Lung Development of Monotremes: Evidence for the Mammalian Morphotype

The reproductive strategies and the extent of development of neonates differ markedly between the three extant mammalian groups: the Monotremata, Marsupialia, and Eutheria. Monotremes and marsupials produce highly altricial offspring whereas the neonates of eutherian mammals range from altricial to precocial. The ability of the newborn mammal to leave the environment in which it developed depends highly on the degree of maturation of the cardio‐respiratory system at the time of birth. The lung structure is thus a reflection of the metabolic capacity of neonates. The lung development in monotremes (Ornithorhynchus anatinus, Tachyglossus aculeatus), in one marsupial (Monodelphis domestica), and one altricial eutherian (Suncus murinus) species was examined. The results and additional data from the literature were integrated into a morphotype reconstruction of the lung structure of the mammalian neonate. The lung parenchyma of monotremes and marsupials was at the early terminal air sac stage at birth, with large terminal air sacs. The lung developed slowly. In contrast, altricial eutherian neonates had more advanced lungs at the late terminal air sac stage and postnatally, lung maturation proceeded rapidly. The mammalian lung is highly conserved in many respects between monotreme, marsupial, and eutherian species and the structural differences in the neonatal lungs can be explained mainly by different developmental rates. The lung structure of newborn marsupials and monotremes thus resembles the ancestral condition of the mammalian lung at birth, whereas the eutherian newborns have a more mature lung structure. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.     

Early postnatal lung development in the eastern quoll (Dasyurus viverrinus)

Early postnatal lung development (1–25 days) in the eastern quoll (Dasyurus viverrinus) was investigated to assess the morphofunctional status of one of the most immature marsupial neonates. Lung volume, surface density, surface area, and parenchymal and nonparenchymal volume proportions were determined using light microscopic morphometry. The lungs of the neonate were at the canalicular stage and consisted of two “balloon-like” airways with few septal ridges. The absolute volume of the lung was only 0.0009 cm³ with an air space surface density of 108.83 cm⁻¹ and a surface area of 0.082 cm². The increase in lung volume in the first three postnatal days was mainly due to airspace expansion. The rapid postnatal development of the lung was indicated by an increase in the septal proportion of the parenchyma around day 4, which was reflected by an increase in the airspace surface density and surface area. By day 5, the lung entered the saccular stage of development with a reduction in septal thickness, expansion of the tubules into saccules and development of a double capillary system. The subsequent saccular period was characterized by repetitive septation steps, which increased the number of airway generations. The lungs of the newborn Dasyurus viverrinus must be considered as structurally and quantitatively insufficient to meet the respiratory requirements at birth. Hence, cutaneous gas exchange might be crucial for the first three postnatal days. The lung has to mature rapidly in the early postnatal period to support the increased metabolic requirements of the developing young.     

On the prenatal initiation of T cell development in the opossum Monodelphis domestica

Thymus‐dependent lymphocytes (T cells) are a critical cell lineage in the adaptive immune system of all jawed vertebrates. In eutherian mammals the initiation of T cell development takes place prenatally and the offspring of many species are born relatively immuno‐competent. Marsupials, in contrast, are born in a comparatively altricial state and with a less well developed immune system. As such, marsupials are valuable models for studying the peri‐ and postnatal initiation of immune system development in mammals. Previous results supported a lack of prenatal T cell development in a variety of marsupial species. In the gray short‐tailed opossum, Monodelphis domestica, however, there was evidence that αβT cells were present on postnatal day 1 and likely initiated development prenatally. Demonstrated here is the presence of CD3ε⁺ lymphocytes in late‐stage embryos at a site in the upper thoracic cavity, the site of an early developing thymus. CD3ε⁺ cells were evident as early as 48 h prior to parturition. In day 14 embryos, where there is clear organogenesis, CD3ε⁺ cells were only found at the site of the early thymus, consistent with no extra‐thymic sites of T cell development in the opossum. These observations are the first evidence of prenatal T cell lineage commitment in any marsupial.     

The neonatal porcine lung: ultrastructural morphology and postnatal development of the terminal airways and alveolar region

Morphology and postnatal development of the porcine lung are described in animals ranging in age from newborn through 60 days. Standardized fixation was accomplished by intratracheal instillation of glutaraldehyde under constant pressure. Light microscopic, scanning, and transmission electron microscopic investigations revealed that the porcine lung follows the common architecture of mammalian lungs, but has certain peculiarities as well: intravascular macrophages, ultrastructurally similar to Kupffer cells, are attached to endothelial cells in pulmonary capillaries and are involved in erythrophagocytosis during the first postnatal weeks. Type II pneumocytes of newborn pigs exhibit signs of cell activation, mainly complex nuclear bodies in the cell nuclei. At the same time high levels of 17-hydroxycorticosteroids are observed in the newborn blood plasma. Terminal airways of the porcine lung are nonalveolarized and are, therefore, of purely conductive function. At birth the porcine lung exhibits a high degree of maturity, and thick-walled primary saccules, as described in newborn rodents, are not seen. Septa appear straight and smooth, owing to rare ramification. Septal buds are discernible, and two capillary networks visible on both sides of septal cross sections are seen. Further subdivision of the airspaces occurs in the first two postnatal weeks. Precociousness and fast postnatal growth of the porcine species are assumed to be the reason of this advanced degree of lung maturity at birth and the following rapid pulmonary development.     

Characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system

The availability of the first marsupial genome sequence has allowed us to characterize the immunome of the gray short-tailed opossum (Monodelphis domestica). Here we report the identification of key immune genes, including the highly divergent chemokines, defensins, cathelicidins, and Natural Killer cell receptors. It appears that the increase in complexity of the mammalian immune system occurred prior to the divergence of the marsupial and eutherian lineages approximately 180 million years ago. Genomes of ancestral mammals most likely contained all of the key mammalian immune gene families, with evolution on different continents, in the presence of different pathogens leading to lineage specific expansions and contractions, resulting in some minor differences in gene number and composition between different mammalian lineages. Gene expansion and extensive heterogeneity in opossum antimicrobial peptide genes may have evolved as a consequence of the newborn young needing to survive without an adaptive immune system in a pathogen laden environment. Given the similarities in the genomic architecture of the marsupial and eutherian immune systems, we propose that marsupials are ideal model organisms for the study of developmental immunology.     

Adaptations of the Marsupial Newborn: Birth as an Extreme Environment

At birth a mammalian neonate enters an extreme environment compared to the intrauterine environment in which it has grown. This transition may be particularly extreme in marsupials because they are born at an exceedingly altricial state, after an exceptionally short gestation. Their stage of development must be considered embryonic by almost any criteria. Yet at this very early stage of development marsupials must travel to the teat, attach and suckle, and have basic functioning of all major physiological systems. In this article, we review the adaptations of the marsupial neonate for survival at an embryonic state, showing that the neonate exhibits a mosaic of accelerations and delays of various tissues and organs as well as several special adaptations to produce the functioning newborn. We then discuss the development of the craniofacial region, the body axis and limbs in order to detail some of the major changes to development leading to this uniquely configured neonate. We show that marsupial development arises out of a variety of heterochronies (changes in relative timing of events) and heterotopies (changes in location of specific developmental events) at the genetic, cellular and organ level. We argue that these data support hypotheses that many of the specific patterns seen in marsupial development arise from the basic constraint of embryonic energetic and tissue resources. Finally ideas on the evolutionary context of the marsupial developmental strategy are briefly reviewed. Anat Rec, 2019. © 2018 Wiley Periodicals, Inc. Anat Rec, 303:235–249, 2020. © 2018 American Association for Anatomy     

Uterine remodelling during pregnancy and pseudopregnancy in the brushtail possum (Trichosurus vulpecula; Phalangeridae)

The formation of a placenta is critical for successful mammalian pregnancy and requires remodelling of the uterine epithelium. In eutherian mammals, remodelling involves specific morphological changes that often correlate with the mode of embryonic attachment. Given the differences between marsupial and eutherian placentae, formation of a marsupial placenta may involve patterns of uterine remodelling that are different from those in eutherians. Here we present a detailed morphological study of the uterus of the brushtail possum (Trichosurus vulpecula; Phalangeridae) throughout pregnancy, using both scanning and transmission electron microscopy, to identify whether uterine changes in marsupials correlate with mode of embryonic attachment as they do in eutherian mammals. The uterine remodelling of T. vulpecula is similar to that of eutherian mammals with the same mode of embryonic attachment (non‐invasive, epitheliochorial placentation). The morphological similarities include development of large apical projections, and a decrease in the diffusion distance for haemotrophes around the period of embryonic attachment. Importantly, remodelling of the uterus in T. vulpecula during pregnancy differs from that of a marsupial species with non‐invasive attachment (Macropus eugenii; Macropodidae) but is similar to that of a marsupial with invasive attachment (Monodelphis domestica; Didelphidae). We conclude that modes of embryonic attachment may not be typified by a particular suite of uterine changes in marsupials, as is the case for eutherian mammals, and that uterine remodelling may instead reflect phylogenetic relationships between marsupial lineages.     

Molecular characterisation and expression of CD4 in two distantly related marsupials: the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii)

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56(lck) binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37-41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.     

Development of the immune system and immunological protection in marsupial pouch young

At birth the tissues of marsupial immune system are underdeveloped. The young animal is not immunocompetent. Histological and immunohistochemical studies of pouch young epithelial tissues provide a clear picture of tissue development but the timing of onset of immunocompetence awaits definition. The survival of the neonatal marsupial in a microbially rich environment is dependent on maternal strategies, including immunoglobulin transfer via milk and, in some species, prenatally via the yolk sac placenta. It is also likely that pouch secretions play a role. This review summarizes our current knowledge of the pathway of immunological development in marsupials and the protection and threats afforded by the pouch environment.     

Histological and immunohistological investigation of the lymphoid tissue in normal and mycobacteria-affected specimens of the Rufous Hare-wallaby (Lagorchestes hirsutus)

The histology of the spleen, lymph nodes, Gut‐associated lymphoid tissue (GALT) and Bronchus‐associated lymphoid tissue (BALT) are described for samples collected opportunistically from healthy and mycobacteria‐affected specimens of the endangered marsupial Lagorchestes hirsutus, the Rufous Hare‐wallaby. The structural elements, organization and distribution of T and B lymphocytes determined by immunohistological techniques using species cross‐reactive antibodies in the lymph nodes, spleen and GALT of this species demonstrated lymphoid cell distributions that were consistent with other marsupial and eutherian mammals. The tissues of animals identified as acid‐fast positive displayed immunopathology consistent with the responses to intracellular bacteria displayed in some eutherian mammals and included the presence of focal lesions, giant cells in the lung and lymphoid aggregations situated adjacent to blood and airway vessels. This is the first study to describe the lymphoid tissue of this rare macropod species and the first to document the tissue bed response to mycobacteria.     

Development of the respiratory system in marsupials

Marsupials at birth are small and relatively undeveloped. At birth, the lung in some species is at the canalicular stage of development and though lung diffusion and metabolic rate are strongly correlated, the allometric exponent suggests that smaller newborns have relatively smaller diffusing capacity with respect to their demand for oxygen. Without improvement in functional or structural parameters newborn marsupials are reliant to varying degrees on skin gas exchange to compensate for the immaturity of the lung. Indeed, in some species there is complete reliance on the skin for gas exchange at birth. Nevertheless, with an early dependence on ventilation, the CNS would appear already to contain neurons with properties and connections that permit rhythmic motor output at birth and pulmonary reflexes mature soon after. Despite appropriate neural control and the presence of surfactant, the highly compliant nature of the newborn chest wall results in substantial chest wall distortion during inspiratory effort which reduce the efficacy of the lung for ventilation. This review explores the morpho-functional development of the respiratory system, including oxygen transport and cardiac shunts, and the establishment of convective requirement in marsupials, a group that places emphasis on extended postnatal development.     

Development of the skin in the eastern quoll (Dasyurus viverrinus) with focus on cutaneous gas exchange in the early postnatal period

A morphological and morphometric study of the skin development in the eastern quoll (Dasyurus viverrinus) was conducted to follow the transition from cutaneous to pulmonary gas exchange in this extremely immature marsupial species. Additionally, the development of the cardiac and respiratory system was followed, to evaluate the systemic prerequisites allowing for cutaneous respiration. The skin in the newborn D. viverrinus was very thin (36 ± 3 µm) and undifferentiated (no hair follicles, no sebaceous and perspiratory glands). Numerous superficial cutaneous capillaries were encountered, closely associated with the epidermis, allowing for gaseous exchange. The capillary volume density was highest in the neonate (0.33 ± 0.04) and decreased markedly during the first 4 days (0.06 ± 0.01). In the same time period, the skin diffusion barrier increased from 9 ± 1 µm to 44 ± 6 µm. From this age on the skin development was characterized by thickening of the different cutaneous layers, formation of hair follicles (day 55) and the occurrence of subcutaneous fat (day 19). The heart of the neonate D. viverrinus had incomplete interatrial, inter-ventricular, and aortico-pulmonary septa, allowing for the possibility that oxygenated blood from the skin mixes with that of the systemic circulation. The fast-structural changes in the systemic circulations (closing all shunts) in the early postnatal period (3 days) necessitate the transition from cutaneous to pulmonary respiration despite the immaturity of the lungs. At this time, the lung was still at the canalicular stage of lung development, but had to be mature enough to meet the respiratory needs of the growing organism. The morphometric results for the skin development of D. viverrinus suggest that cutaneous respiration is most pronounced in neonates and decreases rapidly during the first 3 days of postnatal life. After this time a functional transition of the skin from cutaneous respiration to insulation and protection of the body takes place.     

Histamine in the tissues of the marsupial Setonix brachyurus (the quokka).

Passive cutaneous anaphylactic (PCA) reactions in the marsupial Setonix brachyurus (the quokka) were completely inhibited by the histamine antagonist mepyramine maleate, but were unaffected by disodium cromoglycate or the serotonin antagonist, methysergide. Histological examination of quokka skin indicated that mast cell degranulation occurred during the PCA reaction in this marsupial and animals whose skin was relatively deficient in mast cells were poor PCA recipients. In contrast to many eutherian (placental) species, this marsupial was found to lack histamine in blood leukocytes and platelets. Also, while the peritoneal mast cells of rats and mice contain large quantities of histamine, this amine was not detected to quokka peritoneal washings, even after the induction of a peritoneal exudate or the regular intraperitoneal injection of antigen. Immunologic challenge of quokka blood or peritoneal cells did not induce the synthesis of histamine, but histamine release was elicited from sensitized quokka lung by antigenic challenge.     

Ultrastructure of the olfactory system of three newborn marsupial species

The ultrastructure of the olfactory apparatus of three newborn marsupial species, the northern native cat, the brushtail possum, and the northern brown bandicoot, were examined. The olfactory epithelium and olfactory bulbs were at a similar stage of development in all three species. Receptor cells with cilia were observed, and although the olfactory system undergoes further differentiation during pouch life and although the olfactory epithelium and bulb of the newborn differs from that of the adult, these facts do not preclude the ability of the newborn to detect smell. The presence of touch receptors around the mouth region and of sensory cells within the olfactory epithelium would suggest that touch and smell are two of the senses allowing the newborn marsupial to reach the pouch.     

Prenatal administration of vitamin A alters pulmonary and plasma levels of vascular endothelial growth factor in the developing mouse

Vitamin A and the retinoids play a unique role in mammalian embryonic and foetal development and are essential for both cellular differentiation and the establishment of normal morphogenesis. Vascular endothelial growth factor (VEGF) is a known potent mitogenic factor that plays a key role in lung development and function maintenance. In order to contribute to a better knowledge of the modulating effects of vitamin A in lung development, we investigated the effects of the antenatal administration of vitamin A on VEGF expression in lungs and plasma from foetuses and neonates. Pregnant mice were subjected to subcutaneous administration of vitamin A on the 12th gestational day. The lungs and plasma from foetuses and neonates were collected daily from the 15th gestational day till the day of birth. Our results show that vitamin A modulates VEGF concentrations both in lungs and plasma. Statistically significant differences were observed at gestational days 15 (P = 0.004 for lungs; P < 0.0001 for plasma), 16 (P < 0.0001 for lungs and plasma) and 18 (P < 0.0001 for lungs; P < 0.05 for plasma). Vitamin A tends to increase the expression of this factor in the lung, particularly during the critical period of perinatal adaptation to postnatal life. These effects seem to be spatial and temporally regulated, and point out to the important role of vitamin A during lung development.     

Ontogeny of beta-adrenergic receptors in pulmonary arterial smooth muscle, bronchial smooth muscle, and alveolar lining cells in the rat.

beta-Adrenergic receptors play an integral role in the modulation of cell function in the developing lung. In the rat, there are marked increases in beta receptor density in whole lung during postnatal maturation, but it is now known whether there are differential developmental changes in receptor density in specific cell types. Quantitative light microscopic autoradiography with [125I]iodocyanopindolol ([125I]ICYP) was used to determine maturational changes in beta-adrenergic receptor density in pulmonary arterial smooth muscle (ASM), bronchial smooth muscle (BSM), and alveolar lining cells (ALC) in rat lung during postnatal development (1 day to 6 mo). [125I]ICYP binding to whole lung sections revealed a single class of high-affinity receptors; agonist competitive binding studies suggested that the receptors are primarily of the beta 2 subtype. beta-Adrenergic receptor density in newborn (1 day) lung was lowest in ASM cells and was comparable in BSM cells and ALC. In contrast, in lungs from adult rats (3 mo), receptor density was similar in ASM versus BSM cells and was 2-fold greater in ALC. In addition, the maturational pattern of increasing receptor density differed in ASM compared with BSM and ALC. Receptor density in ASM increased 93% from 1 to 13 days, another 92% from 13 to 20 days, and was unchanged thereafter. In contrast, receptor density in BSM cells did not change from 1 to 13 days, but it increased 65% from 13 to 20 days, rose another 47% from 20 days to 3 mo, and increased an additional 24% from 3 to 6 mo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Posterior pituitary of the newborn marsupial possum,Trichosurus vulpecula

The fetal anterior pituitary-adrenal axis is thought to be involved in the initiation of birth in both eutherian and marsupial mammals. Little is known about the structure and function of the posterior pituitary at birth in the marsupial. Immunocytochemistry, high pressure liquid chromatography, and radioimmunoassay were used to identify vasopressin and mesotocin in the posterior pituitary of a newborn marsupial, the brushtail possum, Trichosurus vulpecula. The concentrations of vasopressin and mesotocin in the head of the newborn possum were 0.34 and 0.28 ng, respectively. The concentration of vasopressin was always greater than that of mesotocin, and the amounts of neuropeptides present in the head increased as the possum developed. © 1993 Wiley-Liss Inc.     

Lung growth response after tracheal occlusion in fetal rabbits is gestational age-dependent.

In utero tracheal occlusion (TO) is a potent stimulus of fetal lung growth, and is currently being applied in clinical trials to treat severe forms of pulmonary hypoplasia. The aim of this study was to examine the effect of timing of TO on pulmonary growth and maturation rates. Fetal rabbits (term = 31 d) were subjected to in utero tracheal clipping at 24 (late pseudoglandular stage) or 27 d of gestation (late canalicular/early terminal sac stage). Sham-operated littermates served as controls (C). Animals were killed at time intervals ranging from 1 to 6 d (early group) or 1 to 3 d (late group) after occlusion. Lung growth was measured by computerized stereologic volumetry and 5'-bromo-2'-deoxyuridine (BrdU) pulse labeling. Pneumocyte II population kinetics were analyzed using a combination of anti-surfactant protein-A and BrdU immunohistochemistry and computer-assisted morphometry. Statistical analysis was performed using unpaired Student's t test. Early TO was followed by an initial 3-d stagnation of growth and subsequently a dramatic acceleration of growth (BrdU-labeling index [LI] 10.1 +/- 0. 6% in TO versus 2.7 +/- 0.5% in C at 29 d, P < 0.001). In contrast, late TO induced an immediate and sustained moderate increase of lung growth (BrdU-LI 2.8 +/- 0.9% in TO versus 1.1 +/- 0.2% in C at 30 d, P < 0.05), associated with relatively more pronounced air-space distension. Whereas late TO caused no significant alterations in type II cell density or proliferation, early TO was followed by a marked increase in type II cell proliferation, paradoxically associated with dramatic reduction of type II cell density after 29 d. The effects of intrauterine TO on fetal lung growth and type II cell kinetics critically depend on the gestational age, and thus on the maturity of the lungs at the time of surgery. These findings have important clinical implications with respect to the timing of fetal interventions aimed at promoting lung growth. The fetal rabbit provides an invaluable model to study the mechanics and age dependency of TO-induced lung growth.     

Comparative ontogeny in humans and chimpanzees: Similarities, differences and paradoxes in postnatal growth and development of the skull

The hypothesis of retarded development is a classic and controversial issue in human evolution. It depends directly on the understanding of ontogenetic trajectories and their basic constituents: timing, rate and associated patterns of maturation. In the present study, we applied geometric morphometrics to investigate postnatal ontogeny in human and chimpanzee skulls (N = 302). We evaluated postnatal ontogenetic rates, based on comparisons of properties of size and shape in adults. At different dental ages the percentage of the adult mean size (growth) and adult mean shape (development) was used to quantify patterns of maturation. We found significantly higher levels of ontogenetic maturity in humans than chimpanzees during pre-M1 and M1 eruption. However, during this ontogenetic period the human increments were lower than those of chimpanzees suggesting lower rates. During and after M2-eruption species did not differ in their ontogenetic trajectories. The results indicate that higher prenatal and lower peri- and postnatal maturation rates characterize human ontogeny when compared with chimpanzees. If mandibular ontogeny is considered alone, a paradox was found. Whereas growth maturation proceeded in an expected trajectory continuously approximating 100% adult mean size, developmental maturity was different. After M1-eruption in both species the morphological distance, which had increased before, became reduced again, and reached adult mean shape in a second developmental peak. Such a tendency was found in humans and chimpanzees. This indicates that both size and shape maturation must be considered to understand the complexity of postnatal mandibular ontogeny.