The protocadherin PAPC establishes segmental boundaries during somitogenesis in xenopus embryos

BACKGROUND: One prominent example of segmentation in vertebrate embryos is the subdivision of the paraxial mesoderm into repeating, metameric structures called somites. During this process, cells in the presomitic mesoderm (PSM) are first patterned into segments leading secondarily to differences required for somite morphogenesis such as the formation of segmental boundaries. Recent studies have shown that a segmental pattern is generated in the PSM of Xenopus embryos by genes encoding a Mesp-like bHLH protein called Thylacine 1 and components of the Notch signaling pathway. These genes establish a repeating pattern of gene expression that subdivides cells in the PSM into anterior and posterior half segments, but how this pattern of gene expression leads to segmental boundaries is unknown. Recently, a member of the protocadherin family of cell adhesion molecules, called PAPC, has been shown to be expressed in the PSM of Xenopus embryos in a half segment pattern, suggesting that it could play a role in restricting cell mixing at the anterior segmental boundary. RESULTS: Here, we examine the expression and function of PAPC during segmentation of the paraxial mesoderm in Xenopus embryos. We show that Thylacine 1 and the Notch pathway establish segment identity one segment prior to the segmental expression of PAPC. Altering segmental identity in embryos by perturbing the activity of Thylacine 1 and the Notch pathway, or by treatment with a protein synthesis inhibitor, cycloheximide, leads to the predicted changes in the segmental expression of PAPC. By disrupting PAPC function in embryos using a putative dominant-negative or an activated form of PAPC, we show that segmental PAPC activity is required for proper somite formation as well as for maintaining segmental gene expression within the PSM. CONCLUSIONS: Segmental expression of PAPC is established in the PSM as a downstream consequence of segmental patterning by Thylacine 1 and the Notch pathway. We propose that PAPC is part of the mechanism that establishes the segmental boundaries between posterior and anterior cells in adjacent segments.     

Activin disrupts somitogenesis in cultured chick embryos

The anatomical and cell biological aspects of somite formation in the chick embryo have been rather well studied. Molecular regulation of somitogenesis in vertebrates is just beginning to be understood. We have studied the effects of human recombinant activin on somitogenesis in gastrulating chick embryos cultured in vitro with a view to assessing the possible role of activin-related molecules in this phenomenon. Activin disrupted somitogenesis in treated embryos, resulting in the formation of abnormal, split or ectopic somites. Light microscopic examination indicated that the ability of activin to interfere with somitogenesis might be partly due to initiation of somite formation at ectopic sites. We show that these cells are indeed somitogenic by their expression of one of the earliest somite-specific marker genes, Pax3. Scanning electron microscopic examination of control and treated embryos revealed direct effects of activin on cell-cell interactions. Cells from treated embryos exhibited disrupted intercellular adhesion leading to large intercellular spaces, altered cell shapes and modification of cell surface protrusions. The effects of activin on somitogenesis appear to be specific, since the neural structures, which are generally more susceptible to chemical insults during gastrulation, were relatively less affected. The results clearly point to a role of activin-related molecules in somitogenesis in the chick embryo.     

Modelling Delta-Notch perturbations during zebrafish somitogenesis

The discovery over the last 15 years of molecular clocks and gradients in the pre-somitic mesoderm of numerous vertebrate species has added significant weight to Cooke and Zeeman's ‘clock and wavefront’ model of somitogenesis, in which a travelling wavefront determines the spatial position of somite formation and the somitogenesis clock controls periodicity (Cooke and Zeeman, 1976). However, recent high-throughput measurements of spatiotemporal patterns of gene expression in different zebrafish mutant backgrounds allow further quantitative evaluation of the clock and wavefront hypothesis. In this study we describe how our recently proposed model, in which oscillator coupling drives the propagation of an emergent wavefront, can be used to provide mechanistic and testable explanations for the following observed phenomena in zebrafish embryos: (a) the variation in somite measurements across a number of zebrafish mutants; (b) the delayed formation of somites and the formation of ‘salt and pepper’ patterns of gene expression upon disruption of oscillator coupling; and (c) spatial correlations in the ‘salt and pepper’ patterns in Delta-Notch mutants. In light of our results, we propose a number of plausible experiments that could be used to further test the model.     

A traction-based mechanism for somitogenesis in the chick

Summary This paper suggests that chick somites form because presomitic cells exert tractional forces on one another. These forces derive from the increase in cell adhesion and density that occurs as N-CAM and N-cadherin are laid down by the motile cells of the presomitic mesoderm, well before the somites form. Harris et al. (1984) have shown that adhesive and motile cells in an appropriate environment in vitro can spontaneously form aggregates under the influence of the tractional forces that they exert. Presomitic mesodermal cells may behave similarly: as CAM production increases local adhesivity, the tractional forces between the cells should become sufficiently strong for groups of cells to segment off the mesenchyme as somites. The successive expression of CAMs down the presomitic mesoderm will thus lead to the formation of an anterior-posterior sequence of somites. This mechanism can explain several aspects of somitogenesis that models generating a repetitive pre-pattern through gating cohorts of cells find hard to explain: first, mesodermal segregation occurs among highly adherent cells; second, that multiple rows of somites can form in embryos cultured on highly adherent substrata; third, that stirred mesoderm will still form normal somites; and, fourth, how somite size can be altered in heat-shocked embryos and elsewhere. Suggestions are given as to how the mechanism may be tested and where else in the embryo it could apply.     

Adhesion molecules during somitogenesis in the avian embryo

In avian embryos, somites constitute the morphological unit of the metameric pattern. Somites are epithelia formed from a mesenchyme, the segmental plate, and are subsequently reorganized into dermatome, myotome, and sclerotome. In this study, we used somitogenesis as a basis to examine tissue remodeling during early vertebrate morphogenesis. Particular emphasis was put on the distribution and possible complementary roles of adhesion-promoting molecules, neural cell adhesion molecule (N-CAM), N-cadherin, fibronectin, and laminin. Both segmental plate and somitic cells exhibited in vitro calcium-dependent and calcium-independent systems of cell aggregation that could be inhibited respectively by anti-N-cadherin and anti-N-CAM antibodies. In vivo, the spatio-temporal expression of N-cadherin was closely associated with both the formation and local disruption of the somites. In contrast, changes in the prevalence of N-CAM did not strictly accompany the remodeling of the somitic epithelium into dermamyotome and sclerotome. It was also observed that fibronectin and laminin were reorganized secondarily in the extracellular spaces after CAM-mediated contacts were modulated. In an in vitro culture system of somites, N-cadherin was lost on individual cells released from somite explants and was reexpressed when these cells reached confluence and established intercellular contacts. In an assay of tissue dissociation in vitro, antibodies to N-cadherin or medium devoid of calcium strongly and reversibly dissociated explants of segmental plates and somites. Antibodies to N-CAM exhibited a smaller disrupting effect only on segmental plate explants. In contrast, antibodies to fibronectin and laminin did not perturb the cohesion of cells within the explants. These results emphasize the possible role of cell surface modulation of CAMs during the formation and remodeling of some transient embryonic epithelia. It is suggested that N-cadherin plays a major role in the control of tissue remodeling, a process in which N-CAM is also involved but to a lesser extent. The substratum adhesion molecules, fibronectin and laminin, do not appear to play a primary role in the regulation of these processes but may participate in cell positioning and in the stabilization of the epithelial structures.     

Fibronectin distribution during somitogenesis in the chick embryo

Somite formation in vertebrates is a multi-stage process. From a relatively homogeneous rod of mesenchyme, the segmental plate, somites are formed in a repeating sequence. Cell-cell adhesion has been proposed as a causal factor in somitogenesis. This led to an analysis of fibronectin in the segmental plate with respect to the initiation of somitogenesis. The pattern of fibronectin distribution can be correlated with the initiation of somitogenesis in the anterior portion of the segmental plate. Fibronectin distribution was determined using a high resolution antibody localization technique. Differences in fibronectin distribution were verified with computer-assisted image analysis. The evidence presented supports the hypothesis that an increase in cell-cell adhesion is a significant factor in the initiation of somitogenesis.     

Uncoupling segmentation and somitogenesis in the chick presomitic mesoderm

Little is known about the tissue interactions and the molecular signals implicated in the sequence of events leading to the subdivision of the somite into its rostral and caudal compartments. It has been demonstrated that rostrocaudal identity of the sclerotome is acquired at the presomitic (PSM) level. However, it is not known whether this compartment specification is fully determined in the PSM or whether it is dependent upon maintenance cues from the surrounding environment, as is the case for somite epithelialization. In this report, we address this issue by examining the expression profiles of C-Delta-1 and C-Notch-1, the avian homologues of mouse Delta-like1 (Delta1) and Notch1 which have been implicated in the specification of the somite rostrocaudal polarity in mouse. In chick, these genes are expressed in distinct but partially overlapping domains in the PSM and subsequently in the caudal regions of the somites. We have used an in vitro assay that consists of culturing PSM explants to examine the regulation of these genes in this tissue. We find that PSM explants cultured without overlying ectoderm continue to lay down stripes of C-Delta-1 expression, although epithelialization is blocked. These results suggest that somite rostrocaudal patterning is an autonomous property of the PSM. In addition, they demonstrate that segmentation is not necessarily coupled with the formation of somites. Dev. Genet. 23:77–85, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Bulk elastic properties of chicken embryos during somitogenesis

We present measurements of the bulk Young's moduli of early chick embryos at Hamburger-Hamilton stage 10. Using a micropipette probe with a force constant k ~0.025 N/m, we applied a known force in the plane of the embryo in the anterior-posterior direction and imaged the resulting tissue displacements. We used a two-dimensional finite-element simulation method to model the embryo as four concentric elliptical elastic regions with dimensions matching the embryo's morphology. By correlating the measured tissue displacements to the displacements calculated from the in-plane force and the model, we obtained the approximate short time linear-elastic Young's moduli: 2.4 ± 0.1 kPa for the midline structures (notocord, neural tube, and somites), 1.3 ± 0.1 kPa for the intermediate nearly acellular region between the somites and area pellucida, 2.1 ± 0.1 kPa for the area pellucida, and 11.9 ± 0.8 kPa for the area opaca.     

The chick embryo: a leading model in somitogenesis studies

The vertebrate body is built on a metameric organization which consists of a repetition of functionally equivalent units, each comprising a vertebra, its associated muscles, peripheral nerves and blood vessels. This periodic pattern is established during embryogenesis by the somitogenesis process. Somites are generated in a rhythmic fashion from the presomitic mesoderm and they subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somitogenesis has been very actively studied in the chick embryo since the 19th century and many of the landmark experiments that led to our current understanding of the vertebrate segmentation process have been performed in this organism. Somite formation involves an oscillator, the segmentation clock whose periodic signal is converted into the periodic array of somite boundaries by a spacing mechanism relying on a traveling threshold of FGF signaling regressing in concert with body axis extension.     

Segmentation and somitogenesis derived from phase dynamics in growing oscillatory media

The formation of spatially repetitive structures along the growth axis of a developing embryo is a common theme in developmental biology. Here we apply the novel flow-distributed oscillator (FDO) mechanism of wave pattern formation to the problem of axial segmentation in general and to somitogenesis in particular. We argue that the conditions for formation of FDO waves are satisfied during somitogenesis in the chick and mouse and that the waves of gene expression observed in these species arise from phase dynamics in a growing oscillatory medium. We substantiate this claim by showing that the FDO mechanism allows the waves to be mimicked by an inorganic experiment and that it predicts a wavelength that coincides with that observed experimentally. To see whether the FDO mechanism is compatible with other aspects of somitogenesis, we construct an FDO-based model of somitogenesis and successfully test it against a number of experimental observations, including the effect of heat shock. Our analysis provides a rigorous physical basis for the hypothesis that the phase dynamics of a segmental clock controls important stages of segmentation during somitogenesis in the chick and mouse as well as in other organisms that undergo segmentation during their axial growth.     

Diisopropylfluorophosphate inhibits acetylcholinesterase activity and disrupts somitogenesis in the zebrafish.

Acetylcholinesterase (AChE) activity, localized histochemically, appeared in the nuclei of presumptive somitic mesodermal cells prior to the onset of somitogenesis. AChE activity appeared in a rostro-caudal sequence, in cells located the equivalent of five somite lengths caudal to the last formed somite. To investigate whether AChE activity was required for somitogenesis, several inhibitors of AChE activity were tested for their ability to block somitogenesis. Diisopropylfluorophosphate (DFP), a broad spectrum inhibitor of serine proteases and related enzymes, was the only AChE inhibitor tested that disrupted somitogenesis. Gastrulae at 50% epiboly exposed continuously to DFP at concentrations between 40 microM and 90 microM completed epiboly, but exhibited a dose-dependent decrease in the number of somites formed, and a parallel decrease in the caudal extent of somite innervation, by 24 hours post-fertilization (h). Fifteen somite (15h) embryos exposed to DFP at the ED50 of 70 microM for 3 hours, followed by recovery to 24h, developed abnormal somites. Approximately five normal somites formed after drug treatment before the first abnormal somite formed. The abnormal somites corresponded in location to that area of the presumptive somitic mesoderm that would have initiated AChE activity while the DFP was present. While exposed to 70 microM DFP, presumptive somites formed and motoneurons extended processes that had initiated AChE activity at the time of treatment with DFP, although at a slower than normal rate. However, embryos exposed to 1 mM DFP for 30 minutes at both the 5 and 15 somite stages, followed by recovery to 24h, developed the normal number of somites but were reduced in the caudal extent of somite innervation, and occasionally developed abnormal primary motoneurons. As with the abnormal somites, the abnormal motoneurons would have initiated AChE activity while the DFP was present. Presumptive somitic mesoderm unable to initiate AChE activity due to inhibition by DFP developed abnormally. While the effects of DFP are not limited to inhibiting AChE, the data support the "clock and wavefront" model proposed for somite formation, and support the hypothesis that AChE activity has a role in somitogenesis in zebrafish.