Compositional patterns in reptilian genomes

Sauropsids form a complex group of vertebrates including squamates (lizards and snakes), turtles, crocodiles, sphenodon and birds (which are often considered as a separate class). Although avian genomes have been relatively well studied, the genomes of the other groups have remained only sparsely characterized. Moreover, the nuclear sequences available in databanks are still very limited. In the present study, we have analysed the compositional patterns, i.e. the GC (molar fraction of guanine and cytosine in DNA) distributions, of 31 reptilian (particularly snake) genomes by analytical ultracentrifugation of DNAs in CsCl gradients. The profiles were characterized by their modal buoyant density rho(o), mean buoyant density < rho>, asymmetry < rho>- rho(o), and heterogeneity H. The modal buoyant density distribution of reptilian DNAs clearly distinguishes two groups. The snakes fall in the same range of modal densities as most mammals, whereas crocodiles, turtles and lizards show higher values (>1.700 g/cm(3)). As far as the more important compositional properties of asymmetry and heterogeneity are concerned, previous studies showed that amphibians and fishes share relatively low values, whereas birds and mammals are characterized by highly heterogeneous and asymmetric patterns (with the exception of Muridae, which have a lower heterogeneity). The present results show that the snake genomes cover a broad range of asymmetry and heterogeneity values, whereas the genomes of crocodiles and turtles cover a narrow range that is intermediate between those of fishes/amphibians and those of mammals/birds.     

Deleterious Mutations of a Claw Keratin in Multiple Taxa of Reptiles

We have recently shown that homologs of mammalian hair keratins are expressed in the claws of the green anole lizard, Anolis carolinensis. To test whether reptilian hair keratin homologs are functionally associated with claws, we investigated the conservation of the prototypical reptilian hair keratin homolog, hard acidic keratin 1 (HA1), in representative species from all main clades of reptiles. A complete cDNA of HA1 was cloned from the claw-forming epidermis of the lacertid lizard Podarcis sicula, and partial HA1 gene sequences could be amplified from genomic DNA of tuatara, lizards, gekkos, turtles, and crocodiles. In contrast, the HA1 gene of the limbless slow worm, Anguis fragilis, and of two species of turtles contained at least one deleterious mutation. Moreover, an HA1 gene was undetectable in the softshell turtle, snakes, and birds. Mapping the presence and absence of HA1 onto the phylogenetic tree of sauropsids suggested that the HA1 gene has been lost independently in several lineages of reptiles. The species distribution of HA1 is compatible with the hypothesis of a primary function of HA1 in claws but also shows that the formation of reptilian claws does not strictly depend on this keratin.     

Technics for blood collection and intravascular infusion of reptiles.

Several methods for external sampling of blood and administration of intravascular fluids are applicable to reptilian species. This paper reviewed some of the technics that have been previously reported and introduced several new procedures applicable to snakes, turtles, alligators, crocodiles, and lizards.     

Cytochemical, biochemical and molecular aspects of the process of keratinization in the epidermis of reptilian scales

The characteristics of scaled skin of reptiles is one of their main features that distinguish them from the other amniotes, birds and mammals. The different scale patterns observed in extant reptiles result from a long evolutive history that allowed each species to adapt to its specific environment. The present review deals with comparative aspects of epidermal keratinization in reptiles, chelonians (turtles and tortoises), lepidosaurian (lizards, snakes, sphenodontids), archosaurians (crocodilians). Initially the morphology and cytology of reptilian scales is outlined to show the diversity in the epidermis among different groups. The structural proteins (alpha-keratins and associated proteins), and enzymes utilized to form the corneous layer of the epidermis are presented. Aside cytokeratins (alpha-keratins), used for making the cytoskeleton, reptilian alpha-keratinocytes produce interkeratin (matrix) and corneous cell envelope proteins. Keratin bundles and degraded cell organelles constitute most of the corneous material of alpha-keratinocytes. Matrix, histidine-rich and sulfur-rich proteins are produced in the soft epidermis and accumulated in the cornified cell envelope. Main emphasis is given to the composition and to the evolution of the hard keratins (beta-keratins). Beta-keratins constitute the hard corneous material of scales. These small proteins are synthesized in beta-keratinocytes and are accumulated into small packets that rapidly merge into a compact corneous material and form densely cornified layers. Beta-keratins are smaller proteins (8-20 kDa) in comparison to alpha-keratins (40-70 kDa), and this size may determine their dense packing in corneocytes. Both glycine-sulfur-rich and glycine-proline-rich proteins have been so far sequenced in the corneous material of scales in few reptilian species. The latter keratins possess C- and N-amino terminal amino acid regions with sequence homology with those of mammalian hard keratins. Also, reptilian beta-keratins possess a central core with homology with avian scale/feather keratins. Multiple genes code for these proteins and their discovery and sequentiation is presently an active field of research. These initial findings however suggest that ancient reptiles already possessed some common genes that have later diversified to produce the specific keratin-associated proteins in their descendants: extant reptiles, birds and mammals. The evolution of these small proteins in lepidosaurians, chelonians and archosaurians represent the next step to understand the evolution of cornification in reptiles and derived amniotes (birds and mammals).     

Parathyroid Glands in Reptiles

The parathyroid glands in reptiles generally develop from thethird and fourth pharyngeal pouches. In lizards and crocodiles,usually only one pair of glands (from the third pouch) persists,while turtles and snakes generally retain both pairs of glandsin adults. The glands consist of cell cords and are similarin structure to those of birds and mammals. A common featureof reptilian parathyroids is the organization of cells arounda lumen in a follicular arrangement. The few physiological studieswhich have been made indicate that parathyroid function in reptilesis in many respects similar to that of mammals. For example,in lizards, parathyroidectomy results in tetanic convulsionsand lowered values of plasma calcium, while administration ofparathyroid extract increases mine phosphate and serum calciumin turtles, and increases the number of osteoclasts in boneof lizards and turtles. However, there are some obvious differencesbetween parathyroid function in reptiles and mammals, on whichmuch more work is needed.     

Distribution and characterization of keratins in the epidermis of the tuatara (Sphenodon punctatus; Lepidosauria, Reptilia)

Reptilian scales are mainly composed of alpha-and beta-keratins. Epidermis and molts from adult individuals of an ancient reptilian species, the tuatara (Sphenodon punctatus), were analysed by immunocytochemistry, mono- and bi-dimensional electrophoresis, and western blotting for alpha- and beta-keratins. The epidermis of this reptilian species with primitive anatomical traits should represent one of the more ancient amniotic epidermises available. Soft keratins (AE1- and AE3-positive) of 40-63 kDa and with isoelectric points (pI) at 4.0-6.8 were found in molts. The AE3 antibody was diffusely localised over the tonofilaments of keratinocytes. The lack of basic cytokeratins may be due to keratin alteration in molts, following corneification or enzymatic degradation of keratins. Hard (beta-) keratins of 16-18 kDa and pI at 6.8, 8.0, and 9.2 were identified using a beta-1 antibody produced against chick scale beta-keratin. The antibody also labeled filaments of beta-cells and of the mature, compact beta-layer. We have shown that beta-keratins in the tuatara resemble those of lizards and snakes, and that they are mainly basic proteins. These proteins replace cytokeratins in the pre-corneoum beta-layers, from which a hard, mechanically resistant corneoum layer is formed over scales. Beta-keratins may have both a fibrous and a matrix role in forming the hard texture of corneoum scales in this ancient species, as well as in more recently evolved reptiles.     

Immunocytochemistry indicates that glycine‐rich beta‐proteins are present in the beta‐layer,while cysteine‐rich beta‐proteins are present in beta‐ and alpha‐layers of snake epidermis

Immunolocalization of glycine‐rich and cysteine–glycine‐medium‐rich beta‐proteins (Beta‐keratins) in snake epidermis indicates a different distribution between beta‐ and alpha‐layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta‐keratin layers alternated with softer and pliable alpha‐keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta‐proteins (formerly beta‐keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine‐rich beta‐proteins of 12–15 kDa, is poorly or not reactive with the beta‐layer of snake epidermis. This suggests that glycine‐rich proteins similar to those present in lizards are altered during maturation of the beta‐layer. Conversely, a glycine–cysteine‐medium‐rich beta‐protein (HgGC10) of 10–12 kDa is present in beta‐ and alpha‐layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta‐ and alpha‐cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta‐proteins rich in glycine mainly accumulate on a scaffold of alpha‐keratin producing a resistant and hydrophobic beta‐layer. Conversely, beta‐proteins lower in glycine but higher in cysteine accumulate on alpha‐keratin filaments present in beta‐ and alpha‐layers producing resistant but more pliable layers.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Molecular packing in the feather keratin filament

Avian feathers have a filament-matrix texture and X-ray diffraction studies show that the filament has a helical structure with four repeating units per turn. Each repeating unit consists of a pair of twisted beta-sheets related by a perpendicular diad, and the twist in the sheets is of opposite hand to that of the helix. Each sheet is believed to comprise a 32-residue segment of the feather keratin molecule, which contains around 100 residues, the remainder constituting the matrix. In the present contribution, the sequence of emu feather is mapped to the low-resolution model derived earlier from X-ray studies. This shows that the inner surface of the "beta-sandwich" is densely populated by hydrophobic residues and that the charged residues and cysteine residues lie on the outer surface. In addition, the inner residues in the repeating unit mesh neatly together in layers oriented perpendicular to the filament axis. Amino acid sequences from a range of avian and reptilian keratins were collected and a 32-residue segment corresponding to the filament framework could be identified in every case, supporting the notion that there is a common plan for the filament framework in all of these materials. The hairpin turns in the beta-sheet were also identified and shown to be unusually rich in proline residues and also of variable composition. Two variants of the mapping were found which have complimentary conformations of the hairpin turns and these are illustrated and discussed. Since feather keratin yields a fiber rather than a crystalline X-ray pattern refinement of the model is restricted to trial-and-error methods and the assumptions made in its derivation are critically examined and some possible modifications discussed.     

Immunocytochemical analysis of beta keratins in the epidermis of chelonians,lepidosaurians, and archosaurians

Beta (beta) keratins are present only in the avian and reptilian epidermises. Although much is known about the biochemistry and molecular biology of the beta keratins in birds, little is known for reptiles. In this study we have examined the distribution of beta keratins in the adult epidermis of turtle, lizard, snake, tuatara, and alligator using light and electron immunocytochemistry with a well-characterized antiserum (anti-beta(1) antiserum) made against a known avian scale type beta keratin. In lizard, snake, and tuatara epidermis this antiserum reacts strongly with the beta-layer, more weakly with the oberhautchen before it merges with the beta-layer, and least intensely with the mesos layer. In addition, the anti-beta(1) antiserum reacts specifically with the setae of climbing pads in gekos, the plastron and carapace of turtles, and the stratum corneum of alligator epidermis. Electron microscopic studies confirm that the reaction of the anti-beta(1) antiserum is exclusively with characteristic bundles of the 3-nm beta keratin filaments in the cells of the forming beta-layer, and with the densely packed electron-lucent areas of beta keratin in the mature bet- layer. These immunocytochemical results suggest that the 3-nm beta keratin filaments of the reptilian integument are phylogenetically related to those found in avian epidermal appendages.     

The role of β-sheets in the structure and assembly of keratins

X-ray diffraction, infrared and electron microscope studies of avian and reptilian keratins, and of stretched wool and hair, have played a central role in the development of models for the β-conformation in proteins. Both α- and β-keratins contain sequences that are predicted to adopt a β-conformation and these are believed to play an important part in the assembly of the filaments and in determining their mechanical properties. Interactions between the small β-sheets in keratins provide a simple mechanism through which shape and chemical complementarity can mediate the assembly of molecules into highly specific structures. Interacting β-sheets in crystalline proteins are often related to one another by diad symmetry and the data available on feather keratin suggest that the filament is assembled from dimers in which the β-sheets are related by a perpendicular diad. The most detailed model currently available is for feather and reptilian keratin but the presence of related β-structural forms in mammalian keratins is also noted.     

More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs

We present the first genomic-scale analysis addressing the phylogenetic position of turtles, using over 1000 loci from representatives of all major reptile lineages including tuatara. Previously, studies of morphological traits positioned turtles either at the base of the reptile tree or with lizards, snakes and tuatara (lepidosaurs), whereas molecular analyses typically allied turtles with crocodiles and birds (archosaurs). A recent analysis of shared microRNA families found that turtles are more closely related to lepidosaurs. To test this hypothesis with data from many single-copy nuclear loci dispersed throughout the genome, we used sequence capture, high-throughput sequencing and published genomes to obtain sequences from 1145 ultraconserved elements (UCEs) and their variable flanking DNA. The resulting phylogeny provides overwhelming support for the hypothesis that turtles evolved from a common ancestor of birds and crocodilians, rejecting the hypothesized relationship between turtles and lepidosaurs.     

Nucleolar structure across evolution: the transition between bi- and tri-compartmentalized nucleoli lies within the class Reptilia

Two types of nucleolus can be distinguished among eukaryotic cells: a tri-compartmentalized nucleolus in amniotes and a bi-compartmentalized nucleolus in all the others. However, though the nucleolus' ultrastructure is well characterized in mammals and birds, it has been so far much less studied in reptiles. In this work, we examined the ultrastructural organization of the nucleolus in various tissues from different reptilian species (three turtles, three lizards, two crocodiles, and three snakes). Using cytochemical and immunocytological methods, we showed that in reptiles both types of nucleolus are present: a bi-compartmentalized nucleolus in turtles and a tri-compartmentalized nucleolus in the other species examined in this study. Furthermore, in a given species, the same type of nucleolus is present in all the tissues, however, the importance and the repartition of those nucleolar components could vary from one tissue to another. We also reveal that, contrary to the mammalian nucleolus, the reptilian fibrillar centers contain small clumps of condensed chromatin and that their surrounding dense fibrillar component is thicker. Finally, we also report that Cajal bodies are detected in reptiles. Altogether, we believe that these results have profound evolutionarily implications since they indicate that the point of transition between bipartite and tripartite nucleoli lies at the emergence of the amniotes within the class Reptilia.     

β-Keratins in crocodiles reveal amino acid homology with avian keratins

The DNA sequences encoding β-keratin have been obtained from Marsh Mugger (Crocodylus palustris) and Orinoco Crocodiles (Crocodylus intermedius). Through the deduced amino acid sequence, these proteins are rich in glycine, proline and serine. The central region of the proteins are composed of two beta-folded regions and show a high degree of identity with β-keratins of aves and squamates. This central part is thought to be the site of polymerization to build the framework of β-keratin filaments. It is believed that the β-keratins in reptiles and birds share a common ancestry. Near the C-terminal, these β-keratins contain a peptide rich in glycine-X and glycine-X-X, and the distinctive feature of the region is some 12-amino acid repeats, which are similar to the 13-amino acid repeats in chick scale keratin but absent from avian feather keratin. From our phylogenetic analysis, the β-keratins in crocodile have a closer relationship with avian keratins than the other keratins in reptiles.     

Unusually high zinc concentrations in snake plasma, with observations on plasma zinc concentrations in lizards, turtles and alligators

We provide evidence that normal plasma zinc levels in snakes are in the same range as the elevated zinc levels associated with haemolytic anaemia and fatal zinc toxicosis in dogs, and with weight loss and anorexia in crocodiles that had ingested coins with high zinc content. Blood plasma samples from large representative groups of snakes, lizards, turtles and alligators were analysed for zinc content by atomic absorption spectrophotometry. Plasma zinc levels in all snake species were five to 50-fold greater than levels reported in mammals. Plasma zinc levels in lizards and turtles were also higher than those of mammals, but significantly lower than those of snakes. Plasma zinc levels in alligators were in the same range as mammals and birds. After prolonged dialysis of snake plasma, 76% of the zinc remains in the retentate, suggesting the presence of a plasma protein with a strong affinity for the metal. Zinc levels vary significantly among the taxonomic groups, emphasizing the common evolutionary origin of crocodiles and birds and their divergence from the other reptiles.     

The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central “rod” region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.     

Keratin intermediate filament chains in the European common wall lizard (Podarcis muralis) and a potential keratin filament crosslinker

On the basis of sequence homology with mammalian α-keratins, and on the criteria that the coiled-coil segments and central linker in the rod domain of these molecules must have conserved lengths if they are to assemble into viable intermediate filaments, a total of 28 Type I and Type II keratin intermediate filament chains (KIF) have been identified from the genome of the European common wall lizard (Podarcis muralis). Using the same criteria this number may be compared to 33 found here in the green anole lizard (Anole carolinensis) and 25 in the tuatara (Sphenodon punctatus). The Type I and Type II KIF genes in the wall lizard fall in clusters on chromosomes 13 and 2 respectively. Although some differences occur in the terminal domains in the KIF chains of the two lizards and tuatara, the similarities between key indicator residues – cysteine, glycine and proline – are significant. The terminal domains of the KIF chains in the wall lizard also contain sequence repeats commonly based on glycine and large apolar residues and would permit the fine tuning of physical properties when incorporated within the intermediate filaments. The H1 domain in the Type II chain is conserved across the lizards, tuatara and mammals, and has been related to its role in assembly at the 2–4 molecule level. A KIF-like chain (K80) with an extensive tail domain comprised of multiple tandem repeats has been identified as having a potential filament-crosslinking role.     

Immunolocalization of beta‐proteins and alpha‐keratin in the epidermis of the soft‐shelled turtle explains the lack of formation of hard corneous material

Immunolocalization of beta‐proteins in the epidermis of the soft‐shelled turtle explains the lack of formation of hard corneous material, Acta Zoologica, Stockholm. The corneous layer of soft‐shelled turtles derives from the accumulation of higher ratio of alpha‐keratins versus beta‐proteins as indicated by gene expression, microscopic, immunocytochemical and Western blotting analysis. Type I and II beta‐proteins of 14–16 kDa, indicated as Tu2 and Tu17, accumulate in the thick and hard corneous layer of the hard‐shelled turtle, but only type II is present in the thinner corneous layer of the soft‐shelled turtle. The presence of proline–proline and proline–cysteine–hinge dipeptides in the beta‐sheet region of all type II beta‐proteins so far isolated from the epidermis of soft‐shelled turtles might impede the formation of beta‐filaments and of the hard corneous material. Western blot analysis suggests that beta‐proteins are low to absent in the corneous layer. The ultrastructural immunolocalization of Tu2 and Tu17 beta‐proteins shows indeed that a diffuse labelling is seen among the numerous alpha‐keratin filaments present in the precorneous and corneous layers of the soft epidermis and that no dense corneous material is formed. Double‐labelling experiments confirm that alpha‐keratin prevails on beta‐proteins. The present observations support the hypothesis that the soft material detected in soft‐shelled turtles derives from the prevalent activation of genes producing type II beta‐proteins and high levels of alpha‐keratins.     

Histochemical characterization of glycoconjugates in the epithelium of the extrapulmonary airways of several vertebrates

Summary The glycoconjugates of the extrapulmonary airways of 11 tetrapode vertebrates have been characterized by means of both conventional and lectin histochemistry. Abundant sialosulphomucins were detected in the secretory cells and periciliary layer of turtles, snakes, birds and mammals while only sialomucins were observed in amphibians. Neutral and traces of acidic mucins were detected in the secretory cells of lizards. The secretory cells of the amphibian airways were reactive to Con-A, DBA and WGA. No -l-fucose residues reactive with UEA-I or LTA were detected in amphibians. The goblet cells of the turtles were stained by DBA, SBA and WGA. Secretory cells of snakes and lizards reacted with Con-A and WGA. The mucous goblet cells of the birds were reactive to Con-A, LTA and WGA. In the chicken, they also showed affinity for PNA and SBA. The ciliated cells ofthe avian species studied were stained by Con-A and WGA. Mammalian goblet cells were reactive to Con-A, UEA-I and WGA. In the rat, affinity for DBA and SBA was also observed. The present results reveal the existence of marked differences in the sugar residues of the glycoconjugates of the extrapulmonary airways of tetrapode vertebrates. Only sialic acid residues appear to be constant constituents of the glycoconjugates of the airways of all species studied.     

The organization of the reptilian brainstem reticular formation: A comparative study using Nissl and Golgi techniques

The brainstem reticular formation has been studied in 16 genera representing 11 families of reptiles. Measurements of Nissl-stained reticular neurons revealed that they are distributed along a continuum, ranging in length from 10 μm to 95 μm. Reticular neurons in crocodilians and snakes tend to be larger than those found in lizards and turtles. Golgi studies revealed that reticular neurons posess long, rectilinear, sparsely branching dendrites. Small reticular neurons ( > 31 μm length) possess fusiform or triangular somata which bear two or three primary dendrites. These dendrites have a somewhat simpler ramification pattern when compared with those of large reticular neurons (< 30 μm length). Large reticular neurons generally possess perikarya which are triangular or polygonal in shape. The somata of large reticular neurons bear an average of four primary dendrites. The dendrites of reptilian reticular neurons ramify predominantly in the transverse plane and are devoid of spines or excrescences. The dendritic ramification patterns observed in the various repitilian reticular nuclei were correlated with known input and output connections of these nuclei. Nissl and Golgi techniques were used to divide the reticular formation into seven nuclei. A nucleus reticularis inferior (RI) is found in the myelencephalon, a reticularis medius (RM) in the caudal two-thirds of the metencephalon, and a reticularis superior (RS) in the rostral metencephalon and caudal mesencephalon. Reticularis inferior can be subdivided into a dorsal portion (RID) and a ventral portion (RIV). All reptilian groups possess RID and RM but RIV is lacking in turtles. Reticularis superior can be subdivided into a large-celled lateral portion (RSL) and a small-celled medial portion (RSM). All reptilian groups possess RSM and RSL, but RSL is quite variable in appearance, being best developed in snakes and crocodilians. The myelencephalic raphe nucleus is also quite variable in its morphology among the different reptilian families. A seventh reticular nucleus, reticularis ventrolateralis (RVL), is found only in snakes and in teiid lizards. It was noted that the reticular formation is simpler (fewer numbers of nuclei) in the representatives of older reptilian lineages and more complex (greater numbers of nuclei) in the more modern lineages. Certain reticular nuclei are present or more extensive in those families which have prominent axial musculature.