The influence of axial structures on chick somite formation

The influence of the axial structures on somite formation was investigated by culturing, on a nutritive agar substrate, segmental plates from chick embryos having 8 to 20 pairs of somites. In the first set of experiments, segmental plate was explanted together with adjacent notochord and approximately the lateral halves of the neural tube and node region. These explants formed 18 to 20 somites within 30 hr. In a second series of experiments, the notochord and neural tube were included as before, but further regression movements in the explants were prevented by removing the node region. These explants formed only 11.9 ± 1.1 somites. Finally, explants of segmental plate that included no neural tube, notochord, or node region were made. These explants had formed 10.7 ± 1.1 somites 14 to 17 hr later. When such explants were cultured for periods longer than 17 hr, there was a marked tendency for the more posterior somites to disperse and for all of the somites to develop a peculiar “hollow” morphology. It was concluded from these results that during the period of development when chick embryos possess 8 to 20 pairs of somites, the segmental plate mesoderm (1) represents about 12 prospective somites, (2) may segment into its full complement of somites without further contact with the axial structures, but (3) requires continued intimate contact with the axial structures for normal somite morphologic differentiation and stability.     

The lateral somitic frontier: dorso-ventral aspects of anterio-posterior regionalization in avian embryos

Patterning events along the anterior-posterior (AP) axis of vertebrate embryos result in the distribution of muscle and bone forming a highly effective functional system. A key aspect of regionalized AP patterning results from variation in the migratory pattern of somite cells along the dorsal-ventral (DV) axis of the body. This occurs as somite cell populations expand around the axis or migrate away from the dorsal midline and cross into the lateral plate. The fate of somitic cells has been intensely studied and many details have been reported about inductive signaling from other tissues that influence somite cell fate and behavior. We are interested in understanding the specific differences between somites in particular AP regions and how these differences contribute to the global pattern of the organism. Using orthotopic transplants of segmental plate between quail and chick embryos, we have mapped the interface of the somitic and lateral plate mesoderm during the formation of the body wall in cervical and thoracic regions. This interface does not change dramatically in the mid-cervical region, but undergoes extensive changes in the thoracic region. Based on this regional mapping and consistent with the extensive literature, we suggest a revised method of classifying regions of the body wall that relies on embryonic cell lineages rather than adult functional criteria.     

Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm

The successful organization of the vertebrate body requires that local information in the embryo be translated into a functional, global pattern. Somite cells form the bulk of the musculoskeletal system. Heterotopic transplants of segmental plate along the axis from quail to chick were performed to test the correlation between autonomous morphological patterning and Hox gene expression in somite subpopulations. The data presented strengthen the correlation of Hox gene expression with axial specification and focus on the significance of Hox genes in specific derivatives of the somites. We have defined two anatomical compartments of the body based on the embryonic origin of the cells making up contributing structures: the dorsal compartment, formed from purely somitic cell populations; and the ventral compartment comprising cells from somites and lateral plate. The boundary between these anatomical compartments is termed the somitic frontier. Somitic tissue transplanted between axial levels retains both original Hox expression and morphological identity in the dorsal compartment. In contrast, migrating lateral somitic cells crossing the somitic frontier do not maintain donor Hox expression but apparently adopt the Hox expression of the lateral plate and participate in the morphology appropriate to the host level. Dorsal and ventral compartments, as defined here, have relevance for experimental manipulations that influence somite cell behavior. The correlation of Hox expression profiles and patterning behavior of cells in these two compartments supports the hypothesis of independent Hox codes in paraxial and lateral plate mesoderm.     

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.     

Cell movement in somite formation and development in the chick: Inhibition of segmentation

The contribution of active cell movement to somite formation (segmentation) and the later dispersal of the somite sclerotome was examined using cytochalasin D (CD). Stage 14–16 chick embryos were grown over liquid medium. After 8 hr in culture, control embryos had an average of six additional pairs of somites while CD (1–2 μg/ml dissolved in DMSO)-treated embryos had no new somites. DMSO alone had no effect on somitogenesis. CD-treated embryos transferred to drug-free medium recovered and segmentation resumed. Normal and CD-treated segmental plates were examined by SEM. Drug-treated segmental plate cells rounded up, consistent with the interaction of CD on contractile microfilaments. Embryos cultured 8 hr with or without CD were fractured through somite pair 20 and examined by SEM. In untreated embryos the sclerotome had dispersed and was migrating toward the notochord. CD stopped sclerotome dispersal. To test whether CD interfered with elaboration of extracellular matrix material associated with somite development, incorporation of [³H]glucosamine and Na₂³⁵SO₄ by somites and segmental plate was determined. There was no difference in total label incorporation. Molecular-weight profiles of proteoglycan obtained using controlled-pore glass-bead columns showed only small proteoglycans for both treated and control tissues. Therefore, the alteration of segmentation and somite morphogenesis by CD was not due to detectable changes in proteoglycan synthesis.     

Determination of somite cells: independence of cell differentiation and morphogenesis

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.     

The somitic level of origin of embryonic chick hindlimb muscles

Studies of avian chimeras made by transplanting groups of quail somites into chick embryos have consistently shown that the muscle cells of the hindlimb are derived from the adjacent somites, however, the pattern of cell distribution from individual somites to individual hindlimb muscles has not been characterized. I have mapped quail cell distribution in the chick hindlimb after single somite transplantation to determine if cells from an individual somite populate discrete limb muscle regions and if there is a spatial correspondence between a muscle's somitic level of origin and the known spinal cord position of its motoneuron pool. At stages 15-18 single chick somites or equivalent lengths of unsegmented somitic mesoderm adjacent to the prospective hindlimb region were replaced with the corresponding tissue from quail embryos. At stages 28-34, quail cell distribution was mapped within individual thigh muscles and shank muscle regions. A quail-specific antiserum and Feulgen staining were used to identify quail cells. Transplants from somite levels 26-33 each gave rise to consistent quail cell labeling in a unique subset of limb muscles. The anteroposterior positions of these subsets corresponded to that of the transplanted somitic tissue. For example, more anterior or anteromedial thigh muscles contained quail cells when more anterior somitic tissue had been transplanted. For the majority of thigh muscles studied and for shank muscle groups, there was also a clear correlation between somitic level of origin and motoneuron pool position. These data are compatible with the hypothesis that motoneurons and the muscle cells of their targets share axial position labels. The question of whether motoneurons from a specific spinal cord segment recognize and consequently innervate muscle cells derived from the same axial level during early axon outgrowth is addressed in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 408-419). Quail cell distribution was also mapped in chick embryos in which quail somites or unsegmented mesoderm had been placed 2-3 somites away from their position of origin. In all cases donor somitic tissues contributed to muscles in accord with their host position. These results indicate that muscle cell precursors within the somites are not specified to migrate to a predetermined target region.     

Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.     

T-cadherin expression alternates with migrating neural crest cells in the trunk of the avian embryo.

Trunk neural crest cells and motor axons move in a segmental fashion through the rostral (anterior) half of each somitic sclerotome, avoiding the caudal (posterior) half. This metameric migration pattern is thought to be caused by molecular differences between the rostral and caudal portions of the somite. Here, we describe the distribution of T-cadherin (truncated-cadherin) during trunk neural crest cell migration. T-cadherin, a novel member of the cadherin family of cell adhesion molecules was selectively expressed in the caudal half of each sclerotome at all times examined. T-cadherin immunostaining appeared graded along the rostrocaudal axis, with increasing levels of reactivity in the caudal halves of progressively more mature (rostral) somites. The earliest T-cadherin expression was detected in a small population of cells in the caudal portion of the somite three segments rostral to last-formed somite. This initial T-cadherin expression was observed concomitant with the invasion of the first neural crest cells into the rostral portion of the same somite in stage 16 embryos. When neural crest cells were ablated surgically prior to their emigration from the neural tube, the pattern of T-cadherin immunoreactivity was unchanged compared to unoperated embryos, suggesting that the metameric T-cadherin distribution occurs independent of neural crest cell signals. This expression pattern is consistent with the possibility that T-cadherin plays a role in influencing the pattern of neural crest cell migration and in maintaining somite polarity.     

Segmental relationship between somites and vertebral column in zebrafish

The segmental heritage of all vertebrates is evident in the character of the vertebral column. And yet, the extent to which direct translation of pattern from the somitic mesoderm and de novo cell and tissue interactions pattern the vertebral column remains a fundamental, unresolved issue. The elements of vertebral column pattern under debate include both segmental pattern and anteroposterior regional specificity. Understanding how vertebral segmentation and anteroposterior positional identity are patterned requires understanding vertebral column cellular and developmental biology. In this study, we characterized alignment of somites and vertebrae, distribution of individual sclerotome progeny along the anteroposterior axis and development of the axial skeleton in zebrafish. Our clonal analysis of zebrafish sclerotome shows that anterior and posterior somite domains are not lineage-restricted compartments with respect to distribution along the anteroposterior axis but support a 'leaky' resegmentation in development from somite to vertebral column. Alignment of somites with vertebrae suggests that the first two somites do not contribute to the vertebral column. Characterization of vertebral column development allowed examination of the relationship between vertebral formula and expression patterns of zebrafish Hox genes. Our results support co-localization of the anterior expression boundaries of zebrafish hoxc6 homologs with a cervical/thoracic transition and also suggest Hox-independent patterning of regionally specific posterior vertebrae.     

Wnt 6 regulates the epithelialisation process of the segmental plate mesoderm leading to somite formation

In higher vertebrates, the paraxial mesoderm undergoes a mesenchymal to epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study, we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. Ectopic expression of Wnt6 leads to sustained expression of markers associated with the epithelial somites and reduced or delayed expression of markers associated with mesenchymally organised somitic tissue. More importantly, we show that Wnt6-producing cells are able to rescue somite formation after ectoderm ablation. Furthermore, injection of Wnt6-producing cells following the isolation of the neural tube/notochord from the segmental plate was able to rescue somite formation at both the structural (epithelialisation) and molecular level, as determined by the expression of marker genes like Paraxis or Pax-3. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists, which result in the segmental plate being unable to form somites. These results show that Wnt6, the only known member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. We propose a model in which the localisation of Wnt6 and its antagonists regulates the process of epithelialisation in the paraxial mesoderm.     

The contribution made by cells from a single somite to tissues within a body segment and assessment of their integration with similar cells from adjacent segments

Somites represent the first visual evidence of segmentation in the developing vertebrate embryo and it is becoming clear that this segmental pattern of the somites is used in the initial stages of development of other segmented systems such as the peripheral nervous system. However, it is not known whether the somites continue to contribute to the maintenance of the segmental pattern after the dispersal of the somitic cells. In particular, the extent to which cells from a single somite contribute to all of the tissues of a single body segment and the extent to which they mix with cells from adjacent segments during their migration is not known. In this study, we have replaced single somites in the future cervical region of 2-day-old chick embryos with equivalent, similarly staged quail somites. The chimerae were then allowed to develop for a further 6 days when they were killed. The cervical region was dissected and serially sectioned. The sections were stained with the Feulgen reaction for DNA to differentiate between the chick and quail cells. The results showed that the cells from a single somite remained as a clearly delimited group throughout their migration. Furthermore, the sclerotome, dermatome and myotome portions from the single somites could always be recognised as being separate from similar cells from other somites. The somitic cells formed all of the tissues within a body segment excluding the epidermis, notochord and neural tissue. There was very little mixing of the somitic cells between adjacent segments. The segmental pattern of the somites is therefore maintained during the migration of the somitic cells and this might be fundamental to a mechanism whereby the segmentation of structures, such as the peripheral nervous system, is also maintained during development.     

Heat shock produces periodic somitic disturbances in the zebrafish embryo.

Environmental influences are known to produce segmental defects in a variety of organisms. In this paper we report upon segmental aberrations produced by brief heat shocks delivered to developing zebrafish embryos. The initial defects in the segmental pattern of somitic boundaries and motoneuron axon outgrowth were usually observed five somites caudal to the somite which was forming at the time of heat shock application. Segmental defects in zebrafish embryos exposed to a single heat shock treatment can occur in a periodic pattern similar to the multiple disturbances observed to occur in chick embryos. These data are discussed with regard to models involving cell cycle synchrony or 'clock and wavefront' schemes in the process of somitogenesis.     

Ethanol treatment induces a delayed segmentation anomaly in the chick embryo

A repeatable somite anomaly is described that results from the incubation of cultured chick embryos in the presence of ethanol. The anomaly comprises a misalignment of approximately five consecutive pairs of somites such that one of each pair is displaced cranially by up to one-half a somite length. The appearance of the malformation is delayed by approximately six somite pairs after the beginning of treatment. These characteristics were shared by embryos treated at the stage of gastrulation (no somites yet present) up to embryos possessing ten pairs of somites at treatment time. The deleterious effect did not appear to result from a disruption in the mechanics of the segmentation process itself, since isolated segmental plates were able to form normal intersomitic clefts in the presence of ethanol. Similarly, there were apparently no alterations in the compaction process that occurs at the cranial end of the segmental plate, since both the contractile and adhesive components were unaffected, as judged by the distributions of actin and fibronectin. The potential mechanisms of the anomaly are discussed with reference to similar segmental defects produced by heat shock. In view of earlier results indicating that cells in the primitive streak at gastrulation are sensitive to the presence of ethanol, it is proposed that this somite anomaly is due to a disruption in the contribution of these mesoderm cells to the segmental plate.     

Regulation of somitogenesis by Ena/VASP proteins and FAK during Xenopus development

The metameric organization of the vertebrate body plan is established during somitogenesis as somite pairs sequentially form along the anteroposterior axis. Coordinated regulation of cell shape, motility and adhesion are crucial for directing the morphological segmentation of somites. We show that members of the Ena/VASP family of actin regulatory proteins are required for somitogenesis in Xenopus. Xenopus Ena (Xena) localizes to the cell periphery in the presomitic mesoderm (PSM), and is enriched at intersomitic junctions and at myotendinous junctions in somites and the myotome, where it co-localizes with beta1-integrin, vinculin and FAK. Inhibition of Ena/VASP function with dominant-negative mutants results in abnormal somite formation that correlates with later defects in intermyotomal junctions. Neutralization of Ena/VASP activity disrupts cell rearrangements during somite rotation and leads to defects in the fibronectin (FN) matrix surrounding somites. Furthermore, inhibition of Ena/VASP function impairs FN matrix assembly, spreading of somitic cells on FN and autophosphorylation of FAK, suggesting a role for Ena/VASP proteins in the modulation of integrin-mediated processes. We also show that inhibition of FAK results in defects in somite formation, blocks FN matrix deposition and alters Xena localization. Together, these results provide evidence that Ena/VASP proteins and FAK are required for somite formation in Xenopus and support the idea that Ena/VASP and FAK function in a common pathway to regulate integrin-dependent migration and adhesion during somitogenesis.     

Two linked hairy/Enhancer of split-related zebrafish genes,her1 and her7, function together to refine alternating somite boundaries

The formation of somites, reiterated structures that will give rise to vertebrae and muscles, is thought to be dependent upon a molecular oscillator that may involve the Notch pathway. hairy/Enhancer of split related [E(spl)]-related (her or hes) genes, potential targets of Notch signaling, have been implicated as an output of the molecular oscillator. We have isolated a zebrafish deficiency, b567, that deletes two linked her genes, her1 and her7. Homozygous b567 mutants have defective somites along the entire embryonic axis. Injection of a combination of her1 and her7 (her1+7) morpholino modified antisense oligonucleotides (MOs) phenocopies the b567 mutant somitic phenotype, indicating that her1 and her7 are necessary for normal somite formation and that defective somitogenesis in b567 mutant embryos is due to deletion of her1 and her7. Analysis at the cellular level indicates that somites in her1+7-deficient embryos are enlarged in the anterior-posterior dimension. Weak somite boundaries are often found within these enlarged somites which are delineated by stronger, but imperfect, boundaries. In addition, the anterior-posterior polarity of these enlarged somites is disorganized. Analysis of her1 MO-injected embryos and her7 MO-injected embryos indicates that although these genes have partially redundant functions in most of the trunk region, her1 is necessary for proper formation of the anteriormost somites and her7 is necessary for proper formation of somites posterior to somite 11. By following somite development over time, we demonstrate that her genes are necessary for the formation of alternating strong somite boundaries. Thus, even though two potential downstream components of Notch signaling are lacking in her1+7-deficient embryos, somite boundaries form, but do so with a one and a half to two segment periodicity.     

Experimental analysis of the mechanisms of somite morphogenesis

Earlier studies have suggested influences on somite morphogenesis by “somite-forming centers,” primitive streak regression, Hensen's node and notochord, and neural plate. Contradictions among these studies were unresolved.Our experiments resolve these conflicts and reveal roles of the primitive streak and notochord in shearing the prospective somite mesoderm into right and left halves and releasing somite-forming capabilities already present. The neural plate appears to be the principal inductor of somites.Embryo fragments containing no somite-forming centers, node, notochord, or streak nevertheless formed somites within 10 hr. Such somites disperse within the next 14–24 hr, which may explain why others failed to see them. In these fragments, an incision alongside the streak substitutes for streak regression in releasing somite formation. All such somites form simultaneously rather than in the normal anteroposterior progression. These fragments contain neural plate, but not notochord. We believe that physical attachment of somites to notochord in normal embryos stabilizes them and prevents dispersal.Pieces of epiblast were rotated 180° putting neural plate over lateral plate mesoderm regions. Somites were induced from the lateral plate by the displaced neural plate region. This is additional evidence of the powerful ability of neuroepithelium to induce somites.     

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.     

Calcium dependence and contraction in somite formation

The existence of a calcium-dependent contractile process in the formation of somites from segmental plate mesoderm was investigated using a Ca2+ agonist and Ca2+ and calmodulin antagonists. The contribution of cell movement and apical constriction in the segmentation process were assessed using SEM of normal and drug-treated somite and segmental plate tissue. Explants that contained segmental plates of stage 14-15 chick embryos were cultured on vitelline membranes in calcium- and magnesium-free (CMF) Hands' solution, liquid culture medium, and medium containing drugs. Ca2+ ionophore A23187 promoted the rapid completion of one new somite pair. CMF halted segmentation. The Ca2+ antagonists verapamil and papaverine reversibly inhibited segmentation. Theophylline did not inhibit segmentation, suggesting that the effects of the Ca2+ antagonists are not due to inhibition of phosphodiesterase activity. These results suggest that somitogenesis is Ca2+-dependent. Two drugs that inhibit the binding of calmodulin, chlorpromazine and trifluoperazine (TFP), halted segmentation. The inhibitory effect of TFP was reversible. The effects of TFP on somites were compared with those of cytochalasin D. The contribution of microtubules to cell shape and movement in somitogenesis was examined by incubation with nocodazole, a reversible inhibitor of tubulin polymerization. Cell elongation and somitogenesis were inhibited.