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.     

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.     

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.     

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.     

Scale structure in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Scales of the Australian lungfish, Neoceratodus forsteri, are secreted within the dermis by a capsule of scleroblasts, and enclosed in a pouch made of collagen fibers, in contact with the epidermis over the posterior third of the scale. Each scale grows from a focus, which represents the first formed part of the scale. On the internal surface of the scale is elasmodin, made of collagen fiber bundles arranged in layers. Elasmodin, unmineralized in N. forsteri, contains cells in the living animal, and the number of layers increases as the scales grow. Squamulin, on the thin external part of the scale, is also laid down in layers, and based on a matrix of fine collagen fibrils, mineralized with a poorly crystalline biogenic calcium hydroxylapatite. Squamulin is divided into separate sections called squamulae, and contains long tubules with cells applied to the wall of the tubule. The anterior and lateral surfaces of the squamulin are ornamented with pediculae, and the posterior surface has longitudinal ridges, from which collagen fibers extend to anchor the scale within the pouch. Elasmodin and squamulin are linked by unmineralized collagen fibrils. The layers, formed at irregular intervals, are connected around the margin of the scale, effectively converting the whole scale into a flat structure resembling a pearl, with the first formed tissues deeply embedded inside the scale, and the youngest on the outer surface. Incremental lines in the hard tissue, and the number of layers in the elasmodin, do not reflect the chronological age of the fish. J. Morphol. 276:1137–1145, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

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.     

The steroidogenic response to angiotensin II in the Australian lungfish,Neoceratodus forsteri

Corticosterone, aldosterone and cortisol were found to be present in lungfish plasma. Plasma levels of these hormones were measured in lungfish following separate single intramuscular injections of three forms of angiotensin II; [Asp¹, Ile⁵], [Asp¹, Val⁵] and [Asn¹, Val⁵]. Aldosterone levels were significantly elevated in response to [Asp¹, Ile⁵] AII and [Asn¹, Val⁵] AII injection. [Asp¹, Val⁵] AII increased plasma corticosterone levels. The difference between these data and the negative results previously reported by Blair-West et al. (1977) are discussed.Abbreviations AII angiotensin II - bw body weight - DOC deoxycorticosterone - RAS renin-angiotensin system - RIA radioimmuno assay     

Cartilage,bone, and intermandibular connective tissue in the australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

The connective tissue that links the bones of the mandible in the Australian lungfish, Neoceratodus forsteri, has been described as an intermandibular cartilage, and as such has been considered important for phylogenetic analyses among lower vertebrates. However, light and electron microscopy of developing lungfish jaws demonstrates that the intermandibular tissue, like the connective tissue that links the bones of the upper jaw, contains fibroblasts and numerous bundles of collagen fibrils, extending from the trabeculae of the bones supporting the tooth plates. It differs significantly in structure and in staining reactions from the cartilage and the bone found in this species. In common with the cladistian Polypterus and with actinopterygians and some amphibians, lungfish have no intermandibular cartilage. The connective tissue linking the mandibular bones has no phylogenetic significance for systematic grouping of lungfish, as it is present in a range of different groups among lower vertebrates. J. Morphol. 274:1085–1089, 2013. © 2013 Wiley Periodicals, Inc.     

Marginal tooth-bearing bones in the lower jaw of the recent australian lungfish,Neoceratodus forsteri (Osteichthyes,Dipnoi)

Developmental studies of the Recent Australian lungfish, Neoceratodus forsteri, show that this species has two sets of functional tooth-bearing bones in the lower jaw of young hatchlings. These coincide with an early stage in the life history when the fish is strictly carnivorous. In N. forsteri, a paired tooth-bearing dentary and an unpaired symphyseal bone and tooth develop slightly later than the permanent vomerine, prearticular, and pterygopalatine tooth plates, which appear at stage 44 of development, and erupt with the permanent dentition between stages 46 and 48, when the hatchling first starts to feed on small aquatic invertebrates. At these stages of development, all of the teeth are long, sharp, and conical and help to retain prey items in the mouth. Disappearance of the transient dentition coincides with complete eruption of the permanent tooth plates and precedes the change to an omnivorous diet. Existence of a transient marginal dentition in this species of lungfish suggests that the presence of an apparently similar marginal dentition in adults of many species of Palaeozoic dipnoans should be considered in phylogenetic analyses of genera within the group, and when analysing the relationships of dipnoans with other primitive animals. © 1995 Wiley-Liss, Inc.     

Formation and structure of scales in the Australian lungfish,neoceratodus forsteri (Osteichthyes: Dipnoi)

The large elasmoid scales of the Australian lungfish, Neoceratodusforsteri, are formed within the dermis by unpigmented scleroblasts, growing within a collagenous dermal pocket below a thick glandular epidermis. The first row of scales, on the trunk of the juvenile lungfish, appears below the lateral line of the trunk, single in this species, at around stage 53. The scales, initially circular in outline, develop anteriorly and posteriorly from the point of initiation in the mid‐trunk region, and rows are added alternately below the line, and above the line, until they reach the dorsal or ventral midline, or the margins of the fins. Scales develop later on the ventral surface of the head, from a separate centre of initiation. Scales consist of three layers, all produced by scleroblasts of dermal origin. The outermost layer of interlocking plates, or squamulae, consists of a mineralised matrix of fine collagen fibrils, covered by unmineralised collagen and a single layer of cells. Squamulae of the anterior and lateral surfaces are ornamented with short spines, and the mineralised tissue of the posterior surface is linked to the pouch by collagen fibrils. The innermost layer, known as elasmodin, consists of bundles of thick collagen fibrils and cells arranged in layers. An intermediate layer, made up of collagen fibrils, links the outer and inner layers. The elasmoid scales of N. forsteri can be compared with scale types among other osteichthyan groups, although the cellsand canaliculi in the mineralised squamulae bear littleresemblance to typical bone. J.Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

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.     

Ultrastructure of the integumental melanophores of the Australian lungfish,Neoceratodus forsteri

The integumental melanophores of Australina lungfish, Neoceratodus forsteri, were examined by light and electron microscopy and found to possess essentially the same structural characteristics observed in other vertebrates. The epidermal melanophores are located in the intermediate epidermis and possess round perikarya and slender dendrites extending into nearby intercellular spaces. The dermal melanophores are found immediately below the basement membrane as well as in the deeper dermis. These cells possess flattened nuclei and dendrites running parallel to the basement membrane. Each melanophore contains numerous oval or elliptical, intensely electron-dense melanosomes, relatively large mitochondria, systems of vacuolar endoplasmic reticulum, groups of free RNP particles, and some microfilaments. Only a few, short microtubules could be demonstrated in the perinuclear cytoplasm of the dermal melanophore, while a relatively large number of late premelanosomes are found both in perikarya and dendritic processes of epidermal melanophores. These premelanosomes exhibit a particulate internal structure in cross section. Both melanosomes and premelanosomes occur singly in the cytoplasm of epidermal cells, thereby confirming the existence of the epidermal melanin unit in the lowest vertebrates thus far examined electron microscopically.     

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.     

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.     

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

Tooth plate formation in the Queensland lungfish, Neoceratodus forsteri, Krefft begins with simple groups of isolated cusps, three in each tooth plate. The cusps fuse in ridges radiating from a point situated posterolingually. During growth, cusps are added to the labial ends of the ridges, and more ridges are added posteriorly, giving a total of seven in each tooth plate. Each tooth grows in thickness by the addition of layers of material, in line with the new cusps, beneath the tooth plate. The tooth plate grows outwards and is resorbed from the inner angle at the same time. The crushing surface is formed by the growth of cusps between the ridges. Angles between the ridges become progressively smaller, and angles between more posterior ridges are consistently less than between more anterior ridges. Similar but less pronounced changes in angles between ridges occur in a fossil genus, Sagenodus inaequalis, examined for comparison.
Vomerine teeth grow in the same way, by fusion of isolated cusps and the addition of new cusps to one end (labial) of the tooth plate. Layers of material are also added beneath the tooth plate. The vomerine tooth plates are initially low-based with long cusps but develop into high-based low cusped incisiform tooth plates in fully grown adults.
The labial dentition of the lower jaw starts to develop like the vomerine teeth, but degenerates by stage (vi) of tooth development. The single medial tooth is resorbed even earlier.
The pattern of tooth plate formation described in this paper is consistent with illustrations published by Semon (1901) and Greil (1908, 1913) but the inferred developmental processes are different.
Implications of the results for the Zahnreihe hypothesis of Edmund and for the phylogeny of Dipnoi are discussed.