Prismatic dentine in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

The Australian lungfish, Neoceratodus forsteri, has a dentition consisting of enamel, mantle dentine and bone, enclosing circumdenteonal, core and interdenteonal dentines. Branching processes from cells that produce interdenteonal dentine leave the cell surface at different angles, with collagen fibrils aligned parallel to the long axis of each process. In the interdenteonal dentine, crystals of calcium hydroxyapatite form within fibrils of collagen, and grow within a matrix of non-collagenous protein. Crystals are aligned parallel to the cell process, as are the original collagen fibrils. Because the processes are angled to the cell surface, the crystals within the core or interdenteonal dentine are arranged in bundles set at angles to each other. Apatite crystals in circumdenteonal dentine are finer and denser than those of the interdenteonal dentine, and form outside the fibrils of collagen. In mature circumdenteonal dentine the crystals of circumdenteonal dentine form a dense tangled mass, linked to interdenteonal dentine by isolated crystals. The functional lungfish tooth plate contains prisms of large apatite crystals in the interdenteonal dentine and masses of fine tangled crystals around each denteon. This confers mechanical strength on a structure with little enamel that is subjected to heavy wear.     

The epithelial sodium channel in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Epithelial sodium channel (ENaC) is a Na⁺-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na⁺ absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, β and γ proteins. The nENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.     

Skin structure in the snout of the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Many fossil lungfish have a system of mineralised tubules in the dermis of the snout, branching extensively and radiating towards the epidermis. The tubules anastomose in the superficial layer of the dermis, forming a plexus consisting of two layers of vessels, with branches that expand into pore canals and flask organs, flanked by cosmine nodules where these are present. Traces of this system are found in the Australian lungfish, Neoceratodus forsteri, consisting of branching tubules in the dermis, a double plexus below the epidermis and dermal papillae entering the epidermis without reaching the surface. In N. forsteri, the tubules, the plexus and the dermal papillae consist of thick, unmineralised connective tissue, enclosing fine blood vessels packed with lymphocytes. Tissues in the epidermis and the dermis of N. forsteri are not associated with deposits of calcium, which is below detectable limits in the skin of the snout at all stages of the life cycle. Canals of the sensory line system, with mechanoreceptors, are separate from the tubules, the plexus and the dermal papillae, as are the electroreceptors in the epidermis. The system of tubules, plexus, dermal papillae and lymphatic capillaries may function to protect the tissues of the snout from infection.     

Ultrastructure of developing tooth plates in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

While the lungfish dentition is partially understood as far as morphology and light microscopic structure is concerned, the ultrastructure is not. Each tooth plate is associated with a dental lamina that develops from the inner layer of endodermal cells that form the oral epithelium. Dentines, bone and cartilage of the jaws differentiate from mesenchyme cells aggregating beneath the oral endothelium. Enamel, in the developing and in the mature form, has similarities to that of other early vertebrates, but unusual characters appear as development proceeds. Ameloblasts are capable of secreting enamel, and, with mononuclear osteoclasts, of remodelling the bone below the tooth plate. The forms of dentine, all based largely on an extracellular matrix of collagen and mineralised with biological apatite, differ from each other and from the underlying bone in the ultrastructure of associated cells and in the mineralised extracellular matrices produced. Cell processes emerging from the odontoblasts and from the osteoblasts vary in length, degree of branching and of anastomoses between the processes, although all of the cell types have large amounts of rough endoplasmic reticulum. Mineralisation of the extracellular matrices varies among the enamel, dentines and bone in the tooth plate. In addition, the development of the hard tissues of the tooth plates indicates that many of the similarities in fine structure of the dentition in lungfish, to tissues in other fish and amphibia, apparent early in development, disappear as the dentition matures.     

Role of epidermal cilia in development of the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

In common with the embryos of other anamniotes, young of the Australian lungfish, Neoceratodus forsteri, have ciliated cells in the epidermis. These first appear at stage 28, ˜ 10 days before hatching, and develop progressively to a peak in numbers and in activity at stage 44, just after hatching. After this point, ciliary action in the epidermal cells slowly declines, and cilia disappear completely from the outer surface of the hatchling by stage 52. Cilia are lost earlier from the oral epithelium, between stages 45 and 46, and from the epithelium covering the gills and lining the operculum at stage 51, although they are retained in the nares and in the cavity of the olfactory organ. To assess possible functions for the ciliated epidermis in lungfish hatchlings, the presence of cilia in the epidermis of young N. forsteri is compared with landmarks of development. The ciliated epidermal cells are not associated with movements of the embryo within the egg capsule, nor are they a part of a feeding mechanism. They are not related to oxygen uptake. The ciliated epidermis appears to function as a mechanism for clearing the animal of particles and settling organisms before hatching, when the egg membranes have developed holes, and after hatching, when the young fish is living among the submerged rootlets of trees growing on the river bank or in dense stands of aquatic plants. The function of a ciliated epidermis in N. forsteri hatchlings in relation to microhabitat is discussed. © 1996 Wiley-Liss, Inc.     

Cranial nerves in the Australian lungfish,Neoceratodus forsteri,and in fossil relatives (Osteichthyes: Dipnoi)

Three systems, two sensory and one protective, are present in the skin of the living Australian lungfish, Neoceratodus forsteri, and in fossil lungfish, and the arrangement and innervation of the sense organs is peculiar to lungfish. Peripheral branches of nerves that innervate the sense organs are slender and unprotected, and form before any skeletal structures appear. When the olfactory capsule develops, it traps some of the anterior branches of cranial nerve V, which emerged from the chondrocranium from the lateral sphenotic foramen. Cranial nerve I innervates the olfactory organ enclosed within the olfactory capsule and cranial nerve II innervates the eye. Cranial nerve V innervates the sense organs of the snout and upper lip, and, in conjunction with nerve IX and X, the sense organs of the posterior and lateral head. Cranial nerve VII is primarily a motor nerve, and a single branch innervates sense organs in the mandible. There are no connections between nerves V and VII, although both emerge from the brain close to each other. The third associated system consists of lymphatic vessels covered by an extracellular matrix of collagen, mineralised as tubules in fossils. Innervation of the sensory organs is separate from the lymphatic system and from the tubule system of fossil lungfish.     

Regionalization of axial skeleton in the lungfish Neoceratodus forsteri (Dipnoi)

Differentiation of the axial skeleton into distinct regions, once thought to be characteristic of the Tetrapoda, also occurs in the actinopterygian Danio rerio. In these taxa, the boundary between the cervical-thoracic regions correlates with Hoxc6 expression and morphological features such as position of the pectoral fin and associated nerves, and the absence of ribs. In the lungfish Neoceratodus, a member of the extant sister taxon to the Tetrapoda, the first vertebral element to chondrify is situated well posterior to the skull, developing from somites 6 and 7 (6/7) and associated with an enlarged cranial rib and nerves innervating the pectoral fin. Two vertebral elements develop later and more anteriorly, associated with somites 4/5 and 5/6. These three elements become incorporated into the occipital region of the skull during Neoceratodus ontogeny, until the cranial rib itself articulates to the rear of the skull. These features of early development indicate a regionalization of the Neoceratodus vertebral column: the cranial rib marks the boundary between the cervical and thoracic regions, the two more anterior vertebrae lacking ribs represent the cervical region, while somites 1-4 (cranial half), lacking any vertebral development, represent the occipital region. However, the cervical region of the vertebral column is effectively lost during ontogeny of Neoceratodus. A recognizable cervical region in the tetrapod vertebral column, as in zebrafish, suggests that cervical vertebrae are not incorporated into the skull but maintained as distinct elements of the column, representing an important shift in relative developmental timing and the influence of heterochrony in this region during the fish-tetrapod transition.     

High resolution transmission electron microscopy of developing enamel in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

The permanent tooth plates of the Australian lungfish, Neoceratodus forsteri, are covered by enamel that develops initially in a similar manner to that of other vertebrates. As the enamel layer matures, it acquires several unusual characteristics. It has radially oriented protoprismatic structures with the long axes of the protoprisms perpendicular to the enamel surface. Protoprisms can be defined as aggregations of hydroxyapatite crystals that lack the highly ordered arrangement of the rods of mammalian enamel but are not without a specific structure of their own. The protoprisms are arranged in layers of variable thickness that are deposited sequentially as the tooth plate grows. They may be confined to the separate layers, or may cross the boundary between each layer. Crystals within the protoprisms are long and thin with hydroxyapatite c-axis dimensions of between 30 and 350 nm, and with typical a-b axis dimensions of 5-10 nm. The hydroxyapatite crystals of lungfish enamel have no centre dark lines of octacalcium phosphate, an unusual character among vertebrates. As each crystal develops, arrays of atoms may change direction, and regions exist where dislocations and extra lattice planes are inserted into the long crystal. The resulting hydroxyapatite crystal is not straight, and has a rough surface. The crystals are arranged in tangled structures with their crystallographic c-axes closely aligned with the long axis of the protoprism. Lungfish enamel differs from the enamel of higher vertebrates in that the hydroxyapatite crystals are of different shape, and, in mature enamel, the protoprisms remain as protoprisms and do not develop into the conventional prismatic structures characteristic of mammalian enamel.     

Homeobox genes in the Australian lungfish, Neoceratodus forsteri.

The aim of the present study was to determine whether the postulated gnathostome duplication from four to eight Hox clusters occurred before or after the split between the actinopterygian and sarcopterygian fish by characterizing Hox genes from the sarcopterygian lungfish, Neoceratodus forsteri. Since lungfish have extremely large genomes, we took the approach of extracting pure high molecular weight (MW) genomic DNA to act as a template for polymerase chain reaction (PCR) of the conserved homeobox domain of the highly conserved Hox genes. The 21 clones thus obtained were sequenced and translated in a BLASTX protein database search to designate Hox gene identity. Fourteen of the clones were from Hox genes, two were Hox pseudogenes, four were Gbx genes, and one most closely resembled the homeobox gene, insulin upstream factor 1. The Hox genes identified were from all four tetrapod clusters A, B, C, and D, confirming their presence in lungfish, and there is no evidence to suggest more than these four functional Hox clusters, as is the case in teleosts. A comparison of Hox group 13 amino acid sequences of lungfish, zebrafish, and mouse provides firm evidence that the expansion of Hox clusters, as seen in zebrafish, occurred after separation of the actinopterygian and sarcopterygian lineages. J. Exp. Zool. (Mol. Dev. Evol.) 285:140-145, 1999.     

Characterization of the hemoglobins of the Australian lungfish Neoceratodus forsteri (Krefft)

We examined for the first time the hemoglobin components of the blood of the Australian lungfish, Neoceratodus forsteri and their functional responses to pH and the allosteric modulators adenosine triphosphate (ATP), guanosine triphosphate (GTP), 2,3-bisphosphoglyceric acid (BPG) and inositol hexaphosphate (IHP) at 25 degrees C. Lysates prepared from stripped, unfractionated hemolysate produced sigmoidal oxygen equilibrium curves with high oxygen affinity (oxygen partial pressure required for 50% hemoglobin saturation, p(50)=5.3 mmHg) and a Hill coefficient of 1.9 at pH 7.5. p(50) was 8.3 and 4.5 mmHg at pH 6 and 8, respectively, which corresponded to a modest Bohr coefficient (Delta log p(50)/Delta pH) of -0.13. GTP increased the pH sensitivity of oxygen binding more than ATP, such that the Bohr coefficient was -0.77 in the presence of 2 mmol L(-1) GTP. GTP was the most potent regulator of hemoglobin affinity, with concentrations of 5 mmol L(-1) causing an increase in p(50) from 5 to 19 mm Hg at pH 7.5, while the order of potency of the other phosphates was IHP>ATP>BPG. Three hemoglobin isoforms were present and each contained both alpha and beta chains with distinct molecular weights. Oxygen affinity and pH-dependence of isoforms I and II were essentially identical, while isoform III had a lower affinity and increased pH-dependence. The functional properties of the hemoglobin system of Neoceratodus appeared consistent with an active aquatic breather adapted for periodic hypoxic episodes.     

The histology of tooth formation in the Australian lungfish, Neoceratodus forsteri Krefft

The histology of developing toothplates of Neoceratodusforsteri from the time of first appearance of the tooth primordia to the adult condition has been investigated. The dentition develops by the formation of a shell of primary epithelial and mesenchymal matrices. Within the shell, secondary mesenchymal matrix and central material, both containing columns of tertiary matrix, are laid down. Primary epidielial matrix appears to contain collagen and is closely associated with the epithelium of the mouth. All other tooth tissues as well as the supporting bone develop in association with mesenchyme. Primary, secondary and tertiary mesenchymal matrices appear to contain collagen. Central dentine contains some fibres, possibly of reticulin or collagen, within a matrix of unknown composition.
The tooth is attached to the underlying bone by a pedestal of bone and this grows with the tooth material.
New tooth tissues are formed in the pulp cavity in layers below the older material, causing the toothplate to grow in every dimension as the animal grows.
An evolutionary pathway is suggested for lungfish with a dentition of cusps arranged in radiating ridges.     

True enamel covering in teeth of the Australian lungfish Neoceratodus forsteri

Lungfish are a unique order of sarcopterygian fish cleidographically positioned between tetrapods and fish. An uninterrupted 400-million-year-old fossil record has documented lungfish skeletal elements to remain virtually unchanged since the Early Devonian. In the current study we investigated the enamel layer of lungfish teeth in order to determine whether there was evidence for higher vertebrate "true" enamel in the Australian lungfish. Juvenile lungfish from the Brisbane River were processed for light and electron microscopy and analyzed for parameters indicative of true enamel formation. Using anti-amelogenin primary antibodies for immunodetection and Western blots, enamel protein epitopes were detected in developing lungfish teeth. Using transmission electron microscopy and electron diffraction analysis, long and parallel-oriented hydroxyapatite crystals were observed in lungfish outer tooth coverings. Our findings indicate that Australian lungfish teeth are covered by a layer of true enamel. Based on the lungfish fossil record we conclude that features of true enamel formation may be as old as 400 million years. Based on taxonomic classification we confirm that true enamel is found not only in tetrapods but also in the sarcopterygian clade of the Gnathostomata.     

Insulin-, glucagonand somatostatin-like immunoreactivity in the endocrine pancreas of the lungfish,Neoceratodus forsteri

Summary The endocrine pancreas of the Australian lungfish,Neoceratodus forsteri, was investigated immunocytochemically for the presence of polypeptide hormone-producing cells. Three cell types were identified, namely insulin-, glucagon-, and somatostatin-immunoreactive elements. The insulin cells are confined solely to the center of the islets. Glucagon and somatostatin cells are distributed peripherally around the central mass of the insulin cells. Isolated cells or clusters of glucagon and somatostatin cells are also dispersed within the exocrine parenchyma. The immunoreactive cell types are compared with those staining with standard histological procedures. The spatial relationships of the different cell populations are examined.