Lynne Selwood

There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development

Abstract Embryos of different species of vertebrate share a common organisation and often look similar. Adult differences among species become more apparent through divergence at later stages. Some authors have suggested that members of most or all vertebrate clades pass through a virtually identical, conserved stage. This idea was promoted by Haeckel, and has recently been revived in the context of claims regarding the universality of developmental mechanisms. Thus embryonic resemblance at the tailbud stage has been linked with a conserved pattern of developmental gene expression – the zootype. Haeckel’s drawings of the external morphology of various vertebrates remain the most comprehensive comparative data purporting to show a conserved stage. However, their accuracy has been questioned and only a narrow range of species was illustrated. In view of the current widespread interest in evolutionary developmental biology, and especially in the conservation of developmental mechanisms, re-examination of the extent of variation in vertebrate embryos is long overdue. We present here the first review of the external morphology of tailbud embryos, illustrated with original specimens from a wide range of vertebrate groups. We find that embryos at the tailbud stage – thought to correspond to a conserved stage – show variations in form due to allometry, heterochrony, and differences in body plan and somite number. These variations foreshadow important differences in adult body form. Contrary to recent claims that all vertebrate embryos pass through a stage when they are the same size, we find a greater than 10-fold variation in greatest length at the tailbud stage. Our survey seriously undermines the credibility of Haeckel’s drawings, which depict not a conserved stage for vertebrates, but a stylised amniote embryo. In fact, the taxonomic level of greatest resemblance among vertebrate embryos is below the subphylum. The wide variation in morphology among vertebrate embryos is difficult to reconcile with the idea of a phyogenetically-conserved tailbud stage, and suggests that at least some developmental mechanisms are not highly constrained by the zootype. Our study also highlights the dangers of drawing general conclusions about vertebrate development from studies of gene expression in a small number of laboratory species.

Forelimb-hindlimb developmental timing changes across tetrapod phylogeny

BackgroundTetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. We aim to examine the potential relation of changes in developmental timing (heterochrony) to the origin of limb morphological diversity in an explicit comparative and quantitative framework. In particular, we studied the relative time sequence of development of the forelimbs versus the hindlimbs in 138 embryos of 14 tetrapod species spanning a diverse taxonomic, ecomorphological and life-history breadth. Whole-mounts and histological sections were used to code the appearance of 10 developmental events comprising landmarks of development from the early bud stage to late chondrogenesis in the forelimb and the corresponding serial homologues in the hindlimb.ResultsAn overall pattern of change across tetrapods can be discerned and appears to be relatively clade-specific. In the primitive condition, as seen in Chondrichthyes and Osteichthyes, the forelimb/pectoral fin develops earlier than the hindlimb/pelvic fin. This pattern is either retained or re-evolved in eulipotyphlan insectivores (= shrews, moles, hedgehogs, and solenodons) and taken to its extreme in marsupials. Although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development. All other species examined, including a bat with its greatly enlarged forelimbs modified as wings in the adult, showed near synchrony in the development of the fore and hindlimbs.ConclusionMajor heterochronic changes in early limb development and chondrogenesis were absent within major clades except Lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. The apparently conservative nature of this trait means that changes in chondrogenetic patterns may serve as useful phylogenetic characters at higher taxonomic levels in tetrapods. Our results highlight the more important role generally played by allometric heterochrony in this instance to shape adult morphology.

Mechanisms underlying the development of pattern in marsupial embryos.

Publisher Summary This chapter describes the marsupial embryonic development during pre-implantation stages up to the trilaminar blastocyst stage. Because the developing polarity of the marsupial embryo is initiated during oogenesis, the review also covers oogenesis and fertilization in marsupials. The chapter discusses the idea that the marsupial embryo acquires a polarized state that is initially related to the position of the nucleus and the pattern of distribution of cytoplasmic organelles, especially yolky storage products, in oocytes and zygotes. The particular nature of this polarized state generates a particular and highly specific pattern of cleavage. The chapter also discusses the potential role of positional signals, the order of cell division and maternal determinants operating at a two-dimensional level in specifying cell fate, and the early steps in determination.

Marsupial Egg and Embryo Coats

Egg and embryo coats of marsupials are reviewed. Marsupial eggs are enclosed by a zona pellucida, mucoid coat and an outer shell coat. An extra-cellular matrix coat that lines the zona pellucida also occurs in some species. The zona pellucida consists of three zona proteins (ZPA, ZPB, ZPC) with considerable similarity to those of eutherian mammals. The zona is thought to be the sperm receptor site and, in some species, but not others, is a barrier to polyspermy. Immunostaining, in situ hybridisation, histochemistry and electron microscopy indicate that the zona originates in the oocyte at the onset of oocyte growth. It changes greatly in shape during ovulation and early development and plays an essential role in blastocyst formation. The mucoid coat is an acid glycoprotein that is produced by non-ciliated cells at all levels of the oviduct and has been implicated in providing a barrier to polyspermy, nourishment of the embryo and an osmotically stable environment for the embryo. Its width varies widely in marsupials. The shell coat is secreted by the epithelia of the utero-tubal junction and upper uterus during oestrus and early cleavage. Evidence is provided for a second wave of secretion at bilaminar and trilaminar blastocyst stages. Shell-free embryos do not survive in vitro during bilaminar and early trilaminar blastocyst stages. Hatching from the shell coat occurs between 65 and 85% of the gestation period usually when the embryo is undergoing early somitogenesis. The extra-cellular matrix is secreted by the oocyte or by the cells of early cleavage stages and initially separates cells from the zona pellucida and from each other. It plays an essential role in blastocyst formation. The zona, mucoid and shell coats provide a framework for blastocyst construction and normal embryos do not form in their absence in some species.

Structural Development in the Newborn Marsupial, the Stripe-Faced Dunnart, Sminthopsis macroura

The structure of various sense organs and endocrine glands was examined in the stripe-faced dunnart, Sminthopsis macroura. This marsupial has the shortest gestation known for all mammals, 10.5-11 days. The morphology of the anterior pituitary, adrenal gland, olfactory epithelium, Merkel cells around the mouth and the utricle of the vestibular system of S. macroura was similar to that observed in other newborn marsupials. These structures are thought to be required by the newborn to transfer from the uterus to the pouch. The stage of development of the urinary system, the semicircular canals of the vestibular system, eyes and lungs was slightly different to that of other newborn marsupials. These structures are thought to have secondary importance in allowing the newborn to reach the teat. Although marsupials display variation in gestation length and produce newborn of differing body weights, there is little difference in morphology between various newborn marsupial species.

Ultrastructure of early cleavage and yolk extrusion in the marsupial Antechinus stuartii

The fertilized egg and the two‐cell stage and four‐cell stage of the marsupial Antechinus stuartii were studied by transmission electron microscopy. The features that make the fertilized egg of Antechinus stuartii different from those of any eutherian mammal are (1) the presence of a shell and (2) the relatively large quantity and polarized distribution of cytoplasmic inclusions, including lipid, protein yolk bodies, and protein fibers. Mitochondria and vesicles of smooth endoplasmic reticulum are also polarized in distribution. Early cleavage differs from that of eutherians in several ways: (1) it occurs in the uterus; (2) there is extrusion of a large, single, membrane‐bound yolk mass at first cleavage; and (3) blastomeres become separated after the second cleavage division and thus do not adhere by cell‐to‐cell contacts. Prior to the second division, blastomeres are connected to each other by remnants of the midbody and to the yolk mass by remnants of a cytoplasmic bridge. The yolk mass after extrusion is surrounded by plasma membrane and contains inclusions of lipid, protein yolk bodies, and fibers, as well as mitochondria and smooth endoplasmic reticulum. The blastomeres of the two‐cell and four‐cell stages also show intracellular polarization in the distribution of retained inclusions and organelles. Vesicles developing at the periphery of blastomeres and discharging their contents extracellularly increase in size and number from the fertilized egg to the four‐cell stage. The discharged contents may be implicated in early development of the blastula cavity.

The influence of incubation temperature on oocyte maturation, parthenogenetic and embryonic development in vitro of the marsupial Monodelphis domestica

Abstract Forty-nine oocytes and 111 embryos from 34 female Monodelphis domestica were used to examine the effect of incubation temperature on oocyte maturation and embryonic development in vitro. An additional 19 females were not used because they were not cycling (7), had unfertilised eggs (7) or had retarded embryos (5). The incubation medium was Dulbeccos modified Eagles medium with high glucose (4.5 g l −1 ) and 10% fetal calf serum held at 5% CO 2 in air. The temperatures used for incubation were 32.6°C, the body temperature of M. domestica , or 37°C, a commonly used temperature for incubation of marsupial cells. At 37°C, fewer oocytes successfully eliminated the first and second polar bodies (63% and 17%, respectively) and successfully underwent the polarisation of yolky cytoplasm characteristic of activation of oocytes (17%), than at 32.6°C. At 32.6°C, 80% eliminated the first polar body, 28% eliminated the second polar body and 60% underwent polarisation of cytoplasm. Two oocytes (8%) developed parthenogenetically to the two-cell stage at 32.6°C. Embryos incubated at 37°C completed fewer divisions, had a slightly increased rate of cleavage and showed an increased tendency to degenerate than embryos cultured at 32.6°C. The zygote had markedly polarised cytoplasm; yellow yolky cytoplasm lay in one hemisphere and darker cytoplasm in the other. The first and second divisions were associated with extrusion of yellow cytoplasm as yolk vesicles and the dark cytoplasm as amorphous material into the perivitelline space. The first division was meridional. The cleavage planes of the second and third divisions varied according to the amount of yolk, from meridional to latitudinal. The fourth and fifth cleavage planes were latitudinal to obliquely latitudinal.

Vertebrate Extracellular Preovulatory and Postovulatory Egg Coats

Abstract Extracellular egg coats deposited by maternal or embryonic tissues surround all vertebrate conceptuses during early development. In oviparous species, the time of hatching from extracellular coats can be considered equivalent to the time of birth in viviparous species. Extracellular coats must be lost during gestation for implantation and placentation to occur in some viviparous species. In the most recent classification of vertebrate extracellular coats, Boyd and Hamilton (Cleavage, early development and implantation of the egg. In: Parkes AS (ed.), Marshalls Physiology of Reproduction, vol. 2, 3rd ed. London: Longmans, Green & Co; 1961:1–126) defined the coat synthesized by the oocyte during oogenesis as primary and the coat deposited by follicle cells surrounding the oocyte as secondary. Tertiary egg coats are those synthesized and deposited around the primary or secondary coat by the maternal reproductive tract. This classification is difficult to reconcile with recent data collected using modern molecular biological techniques that can accurately establish the site of coat precursor synthesis and secretion. We propose that a modification to the classification by Boyd and Hamilton is required. Vertebrate egg coats should be classed as belonging to the following two broad groups: the preovulatory coat, which is deposited during oogenesis by the oocyte or follicle cells, and the postovulatory coats, which are deposited after fertilization by the reproductive tract or conceptus. This review discusses the origin and classification of vertebrate extracellular preovulatory and postovulatory coats and illustrates what is known about coat homology between the vertebrate groups.

An ultrastructural study of the role of an extracellular matrix during normal cleavage in a marsupial, the brushtail possum.

In marsupials, the mechanisms of lineage allocation into pluriblast and trophoblast are related to conceptus polarity and polarized discharge of extracellular matrix (ECM). The brushtail possum, Trichosurus vulpecula, a major pest species in New Zealand, is being intensively studied to develop an immunocontraceptive control method. Of 23 specimens examined, 11 were examined by electron microscopy to study the presence and role of the ECM in lineage allocation in the possum.

Early Cell Lineages in Marsupial Embryos

Publisher Summary Isolated from the constraints of implantation and occurring in simple unilaminar epithelia, the progressive separations of the early marsupial cell lineages show evidence of marsupial evolutionary history. The role of the trophoblast and hypoblast remains nutritional, but nutrient transfer is from the external uterine milieu to the pluriblast and then epiblast rather than from the internal yolk as in the lower amniotes. The yolk has been replaced by a cleavage cavity filled with extracellular matrix. The trophoblast and hypoblast probably play a role in signaling lineage separation, but the nature of this is unknown. Mechanisms to create diversity within developing epithelia also operate in these two lineages. A limited amount of evidence suggests that pluriblast and epiblast cells are pluripotential at least. They have a stem cell–like appearance ultrastructurally and in vitro, but they do not need to be cultured over fibroblast feeder layers to maintain this state. A fate mapping study has confirmed that the neuroectoderm and embryonic ectoderm arise within the epiblast, and histological studies suggest that the epiblast gives rise to all cell lineages of the embryo and extraembryonic mesoderm. Further, fate mapping studies and analysis of cells in vitro should confirm these earlier predictions.