Immunocytochemical localization and characterization of mammalian prolactin- and somatotropin-like material in the pituitary of the Australian lungfish,Neoceratodus forsteri

The aim of the present study was to define at the light-microscopic level expression of prolactin and somatotropin material in the pituitary gland of the Australian lungfish, Neoceratodus forsteri, by use of polyclonal antibodies against ovine prolactin (oPRL) and bovine somatotropin (bSTH). Substances immunologically related to mammalian oPRL as well as bSTH were detected in two morphologically different cell types in the distal lobe, corresponding to the acidophilic cells. The specificity of the antibodies was initially confirmed in a porcine tissue control system. First, our absorption studies confirm that in Neoceratodus the anti-oPRL identifies part of an oPRL-like molecule different from bSTH. Secondly, the anti-bSTH identifies both part of a bSTH-like molecule proper to bovine and Neoceratodus STH, and part of a bSTH-like molecule having antigenic determinants in common with both bSTH and oPRL. This part of the oPRL is, however, not shared with the Neoceratodus PRL as revealed by the anti-oPRL. Altogether these observations support the concepts: (1) that mammalian PRL and STH, or part of those, were established early in evolution, and (2) that dipnoans as living sarcopterygians have an ancestor in common with the early amphibians. The exact nature and physiological functions of the substances detected remain to be defined.     

Vascularization of the pituitary of the Australian lungfish and Neoceratodus forsteri

The vascularization of the brain and the pituitary region of the Australian lungfish, Neoceratodus forsteri is described from serial section reconstruction. The distal lobe has no direct arterial blood supply and receives blood solely from a pituitary portal system basically similar to that of other sarcopterygians. The primary capillary plexus of the median eminence receives its arterial blood from the infundibular arteries, which on their way distribute some small branches to the prechiasmatic region. The primary plexus also receives capillaries from the adjacent pial hypothalamic plexus. The primary capillary plexus of the median eminence comprises a rostral 'uncovered' and caudal 'covered' part which are not sharply delineated. Distinct portal vessels connect the 'uncovered' rostral part of the primary plexus with the secondary capillary plexus supplying the rostral subdivision of the pars distalis. The 'covered' caudal part of the primary plexus merges into the proximal subdivision of the pars distalis, apparently without formation of distinct portal vessels. The primary plexus has some connections with the plexus intermedius via a hypophysial stem capillary plexus. The plexus intermedius has a substantial arterial supply and gives off capillaries to the parenchyma of the pars intermedia. The adenohypophysis is drained into an unpaired hypophysial vein. The significance of the vascular pathways is discussed from comparative, functional, and evolutionary viewpoints.     

Cephalic muscle development in the Australian lungfish,Neoceratodus forsteri

Lungfishes are the extant sister group of tetrapods. As such, they are important for the study of evolutionary processes involved in the water to land transition of vertebrates. The evolution of a true neck, that is, the complete separation of the pectoral girdle from the cranium, is one of the most intriguing morphological transitions known among vertebrates. Other salient changes involve new adaptations for terrestrial feeding, which involves both the cranium and its associated musculature. Historically, the cranium has been extensively investigated, but the development of the cranial muscles much less so. Here, we present a detailed study of cephalic muscle development in the Australian lungfish, Neoceratodus forsteri, which is considered to be the sister taxon to all other extant lungfishes. Neoceratodus shows several developmental patterns previously described in other taxa; the tendency of muscles to develop from anterior to posterior, from their region of origin toward insertion, and from lateral to ventral/medial (outside‐in), at least in the branchial arches. The m.protractor pectoralis appears to develop as an extension of the most posterior m.levatores arcuum branchialium, supporting the hypothesis that the m.cucullaris and its derivatives (protractor pectoralis, levatores arcuum branchialium) are branchial muscles. We present a new hypothesis regarding the homology of the ventral branchial arch muscles (subarcualis recti and obliqui, transversi ventrales) in lungfishes and amphibians. Moreover, the morphology and development of the cephalic muscles confirms that extant lungfishes are neotenic and have been strongly influenced via paedomorphosis during their evolutionary history.     

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.     

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.     

Cranial neural crest cell migration in the Australian lungfish, Neoceratodus forsteri

SUMMARY A crucial role for the cranial neural crest in head development has been established for both actinopterygian fishes and tetrapods. It has been claimed, however, that the neural crest is unimportant for head development in the Australian lungfish ( Neoceratodus forsteri   ), a member of the group (Dipnoi) which is commonly considered to be the living sister group of the tetrapods. In the present study, we used scanning electron microscopy to study cranial neural crest development in the Australian lungfish. Our results, contrary to those of Kemp, show that cranial neural crest cells do emerge and migrate in the Australian lungfish in the same way as in other vertebrates, forming mandibular, hyoid, and branchial streams. The major difference is in the timing of the onset of cranial neural crest migration. It is delayed in the Australian lungfish in comparison with their living sister group the Lissamphibia. Furthermore, the delay in timing between the emergence of the hyoid and branchial crest streams is very long, indicating a steeper anterior-posterior gradient than in amphibians. We are now extending our work on lungfish head development to include experimental studies (ablation of selected streams of neural crest cells) and fate mapping (using fluoresent tracer dyes such as DiI) to document the normal fate as well as the role in head patterning of the cranial neural crest in the Australian lungfish.     

Spawning activity of the Australian lungfish Neoceratodus forsteri in an impoundment

This study assessed the spawning activity of the threatened Australian lungfish Neoceratodus forsteri by measuring egg densities within the artificial habitat of a large impoundment (Lake Wivenhoe, Australia). Eggs were sampled (August to November 2009) from multiple locations across the impoundment, but occurred at highest densities in water shallower than 40 cm along shorelines with a dense cover of submerged terrestrial vegetation. The numbers of eggs declined over the study period and all samples were dominated by early developmental stages and high proportions of unviable eggs. The quality of the littoral spawning habitats declined over the study as flooded terrestrial grasses decomposed and filamentous algae coverage increased. Water temperatures at the spawning site exhibited extreme variations, ranging over 20·4° C in water shallower than 5 cm. Dissolved oxygen concentrations regularly declined to <1 mg l^?1 at 40 and 80 cm water depth. Spawning habitats utilised by N. forsteri within impoundments expose embryos to increased risk of desiccation or excessive submergence through water‐level variations, and extremes in temperature and dissolved oxygen concentration that present numerous challenges for successful spawning and recruitment of N. forsteri in large impoundment environments.     

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.     

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 isolation of multiple forms of beta-endorphin from the intermediate pituitary of the Australian lungfish, Neoceratodus forsteri

The pituitary of the Australian lungfish, Neoceratodus forsteri, was screened immunohistochemically with heterologous antisera specific for either the C-terminal of mammalian beta-endorphin or the acetylated N-terminal of beta-endorphin. Immunopositive cells were only detected with the N-terminal specific antiserum; these cells were restricted to the intermediate pituitary. Acid extracts of the intermediate pituitary were fractionated by Sephadex gel filtration chromatography, CM cation exchange chromatography and reverse phase HPLC. Fractions were analyzed by radioimmunoassay (RIA) with a N-acetyl specific beta-endorphin RIA and by radioreceptor assay for the presence of opiate active forms of beta-endorphin. Both immunoreactive and opiate active forms of beta-endorphin were detected. Of the total beta-endorphin-related material isolated from the intermediate pituitary, approximately 97% was detected with the N-terminal specific RIA and approximately 3% was detected by the radioreceptor assay. The N-acetylated immunoreactive beta-endorphin could be separated into two forms. The major form had an apparent molecular weight of 3.2 Kda. This material had a net charge at pH 2.5 of +5. The minor form of immunoreactive beta-endorphin had an apparent molecular weight of 1.4 Kda and a net charge at pH 2.5 of +1. Neither immunoreactive form exhibited receptor binding activity in the radioreceptor assay. A single peak of opiate active beta-endorphin was detected. This material had an apparent molecular weight of 3.5 Kda and a net charge at pH 2.5 of +7.     

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     

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.     

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 fate of cranial neural crest cells in the Australian lungfish, Neoceratodus forsteri

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.     

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.     

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.     

Development of the vasculature of the pectoral fin in the Australian lungfish, Neoceratodus forsteri

The development of the vasculature of the pectoral fin in the Australian lungfish, Neoceratodus forsteri, was studied by the dye-injection method. Only a single primitive subclavian artery appears from the dorsal aorta for the fin anlage, and it passes laterally through the postaxial region of the structure. The venous channel draining into the posterior cardinal vein is located in the preaxial region medially. As development proceeds, the arteriovenous arrangement in the pectoral fin anlage changes as follows: 1) one artery and one venous plexus, 2) two arteries and one vein, 3) three arteries and one vein, 4) four arteries and one vein, 5) three arteries and two veins, and 6) two arteries (radial and ulnar) and three veins (radial, ulnar, and ulnar marginal). The fin anlage through embryonic first rotation has gradually changed its postaxial margin to face dorsally and its preaxial margin to face ventrally. The second rotation causes the original preaxial margin to become dorsal and the original postaxial margin to become ventral. As a result, the radial and ulnar arteries are observed in the dorsal and ventral regions, respectively, in the medial side of the fin instead of in the lateral side as seen in the previous stage.