J. P. Hill and Katherine Watson's studies of the neural crest in marsupials.

In the early part of the 20th century, J. P. Hill and K. P. Watson embarked on a comprehensive study of the development of the brain in Australian marsupials. Their work included series from three major groups: dasyurids, peramelids, and diprotodonts, covering early primitive streak through brain closure and folding stages. While the major part of the work was on the development of the brain, in the course of this work they documented that cellular proliferations from the neural plate provided much of the mesenchyme of the branchial arches. These proliferations are now known to be the neural crest. However, except for a very brief note, published shortly after Hill's death, this work was never published. In this study, I present Hill and Watson's work on the development of the early neural plate and the neural crest in marsupials. I compare their findings with published work on the South American marsupial, Monodelphis domestica and demonstrate that patterns reported in Monodelphis are general for marsupials. Further, using their data I demonstrate that in dasyurids, which are ultra-altricial at birth, the neural crest migrates early and in massive quantities, even relative to other marsupials. Finally, I discuss the historical context and speculate on reasons for why this work was unpublished. I find little support for ideas that Hill blocked publication because of loyalty to the germ layer theory. Instead, it appears primarily to have been a very large project that was simply orphaned as Watson and Hill pursued other activities.

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.

Comparative skeletal anatomy of neonatal ursids and the extreme altriciality of the giant panda.

Mammalian neonates are born at a wide range of maturity levels. Altricial newborns are born with limited sensory agency and require extensive parental care. In contrast, precocial neonates are relatively mature physically and often capable of independent function shortly after birth. In extant mammals, placental newborns vary from altricial to precocial, while marsupials and monotremes are all extremely altricial at birth. Bears (family Ursidae) have one of the lowest neonatal-maternal mass ratios in placental mammals, and are thought to also have the most altricial placental newborns. In particular, giant pandas (Ailuropoda melanoleuca) are thought to be exceptionally altricial at birth, and possibly marsupial-like. Here we used micro-computer (micro-computed) tomography scanning to visualize the skeletal anatomy of ursid neonates and compare their skeletal maturity with the neonates of other caniform outgroups. Specifically, we asked whether ursid neonates have exceptionally altricial skeletons at birth compared with other caniform neonates. We found that most bear neonates are similar to outgroup neonates in levels of skeletal ossification, with little variation in degree of ossification between ursine bears neonates (i.e. bears of the subfamily Ursinae). Perinatal giant pandas, however, have skeletal maturity levels most similar to a 42-45-day-old beagle fetus (~70% of total beagle gestation period). No bear exhibits the skeletal heterochronies seen in marsupial development. With regards to skeletal development, ursine bears are not exceptionally altricial relative to other caniform outgroups, but characterized largely by the drastic difference between newborn and adult body sizes. A review on the existing hypotheses for ursids' unique reproductive strategy suggests that the extremely small neonatal-maternal mass ratio of ursids may be related to the recent evolution of large adult body size, while life history characteristics retained an ancestral condition. A relatively short post-implantation gestation time may be the proximal mechanism behind the giant panda neonates' small size relative to maternal size and altricial skeletal development at birth.

Heterochrony and developmental timing mechanisms: changing ontogenies in evolution.

Heterochrony, or a change in developmental timing, is an important mechanism of evolutionary change. Historically the concept of heterochrony has focused alternatively on changes in size and shape or changes in developmental sequence, but most have focused on the pattern of change. Few studies have examined changes in the mechanisms that embryos use to actually measure time during development. Recently, evolutionary studies focused on changes in distinct timekeeping mechanisms have appeared, and this review examines two such case studies: the evolution of increased segment number in snakes and the extreme rostral to caudal gradient of developmental maturation in marsupials. In both examples, heterochronic modifications of the somite clock have been important drivers of evolutionary change.

Heterochrony in somitogenesis rate in a model marsupial, Monodelphis domestica.

Marsupial newborns are highly altricial and also show a wide array of shifts in the rate or timing of developmental events so that certain neonatal structures are quite mature. One particularly notable feature is the steep gradient in development along the anterior-posterior axis such that anterior structures are generally well developed relative to posterior ones. Here, we study somitogenesis in the marsupial, Monodelphis domestica, and document two heterochronies that may be important in generating the unusual body plan of the newborn marsupial. First, we demonstrate a 4-fold change in somitogenesis rate along the anterior-posterior axis, which appears to be due to somitogenesis slowing posteriorly. Second, we show that somitogenesis, particularly in the cervical region, initiates earlier in Monodelphis relative to other developmental events in the embryo. The early initiation of somitogenesis may contribute to the early development of the cervical region and forelimbs. Other elements of somitogenesis appear to be conserved. When compared to mouse, we see similar expression of genes involved in the clock and wavefront, and genes of the Wnt, Notch, and fibroblast growth factor (FGF) pathways also cycle in Monodelphis. Further, we could not discern differences in somite maturation rate along the anterior-posterior axis in Monodelphis, and thus rate of maturation of the somites does not appear to contribute to the steep anterior-posterior gradient.

Tempo of trophic evolution and its impact on mammalian diversification.

Mammals are characterized by the complex adaptations of their dentition, which are an indication that diet has played a critical role in their evolutionary history. Although much attention has focused on diet and the adaptations of specific taxa, the role of diet in large-scale diversification patterns remains unresolved. Contradictory hypotheses have been proposed, making prediction of the expected relationship difficult. We show that net diversification rate (the cumulative effect of speciation and extinction), differs significantly among living mammals, depending upon trophic strategy. Herbivores diversify fastest, carnivores are intermediate, and omnivores are slowest. The tempo of transitions between the trophic strategies is also highly biased: the fastest rates occur into omnivory from herbivory and carnivory and the lowest transition rates are between herbivory and carnivory. Extant herbivore and carnivore diversity arose primarily through diversification within lineages, whereas omnivore diversity evolved by transitions into the strategy. The ability to specialize and subdivide the trophic niche allowed herbivores and carnivores to evolve greater diversity than omnivores.

Evolution and development of the mammalian dentition: insights from the marsupial Monodelphis domestica.

To understand developmental mechanisms of evolutionary change, we must first know how different morphologies form. The vast majority of our knowledge on the developmental genetics of tooth formation derives from studies in mice, which have relatively derived mammalian dentitions. The marsupial Monodelphis domestica has a more plesiomorphic heterodont dentition with incisors, canines, premolars, and molars on both the upper and the lower jaws, and a deciduous premolar. The complexity of the M. domestica dentition ranges from simple, unicusped incisors to conical, sharp canines to multicusped molars. We examine the development of the teeth in M. domestica, with a specific focus on the enamel knot, a signaling center in the embryonic tooth that controls shape. We show that the tooth germs of M. domestica express fibroblast growth factor (FGF) genes and Sprouty genes in a manner similar to wild-type mouse molar germs, but with a few key differences.

Developmental origins of precocial forelimbs in marsupial neonates.

Marsupial mammals are born in an embryonic state, as compared with their eutherian counterparts, yet certain features are accelerated. The most conspicuous of these features are the precocial forelimbs, which the newborns use to climb unaided from the opening of the birth canal to the teat. The developmental mechanisms that produce this acceleration are unknown. Here we show that heterochronic and heterotopic changes early in limb development contribute to forelimb acceleration. Using Tbx5 and Tbx4 as fore- and hindlimb field markers, respectively, we have found that, compared with mouse, both limb fields arise notably early during opossum development. Patterning of the forelimb buds is also accelerated, as Shh expression appears early relative to the outgrowth of the bud itself. In addition, the forelimb fields and forelimb myocyte allocation are increased in size and number, respectively, and migration of the spinal nerves into the forelimb bud has been modified. This shift in the extent of the forelimb field is accompanied by shifts in Hox gene expression along the anterior-posterior axis. Furthermore, we found that both fore- and hindlimb fields arise gradually during gastrulation and extension of the embryonic axis, in contrast to the appearance of the limb fields in their entirety in all other known cases. Our results show a surprising evolutionary flexibility in the early limb development program of amniotes and rule out the induction of the limb fields by mature structures such as the somites or mesonephros.

Opossum (Monodelphis domestica): A Marsupial Development Model.

INTRODUCTIONMonodelphis domestica is the most commonly used laboratory marsupial. In addition to the many factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. In this article, we present an overview of aspects of its biology and its use as a model organism. We also discuss basic care, breeding, embryo manipulation, and modifications of common techniques for the study of the development of this species.

Basic Maintenance and Breeding of the Opossum Monodelphis domestica.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. Monodelphis are easily maintained and bred in the lab. To do extensive embryonic work, a reasonably large breeding colony must be maintained. A colony of ~100 animals (~3:1 female:male ratio) allows for sacrifice of up to 12 pregnant females per month for experimental purposes, as well as for replenishment of the colony. However, because adults will fight and often kill one another if kept in the same cage for prolonged periods, we have developed a special breeding protocol that provides high rates of breeding success (75%-90%), with minimal injury due to fighting. Here, we outline this breeding strategy and describe how to successfully maintain a colony of Monodelphis in a laboratory setting.

Monodelphis whole-embryo culture.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. The embryos of Monodelphis, like those of other marsupials, can be cultured in vitro. The length of embryo viability depends in part on the stage at which culture begins, but embryos of different species of marsupials have been cultured for 18 h to almost 72 h. Good culture results for Monodelphis have been obtained using the method presented here. Embryos can be manipulated and then placed in the incubator. We have applied this technique most commonly to embryos at stages 23-25; they have retained viability and normal development through stage 26 when embryos would begin to implant in vivo.

Harvesting monodelphis embryos.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. Monodelphis embryos are easily harvested, as described in this protocol. Depending on the specific use for the embryo, there may be slight differences in euthanasia procedure, fixation, and embryo treatment. Most commonly, specimens will be used for anatomical or molecular (e.g., in situ hybridization) techniques, in which case they will be fixed in standard fixatives appropriate for the particular protocol.

Whole-mount in situ hybridization in monodelphis embryos.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. This protocol details whole-mount in situ hybridization of Monodelphis embryos, but it is broadly applicable to any marsupial. Special conditions have been included throughout the protocol for various stages of marsupial embryos. Nevertheless, whole, preterm embryonic stages (~stage 33 to birth) have proven to be difficult to work with because formation of the cuticle prevents probe and antibody penetration.

Craniofacial development in marsupial mammals: developmental origins of evolutionary change.

Biologists have long studied the evolutionary consequences of the differences in reproductive and life history strategies of marsupial and eutherian mammals. Over the past few decades, the impact of these strategies on the development of the marsupial embryo and neonate has received attention. In this review, the differences in development in the craniofacial region in marsupial and eutherian mammals will be discussed. The review will highlight differences at the organogenic and cellular levels, and discuss hypotheses for shifts in the expression of important regulatory genes. The major difference in the organogenic period is a whole-scale shift in the relative timing of central nervous system structures, in particular those of the forebrain, which are delayed in marsupials, relative to the structures of the oral-facial apparatus. Correlated with the delay in development of nervous system structures, the ossification of the bones of the neurocranium are delayed, while those of the face are accelerated. This study will also review work showing that the neural crest, which provides much of the cellular material to the facial skeleton and may also carry important patterning information, is notably accelerated in its development in marsupials. Potential consequences of these observations for hypotheses on constraint, evolutionary integration, and the existence of developmental modules is discussed. Finally, the implications of these results for hypotheses on the genetic modulation of craniofacial patterning are presented.

Early differentiation and migration of cranial neural crest in the opossum, Monodelphis domestica.

Marsupial mammals are born at a highly altricial state. Nonetheless, the neonate must be capable of considerable functional independence. Comparative studies have shown that in marsupials the morphogenesis of many structures critical to independent function are advanced relative to overall development. Many skeletal and muscular elements in the facial region show particular heterochrony. Because neural crest cells are crucial to forming and patterning much of the face, this study investigates whether the timing of cranial neural crest differentiation is also advanced. Histology and scanning electron microscopy of Monodelphis domestica embryos show that many aspects of cranial neural crest differentiation and migration are conserved in marsupials. For example, as in other vertebrates, cranial neural crest differentiates at the neural ectoderm/epidermal boundary and migrates as three major streams. However, when compared with other vertebrates, a number of timing differences exist. The onset of cranial neural crest migration is early relative to both neural tube development and somite formation in Monodelphis. First arch neural crest cell migration is particularly advanced and begins before any somites appear or regional differentiation exists in the neural tube. Our study provides the first published description of cranial neural crest differentiation and migration in marsupials and offers insight into how shifts in early developmental processes can lead to morphological change.

Time's arrow: heterochrony and the evolution of development.

The concept of heterochrony, which denotes a change in the relative timing of developmental events and processes in evolution, has accompanied attempts to link evolution and development for well over a century. During this time the definition of heterochrony and the application of the concept have varied and by the late 1990's, many questioned the usefulness of the concept. However, in the past decade studies of heterochrony have been revitalized by a new focus on developmental sequence, an examination of heterochrony in explicit phylogenetic contexts and increasing tendencies to examine the heterochrony of many kinds of events, including cellular, molecular and genetic events. Examples of such studies are reviewed in this paper and it is argued that this new application of heterochrony provides an extraordinarily rich opportunity for understanding the developmental basis of evolutionary change.

Sequence heterochrony and the evolution of development.

One of the most persistent questions in comparative developmental biology concerns whether there are general rules by which ontogeny and phylogeny are related. Answering this question requires conceptual and analytic approaches that allow biologists to examine a wide range of developmental events in well-structured phylogenetic contexts. For evolutionary biologists, one of the most dominant approaches to comparative developmental biology has centered around the concept of heterochrony. However, in recent years the focus of studies of heterochrony largely has been limited to one aspect, changes in size and shape. I argue that this focus has restricted the kinds of questions that have been asked about the patterns of developmental change in phylogeny, which has narrowed our ability to address some of the most fundamental questions about development and evolution. Here I contrast the approaches of growth heterochrony with a broader view of heterochrony that concentrates on changes in developmental sequence. I discuss a general approach to sequence heterochrony and summarize newly emerging methods to analyze a variety of kinds of developmental change in explicit phylogenetic contexts. Finally, I summarize a series of studies on the evolution of development in mammals that use these new approaches.

Ontogenetic and phylogenetic transformations of the ear ossicles in marsupial mammals.

This study is based on the examination of histological sections of specimens of different ages and of adult ossicles from macerated skulls representing a wide range of taxa and aims at addressing several issues concerning the evolution of the ear ossicles in marsupials. Three-dimensional reconstructions of the ear ossicles based on histological series were done for one or more stages of Monodelphis domestica, Caluromys philander, Sminthopsis virginiae, Trichosurus vulpecula, and Macropus rufogriseus. Several common trends were found. Portions of the ossicles that are phylogenetically older develop earlier than portions representing more recent evolutionary inventions (manubrium of the malleus, crus longum of the incus). The onset of endochondral ossification in the taxa in which this was examined followed the sequence; first malleus, then incus, and finally stapes. In M. domestica and C. philander at birth the yet precartilaginous ossicles form a supportive strut between the lower jaw and the braincase. The cartilage of Paauw develops relatively late in comparison with the ear ossicles and in close association to the tendon of the stapedial muscle. A feeble artery traverses the stapedial foramen of the stapes in the youngest stages of M. domestica, C. philander, and Sminthopsis virginiae examined. Presence of a large stapedial foramen is reconstructed in the groundplan of the Didelphidae and of Marsupialia. The stapedial foramen is absent in all adult caenolestids, dasyurids, Myrmecobius, Notoryctes, peramelids, vombatids, and phascolarctids. Pouch young of Perameles sp. and Dasyurus viverrinus show a bicrurate stapes with a sizeable stapedial foramen. Some didelphids examined to date show a double insertion of the Tensor tympani muscle. Some differences exist between M. domestica and C. philander in adult ossicle form, including the relative length of the incudal crus breve and of the stapes. Several differences exist between the malleus of didelphids and that of some phalangeriforms, the latter showing a short neck, absence of the lamina, and a ventrally directed manubrium. Hearing starts in M. domestica at an age in which the external auditory meatus has not yet fully developed, the ossicles are not fully ossified, and the middle ear space is partially filled with loose mesenchyme. The ontogenetic changes in hearing abilities in M. domestica between postnatal days 30 and 40 may be at least partially related to changes in middle ear structures.

COMPARATIVE PATTERNS OF CRANIOFACIAL DEVELOPMENT IN EUTHERIAN AND METATHERIAN MAMMALS.

The sequence of differentiation of major elements of the skeletal, muscular and nervous systems of the head is examined in developmental series of five eutherian (placental) and four metatherian (marsupial) mammals. The analysis identifies the elements that are conserved across the Theria, those that are unique to the Metatheria and to the Eutheria, and those that are variable. It is shown that although there are slight shifts in the sequence of development within the somatic tissues of the head, the primary difference between marsupial and placental mammals involves the timing and rate of differentiation of structures of the central nervous system (CNS) relative to a specific subset of structures of the cranial skeleton and musculature. In eutherians, CNS morphogenesis is well underway before the somatic tissues of the head begin differentiation. In metatherians, CNS development is delayed considerably and certain elements of the skeletal and muscular systems are advanced. It is concluded that the developmental differences between marsupial and placental mammals are best explained by the interaction of several processes including neurogenesis as a potential rate-limiting step, the developmental requirements of somatic elements, and the extremely short period of organogenesis of marsupial mammals. Several other issues, including the way that these data may be applied to determine the primitive therian developmental condition, and the use of comparative developmental data to address basic questions on morphogenetic processes, are discussed.

The morphology of the intrinsic tongue musculature in snakes (Reptilia, ophidia): Functional and phylogenetic implications.

Tongue musculature in 24 genera of snakes was examined histologically. In all snakes, the tongue is composed of a few main groups of muscles. The M. hyoglossus is a paired bundle in the center of the tongue. The posterior regions of the tongue possess musculature that surrounds these bundles and is responsible for protrusion. Anterior tongue regions contain hyoglossal bundles, dorsal longitudinal muscle bundles and vertical and transverse bundles, which are perpendicular to the long axis of the tongue. The interaction of the longitudinal with the vertical and horizontal muscles is responsible for bending during tongue flicking. Despite general similarities, distinct patterns of intrinsic tongue musculature characterize each infraorder of snakes. The Henophidia are primitive; the Scolecophidia and Caenophidia are each distinguished by derived characters. These derived characters support hypotheses that these latter taxa are each monophyletic. Cylindrophis (Anilioidea) is in some characters intermediate between Booidea and Colubroidea. The condition in the Booidea resembles the lizard condition; however, no synapomorphies of tongue musculature confirm a relationship with any specific lizard family. Although the pattern of colubroids appears to be the most biomechanically specialized, as yet no behavioral or performance feature has been identified to distinguish them from other snakes.