Getting out of a mammalian egg: the egg tooth and caruncle of the echidna.

In the echidna, after development in utero, the egg is laid in the pouch and incubated for 10 days. During this time, the fetuses develop an egg tooth and caruncle to help them hatch. Using rare and unprecedented access to limited echidna pre- and post-hatching tissues, development of the egg tooth and caruncle were assessed by micro-CT, histology and immunofluorescence. Unlike therian tooth germs that develop by placode invagination, the echidna egg tooth developed by evagination, similar to the first teeth in some reptiles and fish. The egg tooth ankylosed to the premaxilla, rather than forming a tooth root with ligamentous attachment found in other mammals, with loss of the egg tooth associated with high levels of activity odontoclasts and apoptosis. The caruncle formed as a separate mineralisation from the adjacent nasal capsule, and as observed in birds and turtles, the nasal region epithelium on top of the nose expressed markers of cornification. Together, this highlights that the monotreme egg tooth shares many similarities with typical reptilian teeth, suggesting that this tooth has been conserved from a common ancestor of mammals and reptiles.

Transient role of the middle ear as a lower jaw support across mammals.

Mammals articulate their jaws using a novel joint between the dentary and squamosal bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before the bones of the novel mammalian jaw joint form. These mammals need to rely on other mechanisms to allow them to feed. Here, we show that this vital function is carried out by the earlier developing, cartilaginous incus of the middle ear, abutting the cranial base to form a cranio-mandibular articulation. The nature of this articulation varies between monotremes and marsupials, with juvenile monotremes retaining a double articulation, similar to that of the fossil mammaliaform Morganucodon, while marsupials use a versican-rich matrix to stabilise the jaw against the cranial base. These findings provide novel insight into the evolution of mammals and the changing relationship between the jaw and ear.

The defining feature of all mammals is how the jaw works. Fish, reptiles and other animals with backbones have a lower jaw made of many bones fused together, one of which connects to the upper jaw. The lower jaw in mammals, however, is made of a single bone that connects with the upper jaw using a completely unique jaw joint. This new joint emerged as the ancestors of all mammals split from the reptiles around 200 million years ago. The bones that formed the original jaw joint ended up in the middle ear in mammals and switched to a role in hearing. Nowadays, there are three types of mammals: the placentals, marsupials and monotremes (the egg laying mammals). In mice, humans and other placental mammals, the skeleton of the adult jaw joint forms in the embryo before birth. However, marsupials (such as kangaroos and opossums) and monotremes (platypuses and echidnas) are born at a much earlier embryonic stage, before the adult jaw joint has formed. It is therefore unclear how newborn marsupials and monotremes are able to move their jaws to feed on milk from their mother. Anthwal et al. compared how the jaw develops in mice, opossums, platypuses and echidnas before and after the adult jaw joint becomes functional. The experiments showed that young echidnas, platypuses and opossums use their middle ear bones to articulate the lower jaw with the head before the adult jaw joint forms. In young opossums, the ear bones form a cushion to support the jaw. In juvenile platypuses a double joint is evident, with the ear bones forming a joint at the same time as the newly formed adult jaw joint, similar to the situation observed in fossils of mammal ancestors. The experiments also indicated that mice and other placental mammals may potentially use their ear bones to support the jaw before birth. These findings shed light on why the ear and jaw have such a close connection in mammals. In humans, the ear and jaw bones are still connected by ligaments, explaining why trauma to the jaw joint can cause dislocation of the ear bones. Similarly, defects in the development of the jaw can impact the ear, such as in Treacher Collins Syndrome, where in some cases the jaw joint fails to form and the ear bones appear to try and take this role. Understanding how the ear and jaw evolved will help us understand why they look like they do and why a defect in one can have knock-on consequences for the other.

Three-dimensional mammalian tooth development using diceCT.

OBJECTIVE: This study aims to develop the Diffusible Iodine-based Contrast-Enhanced CT (diceCT) method for non-destructive imaging of both soft and mineralised tissues. We sought to document the 3D spatio-temporal pattern of mammalian tooth development including multiple tooth classes and generations, using the tammar wallaby (Macropus eugenii) as a model species. DESIGN: We took microCT scans of developing fetuses and pouch young stained using Lugol's Iodine (I2KI) contrast agent. Stained versus unstained specimen comparisons were then made to investigate whether staining had improved visualisation of structures. Scan slices were compared to histological sections to confirm the identity of tissues and structures. Tissue layers were digitally segmented to create 3D models. RESULTS: DiceCT dramatically enhanced visual contrast of soft tissues, allowing differentiation between epithelial and mesenchymal layers. Subvolume scans at higher magnification achieved single-cell layer resolution within relatively large intact heads. We observed in-situ initiating teeth, which progressed through major stages of tooth development including morphogenesis and mineralisation. In addition, we traced the development of other mineralized and unmineralised tissues, such as the cranial bones and the brain, eye and olfactory system. CONCLUSIONS: DiceCT was time- and cost-effective in producing complex 3D models of the entire dentition of the tammar wallaby at each developmental stage with tissue-level resolution. The 3D view of soft and mineralised tooth structures allowed us to define tooth class and generation from a developmental perspective. Additionally, the development of other organs can also be documented using the same scans, demonstrating the efficiency and versatility of this technique.

Administration of 5alpha-androstane-3alpha,17beta-diol to female tammar wallaby pouch young causes development of a mature prostate and male urethra.

Secretion of 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) by the testes of the tammar wallaby is responsible for initiation of prostatic development after d 20 in male pouch young. To ascertain the role of this hormone in the subsequent growth and differentiation of the prostate and in the development of the male phallus, 5alpha-adiol was administered to tammar female pouch young in two regimens. Administration of the hormone by mouth (8 microg/g body weight.wk) between d 70 and 150 of pouch life caused prostate development equivalent to that in d 150 males and promoted growth and differentiation of the penis, but not masculinization of the urethra. Treatment with a small dose of 5alpha-adiol enanthate (1 microg/g body weight.wk) from d 20-150 produced similar results. However, administration of larger doses of 5alpha-adiol enanthate (10 or 100 microg/g body weight.wk) from d 20-150 caused supraphysiological growth of the prostate, development of a male-type urethra, and penile growth. These results indicate that prostatic development and penile growth can be initiated over a wide time period, but that formation of a male urethra requires androgen action before d 70, when male penile differentiation begins. This further strengthens the hypothesis that 5alpha-adiol is the circulating androgen responsible in this species for virilization during development.

The vomeronasal organ of the tammar wallaby.

The vomeronasal organ is the primary olfactory organ that detects sexual pheromones in mammals. We investigated the anatomy of the vomeronasal organ of the tammar wallaby (Macropus eugenii), a small macropodid marsupial. Pheromones may be important for activation of the hypothalamo-pituitary axis of tammar males at the start of the breeding season because plasma testosterone and luteinizing hormone concentration in males rise concurrently with pregnancy and the post-partum ovulation in females. The gross anatomy and the connection to the brain of the vomeronasal organ were examined by light and electron microscopy in adult male and female tammars. The vomeronasal organ was well developed in both sexes. The vomeronasal organ is a tubular organ connected at the rostral end via the nasopalatine duct (incisive duct) to the mouth and nasal cavity. At the rostral end the lumen of the vomeronasal organ was crescent shaped, changing to a narrow oval shape caudally. Glandular tissue associated with the vomeronasal organ increased towards the blind end of the organ. The tammar has the typical pattern of mammalian vomeronasal organs with electron-dense supporting cells and electron-lucent receptor cells. Microvilli were present on the surface of both epithelia while cilia were only found on the surface of the non-receptor epithelium. Some non-receptor epithelial cells appeared to secrete mucus into the vomeronasal organ lumen. The vomeronasal organ shows a high degree of structural conservation compared with eutherian mammals. The degree of vomeronasal organ development makes it likely that, as in other mammals, pheromones are important in the reproduction of the tammar.