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.     

Off limits--integrins holding boundaries in somitogenesis

Borders are essential for demarcating repeated structures such as somites during vertebrate development. Two recent articles describe roles for Integrinalpha5 and its ligand Fibronectin1 in zebrafish anterior intersomitic boundary formation and link them to Notch and Eph-Ephrin pathways in epithelialization of somite boundary cells. Together with these pathways, Integrinalpha5 and Fibronectin1 orchestrate the orderly formation of somite and later myotome borders. These studies shed light on components downstream of the periodic segmentation mechanism - the 'segmentation clock' - in somitogenesis.     

Regulation of segmental patterning by retinoic acid signaling during Xenopus somitogenesis

Somites, the segmented building blocks of the vertebrate embryo, arise one by one in a patterning process that passes wavelike along the anteroposterior axis of the presomitic mesoderm (PSM). We have studied this process in Xenopus embryos by analyzing the expression of the bHLH gene, Thylacine1, which is turned on in the PSM as cells mature and segment, in a pattern that marks both segment boundaries and polarity. Here, we show that this segmental gene expression involves a PSM enhancer that is regulated by retinoic acid (RA) signaling at two levels. RA activates Thylacine1 expression in rostral PSM directly. RA also activates Thylacine1 expression in the caudal PSM indirectly by inducing the expression of MKP3, an inhibitor of the FGF signaling pathway. RA signaling is therefore a major contributor to segmental patterning by promoting anterior segmental polarity and by interacting with the FGF signaling pathway to position segmental boundaries.     

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.     

The studies of temporal and spatial characteristics of somitogenesis in fish embryos

New data were obtained corroborating that somitogenesis is a rhythmic process, in which the time of somite formation is strictly constant. This constant (tau s) can be considered as a natural unit of developmental "biological clock" characterizing rhythmic processes. The constant tau s can be determined with an exceptional accuracy that has no analogs among the known biological processes. This fact alone suggests that the accuracy of developmental clock is very high. In addition to the constancy of tau s, all forming somites have equal linear size along the notochord axis. In the process with strictly constant temporal and spatial factors, time plays the leading role in triggering the formation of new somite. This became clear in studies of twin embryos. Both embryos had the same number of somites but they were shorter than in the normal embryos. Also, we measured the length of head and both segmented and unsegmented caudal parts of the trunk. Combined with the published data on somitogenesis, our results suggest that the previously proposed scheme for the role of developmental clock in embryogenesis predicts: (1) a possible loss of some embryonic stages without serious consequences for subsequent development and (2) periodic switching on/off any embryonic processes (at the molecular, cellular, or supercellular level) with intervals multiple to tau s.     

Development of xenopus laevis embryos in a static magnetic field

Xenopus laevis embryos were exposed to a DC magnetic field (2.5 kG) for periods up to 1 week. The previously reported stabilization of cell membranes by stationary magnetic fields could not be demonstrated.     

Temperature and somitogenesis in embryos of the plaice (Pleuronectes platessa)

The rate of somitogenesis was observed in plaice embryos reared at 5 and 12°C. Somite formation occurred relatively later in development at 12 than at 5°C. At any given stage of development 5°C plaice embryos had significantly more somites than embryos reared at 12°C.     

Expression of protocadherin 18 in the CNS and pharyngeal arches of zebrafish embryos

Here, we report the results of molecular cloning and expression analyses of a non-clustered protocadherin (pcdh), pcdh18 in zebrafish embryos. The predicted zebrafish pcdh18 protein shows 6566% identity and 7879% homology with its mammalian and Xenopus counterparts. It has a Disabled-1 binding motif in its cytoplasmic domain, which is characteristic of pcdh18. Zebrafish embryos expressed pcdh18 by the early gastrula stage, 6 h post-fertilization (hpf), in their animal cap but not in the germ ring or the shield. pcdh18 was expressed in the neural tube and the central nervous system (CNS) from 12 hpf. Some populations of cells in the lateral neural tube and spinal cord of 1218 hpf embryos expressed pcdh18, but expression in these cells disappeared by 24 hpf. The hindbrain of embryos at 2456 hpf expressed pcdh18 in cells closely adjacent to the rostral and caudal rhombomeric boundaries in a thread-like pattern running in the dorsoventral direction. The pcdh18-positive cells were localized in the ventral part of the hindbrain at 24 hpf and in the dorsal part from 36 hpf. pcdh18 was also expressed in the telencephalon, diencephalon, tectum, upper rhombic lip, retina and otic vesicle. Expression in the CNS decreased markedly before hatching. Pharyngeal arch primordia, arches, jaws and gills expressed pcdh18, and the molecule was also expressed in some endodermal cells in late embryos.     

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.     

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.     

The effects of collagen synthesis inhibitory drugs on somitogenesis and myogenin expression in cultured chick and mouse embryos

The role of fibrillar collagen on myogenic differentiation has previously been studied in tissue culture cell lines but has not been studied in situ. We treated cultured chick and mouse embryos with collagen synthesis inhibitors to determine the role of fibrillar collagen on somitogenesis and on myogenic differentiation in vivo. Stage 12 chick embryos and 8.7 dpc mouse embryos were cultured in control medium or a range of concentrations of the collagen synthesis inhibitors ethyl-3,4-dihydroxybenzoate (EDHB) or cis-hydroxy-proline (CHP). Chick embryos were cultured for 24 h and mouse embryos were cultured for 30 h. Both collagen synthesis inhibitors produced a range of somite abnormalities including formation of fewer and irregular somites in both chick and mouse at high drug concentrations, as well as formation of double somites in EDHB-treated chick embryos. Examination of EDHB-treated mouse embryos by scanning electron microscopy demonstrated a dosage-dependent loss of fibrillar collagen and associated extracellular matrix. Expression of myogenin in EDHB-treated mouse embryos, examined by whole-mount in situ hybridization, was suppressed at higher dosage levels. This study suggests that inhibition of fibrillar collagen production and/or loss of fibrillar collagen in the developing avian and mammalian embryo results in abnormal somite formation and perturbed myogenic differentiation.     

The expression of slow myosin during mammalian somitogenesis and limb bud differentiation

The developmental pattern of slow myosin expression has been studied in mouse embryos from the somitic stage to the period of secondary fiber formation and in myogenic cells, cultured from the same developmental stages. The results obtained, using a combination of different polyclonal and monoclonal antibodies, indicate that slow myosin is coexpressed in virtually all the cells that express embryonic (fast) myosin in somites and limb buds in vivo as well as in culture. On the contrary fetal or late myoblasts (from 15-d-old embryos) express in culture only embryonic (fast) myosin. At this stage, muscle cells in vivo, as already shown (Crow, M.T., and F.A. Stockdale. 1986. Dev. Biol. 113:238-254; Dhoot, G.K. 1986. Muscle & Nerve. 9:155-164; Draeger, A., A.G. Weeds, and R.B. Fitzsimons. 1987. J. Neurol. Sci. 81:19-43; Miller, J.B., and F.A. Stockdale. 1986. J. Cell Biol. 103:2197-2208), consist of primary myotubes, which express both myosins, and secondary myotubes, which express preferentially embryonic (fast) myosin. Under no circumstance neonatal or adult fast myosins were detected. Western blot analysis confirmed the immunocytochemical data. These results suggest that embryonic myoblasts in mammals are all committed to the mixed embryonic-(fast) slow lineage and, accordingly, all primary fibers express both myosins, whereas fetal myoblasts mostly belong to the embryonic (fast) lineage and likely generate fibers containing only embryonic (fast) myosin. The relationship with current models of avian myogenesis are discussed.     

The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis

Canonical Wnt/beta-catenin signaling regulates the activation of the myogenic determination gene Myf5 at the onset of myogenesis, but the underlying molecular mechanism is unknown. Here, we report that the Wnt signal is transduced in muscle progenitor cells by at least two Frizzled (Fz) receptors (Fz1 and/or Fz6), through the canonical beta-catenin pathway, in the epaxial domain of newly formed somites. We show that Myf5 activation is dramatically reduced by blocking the Wnt/beta-catenin pathway in somite progenitor cells, whereas expression of activated beta-catenin is sufficient to activate Myf5 in somites but not in the presomitic mesoderm. In addition, we identified Tcf/Lef sequences immediately 5' to the Myf5 early epaxial enhancer. These sites determine the correct spatiotemporal expression of Myf5 in the epaxial domain of the somite, mediating the synergistic action of the Wnt/beta-catenin and the Shh/Gli pathways. Taken together, these results demonstrate that Myf5 is a direct target of Wnt/beta-catenin, and that its full activation requires a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells.     

The role of Suppressor of Hairless in Notch mediated signalling during zebrafish somitogenesis

Suppressor of Hairless (Su(H)) codes for a protein that interacts with the intracellular domain of Notch to activate the target genes of the Delta-Notch signalling pathway. We have cloned the zebrafish homologue of Su(H) and have analysed its function by morpholino mediated knockdown. While there are at least four notch and four delta homologues in zebrafish, there appears to be only one complete Su(H) homologue. We have analysed the function of Su(H) in the somitogenesis process and its influence on the expression of notch pathway genes, in particular her1, her7, deltaC and deltaD. The cyclic expression of her1, her7 and deltaC in the presomitic mesoderm is disrupted by the Su(H) knockdown mimicking the expression of these genes in the notch1a mutant deadly seven. deltaD expression is similarly affected by Su(H) knockdown like deltaC but shows in addition an ectopic expression in the developing neural tube. The inactivation of Su(H) in a fss/tbx24 mutant background leads furthermore to a clear breakdown of cyclic her1 and her7 expression, indicating that the Delta-Notch pathway is required for the creation of oscillation and not only for the synchronisation between neighbouring cells. The strongest phenotypes in the Su(H) knockdown embryos show a loss of all somites posterior to the first five to seven ones. This phenotype is stronger than the known amorphic phenotypes for notch1 (des) or deltaD (aei) in zebrafish, but mimicks the knockout phenotype of RBP-Jkappa gene in the mouse, which is the homologue of Su(H). This suggests that there is some functional redundancy among the Notch and Delta genes. This fact that the first five to seven somites are only weakly affected by Su(H) knockdown indicates that additional genetic pathways may be active in the specification of the most anterior somites.     

A role for programmed cell death during early neurogenesis in xenopus

In vertebrates, little is known on the role of programmed cell death (PCD) occurring within the population of dividing neural precursors and newly formed neuroblasts during early neural development. During primary neurogenesis, PCD takes place within the neuroectoderm of Xenopus embryos in a reproducible stereotypic pattern, suggesting a role for PCD during the early development of the CNS. We find that the spatio-temporal pattern of PCD is unaffected in embryos in which cell proliferation has been blocked and whose neuroecotoderm contains half the normal number of cells. This shows that PCD is not dependent on cell division. It further suggests that PCD does not solely function to regulate absolute cell numbers within the neuroectoderm. We demonstrate that PCD can be reproducibly inhibited in vivo during primary neurogenesis by the overexpression of human Bcl-2. Following PCD inhibition, normal neurogenesis is disrupted, as seen by the expansion of the expression domains of XSox-2, XZicr-2, XNgnr-1, XMyT-1, and N-Tubulin, XNgnr-1 being the most affected. PCD inhibition, however, did not affect the outcome of lateral inhibition. We propose, then, that PCD regulates primary neurogenesis at the level of neuronal determination.