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 Finally, I discuss the historical context and speculate on reasons for why this work was unpublished.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.     

The effects of the glucocorticoid,dexamethasone, on the development of the Drosophila embryo

We have investigated the effects of the glucocorticoid, dexamethasone, and five structural analogs on Drosophila development in an effort to identify steroid ligands that may play a role in the embryogenesis of this organism. Embryos were exposed to glucocorticoids either by direct culture in supplemented medium, or by examining embryos from adult flies raised on supplemented fly food. After exposure, embryos were examined for developmental defects. At a morphological level, exposure to dexamethasone disrupts the dorsolateral folding of the amnioserosa during germ band extension. In addition, germ band retraction and dorsal closure is also disrupted. The phenocritical period of these effects is within the first 4 h of embryogenesis. This response is dosage sensitive, with embryos responding to concentrations of dexamethasone ranging from 10^–6 to 10^–3M. Furthermore, glucocorticoids which are closely related structural analogs of dexamethasone also disrupt germ band retraction and dorsal closure, while other tested steroids had no effect on embryonic development. At a molecular level, expression of the gene, Krüppel, is absent from the amnioserosa of dexamethasone-treated embryos. The cuticular phenocopy resulting from exposure to dexamethasone and related glucocorticoids is morphologically similar to the mutant phenotype associated with four genes required for germ band retraction, namely hindsight, serpent, tail-up and u-shaped. The results of this study represent the first association of a glucocorticoid with dose, stage and tissue specific effects on Drosophila development at both morphological and molecular levels.     

Sequence and analysis of zona pellucida 2 cDNA (ZP2) from a marsupial,the brushtail possum,Trichosurus vulpecula

All mammalian eggs are surrounded by the zona pellucida, an extracellular coat involved in vital functions during fertilization and early development. The zona pellucida glycoproteins are promising antigenic targets for development of contraceptive vaccines to control pest populations of marsupials in Australia and New Zealand. Our current understanding of the function of the zona pellucida glycoproteins is based almost entirely on the mouse and may not be representative of gamete interactions in all eutherian or marsupial mammals. This study reports the isolation and characterization of the ZP2 gene from the brushtail possum (Trichosurus vulpecula). The brushtail possum ZP2 mRNA is 2,182 nucleotides long with an open reading frame coding for a polypeptide chain of 712 amino acids with a molecular mass of 79,542 d. The deduced amino acid sequence of possum ZP2 is 48 to 55% identical to that of eutherian mammals. It shares several structural characteristics including N-linked glycosylation sites, location and number of cysteine residues, and hydropathy profile. The brushtail possum ZP2gene is expressed exclusively in the ovary. Further studies are planned to elucidate the specific site of ZP2 expression within the ovary and its function during fertilization in marsupials. Mol. Reprod. Dev. 51:322–329, 1998. © 1998 Wiley-Liss, Inc.     

Brief communication: Forelimb compliance in arboreal and terrestrial opossums

Primates display high forelimb compliance (increased elbow joint yield) compared to most other mammals. Forelimb compliance, which is especially marked among arboreal primates, moderates vertical oscillations of the body and peak vertical forces and may represent a basal adaptation of primates for locomotion on thin, flexible branches. However, Larney and Larson (Am J Phys Anthropol 125 [2004] 42–50) reported that marsupials have forelimb compliance comparable to or greater than that of most primates, but did not distinguish between arboreal and terrestrial marsupials. If forelimb compliance is functionally linked to locomotion on thin branches, then elbow yield should be highest in marsupials relying on arboreal substrates more often. To test this hypothesis, we compared forelimb compliance between two didelphid marsupials, Caluromys philander (an arboreal opossum relying heavily on thin branches) and Monodelphis domestica (an opossum that spends most of its time on the ground). Animals were videorecorded while walking on a runway or a horizontal 7‐mm pole. Caluromys showed higher elbow yield (greater changes in degrees of elbow flexion) on both substrates, similar to that reported for arboreal primates. Monodelphis was characterized by lower elbow yield that was intermediate between the values reported by Larney and Larson (Am J Phys Anthropol 125 [2004] 42–50) for more terrestrial primates and rodents. This finding adds evidence to a model suggesting a functional link between arboreality—particularly locomotion on thin, flexible branches—and forelimb compliance. These data add another convergent trait between arboreal primates, Caluromys, and other arboreal marsupials and support the argument that all primates evolved from a common ancestor that was a fine‐branch arborealist. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc.     

Impact of Pleistocene aridity oscillations on the population history of a widespread,vagile Australian mammal,Macropus fuliginosus

Aim Climatic fluctuations during the Pleistocene have shaped the population structure of many extant taxa. However, few studies have examined widespread species inhabiting the Australian continent, where periods of increased aridity characterized the Pleistocene. Here we investigate the phylogeography and population history of a widespread and vagile southern Australian marsupial, the western grey kangaroo (Macropus fuliginosus). Location Southern Australia. Methods We examined the variation of the mitochondrial DNA (mtDNA) control region from 511 individuals of M. fuliginosus sampled throughout their transcontinental distribution. Maximum likelihood and Bayesian analyses were used to investigate the phylogeography and coalescence analyses were then used to test hypothesized biogeographical scenarios. Results The combined results of the phylogeographical and coalescence analyses revealed a complex evolutionary history. Macropus fuliginosus originated in the south‐west of the continent, with north‐western and south‐western populations subsequently diverging as a result of vicariance events during the mid‐Pleistocene. Subsequent arid phases affected these populations differently. In the north‐west, the expansion and contraction of the arid zone resulted in repeated vicariance events and multiple divergent north‐western mtDNA subclades. In contrast, the south‐western population was less impacted by climatic oscillations but gave rise to a major transcontinental eastward expansion. Main conclusions Macropus fuliginosus exhibits the genetic signature of divergence due to unidentified barriers in south‐western Western Australia, while previously identified barriers across southern Australia appear to have had little impact despite evidence of a broad‐scale range expansion prior to the Last Glacial Maximum (LGM). This pattern of localized expansion and contraction is comparable to unglaciated regions in both the Northern and Southern Hemispheres. Furthermore, this study indicates that despite the potential similarities between Northern Hemisphere glaciation and the activation of dune systems in the Australian arid zone, both of which rendered large areas inhospitable, the biotic responses and resultant phylogeographical signatures are dissimilar. Whereas a limited number of major geographically concordant refugia are observed in glaciated areas, the Southern Hemisphere arid zone appears to be associated with multiple species‐specific idiosyncratic refugia.     

Suspensor,gibberellin and in vitro development of Phaseolus coccineus embryos

Summary Embryos of Phaseolus coccineus in different stages of development (from 0.5 to 5 mm in length) were grown in vitro. Both intact embryos (with suspensor) and embryos deprived of suspensor were studied. It was found that removal of the suspensor has no effect on the development of embryos which have reached a length of 5 mm. With younger embryos, removal of the suspensor reduces embryo development, the negative effect being the greater the younger the embryo. It was shown that gibberellic acid (GA₃) concentrations of 10^-8 to 10^-6M can replace the suspensor in heart-shaped and early cotyledonary embryos (0.5 to 1.5 mm in length), whereas they reduce the development of suspensor-deprived embryos of later stages (embryos 2 to 3 mm in length) as compared with intact embryos of similar size grown on hormone-free medium. GA₃ concentrations of 10^-5 and 10^-4M are generally inhibitory and may stimulate callus formation in some embryos. The present data and those of Alpi et al. (1975) concur in ascribing a major role to gibberellins in characterizing the physiological function of the suspensor in early embryogenesis in Phaseolus coccineus.Abbreviation GA gibberellic aid     

Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo

Summary Mutations in seven different maternal-effect loci on the second chromosome of Drosophila melanogaster all cause alterations in the anterior-posterior pattern of the embryo. Mutations in torso (tor) and trunk (trk) delete the anterior- and posterior-most structures of the embryo. At the same time they shift cellular fates which are normally found in the subterminal regions of the embryo towards the poles. Mutations in vasa (vas), valois (vls), staufen (stau) and tudor (tud) cause two embryonic defects. For one they result in absence of polar plasm, polar granules and pole cells in all eggs produced by mutant females. Secondly, embryos developing inside such eggs show deletions of abdominal segments. In addition, embryos derived from staufen mothers lack anterior head structures, embryos derived from valois mothers frequently fail to cellularize properly. Mutations in exuperantia (exu) cause deletions of anterior head structures, similar to torso, trunk and staufen. However in exu, these head structures are replaced by an inverted posterior end which comprises posterior midgut, proctodeal region, and often malpighian tubules.The effects of all mutations can be traced back to the beginning stages of gastrulation, indicating that the alterations in cellular fates have probably taken place by that time. Analysis of embryos derived from double mutant mothers suggests that these three phenotypic groups of mutants interfere with three different, independent pathways. All three pathways seem to act additively on the system which specifies anterior-posterior cellular fates within the egg.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

Efficient embryonic culture method for the Japanese striped snake,Elaphe quadrivirgata,and its early developmental stages

The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named “sausage‐style (SS) culture”, allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9 min, between 60/70 and 80/91 somite stages 42.4 min, and between 113–115 and 126/127 somite stages 71 min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E. quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full‐color images with high‐resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo.     

Studies on Anoplotaenia dasyuri Beddard, 1911 (Cestoda: Taeniidae), a parasite of the Tasmanian devil: Observations on the egg and metacestode

The ultrastructure and hatching mechanisms of the egg of Anoplotaenia dasyuri showed minor differences from those of other taeniid species. The embryophore disintegrated under the action of pepsin, but activity of the embryo did not occur until placed in an alkaline solution containing pancreatin. The addition of bile from various host species did not affect the hatching of eggs of A. Daysuri although it did increase the rate of hatching of eggs of Taenia pisiformis. Metacestodes were recovered from the hearts of experimentally infected possums (Trichosurus vulpeculd), kangaroos (Macropus giganteus and M.fuliginosus) and from the livers of mice and guinea pigs fed eggs of A. Daysuri. A wombat (Vombatus ursinus), rats and rabbits could not be infected orally. Hatched and activated embryos, injected intravenously, developed in the hearts of kangaroos and wallabies (M. rufogriseus), the livers, lungs, kidneys and hearts of mice, but not at all in rats and rabbits. Similarly, injections of embryos into the portal veins of mice, rats and rabbits resulted in development of metacestodes in the lungs and liver of mice only. The development of some stages of the metacestode are briefly described and the possible phylogeny of the parasite discussed.     

Cardiovascular characteristics of two resting marsupials: An insight into the cardio-respiratory allometry of marsupials

Summary Minimum resting values for several cardiovascular and respiratory characteristics were established for two marsupial species,Trichosurus vulpecula andMacropus eugenii. Certain characteristics including heart rate, stroke volume and cardiac output varied significantly with body mass and allometric equations of the formy=aM ^b were derived to describe the relationships. The exponents of body mass,b, were generally similar to those for eutherian mammals, but in the marsupials they intercept,a, differed significantly from reported eutherian values.Although resting cardiac output in the marsupials appeared reduced in proportion to their lower resting oxygen consumption this pattern was not repeated for other variables. The stroke volume of the marsupials was 156% of eutherian levels while heart rate was less than 50% of the eutherian values.Initial data for respiratory variables also indicated comparable differences in this aspect of oxygen transport between marsupials and eutherians. Minimum respiratory rates of the marsupials were much lower than those of eutherians and tidal volumes appear larger in marsupials. The results are interpreted as suggesting that marsupials may have a large aerobic capacity.     

The complete primary structure of late lactation protein from quokka (Setonix brachyurus)

The complete primary structure of the late lactation protein from the milk of quokka (Setonix brachyurus) is presented. The amino acid sequence was established by N-terminal sequence analysis of high-performance liquid chromatography purified intact protein and peptides isolated from chemical and enzymatic digests of the protein. The protein contains 158 residues including four cysteines. The sequence comparison with the tamar wallaby (Macropus eugenii) late lactation protein shows only five differences. The protein is identified as a new member of a novel late lactation protein family present in the milk of marsupials.     

Transplantation of a polyembryonic wasp embryo: a technique for transferring endoparasitic embryo into the host egg

Concealed development of many animal embryos prevents examination of development and limits the application of embryo manipulation techniques aimed at understanding developmental processes. In embryos developing in utero, such as in mammals, it is necessary to dissect embryos from the mother and, upon manipulative intervention, to implant them back into the recipient. Parasitic wasps present a promising system for understanding the evolution of early developmental processes. In basal ectoparasitic species that lay eggs on the surface of the host, it is possible to adapt embryo manipulation techniques developed in Drosophila. However, their derived endoparasitic relatives, which exhibit various modifications of developmental programs, undergo concealed development within the host body. For example, the parasitic polyembryonic wasp Copidosoma floridanum oviposits an egg into the egg of the host moth Trichoplusia ni. The host larva emerges and the parasite undergoes development within the host body, preventing embryo manipulation as a means of examining developmental regulation. Here we present a protocol for embryo transfer that allows the transplantation of C. floridanum egg into the host egg. This approach opens a new avenue in the application of various embryo manipulation techniques aimed at understanding the evolution of embryogenesis in endoparasitic Hymenoptera. In addition, this approach has potential for the development of other tools in C. floridanum, such as transgenesis and reverse genetics, which can also be extended to other endoparasitic species.     

Genetic evidence for a complex of species within Rugopharynx australis (Mönnig, 1926) (Nematoda: Stronglyloidea) from macropodid marsupials

An electrophoretic analysis using 17 enzyme loci was carried out on specimens of the gastric nematode of macropodid marsupials, Rugopharynx australis (Mönnig, 1926), collected from Macropus eugenii (Desmarest), M. fuliginosus (Desmarest), M. giganteus Shaw, M. robustus Gould, M. rufogriseus (Desmarest), M. rufus (Desmarest), Thylogale billardierii (Desmarest) and Wallabia bicolor (Desmarest) from south-eastern Australia. The extent of fixed genetic differences between nematodes from different host species ranged from 0–53%. The two distinct morphological forms of the parasite found in M. rufogriseus differed at 50% of loci. Specimens present in M. fuliginosus and M. giganteus were indistinguishable genetically, as were nematodes from M. rufus and M. robustus. Of the two morphologically distinct congeners included in the analysis as controls, Rugopharynx epsilon (Johnston & Mawson, 1939) was genetically distinct (46–69% fixed genetic differences) from all specimens of the R. australis complex while R. rufogrisea Magzoub, 1964 was closely related to one of the two species occuring in M. rufogriseus. It was concluded that R. australis is a species complex, with a genetically distinct species present in M. eugenii, M. fuliginosus/M. giganteus, M. robustus/M. rufus, W. bicolor and T. billardierii, and two species in M. rufogriseus.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

External features of the developing embryo of the stonefly,Kamimuria tibialis (pictet) (plecoptera,perlidae)

External features of the egg, developing embryo, and first instar nymph of Kamimuria tibialis are described. The embryonic development from the germ disc to the full-grown embryo is divided into 12 stages. The saclike embryonic rudiment is formed by the bending and folding of the germ disc. The embryo first elongates at the egg surface and then sinks into the yolk due to caudal flexure. In the head, four paired protocerebral lobes differentiate and the fourth lobes are thought to be the rudiments of preantennal ganglia. The columnar serosal cells appear at the posterior pole of the egg and they disappear before katatrepsis. The coniform chloride cells occur at the hind margins of the first nine abdominal segments in the full-grown embryo and first instar nymph. Amnion formation in K. tibialis is very similar to that of Allonarcys proteus and the Isoptera. It is proposed that the immersed type of growth pattern of embryos is divided into two subtypes in hemimetabolous insects; one is in the Palaeoptera and Paraneoptera, and the other is in the Plecoptera, Orthoptera, Notoptera, Isoptera, Embioptera, and the blattarian, Periplaneta americana.