Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus)

Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes. We have used chromosome paints and DAPI banding to identify and classify pairs of autosomes and sex chromosomes. We have established an agreed nomenclature and identified anchor BAC clones for each chromosome that will ensure unambiguous gene localizations.     

Intestinal mucosa of the platypus,Ornithorhynchus anatinus

The intestinal mucosa of the platypus takes the form of numerous transverse surface folds. These folds are made up of a lamina propria covered by pseudostratified epithelium which lies on a thick modified basement membrane. The cells of the intestinal epithelium consist of columnar cells which generally resemble typical intestinal epithelium and cuboidal cells, which are undifferentiated in appearance, show few organelles and possess an electron lucent cytoplasm. Numerous desmosomes are found between the adjacent cell membranes of both cell types. Villi are absent and appear to be represented by the large surface folds. Intestinal glands are composed of columnar epithelium similar to that found in the intestinal glands of other mammalian species. Groups of these glands drain into common tubular ducts which follow a tortuous course and empty into the intestinal lumen between the surface folds. The peculiar morphological features of the platypus intestinal mucosa raise questions concerning traditional concepts of intestinal gland formation as well as the origin and migration of intestinal epithelium with regard to this particular species.     

Morphology of the kidney of the platypus (Ornithorhynchus anatinus: Monotremata)

The platypus kidney shows morphological similarities to those of other mammals. Macroscopically, the cortex is easily distinguishable from the fairly wide medulla. Within the medulla, no clear border is observed between the inner and outer zones. Light and transmission electron microscopically, the glomeruli show quite similar architecture to those of other mammals; however, the glomerular lobulation is very clear. The glomerular tufts are rather simple, but capillary lumen varies widely in size, which is one of the unique features of the platypus kidney. The urinary tubule is generally similar to that of human and other mammals in shape and segmentation; however, the staining specificities of histochemical reactions and the shape of epithelial cells of the Henle's loop differ from those ofother mammals. The most conspicuous features are: 1) although no protein casts are found in the tubular lumina, epithelial cells of the proximal convoluted tubule (PCT) have numerous electron-dense vesicles as in human nephrotic kidneys; and 2) the platypus Henle's loop consists of the thick epithelial cells similar to the mammalian type nephron of birds. As compared to those of other mammals such as humans and rats, our observations suggest that the platypus kidney is less developed, in terms of evolution.© Willey-Liss, Inc.     

Histological and immunohistological investigation of lymphoid tissue in the platypus (Ornithorhynchus anatinus)

The gross and histological appearance and the distribution of T and B lymphocytes and plasma cells are described for lymphoid tissues obtained from 15 platypuses. The spleen was bilobed and surrounded by a thick capsule of collagen, elastic fibres and little smooth muscle. White pulp was prominent and included germinal centres and periarterial lymphoid sheaths. Red pulp contained haematopoietic tissue. A thin lobulated thymus was located within the mediastinum overlying the heart. The cortex of lobules consisted of dense aggregates of small and medium lymphocytes, scattered macrophages and few reticular epithelial cells. In the medulla, Hassall's corpuscles were numerous, lymphocytes were small and less abundant, and reticular cells were more abundant than in the cortex. Lymphoid nodules scattered throughout loose connective tissue in cervical, pharyngeal, thoracic, mesenteric and pelvic sites measured 790±370 μm (mean±S.D. , n = 39) in diameter, the larger of which could be observed macroscopically. These consisted of single primary or secondary follicles supported by a framework of reticular fibres. Macrophages were common in the germinal centres. The platypus had a full range of gut-associated lymphoid tissue. No tonsils were observed macroscopically but histologically they consisted of submucosal follicles and intraepithelial lymphocytes. Peyer's patches were not observed macroscopically but histologically they consisted of several prominent submucosal secondary follicles in the antimesenteric wall of the intestine. Caecal lymphoid tissue consisted of numerous secondary follicles in the submucosa and densely packed lymphocytes in the lamina propria. Bronchus-associated lymphoid tissue was not observed macroscopically but was identified in 7 of 11 platypus lungs assessed histologically. Lymphoid cells were present as primary follicles associated with bronchi, as aggregates adjacent to blood vessels and as intraepithelial lymphocytes. The distribution of T lymphocytes, identified with antihuman CD3 and CD5, and B lymphocytes and plasma cells, identified with antihuman CD79a and CD79b and antiplatypus immunoglobulin, within lymphoid tissues in the platypus was similar to that described in therian mammals except for an apparent relative paucity of B lymphocytes. This study establishes that the platypus has a well-developed lymphoid system which is comparable in histological structure to that in therian mammals. It also confirms the distinctiveness of its peripheral lymphoid tissue, namely lymphoid nodules. Platypus lymphoid tissue has all the essential cell types, namely T and B lymphocytes and plasma cells, to mount an effective immune response against foreign antigens.     

TCR gamma chain diversity in the spleen of the duckbill platypus (Ornithorhynchus anatinus)

TCR gamma (TRG) chain diversity in splenic gammadelta T cells was determined for an egg-laying mammal (or monotreme), the duckbill platypus. Three distinct V subgroups were found in the expressed TRG chains and these three subgroups are members of a clade not found so far in eutherian mammals or birds. Each subgroup contains approximately five V gene segments, and their overall divergence is much less than is found in eutherians and birds, consistent with their recent evolution from an ancestral V gene segment. The platypus TRG locus also contains three C region genes and many of the residues involved in TCR function, such as interactions with CD3, were conserved in the monotreme C regions. All non-eutherian mammals (monotremes and marsupials) lacked the second cysteine residue necessary to form the intradomain disulfide bond in the C region, a loss apparently due to independent mutations in marsupials and monotremes. Monotreme TRGC regions also had among the most variation in the length of the connecting peptide region described for any species due to repeated motifs.     

Intestinal mucosa and intra-abdominal lymphoid tissues of the platypus, Ornithorhynchus anatinus

The morphological features of the intestinal mucosa and intra-abdominal lymphoid tissues of the platypus were examined. The mucosal surface of the intestine was characterized by the formation of large folds instead of the finger-like villi found in placental mammals. The lamina propria of the mucosal fold was well developed and contained numerous lymphocytes, expressing the lymphoid nature which is characteristic of the lamina propria of mammalian intestines. Although numerous well-developed Peyer's patches were observed in the ileum, solitary lymphoid nodules could not be found anywhere in the small intestine. Other intra-abdominal lymphoid tissues, particularly mesenteric lymphoid nodules, were well developed. However, each nodule represented a single follicle in contrast to the mammalian mesenteric lymph node which is composed of numerous follicles fused together. On the basis of the above findings, the tissues in question are considered to be at an evolutionary level preceding that of placental mammals.     

Development of the hypothalamus and pituitary in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus)

The living monotremes (platypus and echidnas) are distinguished by the development of their young in a leathery-shelled egg, a low and variable body temperature and a primitive teat-less mammary gland. Their young are hatched in an immature state and must deal with the external environment, with all its challenges of hypothermia and stress, as well as sourcing nutrients from the maternal mammary gland. The Hill and Hubrecht embryological collections have been used to follow the structural development of the monotreme hypothalamus and its connections with the pituitary gland both in the period leading up to hatching and during the lactational phase of development, and to relate this structural maturation to behavioural development. In the incubation phase, development of the hypothalamus proceeds from closure of the anterior neuropore to formation of the lateral hypothalamic zone and putative medial forebrain bundle. Some medial zone hypothalamic nuclei are emerging at the time of hatching, but these are poorly differentiated and periventricular zone nuclei do not appear until the first week of post-hatching life. Differentiation of the pituitary is also incomplete at hatching, epithelial cords do not develop in the pars anterior until the first week, and the hypothalamo-neurohypophyseal tract does not appear until the second week of post-hatching life. In many respects, the structure of the hypothalamus and pituitary of the newly hatched monotreme is similar to that seen in newborn marsupials, suggesting that both groups rely solely on lateral hypothalamic zone nuclei for whatever homeostatic mechanisms they are capable of at birth/hatching.     

Development of the dorsal and ventral thalamus in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus)

The living monotremes (platypus and echidnas) are distinguished from therians as well as each other in part by the unusual structure of the thalamus in each. In particular, the platypus has an enlarged ventral posterior (VP) nucleus reflecting the great behavioural importance of trigeminosensation and electroreception. The embryological collections of the Museum für Naturkunde in Berlin were used to analyse the development of the dorsal thalamus and ventral thalamus (prethalamus) in both species. Prosomeric organization of the forebrain emerged at 6 mm crown-rump length (CRL), but thalamic neurogenesis did not commence until about 8–9 mm CRL. Distinctive features of the dorsal thalamus in the two species began to emerge after hatching (about 14–15 mm CRL). During the first post-hatching week, dense clusters of granular cells aggregated to form the VP of the platypus, whereas the VP complex of the echidna remained smaller and divided into distinct medial and lateral divisions. At the end of the first post-hatching week, the thalamocortical tract was much larger in the platypus than the echidna. The dorsal thalamus of the platypus is essentially adult-like by the sixth week of post-hatching life. The similar appearance of the dorsal thalamus in the two species until the time of hatching, followed by the rapid expansion of the VP in the platypus, is most consistent with ancestral platypuses having undergone changes in the genetic control of thalamic neurogenesis to produce a large VP for trigeminal electroreception after the divergence of the two lineages of monotreme.     

Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.)

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.     

On the morphology of the brachial plexus of the platypus (Ornithorhynchus anatinus) and the echidna (Tachyglossus aculeatus)

Four forelimbs of 3 platypuses and 3 forelimbs of 2 echidnas were examined to study the precise form of the brachial plexus and to clarify the structural characteristics of the brachial plexus in phylogeny. The spinal components contributing to the plexus (C4–T2) and the formation patterns of the 3 trunks of the plexus were the same as those generally observed in mammals. In the cranial half of the brachial plexus from C4, 5 and 6 in monotremes, division into the ventral bundle (lateral cord) and dorsal bundle (axillary nerve) is clear, as in other mammals. However, for monotremes, in the caudal half of the plexus from C7 and T1 (+T2) and the nerves arising from the caudal plexus there is no definite division into the ventral and dorsal bundles, which distribute to the flexor and extensor parts of the forelimbs, respectively. The lower trunk of the monotreme brachial plexus forms a cord which contains both ventral and dorsal components. This characteristic diverges from the generally accepted idea that the tetrapod limb plexus is divided clearly into 2 layers: a dorsal layer for extensors and a ventral layer for flexors of the limb. Considering the incomplete dorsoventral division of forelimb nerves in some reptiles and urodeles, the caudal half of the monotreme brachial plexus has characteristics in common with those of lower tetrapods.     

Spermiogenesis and spermiation in a monotreme mammal, the platypus, Ornithorhynchus anatinus

Spermatogenesis in the platypus ( Ornithorhynchus anatinus ) is of considerable biological interest as the structure of its gametes more closely resemble that of reptiles and birds than marsupial or eutherian mammals. The ultrastructure of 16 steps of spermatid development is described and provides a basis for determining the kinetics of spermatogenesis. Steps 1–3 correspond to the Golgi phase of spermatid development, steps 4–8 correspond to the cap phase, steps 9–12 are the acrosomal phase, and steps 13–16 are the maturation phase. Acrosomal development follows the reptilian model and no acrosomal granule is formed. Most other features of spermiogenesis are similar to processes in reptiles and birds. However, some are unique to mammals. For example, a thin, lateral margin of the acrosome of platypus sperm expands over the nucleus as in other mammals, and more than in reptiles and birds. Also, a tubulobulbar complex develops around the spermatid head, a feature which appears to be unique to mammals. Further, during spermiation the residual body is released from the caudal end of the nucleus of platypus sperm leaving a cytoplasmic droplet located at the proximal end of the middle piece as in marsupial and eutherian mammals. Other features of spermiogenesis in platypus appear to be unique to monotremes. For example, nuclear condensation involves the formation of a layer of chromatin granules under the nucleolemma, and development of the fibrous sheath of the principal piece starts much later in the platypus than in birds or eutherian mammals.     

The pretectal nuclei in two monotremes: the short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus)

We have examined the organization of the pretectal area in two monotremes (the short beaked echidna—Tachyglossus aculeatus, and the platypus—Ornithorhynchus anatinus) and compared it to that in the Wistar strain rat, using Nissl staining in conjunction with enzyme histochemistry (acetylcholinesterase and NADPH diaphorase) and immunohistochemistry for parvalbumin, calbindin, calretinin and non-phosphorylated neurofilament protein (SMI-32 antibody). We were able to identify distinct anterior, medial, posterior (now called tectal gray) and olivary pretectal nuclei as well as a nucleus of the optic tract, all with largely similar topographical and chemoarchitectonic features to the homologous regions in therian mammals. The positions of these pretectal nuclei correspond to the distributions of retinofugal terminals identified by other authors. The overall size of the pretectum in both monotremes was found to be at least comparable in size, if not larger than, the pretectum of representative therian mammals of similar brain and body size. Our findings suggest that the pretectum of these two monotreme species is comparable in both size and organization to that of eutherian mammals, and is more than just an undifferentiated area pretectalis. The presence of a differentiated pretectum with similar chemoarchitecture to therians in both living monotremes lends support to the idea that the stem mammal for both prototherian and therian lineages also had a differentiated pretectum. This in turn indicates that a differentiated pretectum appeared at least 125 million years ago in the mammalian lineage and that the stem mammal for proto- and eutherian lineages probably had similar pretectal nuclei to those identified in its descendants.     

Splenic hemopoiesis of the platypus (Ornithorhynchus anatinus): Evidence of primary hemopoiesis in the spleen of a primitive mammal

The generation of blood cells has been observed in the spleen and in the bone marrow of the platypus. Hemopoiesis was found to be far more active in the spleen than in the bone marrow judging by the number of proliferating hemopoietic elements within a unit area of tissue from each organ. Granulocytes, erythroblasts, and megakaryocytes, with the related immature forms for each cell line, were noted in the spleen. In contrast, there were very few examples of immature forms of these cell lines and a complete absence of mature megakaryocytes in the bone marrow. These findings suggest that the spleen is the primary hemopoietic organ in the platypus. Since the platypus is one of two species representing the most primitive existing mammals, it seems likely that the spleen may be the primary hemopoietic organ in mammalian evolution.     

Venom from the platypus, Ornithorhynchus anatinus, induces a calcium-dependent current in cultured dorsal root ganglion cells

The platypus (Ornithorhynchus anatinus), a uniquely Australian species, is one of the few living venomous mammals. Although envenomation of humans by many vertebrate and invertebrate species results in pain, this is often not the principal symptom of envenomation. However, platypus envenomation results in an immediate excruciating pain that develops into a very long-lasting hyperalgesia. We have previously shown that the venom contains a C-type natriuretic peptide that causes mast cell degranulation, and this probably contributes to the development of the painful response. Now we demonstrate that platypus venom has a potent action on putative nociceptors. Application of the venom to small to medium diameter dorsal root ganglion cells for 10 s resulted in an inward current lasting several minutes when the venom was diluted in buffer at pH 6.1 but not at pH 7.4. The venom itself has a pH of 6.3. The venom activated a current with a linear current-voltage relationship between -100 and -25 mV and with a reversal potential of -11 mV. Ion substitution experiments indicate that the current is a nonspecific cationic current. The response to the venom was blocked by the membrane-permeant Ca(2+)-ATPase inhibitor, thapsigargin, and by the tyrosine- and serine-kinase inhibitor, k252a. Thus the response appears to be dependent on calcium release from intracellular stores. The identity of the venom component(s) that is responsible for the responses we have described is yet to be determined but is probably not the C-type natriuretic peptide or the defensin-like peptides that are present in the venom.     

Prenatal development of the human Brunner's glands

The prenatal development of the human Brunner's glands has been investigated in 23 fetuses from the 10th week of gestation to full-term. At 12 weeks, a few cords of epithelial cells were seen budding from the duodenal mucosa immediately beyond the pyloric sphincter. They represent the initial stage of the development of Brunner's glands. At 16 weeks, Brunner's glands originated as simple tubular downgrowths from the bottoms of the most proximal crypts of the duodenum. The secretory products of the component cells of these primitive tubules contained periodic acid schiff (PAS) positive material which was largely supranuclear in position and resisted digestion by diastase. From 20 weeks to full term, the Brunner's glands developed in a progressive fashion starting in the proximal part of the duodenum near the pyloroduodenal junction. Further tubular downgrowths were added distally, leading to an increase in length of the glandular tissue. The gland showed an increase in size proximally due to elongation and branching of the tubules. At birth, the glandular cells of Brunner's glands resembled those of normal adult in structure and staining reactions. The PAS staining of the cells of the early developed glands (at 12 weeks) was as intense as those of the full-term. The secretory materials of the developed Brunner's glands showed negative reaction with Alcian blue (AB) at pH 2.5 at any stage of development. These results suggest that the mucin secreted by the developed Brunner's glands of human is neutral mucopolysaccharide in nature.     

Isolation of Mucor circinelloides from a case of ulcerative mycosis of platypus (Ornithorhynchus anatinus), and a comparison of the response of Mucor circinelloides and Mucor amphibiorum to different culture temperatures.

The fungus Mucor circinelloides was isolated from a platypus (Ornithorhynchus anatinus) suffering from ulcerative mycosis. On horse blood agar at 20, 25 and 30 degrees C, the fungus formed sphaerule-like bodies, a morphology previously associated with Mucor amphibiorum, the species thought to be responsible for the disease in platypus. A biopsy taken from the ulcer was fixed, cut and stained. The sections were compared with sections taken from other platypuses suffering from ulcerative mycosis, and from which M. amphibiorum had been isolated. There were no discernible differences between the sphaerule-like bodies found in any of the sections. The presence of sphaerule-like bodies in tissues of ulcerated animals can, therefore, probably no longer be relied upon as a definitive method for the diagnosis of M. amphibiorum infection. It is possible that M. circinelloides is either a primary or a secondary pathogen of platypuses, and further work is required to resolve this point. The isolate of M. circinelloides grew at temperatures up to 38 degrees C, with an optimum temperature for growth of 30 degrees C. Of six isolates of M. amphibiorum derived from both platypus and amphibians, two grew well at 38 degrees C. The growth of one of these isolates at elevated temperatures may be explained by the hot climate of the area in Queensland in which it was found. All of the isolates tested had maximum temperatures for growth in excess of the body temperature of platypuses (32 degrees C).     

Evidence for an early appearance of modern post-switch immunoglobulin isotypes in mammalian evolution (II); cloning of IgE,IgG1 and IgG2 from a monotreme,the duck-billed platypus,Ornithorhynchus anatinus

To trace the emergence of the modern post-switch immunoglobulin (Ig) isotypes in vertebrate evolution we have studied Ig expression in mammals distantly related to eutherians. We here present an analysis of the Ig expression in an egg-laying mammal, a monotreme, the duck-billed platypus (Ornithorhynchus anatinus). Fragments of platypus IgG and IgE cDNA were obtained by a PCR-based screening using degenerate primers. The fragments obtained were used as probes to isolate full-length cDNA clones of three platypus post-switch isotypes, IgG1, IgG2, and IgE. Comparative amino acid sequence analysis against IgY, IgE and IgG from various animal species revealed that platypus IgE and IgG form branches that are clearly separated from those of their eutherian (placental) counterparts. However, the platypus IgE and IgG still conform to the general structure displayed by the respective Ig isotypes of eutherian and marsupial mammals. According to our findings, all of the major evolutionary changes in the expression array and basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the separation of monotremes from marsupial and placental mammals. Hence, our results indicate that the modern post-switch isotypes appeared very early in the mammalian lineage, possibly already 310-330 million years ago.