Segmentation and moral status in vivo and in vitro: a scientific perspective

A basic consideration in research on human embryos is the controversy about when the embryo acquires moral status. The author refutes the contention that segmentation is the determinant of moral status. She notes that segmentation, as a stage in embryonic development, does not coincide with the development of "irreversible individuality" upon which the segmentation argument depends. Dawson also finds a lack of clarity in the meaning of "individuality." These problems, she maintains, prevent segmentation from being morally important and render the proposed 14-day limit on embryo research unnecessary. Dawson concludes that to introduce a time restriction on embryo research is premature because it is based on an inadequate philosophical argument.     

Molecular clocks underlying vertebrate embryo segmentation: A 10-year-old hairy-go-round

Segmentation of the vertebrate embryo body is a fundamental developmental process that occurs with strict temporal precision. Temporal control of this process is achieved through molecular segmentation clocks, evidenced by oscillations of gene expression in the unsegmented presomitic mesoderm (PSM, precursor tissue of the axial skeleton) and in the distal limb mesenchyme (limb chondrogenic precursor cells). The first segmentation clock gene, hairy1, was identified in the chick embryo PSM in 1997. Ten years later, chick hairy2 expression unveils a molecular clock operating during limb development. This review revisits vertebrate embryo segmentation with special emphasis on the current knowledge on somitogenesis and limb molecular clocks. A compilation of human congenital disorders that may arise from deregulated embryo clock mechanisms is presented here, in an attempt to reconcile different sources of information regarding vertebrate embryo development. Challenging open questions concerning the somitogenesis clock are presented and discussed, such as When?, Where?, How?, and What for? Hopefully the next decade will be equally rich in answers. Birth Defects Research (Part C) 81:65–83, 2007. © 2007 Wiley‐Liss, Inc.     

Application of convolutional neural network on early human embryo segmentation during in vitro fertilization

Selection of the best quality embryo is the key for a faithful implantation in in vitro fertilization (IVF) practice. However, the process of evaluating numerous images captured by time-lapse imaging (TLI) system is time-consuming and some important features cannot be recognized by naked eyes. Convolutional neural network (CNN) is used in medical imaging yet in IVF. The study aims to apply CNN on day-one human embryo TLI. We first presented CNN algorithm for day-one human embryo segmentation on three distinct features: zona pellucida (ZP), cytoplasm and pronucleus (PN). We tested the CNN performance compared side-by-side with manual labelling by clinical embryologist, then measured the segmented day-one human embryo parameters and compared them with literature reported values. The precisions of segmentation were that cytoplasm over 97%, PN over 84% and ZP around 80%. For the morphometrics data of cytoplasm, ZP and PN, the results were comparable with those reported in literatures, which showed high reproducibility and consistency. The CNN system provides fast and stable analytical outcome to improve work efficiency in IVF setting. To conclude, our CNN system is potential to be applied in practice for day-one human embryo segmentation as a robust tool with high precision, reproducibility and speed.     

Insect segmentation: Genes, stripes and segments in "Hoppers".

Recent work has revealed that orthologues of several segmentation genes are expressed in the grasshopper embryo, in patterns resembling those shown in Drosophila. This suggests that, despite great differences between the embryos, a hierarchy of gap/pair-rule/segment polarity gene function may be a shared and ancestral feature of insect segmentation.     

Patterning embryos with oscillations: structure, function and dynamics of the vertebrate segmentation clock

The segmentation clock is an oscillating genetic network thought to govern the rhythmic and sequential subdivision of the elongating body axis of the vertebrate embryo into somites: the precursors of the segmented vertebral column. Understanding how the rhythmic signal arises, how it achieves precision and how it patterns the embryo remain challenging issues. Recent work has provided evidence of how the period of the segmentation clock is regulated and how this affects the anatomy of the embryo. The ongoing development of real-time clock reporters and mathematical models promise novel insight into the dynamic behavior of the clock.     

Segmentation in vertebrates: a molecular clock linked to periodic somite formation]

In the vertebrate embryo, somites constitute the basis of the segmental body pattern. They give rise to the axial skeleton, the dermis of the back and all striated muscles of the body. In the chick embryo, a pair of somites buds off, in a highly coordinated fashion, every 90 minutes, from the cranial end of the presomitic mesoderm (PSM) while new mesenchymal cells enter the paraxial mesoderm as a consequence of gastrulation. The processes leading to the segmentation of the somite are not yet understood. We have identified and characterised c-hairy1, an avian homologue of the Drosophila segmentation gene, hairy. c-hairy1 is strongly expressed in the presomitic mesoderm where its mRNA exhibits a cyclic posterior-to-anterior wave of expression whose periodicity corresponds to the formation time of one somite (90 min). Fate mapping of the rostral half of the PSM using the quail-chick chimera technique supports a model of cryptic segmentation within the presomitic mesoderm, and indicates that c-hairy1 expression dynamics are not due to massive cell displacement. Analysis of in vitro cultures of isolated presomitic mesoderm demonstrates that rhythmic c-hairy1 mRNA production and degradation is an autonomous property of the paraxial mesoderm. Rather than resulting from the caudal-to-rostral propagation of an activating signal, it arises from pulses of c-hairy1 expression that are coordinated in time and space. Blocking protein synthesis does not alter the propagation of c-hairy1 expression, indicating that negative autoregulation of c-hairy1 expression is unlikely to control its periodic expression. Most of the segmentation models proposed for somite formation rely on the existence of an internal clock coordinating the cells to segment together to form a somite. These results provide the first molecular evidence of a developmental clock linked to segmentation and somitogenesis of the paraxial mesoderm, and support the possibility that segmentation mechanisms used by invertebrates and vertebrates have been conserved.     

Trunk segment numbers and sequential segmentation in myriapods

Sequential segmentation from a posterior "proliferative zone" is considered to be the primitive mechanism of segmentation in arthropods. Several studies of embryonic and post-embryonic development and gene expression suggest that this occurs in all major arthropod taxa. Sequential segmentation is often associated with the idea of posterior production of body units that accumulate along the main body axis. However, the precise mechanism of sequential segmentation has not been identified yet, and, while searching for the genetic circuitry able to generate a first periodic pattern in the embryo, we can at least outline the distinctive role in segmentation of a proliferative zone. A perusal of myriapod segmentation patterns suggests that these patterns result from multi-layered developmental processes, where gene expression and epigenetic mechanisms interact in a nonstrictly hierarchical way. The posterior zone is possibly a zone of periodic signal production, but, in general, the resulting segmental pattern is not completely attributable to the activity of the signal generator. In this sense, a posterior proliferative zone would be more a "segmental organizer" than a "segment generator."     

Drosophila segmentation genes and blastoderm cell identities

The formation of the segmentation pattern in Drosophila embryos provides an excellent model for investigating the process of pattern formation in multicellular organisms. Several genes required in an embryo for normal segmentation have been analyzed by classical and molecular genetic and morphological techniques. A detailed consideration of these results suggests that these segmentation genes are combinatorially involved in translating the positional identities of individual cells at an early stage in Drosophila development.     

Turkey embryo staging from cleavage through hypoblast formation

The progressive development of the turkey embryo from first cleavage through hypoblast formation was examined in order to determine the applicability of a chicken embryo staging procedure. It was concluded that the temporal and spatial events associated with the development of the early turkey embryo are sufficiently different from those of the chicken embryo to warrant a separate staging procedure. Cleavage is asynchronous and often results in asymmetrical segmentation. Unlike the chicken embryo, which at oviposition has already formed the area pellucida and area opaca and is classified as a Stage X embryo, the turkey embryo at oviposition is only at the beginning of area pellucida formation and is classified as a Stage VII embryo. After about 3 hr of incubation and prior to completion of the area pellucida, hypoblast formation begins at the posterior end, thereby establishing the bilaterally symmetrical pattern of the embryo. When viewed from the dorsal surface, an opaque region is observed at the center of the area pellucida. This opacity is unique to the turkey embryo and is referred to as the area alba. When viewed from the ventral surface, the area alba appears to be composed of large whitish cells. To conclude, the rate of turkey embryo development through the completion of hypoblast formation, which consists of 11 stages, lags behind that of the chicken. Furthermore, the organization as well as origin of the area pellucida and hypoblast observed in the turkey embryo differ from that of the chicken embryo. © 1993 Wiley-Liss, Inc.     

Pipeline for acquisition of quantitative data on segmentation gene expression from confocal images

We describe a data pipeline developed to extract the quantitative data on segmentation gene expression from confocal images of gene expression patterns in Drosophila. The pipeline consists of five steps: image segmentation, background removal, temporal characterization of an embryo, data registration and data averaging. This pipeline was successfully applied to obtain quantitative gene expression data at cellular resolution in space and at the 6.5-minute resolution in time, as well as to construct a spatiotemporal atlas of segmentation gene expression. Each data pipeline step can be easily adapted to process a wide range of images of gene expression patterns.     

And the segmentation clock keeps ticking

The vertebrate body is organized in segments, easily visible in the consecutive vertebrae of the skeleton. These are first defined in the embryo by the formation of somites. Somites are generated at regular intervals from the presomitic mesoderm by a combination of oscillating signals, known as the segmentation clock, which establish the pace at which new somites are formed, and signaling gradients that set the location of new intersomitic borders. Using a microarray approach, Dequéant et al. have now shown that the segmentation clock is more complex than previously thought and includes oscillating expression of genes from at least three signaling pathways organized in coordinated networks.     

Segmentation (and eve) in very odd insect embryos

The formation of segments in the Drosophila early embryo is understood in greater detail than any other complex developmental process. Now, by studying other types of insect embryo, we can hope to deduce something of the ancestral mechanism of segmentation and the ways in which it has been modified in evolution. The parasitic wasp, Copidosoma floridanum, is spectacularly atypical of insects in that the small egg cell divides extensively, with no initial syncytial phase, and forms eventually some 2000 embryos⁽¹⁾. This process raises intriguing questions about the control of embryonic polarity and segmentation.     

Pipeline for acquisition of quantitative data on segmentation gene expression from confocal images

We describe a data pipeline developed to extract the quantitative data on segmentation gene expression from confocal images of gene expression patterns in Drosophila. The pipeline consists of 5 steps: image segmentation, background removal, temporal characterization of an embryo, data registration and data averaging. This pipeline was successfully applied to obtain quantitative gene expression data at cellular resolution in space and at the 6.5 minute resolution in time, as well as to construct a spatiotemporal atlas of segmentation gene expression. Each data pipeline step can be easily adapted to process a wide range of images of gene expression patterns.     

Oscillations of the snail genes in the presomitic mesoderm coordinate segmental patterning and morphogenesis in vertebrate somitogenesis

The segmented body plan of vertebrate embryos arises through segmentation of the paraxial mesoderm to form somites. The tight temporal and spatial control underlying this process of somitogenesis is regulated by the segmentation clock and the FGF signaling wavefront. Here, we report the cyclic mRNA expression of Snail 1 and Snail 2 in the mouse and chick presomitic mesoderm (PSM), respectively. Whereas Snail genes' oscillations are independent of NOTCH signaling, we show that they require WNT and FGF signaling. Overexpressing Snail 2 in the chick embryo prevents cyclic Lfng and Meso 1 expression in the PSM and disrupts somite formation. Moreover, cells mis-expressing Snail 2 fail to express Paraxis, remain mesenchymal, and are thereby inhibited from undergoing the epithelialization event that culminates in the formation of the epithelial somite. Thus, Snail genes define a class of cyclic genes that coordinate segmentation and PSM morphogenesis.     

Cell coherence during production of the presomitic mesoderm and somitogenesis in the mouse embryo

In this study, we investigated (in the early mouse embryo) the clonal properties of precursor cells which contribute to the segmented myotome, a structure derived from the somites. We used the laacZ method of single cell-labelling to visualise clones born before segmentation and bilateralisation. We found that clones which contribute to several segments both unilateral and bilateral were regionalised along the mediolateral axis and that their mediolateral position was maintained in successive adjacent segments. Furthermore, clones contributed to all segments, from their most anterior to their most posterior borders. Therefore, it appears that mediolateral regionalisation of myotomal precursor cells is a property established before bilateralisation of the presomitic mesoderm and that coherent clonal growth accompanies cell dispersion along both the mediolateral and anteroposterior axes. These findings in the mouse correlate well with what is known in the chick, suggesting conservation of the mode of production and distribution of the cells of the presomitic mesoderm. However, in addition, we also found that the mediolateral contribution of a clone is already determined in the pool of self-renewing cells that produces the myotomal precursor cells and thus that this pool is itself regionalised. Finally, we found that bilateral clones exhibit symmetry in right and left sides in the embryo at all levels of the mediolateral axis of the myotome. All these properties indicate synchrony and symmetry of formation of the presomitic mesoderm on both sides of the embryo leading to formation of a static embryonic structure with few cell movements. We suggest that sequential production of groups of cells with an identical clonal origin for both sides of the embryo from a single pool of self-renewing cells, coupled with acquisition of static cell behaviour, could play a role in colinearity of expression of Hox genes and in the segmentation system of higher vertebrates.     

Segmentation,neurogenesis and formation of early axonal pathways in the centipede,Ethmostigmus rubripes (Brandt)

Summary We have examined the embryo of the centipedeEthmostigmus rubripes to determine the degree of evolutionary conservatism in the developmental processes of segmentation, neurogenesis and axon formation between the insects and the myriapods. A conspicuous feature of centipede embryogenesis is the early separation of the left and right sides of the ganglionic primordia by extra-embryonic ectoderm. An antibody to the protein encoded by theDrosophila segmentation geneengrailed binds to cells in the posterior margin of the limb buds in the centipede embryo, in common with insect and crustacean embryos. However, whereas in insects and crustaceans this protein is also expressed in a subset of cells in the neuroectoderm, the anti-engrailed antibody did not bind to cells in the ganglionic primordia of the centipede embryo. Use of the BrdU labelling technique to mark mitotically active cells revealed that neuroblasts, the ubiquitous neuron stem cell type in insects, are not present in the centipede. The earliest central axon pathways in the centipede embryo do not arise from segmentally repeated neurons, as is the case in insects, but rather by the posteriorly directed growth of axons originating from neurons located in the brain. Axonogenesis by segmental neurons begins later in development; the pattern of neurons involved is not obviously homologous to the conservative set of central pioneering neurons found in insects. Our observations point to considerable differences between the insects and the myriapods in mechanisms for neurogenesis and the formation of central axon pathways, suggesting that these developmental processes have not been strongly conserved during arthropod evolution.     

The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

Evidence for medial/lateral specification and positional information within the presomitic mesoderm.

In the vertebrate embryo, segmentation is built on repetitive structures, named somites, which are formed progressively from the most rostral part of presomitic mesoderm, every 90 minutes in the avian embryo. The discovery of the cyclic expression of several genes, occurring every 90 minutes in each presomitic cell, has shown that there is a molecular clock linked to somitogenesis. We demonstrate that a dynamic expression pattern of the cycling genes is already evident at the level of the prospective presomitic territory. The analysis of this expression pattern, correlated with a quail/chick fate-map, identifies a 'wave' of expression travelling along the future medial/lateral presomitic axis. Further analysis also reveals the existence of a medial/lateral asynchrony of expression at the level of presomitic mesoderm. This work suggests that the molecular clock is providing cellular positional information not only along the anterior/posterior but also along the medial/lateral presomitic axis. Finally, by using an in vitro culture system, we show that the information for morphological somite formation and molecular segmentation is segregated within the medial/lateral presomitic axis. Medial presomitic cells are able to form somites and express segmentation markers in the absence of lateral presomitic cells. By contrast, and surprisingly, lateral presomitic cells that are deprived of their medial counterparts are not able to organise themselves into somites and lose the expression of genes known to be important for vertebrate segmentation, such as Delta-1, Notch-1, paraxis, hairy1, hairy2 and lunatic fringe.