—Supplemental materials are available for this article for free at www.tandfonline.com/UJVPCitation for this article: Liebrecht, T., J. Fortuny, À. Galobart, J. Müller, and P. Martin Sander. 2016. A large, multiple-tooth-rowed captorhinid reptile (Amniota: Eureptilia) from the Upper Permian of Mallorca (Balearic Islands, western Mediterranean). Journal of Vertebrate Paleontology. DOI: 10.1080/02724634.2017.1251936.

Zebrin II / Aldolase C Expression in the Cerebellum of the Western Diamondback Rattlesnake (Crotalus atrox)

Aldolase C, also known as Zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+), alternating with stripes of Purkinje cells with little or no expression (ZII-). The patterns of ZII+ and ZII- stripes in the cerebellum of birds and mammals are strikingly similar, suggesting that it may have first evolved in the stem reptiles. In this study, we examined the expression of ZII in the cerebellum of the western diamondback rattlesnake (Crotalus atrox). In contrast to birds and mammals, the cerebellum of the rattlesnake is much smaller and simpler, consisting of a small, unfoliated dome of cells. A pattern of alternating ZII+ and ZII- sagittal stripes cells was not observed: rather all Purkinje cells were ZII+. This suggests that ZII stripes have either been lost in snakes or that they evolved convergently in birds and mammals.

Crocodylians evolved scattered multi-sensory micro-organs.

BackgroundDuring their evolution towards a complete life cycle on land, stem reptiles developed both an impermeable multi-layered keratinized epidermis and skin appendages (scales) providing mechanical, thermal, and chemical protection. Previous studies have demonstrated that, despite the presence of a particularly armored skin, crocodylians have exquisite mechanosensory abilities thanks to the presence of small integumentary sensory organs (ISOs) distributed on postcranial and/or cranial scales.ResultsHere, we analyze and compare the structure, innervation, embryonic morphogenesis and sensory functions of postcranial, cranial, and lingual sensory organs of the Nile crocodile (Crocodylus niloticus) and the spectacled caiman (Caiman crocodilus). Our molecular analyses indicate that sensory neurons of crocodylian ISOs express a large repertoire of transduction channels involved in mechano-, thermo-, and chemosensory functions, and our electrophysiological analyses confirm that each ISO exhibits a combined sensitivity to mechanical, thermal and pH stimuli (but not hyper-osmotic salinity), making them remarkable multi-sensorial micro-organs with no equivalent in the sensory systems of other vertebrate lineages. We also show that ISOs all exhibit similar morphologies and modes of development, despite forming at different stages of scale morphogenesis across the body.ConclusionsThe ancestral vertebrate diffused sensory system of the skin was transformed in the crocodylian lineages into an array of discrete multi-sensory micro-organs innervated by multiple pools of sensory neurons. This discretization of skin sensory expression sites is unique among vertebrates and allowed crocodylians to develop a highly-armored, but very sensitive, skin.

Reptile scale paradigm: Evo-Devo, pattern formation and regeneration.

The purpose of this perspective is to highlight the merit of the reptile integument as an experimental model. Reptiles represent the first amniotes. From stem reptiles, extant reptiles, birds and mammals have evolved. Mammal hairs and feathers evolved from Therapsid and Sauropsid reptiles, respectively. The early reptilian integument had to adapt to the challenges of terrestrial life, developing a multi-layered stratum corneum capable of barrier function and ultraviolet protection. For better mechanical protection, diverse reptilian scale types have evolved. The evolution of endothermy has driven the convergent evolution of hair and feather follicles: both form multiple localized growth units with stem cells and transient amplifying cells protected in the proximal follicle. This topological arrangement allows them to elongate, molt and regenerate without structural constraints. Another unique feature of reptile skin is the exquisite arrangement of scales and pigment patterns, making them testable models for mechanisms of pattern formation. Since they face the constant threat of damage on land, different strategies were developed to accommodate skin homeostasis and regeneration. Temporally, they can be under continuous renewal or sloughing cycles. Spatially, they can be diffuse or form discrete localized growth units (follicles). To understand how gene regulatory networks evolved to produce increasingly complex ectodermal organs, we have to study how prototypic scale-forming pathways in reptiles are modulated to produce appendage novelties. Despite the fact that there are numerous studies of reptile scales, molecular analyses have lagged behind. Here, we underscore how further development of this novel experimental model will be valuable in filling the gaps of our understanding of the Evo-Devo of amniote integuments.

Hard cornification in reptilian epidermis in comparison to cornification in mammalian epidermis

The structure of reptilian hard (beta)-keratins, their nucleotide and amino acid sequence, and the organization of their genes are presented. These 13-19 kDa proteins are basic, rich in glycine, proline and serine, and different from cytokeratins. Their mRNAs are expressed in beta-cells. The central part of beta-keratins (this region has been previously termed 'core-box' and is peculiar of all sauropsid proteins) is composed of two beta-folded regions and shows a high identity with avian beta-keratins. This central part present in all beta-keratins, including feather keratins, is the site of polymerization to build the framework of beta-keratin filaments. Beta-keratins appear cytokeratin-associated proteins. Their central region might have originated in an ancestral glycine-rich protein present in stem reptiles from which beta-keratins evolved and diversified into reptiles and birds. Stem reptiles of the Carboniferous period might have possessed glycine-rich proteins derived from exons/domains corresponding to the variable, glycine-rich region of cytokeratins. Beta-keratins might have derived from a gene coding for small glycine-rich keratin-associated proteins. The glycine-rich regions evolved differently in the lineage leading to modern reptiles and birds versus that leading to mammals. In the reptilian lineage some amino acid regions produced by point mutations and amino acid changes might have given rise to originate the central beta-pleated region. The latter allowed the formation of filamentous proteins (beta-keratins) associated with intermediate filament keratins and replaced them in beta-keratin cells. In the mammalian lineage no beta-pleated region was generated in their matrix proteins, the glycine-rich keratin-associated proteins. The latter evolved as glycine-tyrosine-rich, sulphur-rich, and ultra-sulphur-rich proteins that are used for building hairs, horns and nails.

The origin of parental care in birds: a reassessment

The origin of parental care in birds: a reassessment

Glial fibrillary acidic protein-immunopositive structures in the brain of a Crocodilian, Caiman crocodilus, and its bearing on the evolution of astroglia.

Caiman crocodilus, as a representative of the order Crocodilia, was used in immunohistochemical studies. Immunohistochemical procedures were performed on free-floating sections using a monoclonal antibody against porcine glial fibrillary acidic protein (GFAP) and employing standard avidin-biotin complex methodology. The astroglia of Caiman exhibited robust immunoreactivity to the antibodies raised against mammalian GFAP. In Caiman, the predominant GFAP-immunopositive elements are the radial ependymoglia, similar to other reptiles. The regional variability of glial architecture in Caiman, however, seems greater than in other reptiles so far examined, although it is less compared with chickens. We suggest that this finding corresponds to a more advanced "regional adaptation" of the glial structure in Caiman compared with other reptiles. The main feature that distinguishes the astroglia of Caiman from those of other reptiles is the widespread occurrence of GFAP-immunopositive astrocytes. These cells are limited in lizards and snakes, are not present in turtles, but are found in every major brain area in Caiman. However, even in Caiman, astrocytes are only intermingled with radial glia and are not the predominant glial element of any brain area. The occurrence of astrocytes does not correlate with brain wall thickness. Despite their origin from different ancestral groups of stem reptiles (synapsid or diapsid), mammals and birds exhibit some common general features in their glial architecture and GFAP distribution: 1) predominance of astrocytes and 2) absent or limited GFAP immunopositivity of several brain areas. The present study demonstrates that, even in Caiman, a representative of the reptilian group most closely related to birds, these features are present only in part, suggesting that, in mammals and birds, they have evolved independently.

Glial fibrillary acidic protein-immunopositive structures in the brain of a Crocodilian,Caiman crocodilus, and its bearing on the evolution of astroglia

Caiman crocodilus, as a representative of the order Crocodilia, was used in immunohistochemical studies. Immunohistochemical procedures were performed on free-floating sections using a monoclonal antibody against porcine glial fibrillary acidic protein (GFAP) and employing standard avidin-biotin complex methodology. The astroglia of Caiman exhibited robust immunoreactivity to the antibodies raised against mammalian GFAP. In Caiman, the predominant GFAP-immunopositive elements are the radial ependymoglia, similar to other reptiles. The regional variability of glial architecture in Caiman, however, seems greater than in other reptiles so far examined, although it is less compared with chickens. We suggest that this finding corresponds to a more advanced “regional adaptation” of the glial structure in Caiman compared with other reptiles. The main feature that distinguishes the astroglia of Caiman from those of other reptiles is the widespread occurrence of GFAP-immunopositive astrocytes. These cells are limited in lizards and snakes, are not present in turtles, but are found in every major brain area in Caiman. However, even in Caiman, astrocytes are only intermingled with radial glia and are not the predominant glial element of any brain area. The occurrence of astrocytes does not correlate with brain wall thickness. Despite their origin from different ancestral groups of stem reptiles (synapsid or diapsid), mammals and birds exhibit some common general features in their glial architecture and GFAP distribution: 1) predominance of astrocytes and 2) absent or limited GFAP immunopositivity of several brain areas. The present study demonstrates that, even in Caiman, a representative of the reptilian group most closely related to birds, these features are present only in part, suggesting that, in mammals and birds, they have evolved independently. J. Comp. Neurol. 431:460–480, 2001. © 2001 Wiley-Liss, Inc.

Cochlear mechanisms from a phylogenetic viewpoint.

The hearing organ of the inner ear was the last of the paired sense organs of amniotes to undergo formative evolution. As a mechanical sensory organ, the inner-ear hearing organ's function depends highly on its physical structure. Comparative studies suggest that the hearing organ of the earliest amniote vertebrates was small and simple, but possessed hair cells with a cochlear amplifier mechanism, electrical frequency tuning, and incipient micromechanical tuning. The separation of the different groups of amniotes from the stem reptiles occurred relatively early, with the ancestors of the mammals branching off first, approximately 320 million years ago. The evolution of the hearing organ in the three major lines of the descendents of the stem reptiles (e.g., mammals, birds-crocodiles, and lizards-snakes) thus occurred independently over long periods of time. Dramatic and parallel improvements in the middle ear initiated papillar elongation in all lineages, accompanied by increased numbers of sensory cells with enhanced micromechanical tuning and group-specific hair-cell specializations that resulted in unique morphological configurations. This review aims not only to compare structure and function across classification boundaries (the comparative approach), but also to assess how and to what extent fundamental mechanisms were influenced by selection pressures in times past (the phylogenetic viewpoint).

Structure and expression of the transthyretin gene in the choroid plexus: a model for the study of the mechanism of evolution.

Thyroid hormones are key regulators of brain differentiation and function. They permeate strongly into lipid membranes. However, a substantial portion of thyroid hormone is retained in the intravascular/extracellular compartments by binding to plasma proteins. In the brain, transthyretin is the most important of these proteins. This transthyretin is synthesized in the epithelial cells of the choroid plexus and exclusively secreted towards the brain. A net movement of thyroid hormones from the blood to the brain ensues. During evolution, transthyretin synthesis in the choroid plexus and the beginnings of a neocortex first appeared at the stage of the stem reptiles. The affinity of transthyretin for thyroxine increased and that for triiodothyronine decreased during evolution. This could augment the importance of deiodination for regulation of metabolism and gene expression by thyroid hormones in the brain. Successive shifts of the splice site at the 5' end of exon 2 of transthyretin precursor mRNA in the 3' direction led to a shortening of the N-terminal sections and to an increase in hydrophilicity of the N-terminal regions of transthyretin. This shift can be explained by a sequence of single base mutations. It could be an example for a molecular mechanism of positive Darwinian evolution. The selection pressure, which led to the expression of the transthyretin gene in the choroid plexus during evolution, might have been the maintenance of thyroid hormone homeostasis in the extracellular compartment of the brain in the presence of the greatly increasing volume of the lipid phase.

Reelin mRNA expression during embryonic brain development in the turtle Emys orbicularis.

The expression of reelin messenger ribonucleic acid (mRNA) was studied during embryonic brain development in the turtle Emys orbicularis, by using radioactive in situ hybridization. A high expression was consistently found in the olfactory bulb and in a few neurons in the marginal zone and, to a lesser extent, in the subplate of the dorsal and medial cortical sectors. In the diencephalon, the ventral division of lateral geniculate nuclei and the prospective reticular thalamic nuclei were strongly positive. High reelin signal was also associated with some layers of the tectum and with the external granule cell layer of the cerebellum. A more moderate signal was detected in the septal nuclei, striatum, dorsal ventricular ridge, retina, habenular nuclei, and hypothalamus, and in some reticular nuclei of the midbrain and hindbrain and in ventral spinal cord. The cortical plate, basal forebrain, amygdala, and tegmentum were weakly labeled. When they are compared to reelin expression during mammalian brain development, our data reveal an evolutionarily conserved canvas of reelin expression and significant differences, particularly in developing cortical fields. Most significantly, the developing turtle cortex does not display the heavy reelin expression in subpial Cajal-Retzius cells that is so typical of its mammalian counterpart. Given the key role of reelin in laminar cortical development, our data suggest that the increase in the number of reelin-producing cells and/or the amplification of reelin expression in the cortical marginal zone might have been a driving factor during the evolution of the laminated cerebral cortex from stem reptiles to mammals, as indicated in previous comparative analyses.

Evidence for a possible avian dorsal thalamic region comparable to the mammalian ventral anterior, ventral lateral, and oral ventroposterolateral nuclei

In the present study, we investigated whether a dorsal thalamic region comparable to the motor part of the mammalian ventral tier (the ventral anterior nucleus, the ventral lateral nucleus, and the oral ventroposterolateral nucleus) exists in pigeon. With this aim, we reinvestigated the projections of the pigeon dorsal pallidum to the dorsal thalamus by using 1) injections of the anterogradely transported form of biotinylated dextran amine (BDA; 10,000 molecular weight) in the pigeon dorsal pallidum (paleostriatum primitivum) and 2) injections of the retrogradely transported form of BDA (3,000 molecular weight) in the pigeon dorsal thalamus. Our results indicate that the dorsal pallidum in pigeons projects to three areas of the dorsal thalamus: the dorsointermediate posterior nucleus, the ventrointermediate area, and the nucleus subrotundus. Only the projection to the dorsointermediate posterior nucleus was described previously (Karten and Dubbeldam [1973] J. Comp. Neurol. 148:61-90; Kitt and Brauth [1982] Neuroscience 6:1551-1566). To investigate whether any of the dorsal thalamic nuclei receiving pallidal input project to a motor cortical field, injections of the retrograde tracer Fluoro-Gold were placed into the rostral Wulst. This is an avian cortical field that appears to contain a region comparable to mammalian primary somatomotor cortex (Karten [1971] Anat. Rec. 169:353; Wild [1992] J. Comp. Neurol. 287:1-18). Our results indicate that neurons in the rostral ventrointermediate area, but not in the nucleus subrotundus, the dorsointermediate posterior nucleus, or the intermediate or caudal parts of the ventrointermediate area, project to the rostral Wulst. In addition to the input from the dorsal pallidum, the avian ventrointermediate area also receives input from the lateral substantia nigra and the lateral and internal cerebellar nuclei (present results). Our results suggest the existence in birds of a pallidothalamocortical loop similar to the pallidoventral tier-motor cortex loop of mammals and suggest that the avian ventrointermediate area is comparable to the motor part of the mammalian ventral tier in both location and connections. If this is confirmed by physiological experiments, then it would indicate that basal ganglia control of movement mediated by a pallidothalamocortical loop may have evolved with the stem reptiles.

Evidence for a possible avian dorsal thalamic region comparable to the mammalian ventral anterior, ventral lateral, and oral ventroposterolateral nuclei

In the present study, we investigated whether a dorsal thalamic region comparable to the motor part of the mammalian ventral tier (the ventral anterior nucleus, the ventral lateral nucleus, and the oral ventroposterolateral nucleus) exists in pigeon. With this aim, we reinvestigated the projections of the pigeon dorsal pallidum to the dorsal thalamus by using 1) injections of the anterogradely transported form of biotinylated dextran amine (BDA; 10,000 molecular weight) in the pigeon dorsal pallidum (paleostriatum primitivum) and 2) injections of the retrogradely transported form of BDA (3,000 molecular weight) in the pigeon dorsal thalamus. Our results indicate that the dorsal pallidum in pigeons projects to three areas of the dorsal thalamus: the dorsointermediate posterior nucleus, the ventrointermediate area, and the nucleus subrotundus. Only the projection to the dorsointermediate posterior nucleus was described previously (Karten and Dubbeldam [1973] J. Comp. Neurol. 148:61-90; Kitt and Brauth [1982] Neuroscience 6:1551-1566). To investigate whether any of the dorsal thalamic nuclei receiving pallidal input project to a motor cortical field, injections of the retrograde tracer Fluoro-Gold were placed into the rostral Wulst. This is an avian cortical field that appears to contain a region comparable to mammalian primary somatomotor cortex (Karten [1971] Anat. Rec. 169:353; Wild [1992] J. Comp. Neurol. 287:1-18). Our results indicate that neurons in the rostral ventrointermediate area, but not in the nucleus subrotundus, the dorsointermediate posterior nucleus, or the intermediate or caudal parts of the ventrointermediate area, project to the rostral Wulst. In addition to the input from the dorsal pallidum, the avian ventrointermediate area also receives input from the lateral substantia nigra and the lateral and internal cerebellar nuclei (present results). Our results suggest the existence in birds of a pallidothalamocortical loop similar to the pallidoventral tier-motor cortex loop of mammals and suggest that the avian ventrointermediate area is comparable to the motor part of the mammalian ventral tier in both location and connections. If this is confirmed by physiological experiments, then it would indicate that basal ganglia control of movement mediated by a pallidothalamocortical loop may have evolved with the stem reptiles. J. Comp. Neurol. 384:86-108, 1997. © 1997 Wiley-Liss, Inc.

Abundant Synthesis of Transthyretin in the Brain, but not in the Liver, of Turtles

The binding of thyroxine to proteins in the blood plasma of the turtle, Trachemys scripta, was analyzed by incubation with radioactive thyroxine, electrophoresis and autoradiography. Albumin and an α-globulin were found to bind thyroxine; no thyroxine-binding transthyretin was detected in the prealbumin region. In contrast to blood plasma, a thyroxine-binding prealbumin was observed in medium from T. scripta choroid plexus incubated in vitro. RNA was extracted from brain tissue containing choroid plexus and from liver of T. scripta and Chelydra serpentina and analyzed by hybridization with transthyretin cDNA from the lizard Tiliqua rugosa. The brain RNAs contained substantial amounts of transthyretin mRNA, whereas only trace amounts of transthyretin mRNA were detected in RNA from liver. No transthyretin mRNA was observed in RNA from kidney. The results support the hypothesis that the expression of the transthyretin gene first evolved in the choroid plexus of the brain at the stage of the stem reptiles, whereas abundant transthyretin synthesis in liver evolved much later, and independently, in mammals and birds.

The Relation of the Dual Thyroxine/Vitamin D-Binding Protein (TBP/DBP) of Emydid Turtles to Vitamin D-Binding Proteins of Other Vertebrates

The relation of a dual binding protein, involved in the transport of both thyroxine (T 4) and vitamin D 3, in the blood of emydid turtles to blood proteins in other vertebrates was examined immunologically. Binding studies with 25-OH-[ 3H]cholecalciferol (D 3) confirmed the presence of a D 3-binding protein (DBP) in the plasma of a wide variety of chelonian species representing both major suborders, as well as other species of reptiles, amphibians, birds, and mammals. Analysis by polyacrylamide gel electrophoresis (PAGE) showed that these binding proteins exhibited variable electrophoretic mobilities and in some species multiple DBPs were observed. Western blot analysis of these gels and additional tests with SDSPAGE showed that an antiserum against the dual T 4/D 3-binding protein (TBP/DBP) from the turtle Trachemys scripta cross-reacted with the DBPs of diverse turtles, one crocodilian ( Alligator), and birds; however, there was little or no cross-reaction with plasma from squamate reptiles (three snakes, four lizards), one crocodilian ( Osteolaemus), amphibians (two anurans, one urodele), or mammals (six eutherians, one metatherian). The cross-reacting proteins (DBPs) all exhibit a similar M r (≈60 K). Adsorption of plasma by TBP/DBP-affinity chromatography confirmed this phylogenetic pattern of cross-reactions between the anti-TBP/DBP serum and functional DBPs in chicken, turtle, and alligator blood (affinity adsorption effectively eliminated D 3 binding); there was only weak cross-reaction with DBP in a lizard and the second crocodilian (50% reduction in D 3 binding) and none with DBP of snakes or mammals (D 3 binding was unaffected). Results suggest that the plasma protein (TBP/DBP) that exhibits dual, high-affinity binding of T 4 and D 3 in one turtle family evolved from the more "primitive" vitamin D-binding protein of stem reptiles; thus, the high-affinity T 4-binding site on this molecule is probably a derived characteristic of DBP in the Emydidae.

Evolution of marsupial and other vertebrate thyroxine-binding plasma proteins

Binding of radioactive thyroxine to proteins in the plasma of vertebrates was studied by electrophoresis followed by autoradiography. Albumin was found to be a thyroxine carrier in the blood of all studied fish, amphibians, reptiles, monotremes, marsupials, eutherians (placental mammals), and birds. Thyroxine binding to transthyretin was detected in the blood of eutherians, diprotodont marsupials, and birds, but not in blood from fish, toads, reptiles, monotremes, and Australian polyprotodont marsupials. Globulins binding thyroxine were only observed in the plasma of some mammals. Apparently, albumin is the phylogenetically oldest thyroxine carrier in vertebrate blood. Transthyretin gene expression in the liver developed in parallel, and independently, in the evolutionary lineages leading to eutherians, to diprotodont marsupials, and to birds. In contrast, high transthyretin mRNA levels, strong synthesis, and secretion of transthyretin in choroid plexus from reptiles and birds indicate that transthyretin gene expression in the choroid plexus evolved much earlier than in the liver, probably at the stage of the stem reptiles. NH2-terminal sequence analysis suggests a change of transthyretin pre-mRNA splicing during evolution.