Notch signalling and the synchronization of the somite segmentation clock

In vertebrates with mutations in the Notch cell-cell communication pathway, segmentation fails: the boundaries demarcating somites, the segments of the embryonic body axis, are absent or irregular. This phenotype has prompted many investigations, but the role of Notch signalling in somitogenesis remains mysterious. Somite patterning is thought to be governed by a "clock-and-wavefront" mechanism: a biochemical oscillator (the segmentation clock) operates in the cells of the presomitic mesoderm, the immature tissue from which the somites are sequentially produced, and a wavefront of maturation sweeps back through this tissue, arresting oscillation and initiating somite differentiation. Cells arrested in different phases of their cycle express different genes, defining the spatially periodic pattern of somites and controlling the physical process of segmentation. Notch signalling, one might think, must be necessary for oscillation, or to organize subsequent events that create the somite boundaries. Here we analyse a set of zebrafish mutants and arrive at a different interpretation: the essential function of Notch signalling in somite segmentation is to keep the oscillations of neighbouring presomitic mesoderm cells synchronized.     

Retinoic acid signalling links left-right asymmetric patterning and bilaterally symmetric somitogenesis in the zebrafish embryo

During embryogenesis, cells are spatially patterned as a result of highly coordinated and stereotyped morphogenetic events. In the vertebrate embryo, information on laterality is conveyed to the node, and subsequently to the lateral plate mesoderm, by a complex cascade of epigenetic and genetic events, eventually leading to a left-right asymmetric body plan. At the same time, the paraxial mesoderm is patterned along the anterior-posterior axis in metameric units, or somites, in a bilaterally symmetric fashion. Here we characterize a cascade of laterality information in the zebrafish embryo and show that blocking the early steps of this cascade (before it reaches the lateral plate mesoderm) results in random left-right asymmetric somitogenesis. We also uncover a mechanism mediated by retinoic acid signalling that is crucial in buffering the influence of the flow of laterality information on the left-right progression of somite formation, and thus in ensuring bilaterally symmetric somitogenesis.     

Retinoic acid coordinates somitogenesis and left-right patterning in vertebrate embryos

A striking feature of the body plan of a majority of animals is bilateral symmetry. Almost nothing is known about the mechanisms controlling the symmetrical arrangement of the left and right body sides during development. Here we report that blocking the production of retinoic acid (RA) in chicken embryos leads to a desynchronization of somite formation between the two embryonic sides, demonstrated by a shortened left segmented region. This defect is linked to a loss of coordination of the segmentation clock oscillations. The lateralization of this defect led us to investigate the relation between somitogenesis and the left-right asymmetry machinery in RA-deficient embryos. Reversal of the situs in chick or mouse embryos lacking RA results in a reversal of the somitogenesis laterality defect. Our data indicate that RA is important in buffering the lateralizing influence of the left-right machinery, thus permitting synchronization of the development of the two embryonic sides.     

Segmentation in the vertebrate nervous system

Although there is good evidence that growing axons can be guided by specific cues during the development of the vertebrate peripheral nervous system, little is known about the cellular mechanisms involved. We describe here an example where axons make a clear choice between two neighbouring groups of cells. Zinc iodide-osmium tetroxide staining of chick embryos reveals that motor and sensory axons grow from the neural tube region through the anterior (rostral) half of each successive somite. 180 degrees antero-posterior rotation of a portion of the neural tube relative to the somites does not alter this relationship, showing that neural segmentation is not intrinsic to the neural tube. Furthermore, if the somitic mesoderm is rotated 180 degrees about an antero-posterior axis, before somite segmentation, axons grow through the posterior (original anterior) half of each somite. Some difference therefore exists between anterior and posterior cells of the somite, undisturbed by rotation, which determines the position of axon outgrowth. It is widespread among the various vertebrate classes.     

Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock

The segmented aspect of the vertebrate body plan first arises through the sequential formation of somites. The periodicity of somitogenesis is thought to be regulated by a molecular oscillator, the segmentation clock, which functions in presomitic mesoderm cells. This oscillator controls the periodic expression of 'cyclic genes', which are all related to the Notch pathway. The mechanism underlying this oscillator is not understood. Here we show that the protein product of the cyclic gene lunatic fringe (Lfng), which encodes a glycosyltransferase that can modify Notch activity, oscillates in the chick presomitic mesoderm. Overexpressing Lfng in the paraxial mesoderm abolishes the expression of cyclic genes including endogenous Lfng and leads to defects in segmentation. This effect on cyclic genes phenocopies inhibition of Notch signalling in the presomitic mesoderm. We therefore propose that Lfng establishes a negative feedback loop that implements periodic inhibition of Notch, which in turn controls the rhythmic expression of cyclic genes in the chick presomitic mesoderm. This feedback loop provides a molecular basis for the oscillator underlying the avian segmentation clock.     

Somite-specific expression of a novel fibronectin variant FN3 is negatively regulated by SHH

Fibronectins (FNs) are major extracellular proteins in blood plasma and many tissues of vertebrates, and play important roles in adhesion, migration and differentiation of cells. We have identified a novel variant (FN3) of fibronectin in zebrafish. FN3 mRNA is abundant, as detected by whole-mount in situ hybridization, in the presomitic mesoderm and the newly formed somites, but less abundant in mature somites. Ectopic expression of Sonic Hedgehog (SHH) results in a decrease of FN3 expression, whereas the expression level of FN3 increases in the flh mutants that lack the notochord. Our results suggest that FN3 may be involved in the formation of somites, but during somite differentiation its expression needs to be downregulated by signals derived from the axial tissues.     

The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity

The serially segmented (metameric) structures of vertebrates are based on somites that are periodically formed during embryogenesis. A 'clock and wavefront' model has been proposed to explain the underlying mechanism of somite formation, in which the periodicity is generated by oscillation of Notch components (the clock) in the posterior pre-somitic mesoderm (PSM). This temporal periodicity is then translated into the segmental units in the 'wavefront'. The wavefront is thought to exist in the anterior PSM and progress backwards at a constant rate; however, there has been no direct evidence as to whether the levels of Notch activity really oscillate and how such oscillation is translated into a segmental pattern in the anterior PSM. Here, we have visualized endogenous levels of Notch1 activity in mice, showing that it oscillates in the posterior PSM but is arrested in the anterior PSM. Somite boundaries formed at the interface between Notch1-activated and -repressed domains. Genetic and biochemical studies indicate that this interface is generated by suppression of Notch activity by mesoderm posterior 2 (Mesp2) through induction of the lunatic fringe gene (Lfng). We propose that the oscillation of Notch activity is arrested and translated in the wavefront by Mesp2.     

A common somitic origin for embryonic muscle progenitors and satellite cells

In the embryo and in the adult, skeletal muscle growth is dependent on the proliferation and the differentiation of muscle progenitors present within muscle masses. Despite the importance of these progenitors, their embryonic origin is unclear. Here we use electroporation of green fluorescent protein in chick somites, video confocal microscopy analysis of cell movements, and quail-chick grafting experiments to show that the dorsal compartment of the somite, the dermomyotome, is the origin of a population of muscle progenitors that contribute to the growth of trunk muscles during embryonic and fetal life. Furthermore, long-term lineage analyses indicate that satellite cells, which are known progenitors of adult skeletal muscles, derive from the same dermomyotome cell population. We conclude that embryonic muscle progenitors and satellite cells share a common origin that can be traced back to the dermomyotome.     

Evolutionary origin and development of snake fangs

Many advanced snakes use fangs-specialized teeth associated with a venom gland-to introduce venom into prey or attacker. Various front- and rear-fanged groups are recognized, according to whether their fangs are positioned anterior (for example cobras and vipers) or posterior (for example grass snakes) in the upper jaw. A fundamental controversy in snake evolution is whether or not front and rear fangs share the same evolutionary and developmental origin. Resolving this controversy could identify a major evolutionary transition underlying the massive radiation of advanced snakes, and the associated developmental events. Here we examine this issue by visualizing the tooth-forming epithelium in the upper jaw of 96 snake embryos, covering eight species. We use the sonic hedgehog gene as a marker, and three-dimensionally reconstruct the development in 41 of the embryos. We show that front fangs develop from the posterior end of the upper jaw, and are strikingly similar in morphogenesis to rear fangs. This is consistent with their being homologous. In front-fanged snakes, the anterior part of the upper jaw lacks sonic hedgehog expression, and ontogenetic allometry displaces the fang from its posterior developmental origin to its adult front position-consistent with an ancestral posterior position of the front fang. In rear-fanged snakes, the fangs develop from an independent posterior dental lamina and retain their posterior position. In light of our findings, we put forward a new model for the evolution of snake fangs: a posterior subregion of the tooth-forming epithelium became developmentally uncoupled from the remaining dentition, which allowed the posterior teeth to evolve independently and in close association with the venom gland, becoming highly modified in different lineages. This developmental event could have facilitated the massive radiation of advanced snakes in the Cenozoic era, resulting in the spectacular diversity of snakes seen today.     

Generation of mutants with developmental defects in zebrafish by ENU mutagenesis

As a good model for studying early development of vertebrates, zebrafish (Danio rerio) is attracting more and more attention. Following ENU mutagenesis, 320 F2 families were established. Mutants, which showed defects in epiboly, axis, somite, head, and cardiac and blood systems, were identified by observing morphological changes in F3 embryos. So far, 35 mutant lines have been established, the majority of which showed anomalies in axis and somite formation. These mutant lines provide useful genetic resources for cloning of the mutant genes and for studying mechanisms of early development of vertebrate embryos.     

Serial nuclear transfer improves the development of interspecies reconstructed giant panda (Aluropoda melanoleuca) embryos

Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supports early development of reconstructed embryos in the ooplasm from another species. In this study, we transferred nonquiescent somatic cells from a giant panda into the perivitelline space of the enucleated rabbit oocytes. After electrofusion (at the rate of 71.6%) and electrical activation, 4.2% of the panda-rabbit reconstructed embryos developed to blastocyst in vitro. For improving the development rate of reconstructed embryos, we used serial NT in this study, i.e. blastomeres from reconstructed morulae were transferred into the perivitelline space of the enucleated rabbit oocytes. The fusion rates in the groups of serial I, serial II and serial III were 79.5%, 84.1% and 78.0%, respectively, having no difference with that of somatic group. And the blastocyst rates in serial NT groups were 19.4%, 13.5% and 10.3%, respectively, which are significantly higher than that in somatic NT group. These results indicate that the nuclei from nonquiescent somatic cells can support early development of reconstructed embryos and serial NT can improve the development rate of interspecies reconstructed embryos. These authors contributed equally to this work.     

Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo

The widespread use of clomiphene citrate and exogenous gonadotrophins for in vitro fertilization (IVF) in human frequently results in the production of multiple embryos. Replacement of more than two embryos increases pregnancy rate but may result in multiple pregnancies with increased pre- and post-natal abnormality. Preservation of embryos for a limited time allows fewer embryos to be replaced on several different occasions and thus the problems of multiple pregnancy can be minimized, the effectiveness of a single IVF procedure increased and embryo replacement in adverse maternal conditions avoided. Preimplantation embryos have been successfully cryopreserved in many animal species. The sensitivity of embryos to cooling and freezing varies between species and stages of embryo development. We report here the cryopreservation procedures that allow a high survival rate of four- and eight-cell human embryos and the establishment of a pregnancy following the freezing and storage of an eight-cell embryo for 4 months in liquid nitrogen. The pregnancy terminated at 24 weeks' gestation due to development of a septic Streptomyces agalactiae chorion amnionitis after premature membrane rupture.     

Neural crest regulates myogenesis through the transient activation of NOTCH

How dynamic signalling and extensive tissue rearrangements interact to generate complex patterns and shapes during embryogenesis is poorly understood. Here we characterize the signalling events taking place during early morphogenesis of chick skeletal muscles. We show that muscle progenitors present in somites require the transient activation of NOTCH signalling to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. Our results indicate that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool.     

Information for the dorsal--ventral pattern of the Drosophila embryo is stored as maternal mRNA

Maternal-effect mutations in 10 loci in Drosophila produce totally 'dorsalized' embryos. Injection of RNA isolated from wild-type embryos into mutants at six loci partially restores dorsal-ventral polarity. For the mutant snake, injection of poly(A)+ RNA restores a complete dorsal-ventral pattern.     

Developmental basis of limblessness and axial patterning in snakes.

The evolution of snakes involved major changes in vertebrate body plan organization, but the developmental basis of those changes is unknown. The python axial skeleton consists of hundreds of similar vertebrae, forelimbs are absent and hindlimbs are severely reduced. Combined limb loss and trunk elongation is found in many vertebrate taxa, suggesting that these changes may be linked by a common developmental mechanism. Here we show that Hox gene expression domains are expanded along the body axis in python embryos, and that this can account for both the absence of forelimbs and the expansion of thoracic identity in the axial skeleton. Hindlimb buds are initiated, but apical-ridge and polarizing-region signalling pathways that are normally required for limb development are not activated. Leg bud outgrowth and signalling by Sonic hedgehog in pythons can be rescued by application of fibroblast growth factor or by recombination with chick apical ridge. The failure to activate these signalling pathways during normal python development may also stem from changes in Hox gene expression that occurred early in snake evolution.     

蛇形图的消圈公式

本文讨论了任意形状蛇形图的消圈数,给出每节都是4-圈情形的蛇形图别名函数C(H),并证明每节为4-圈的蛇形图的消圈数等于它的别名函数的势.另外,在保持消圈数不变的情况下,通过简单的收缩、剖分运算把求解任意情形的蛇形图的消圈数问题归为求解每节都是4-圈特殊情形下的蛇形图消圈数问题.     

A new secreted protein that binds to Wnt proteins and inhibits their activities

The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. We use Xenopus embryos to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8 produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.     

Noise-resistant and synchronized oscillation of the segmentation clock

Periodic somite segmentation in vertebrate embryos is controlled by the 'segmentation clock', which consists of numerous cellular oscillators. Although the properties of a single oscillator, driven by a hairy negative-feedback loop, have been investigated, the system-level properties of the segmentation clock remain largely unknown. To explore these characteristics, we have examined the response of a normally oscillating clock in zebrafish to experimental stimuli using in vivo mosaic experiments and mathematical simulation. We demonstrate that the segmentation clock behaves as a coupled oscillator, by showing that Notch-dependent intercellular communication, the activity of which is regulated by the internal hairy oscillator, couples neighbouring cells to facilitate synchronized oscillation. Furthermore, the oscillation phase of individual oscillators fluctuates due to developmental noise such as stochastic gene expression and active cell proliferation. The intercellular coupling was found to have a crucial role in minimizing the effects of this noise to maintain coherent oscillation.