A protocol for high-resolution episcopic microscopy and 3D volumetric analyses of the adult mouse brain.

The rapid evolution of different imaging modalities in the last two decades has enabled the investigation of the role of different genes in development and disease to be studied in a range of model organisms. However, selection of the appropriate imaging technique depends on a number of constraints, including cost, time, image resolution, size of the sample, computational complexity and processing power. Here, we use the adult mouse central nervous system to investigate whether High-Resolution Episcopic Microscopy (HREM) can provide an effective means to study the volume of individual subregions within the brain. We find that HREM can provide precise volume quantification of different structures within the mouse brain, albeit with limitations regarding the time involved for analysis and the necessity of some estimations.

Enhanced study of facial soft tissues using a novel large scale histology technique.

The safety and effectiveness of facial cosmetic surgery procedures are dependent on detailed 3D understanding of the complex surgical anatomy of the face. Traditional, small sample size anatomical dissection studies have limitations in providing definitive clarification of the fascial layers of the face, and especially in their relationship with the facial nerve, and their reaction to surgical manipulation. The objective study of large tissue areas is required to effectively demonstrate the broader architecture. Conventional histology techniques were modified to handle extraordinarily large tissue samples to fulfill this requirement. Full-thickness soft tissue samples (skin to bone) of maximum length 18 cm, width 4 cm, and tissue thickness 1 cm, were harvested from 20 hemifaces of 15 fresh human cadavers (mean age at death = 81 years). After fixation, the samples were processed with an automated processor using paraffin wax for 156 h, sectioned at 30 µm, collected on gelatin-chromium-coated glass slides, stained with a Masson's Trichrome technique and photographed. Using this technique, excellent visualization was obtained of the fascial connective tissue and its relationship with the facial mimetic muscles, muscles of mastication and salivary glands in 73 large histological slides. The resulting slides improved the study of the platysma and superficial musculo-aponeurotic system (SMAS), the spaces and ligaments, the malar fat pad, and the facial nerve in relations to the deep fascia. Additionally, surgically induced changes in the soft-tissue organization were successfully visualized. This technique enables improved insight into the broad structural architecture and histomorphology of large-scale facial tissues.

Comparative morphology of the electrosensory system of the epaulette shark Hemiscyllium ocellatum and brown-banded bamboo shark Chiloscyllium punctatum.

We compared the electrosensory system of two benthic elasmobranchs Hemiscyllium ocellatum and Chiloscyllium punctatum. The distribution of the ampullary pores on the head was similar for both species, with a higher density of pores anteriorly and a lower density posteriorly, although C. punctatum generally possessed larger pores. Ampullary canals of the mandibular cluster were quasi-sinusoidal in H. ocellatum, a shape previously found in benthic rays only, whereas ampullary canals in C. punctatum were of a linear morphology as reported for many shark and ray species previously. The ampullae proper were of the lobular type, as occurs in most galean sharks. Chiloscyllium punctatum had six sensory chambers compared with the five per ampulla in H. ocellatum, which were generally smaller than those of C. punctatum. The sensory epithelium comprised flattened receptor cells, compared with the usual pear-shaped receptor cells encountered in other elasmobranchs and their apically nucleated supportive cells did not protrude markedly into the ampullary lumen, unlike those in benthic rays.

Developmental asynchrony and antagonism of sex determination pathways in a lizard with temperature-induced sex reversal.

Vertebrate sex differentiation follows a conserved suite of developmental events: the bipotential gonads differentiate and shortly thereafter sex specific traits become dimorphic. However, this may not apply to squamates, a diverse vertebrate lineage comprising of many species with thermosensitive sexual development. Of the three species with data on the relative timing of gonad differentiation and genital dimorphism, the females of two (Niveoscincus ocellatus and Barisia imbricata) exhibit a phase of temporary pseudohermaphroditism or TPH (gonads have differentiated well before genital dimorphism). We report a third example of TPH in Pogona vitticeps, an agamid with temperature-induced male to female sex reversal. These findings suggest that for female squamates, genital and gonad development may not be closely synchronised, so that TPH may be common. We further observed a high frequency of ovotestes, a usually rare gonadal phenotype characterised by a mix of male and female structures, exclusively associated with temperature-induced sex reversal. We propose that ovotestes are evidence of a period of antagonism between male and female sex-determining pathways during sex reversal. Female sexual development in squamates is considerably more complex than has been appreciated, providing numerous avenues for future exploration of the genetic and hormonal cues that govern sexual development.

Sex determination mode does not affect body or genital development of the central bearded dragon (Pogona vitticeps).

BACKGROUND: The development of male- or female-specific phenotypes in squamates is typically controlled by either temperature-dependent sex determination (TSD) or chromosome-based genetic sex determination (GSD). However, while sex determination is a major switch in individual phenotypic development, it is unknownhow evolutionary transitions between GSD and TSD might impact on the evolution of squamate phenotypes, particularly the fast-evolving and diverse genitalia. Here, we take the unique opportunity of studying the impact of both sex determination mechanisms on the embryological development of the central bearded dragon (Pogona vitticeps). This is possible because of the transitional sex determination system of this species, in which genetically male individuals reverse sex at high incubation temperatures. This can trigger the evolutionary transition of GSD to TSD in a single generation, making P. vitticeps an ideal model organism for comparing the effects of both sex determination processes in the same species. RESULTS: We conducted four incubation experiments on 265 P. vitticeps eggs, covering two temperature regimes ("normal" at 28 °C and "sex reversing" at 36 °C) and the two maternal sexual genotypes (concordant ZW females or sex-reversed ZZ females). From this, we provide the first detailed staging system for the species, with a focus on genital and limb development. This was augmented by a new sex chromosome identification methodology for P. vitticeps that is non-destructive to the embryo. We found a strong correlation between embryo age and embryo stage. Aside from faster growth in 36 °C treatments, body and external genital development was entirely unperturbed by temperature, sex reversal or maternal sexual genotype. Unexpectedly, all females developed hemipenes (the genital phenotype of adult male P. vitticeps), which regress close to hatching. CONCLUSIONS: The tight correlation between embryo age and embryo stage allows the precise targeting of specific developmental periods in the emerging field of molecular research on P. vitticeps. The stability of genital development in all treatments suggests that the two sex-determining mechanisms have little impact on genital evolution, despite their known role in triggering genital development. Hemipenis retention in developing female P. vitticeps, together with frequent occurrences of hemipenis-like structures during development in other squamate species, raises the possibility of a bias towards hemipenis formation in the ancestral developmental programme for squamate genitalia.

Ultrastructure of the ampullary organs of Plicofollis argyropleuron (Siluriformes: Ariidae).

The morphology of ampullary organs in Plicofollis argyropleuron, collected from a southeast Queensland estuary, was examined by light and electron microscopy to assess the morphological characteristics of teleost ampullary organs in environments with fluctuating salinities. This catfish possesses both macroampullae and microampullae. Both have the typical teleost arrangement of an ampullary pore linked by a canal to a single ampulla that is lined with receptor and supportive cells. The canal wall of macroampullae consists of a collagen sheath, a basement membrane, and two layers of squamous epithelial cells adjacent to the lumen, joined by desmosomes and tight junctions near the surface of the epithelium. Ampullary pore diameters are similar in range for both the macroampullae and the microampullae, with microampullae always arising from the larger pores within a single region of the head. Canal length of the macroampullae is longer than those of the microampullae. Macroampullae also contain approximately 10 times as many receptor cells compared with the microampullae. In both organs, these pear-shaped receptor cells alternate with supportive cells along the entire luminal surface of the ampulla. The apical region of receptor cells extends into the lumen and bears numerous microvilli. The basal region of receptor cells adjoins to either individual or multiple unmyelinated neural terminals. The coexistence of two markedly different ampullary organ morphologies within a single species support theories concerning the possible multifunctionality of these sensory organs.

Morphology of the teleost ampullary organs in marine salmontail catfish Neoarius graeffei (Pisces: Ariidae) with comparative analysis to freshwater and estuarine conspecifics.

We hypothesized that due to the relative conductivity of the environment, and to maintain sensory function, ampullary organs of marine Neoarius graeffei would differ morphologically from those described previously for estuarine and freshwater conspecifics. Unlike the ampullary systems of N. graeffei from freshwater and estuarine habitats, the ampullary pores of marine specimens occur in two distinct patterns; numerous pores seemingly randomly scattered on the head and ventro-lateral regions of the body, and pores arranged in distinctive vertical lines above the lateral line on the dorso-lateral body of the fish. Light and electron microscopy revealed that the ampullary organs also differed morphologically from estuarine and freshwater specimens in the presence of longer ampullary canals, a hitherto unreported canal wall composition, and in the collagen sheath surrounding both the canal and the ampulla proper within dermal connective tissues. Ampullary pores were wider in marine individuals and opened to the longest ampullary canals reported for this species. The canal wall was lined by cuboidal and squamous epithelial cells. Each ampullary canal opened into a single ampulla proper containing significantly more receptor cells than estuarine and freshwater conspecifics. The distribution of ampullary pores as well as the microstructure of the ampullary organs indicates that the electrosensory system of marine N. graeffei differs from those of estuarine and freshwater specimens in ways that would be expected to maintain the functionality of the system in a highly conductive, fully marine environment, and reveals the remarkable plasticity of this species' ampullary system in response to habitat conductivity.

Firing the sting: chemically induced discharge of cnidae reveals novel proteins and peptides from box jellyfish (Chironex fleckeri) venom.

Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or "venom" recovery, we utilized both top-down and bottom-up transcriptomics-proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.

Morphology of the ampullae of Lorenzini in juvenile freshwater Carcharhinus leucas.

Ampullae of Lorenzini were examined from juvenile Carcharhinus leucas (831-1,045 mm total length) captured from freshwater regions of the Brisbane River. The ampullary organ structure differs from all other previously described ampullae in the canal wall structure, the general shape of the ampullary canal, and the apically nucleated supportive cells. Ampullary pores of 140-205 µm in diameter are distributed over the surface of the head region with 2,681 and 2,913 pores present in two sharks that were studied in detail. The primary variation of the ampullary organs appears in the canal epithelial cells which occur as either flattened squamous epithelial cells or a second form of pseudostratified contour-ridged epithelial cells; both cell types appear to release material into the ampullary lumen. Secondarily, this ampullary canal varies due to involuted walls that form a clover-like canal wall structure. At the proximal end of the canal, contour-ridged cells abut a narrow region of cuboidal epithelial cells that verge on the constant, six alveolar sacs of the ampulla. The alveolar sacs contain numerous receptor and supportive cells bound by tight junctions and desmosomes. Pear-shaped receptor cells that possess a single apical kinocilium are connected basally by unmyelinated neural boutons. Opposed to previously described ampullae of Lorenzini, the supportive cells have an apical nucleus, possess a low number of microvilli, and form a unique, jagged alveolar wall. A centrally positioned centrum cap of cuboidal epithelial cells overlies a primary afferent lateral line nerve.

Effects of bound versus soluble pentosan polysulphate in PEG/HA-based hydrogels tailored for intervertebral disc regeneration.

Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFß). In this present study, we aimed to assess whether a 'bound' PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in increased matrix formation when compared to the addition of soluble PPS to the hydrogel, or the hydrogel alone. We believe that this new generation injectable, degradable hydrogel, incorporating now a covalently bound-PPS, when combined with MPCs, has the potential to assist cartilage regeneration in a multitude of therapeutic targets, including for intervertebral disc (IVD) degeneration.

An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration.

Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-ß. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.

Squeezers and leaf-cutters: differential diversification and degeneration of the venom system in toxicoferan reptiles.

Although it has been established that all toxicoferan squamates share a common venomous ancestor, it has remained unclear whether the maxillary and mandibular venom glands are evolving on separate gene expression trajectories or if they remain under shared genetic control. We show that identical transcripts are simultaneously expressed not only in the mandibular and maxillary glands, but also in the enigmatic snake rictal gland. Toxin molecular frameworks recovered in this study were three-finger toxin (3FTx), CRiSP, crotamine (beta-defensin), cobra venom factor, cystatin, epididymal secretory protein, kunitz, L-amino acid oxidase, lectin, renin aspartate protease, veficolin, and vespryn. We also discovered a novel low-molecular weight disulfide bridged peptide class in pythonid snake glands. In the iguanian lizards, the most highly expressed are potentially antimicrobial in nature (crotamine (beta-defensin) and cystatin), with crotamine (beta-defensin) also the most diverse. However, a number of proteins characterized from anguimorph lizards and caenophidian snakes with hemotoxic or neurotoxic activities were recruited in the common toxicoferan ancestor and remain expressed, albeit in low levels, even in the iguanian lizards. In contrast, the henophidian snakes express 3FTx and lectin toxins as the dominant transcripts. Even in the constricting pythonid and boid snakes, where the glands are predominantly mucous-secreting, low-levels of toxin transcripts can be detected. Venom thus appears to play little role in feeding behavior of most iguanian lizards or the powerful constricting snakes, and the low levels of expression argue against a defensive role. However, clearly the incipient or secondarily atrophied venom systems of these taxa may be a source of novel compounds useful in drug design and discovery.

Structural and molecular diversification of the Anguimorpha lizard mandibular venom gland system in the arboreal species Abronia graminea.

In the past, toxinological research on reptiles has focused principally on clinically important species. As a result, our understanding of the evolution of the reptile venom system is limited. Here, for the first time, we describe the structural and molecular evolutionary features of the mandibular toxin-secreting gland of Abronia graminea, a representative of one of the poorly known and entirely arboreal lineages of anguimorph lizards. We show that the mandibular gland is robust and serous, characters consistent with those expected of a toxin-secreting gland in active use. A wide array of transcripts were recovered that were homologous to those encoded by the indisputably venomous helodermatid lizards. We show that some of these toxin transcripts are evolving under active selection and show evidence of rapid diversification. Helokinestatin peptides in particular are revealed to have accumulated residues that have undergone episodic diversifying selections. Conversely, the natriuretic peptides have evolved under tremendous evolutionary constraints despite being encoded in tandem with helokinestatins by the same gene precursor. Of particular note is the sequencing for the first time of kunitz peptides from a lizard toxin-secreting gland. Not only are kunitz peptides shown to be an ancestral toxicoferan toxin, the ancestral state of this peptide is revealed to be a dual domain encoding precursor. This research provides insight into the evolutionary history of the ancient toxicoferan reptile venom system. In addition, it shows that even 'clinically irrelevant' species can be a rich source of novel venom components, worthy of investigation for drug design and biomedical research.

Microampullary organs of a freshwater eel-tailed catfish, Plotosus (tandanus) tandanus.

Whole body studies of Plotosus tandanus revealed that ampullary pores occur over the entire body of the fish, but are in higher concentrations in the head region. These pores give rise to a short canal (50-60 microm) produced by columnar epithelial cells bound together by tight junctions and desmosomes. At the junction of the canal and the ampulla, cuboidal epithelial cells make up the wall. The ampulla consists of layers of collagen fibers that surround flattened epithelial cells in the lateral regions and give rise to supportive cells that encase a small number of receptor cells (10-15). The ampullary wall comprises several types of cells that are adjoined via tight junctions and desmosomes between cell types. The ovoid receptor cells possess microvilli along the luminar apical area. Beneath this area, the cells are rich in mitochondria and rough endoplasmic reticulum. An unmyelinated neuron adjoins with each receptor cell opposite multiple presynaptic bodies. This form of microampulla has not been previously described within the Family Plotosidae.

Ampullary organs and electroreception in freshwater Carcharhinus leucas.

The ampulla of Lorenzini of juvenile Carcharhlinus leucas differ histologically from those previously described for other elasmobranchs. The wall of the ampullary canal consists of protruding hillock-shaped epidermal cells that appear to secrete large quantities of a mucopolysaccharide gel. The ampullary organs comprise a long canal sheathed in collagen terminating in an ampulla. Each ampulla contains six alveolar sacs, with each sac containing hundreds of receptor cells. The receptor cells are characteristic of others described for elasmobranchs being pear-shaped cells with a central nucleus and bearing a single kinocilium in the exposed apical region of the cell. The supportive cells differ from general elasmobranch ampullary histology in that some have an apical nucleus. These ampullary structures allow Carcharhinus leucas to detect and respond to artificial electrical fields. Carcharhinus leucas from freshwater habitats respond to electrical signals supplied in freshwater aquaria by abruptly turning towards low voltage stimuli (< or = 10 microA) and either swimming over or biting at the origin of the stimulus.

Distribution and morphology of the ampullary organs of the salmontail catfish, Arius graeffei.

Whole body staining of Arius graeffei revealed that ampullary pores cover the body with their highest densities occurring on the head and lowest densities on the mid-ventral surface. Each ampullary organ consists of a long canal (0.2-1.75 mm) passing perpendicular to the basement membrane, through the epidermis into underlying dermal connective tissues, curving thereafter to run roughly parallel to the epidermis. Histochemical staining techniques (Alcian blue and Lillie's allochrome) indicate that the canals contain a neutral to acidic glycoprotein-based mucopolysaccharide gel that varies in composition along the length of the canal. Collagen fibers, arranged in a sheath, surround a layer of squamous epithelium that lines each ampullary canal. At the proximal end of the canal, squamous cells are replaced by cuboidal epithelial cells that protrude into the lumen, thus constricting the lumen to form a small pore into the ampulla. The ampulla is lined with receptor and supportive cells. The numerous (60-120) pear-shaped receptor cells bear microvilli on their luminal surface. Two forms of receptor cells exist in each ampullary organ: basal and equatorial receptor cells. Each receptor cell is connected to an unmyelinated nerve. Each receptor cell is surrounded by supportive cells on all but the apex. Tight junctions and underlying desmosomes occur between adjacent receptor and supportive cells. This form of ampullary organ has not previously been described for teleosts. J. Morphol. 239:97-105, 1999. © 1999 Wiley-Liss, Inc.