The effect of somite manipulation on the development of motoneuron projection patterns in the embryonic chick hindlimb

Although the formation of motoneuron projections to individual muscles in the embryonic chick hindlimb has been shown to involve the specific recognition of environmental cues, the source of these cues and their mode of acquisition are not known. I show in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 394-407) that there is a correlation between the segmental level of origin of motoneurons and the somitic level of origin of the muscle cells of their targets in the chick hindlimb. These data are compatible with the hypothesis that the developmental basis for specific recognition is a positional one. Motoneurons and myogenic cells may be uniquely labeled in accord with their axial level of origin early in development and subsequently matched on the basis of these labels. To test this hypothesis, I have assessed motoneuron projection patterns in the embryonic chick hindlimb after somitic tissue manipulations. In one series of embryos, somitic mesoderm at levels 26-29 or 27-29 was reversed about the anteroposterior axis prior to myogenic cell migration and axon outgrowth. Since previous studies have shown that cells migrate from the somites in accord with their position and that somites 26-29 populate anterior thigh musculature, this operation will have reversed the somitic level of origin of anterior thigh muscles. Retrograde HRP labeling of projections to anterior thigh muscles at stage (st) 30 and st 35-38 showed that motoneuron projections were largely normal. This finding suggests that limb muscle cells or their source, the somites, do not contain the cues responsible for specific recognition prior to myogenic cell migration and axon outgrowth. To confirm that specific guidance cues were still intact after somitic mesoderm reversal, I also assessed motoneuron projections in embryos where somitic tissue plus adjacent spinal cord segments at levels 26-29 were reversed in a similar manner. Analyses of the distribution of retrogradely labeled motoneurons in reversed cord segments at st 35-36 indicated that motoneuron projections were reversed. This finding suggests that motoneurons have altered their course to project to correct targets despite the altered somitic origin of their targets and, thus, that specific guidance cues were intact. I conclude that if cues governing target or pathway choice are encoded positionally then they must be associated with other embryonic tissues such as the connective tissues or that guidance cues are acquired by myogenic cells after the onset of migration and motoneuron specification.     

A novel signal induces a segmentation fissure by acting in a ventral-to-dorsal direction in the presomitic mesoderm

We describe here a novel inductive action that operates during somitic segmentation in chicken embryos. We previously reported that the posterior border cells located at a next-forming boundary in the anterior end of the presomitic mesoderm (PSM) exhibit an inductive activity that acts on the anterior cells to cause the formation of a somitic fissure (Sato, Y., Yasuda, K., Takahashi, Y., 2002. Morphological boundary forms by a novel inductive event mediated by Lunatic fringe and Notch during somitic segmentation. Development 129, 3633-3644). In this study, we have found a second inductive action along the dorso-ventral (D-V) axis during fissure formation. When relocated into a non-segmenting region of PSM, the ventral-most cells taken from the presumptive boundary are sufficient to induce an ectopic fissure in host cells. The ventrally derived signal acts in a ventral-to-dorsal direction but not ventrally, regardless of where the ventral cells are placed. This directional signaling is governed, at least in part, by the signal-receiving cells of the PSM, which we found to be polarized along the D-V axis, and also by intimate cell-cell interactions. Finally, we have observed that morphological segmentation is able to rearrange the anterior and posterior regionalization of individual somites. These findings suggest that discrete unidirectional signals along both the antero-posterior and the D-V axes act coordinately to achieve the formation of the intersomitic fissure, and also that fissure formation is important for the fine-tuning of A-P regionalization in individual somites.     

Extrinsic influences on limb muscle organisation

Skeletal muscle within the vertebrate limb originates from the somite. Much work has focussed upon the role of secreted signalling molecules of the Hedgehog, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and Wnt families plus their associated antagonists in establishing somitic cell types, yet there is no consensus on how these signals combine to influence muscle patterning. When somitic cells migrate into the limb bud, they become subject to a new set of guidance and patterning cues. Here we discuss the possible roles played by signalling proteins, particularly Hedgehogs, in guiding the cells of the limb musculature to their fate.     

Cell morphogenesis in Arabidopsis

Cell morphogenesis encompasses all processes required to establish a three-dimensional cell shape. Cells acquire the architecture specific to their developmental context by using the spatial information provided by internal or external cues. As a response to these signals, cells become reorganized and establish functionally distinct subcellular domains that ultimately lead to morphological changes. In its simplest form, cell morphogenesis results in the establishment of asymmetry along one axis, a cell polarity. Although cell polarity has been studied intensively in budding yeast and epithelial cells, little is known about more complex modes of cell morphogenesis involving multiple axes. In this review we compare the regulation of cell morphogenesis of different genetically well-characterized cell types in Arabidopsis thaliana. BioEssays 20:20–29, 1998. © 1998 John Wiley & Sons, Inc.     

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.     

Making heads from tails: Development of a reversed anterior-posterior axis during budding in an acoel

The anterior-posterior axis is a key feature of the bilaterian body plan. Although axis specification during embryogenesis has been studied extensively, virtually nothing is known about how this axis can be established post-embryonically, as occurs in budding animals. We investigated bud formation in the acoel Convolutriloba retrogemma, which reproduces by a remarkable process involving the formation of animals with linked but completely opposite body axes. Reverse axes are established anew during each round of budding and manifestations of the bud's new axis develop gradually, with regionalization of axial patterning genes (Hox and otx) and the establishment of organized musculature occurring secondarily, after bud initiation. A swath of tissue at the parent-bud boundary has no regenerative potential and appears devoid of inherent axial polarity. GSK-3 inhibitor trials suggest that Wnt/β-catenin or Hedgehog signalling may mediate the establishment of this unpolarized zone. Formation of unpolarized tissue may provide a buffer between opposing polarity cues and be a general mechanism by which budding animals establish and maintain linked body axes. In addition to elucidating the developmental basis of budding in a bilaterian, this study provides insight into convergence in animal budding mechanisms, redeployment of embryonic gene expression during budding, and Hox gene evolution.     

Axis specification in animal development

Axis specification is the first step in defining specific regions of the developing embryo. Embryos exploit asymmetries, either pre-existing in the egg or triggered by external cues, to establish embryonic axes. The axial information is then used to generate regional differences within the embryo. In this review, we discuss experiments in animals which address three questions: whether the unfertilized egg is constructed with pre-determined axes, what cues are used to specify the embryonic axes, and how these cues are interpreted to generate the initial regional differences within the embryo. Based on mapping the data onto an animal phylogeny, we then propose a scenario for how this primary developmental decision occurred in ancestral metazoans.     

Assembly of trunk and limb blood vessels involves extensive migration and vasculogenesis of somite-derived angioblasts

Vascular development requires the assembly of precursor cells into blood vessels, but how embryonic vessels are assembled is not well understood. To determine how vascular cells migrate and assemble into vessels of the trunk and limb, marked somite-derived angioblasts were followed in developing embryos. Injection of avian somites with the cell-tracker DiI showed that somite-derived angioblasts in unperturbed embryos migrated extensively and contributed to trunk and limb vessels. Mouse-avian chimeras with mouse presomitic mesoderm grafts had graft-derived endothelial cells in blood vessels at significant distances from the graft, indicating that mouse angioblasts migrated extensively in avian hosts. Mouse graft-derived endothelial cells were consistently found in trunk vessels, such as the perineural vascular plexus, the cardinal vein, and presumptive intersomitic vessels, as well as in vessels of the limb and kidney rudiment. This reproducible pattern of graft colonization suggests that avian vascular patterning cues for trunk and limb vessels are recognized by mammalian somitic angioblasts. Mouse-quail chimeras stained with both the quail vascular marker QH1 and the mouse vascular marker PECAM-1 had finely chimeric vessels, with graft-derived mouse cells interdigitated with quail vascular cells in most vascular beds colonized by graft cells. Thus, diverse trunk and limb blood vessels have endothelial cells that developed from migratory somitic angioblasts, and assembly of these vessels is likely to have a large vasculogenic component.     

Expression of the cardiac actin gene in axolotl embryos

Axolotis are an important model system for studying heart development. Patterning of the somitic mesoderm occurs in axolotis in a manner that is much more similar to the pattern observed in higher vertebrates than in Xenopus. For these reasons we cloned the axolotl cardiac actin gene, since this gene is expressed during the development of both somitic and cardiac muscle in other vertebrates. In this paper we characterize its expression. Expression of cardiac actin RNA is switched on during gastrula stages and appears in the somitic mesoderm when it is formed; expression is later activated in the embryonic heart. In adults the gene is expressed only in the heart. The results demonstrate that the clone encoding cardiac actin provides a useful marker for studying development of both skeletal and cardiac muscle development in axolotls.     

Intrinsic,Hox-dependent cues determine the fate of skeletal muscle precursors

It is generally held that vertebrate muscle precursors depend totally on environmental cues for their development. We show that instead, somites are predisposed toward a particular myogenic program. This predisposition depends on the somite's axial identity: when flank somites are transformed into limb-level somites, either by shifting somitic boundaries with FGF8 or by overexpressing posterior Hox genes, they readily activate the program typical for migratory limb muscle precursors. The intrinsic control over myogenic programs can only be overridden by FGF4 signals provided by the apical ectodermal ridge of a developing limb.     

The cerberus-related gene, Cerr1, is not essential for mouse head formation

The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus-related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells. We found that head formation is normal in Cerr1(-/-) embryos and we detected no obvious phenotypic defects in adult Cerr1(-/-) mice. However, in embryonic tissue layer recombination assays, Cerr1(-/-) presomitic/somitic mesoderm, unlike Cerr1-expressing wild-type presomitic/somitic mesoderm, was unable to maintain expression of the anterior neural marker gene Otx2 in ectoderm explants. These findings suggest that establishment of anterior identity in the mouse may involve the action of multiple functionally redundant factors.     

Embryonic cell motility can be guided by physiological electric fields

Migratory embryonic quail somitic fibroblasts display a striking sensitivity to small, steady electric fields. There are three components to their response. They begin to orient their long axes perpendicular to the field lines within 5 min of current application at the optimal field strength of 600 mV/mm. The threshold field for significant orientation in 90 min is 150 mV/mm (only 3 mV/cell width). The cells migrate toward the cathode with a similar low threshold. At field strengths greater than 400 mV/mm, the cells also elongate beginning about 1 h after field application. The importance of this embryonic cell galvanotaxis and orientation by electric fields lies in the possible utilization of this behavior both by the embryo in the guidance of embryonic cell migration in vivo and by the investigator to control cell morphology and directionality of movement in vitro in order to study mechanisms of motility.     

Mediolateral patterning of somites: multiple axial signals, including Sonic hedgehog, regulate Nkx-3.1 expression

The axial structures, the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. Here, we investigated the role of the axial structures in the mediolateral patterning of the somite by using a newly identified murine homeobox gene, Nkx-3.1, as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. We found that the expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. We showed that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures by exposing explants to either COS cells transfected with a Shh expression construct or to recombinant SHH. Shh could induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites but not in more mature, differentiated ones. The effects of Shh on Nkx-3.1 expression were antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. Additionally, we confirmed that the expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial strucutres but that Shh by itself is not capable of inducing or maintaining it. We suggest that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites.     

Novel as1 and as2 defects in leaf adaxial-abaxial polarity reveal the requirement for ASYMMETRIC LEAVES1 and 2 and ERECTA functions in specifying leaf adaxial identity

The shoot apical meristem (SAM) of seed plants is the site at which lateral organs are formed. Once organ primordia initiate from the SAM, they establish polarity along the adaxial-abaxial, proximodistal and mediolateral axes. Among these three axes, the adaxial-abaxial polarity is of primary importance in leaf patterning. In leaf development, once the adaxial-abaxial axis is established within leaf primordia, it provides cues for proper lamina growth and asymmetric development. It was reported previously that the Arabidopsis ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) genes are two key regulators of leaf polarity. In this work, we demonstrate a new function of the AS1 and AS2 genes in the establishment of adaxial-abaxial polarity by analyzing as1 and as2 alleles in the Landsberg erecta (Ler) genetic background. We provide genetic evidence that the Arabidopsis ERECTA (ER) gene is involved in the AS1-AS2 pathway to promote leaf adaxial fate. In addition, we show that AS1 and AS2 bind to each other, suggesting that AS1 and AS2 may form a complex that regulates the establishment of leaf polarity. We also report the effects on leaf polarity of overexpression of the AS1 or AS2 genes under the control of the cauliflower mosaic virus (CAMV) 35S promoter. Although plants with as1 and as2 mutations have very similar phenotypes, 35S::AS1/Ler and 35S::AS2/Ler transgenic plants showed dramatically different morphologies. A possible model of the AS1, AS2 and ER action in leaf polarity formation is discussed.     

Spatial and temporal contribution of somitic myoblasts to avian hind limb muscles

Skeletal muscles of the avian limb are derived from mononucleated myogenic precursor cells (myoblasts) that migrate into the somatopleural mesoderm of the developing limb bud from the ventrolateral dermomyotome of limb adjacent somites. In the present study, we utilized replication-deficient lacZ-encoding retroviruses to elucidate the source of myoblasts for all hind limb muscles in the chick and define the distinct patterns of myoblast distribution within the limb. We also examined, using the same marker, whether the time of migration from the somites into the limb dictates the spatial contribution the myoblasts make to the developing musculature, particularly in relation to the proximodistal and dorsovental axes. Finally, we used these investigations to examine whether the precursors of both primary and secondary myotubes are derived from somitic mesoderm, a presumption, which up until now, has not been demonstrated in vivo. Overall, the results of our studies demonstrate that individual somites have a selective spatial pattern of participation in the development of the avian hind limb musculature and contribute to both primary and secondary myotubes. We also show that both early and later migrating myoblasts can contribute fully to the formation of the appendicular muscles.     

3D reconstructions of quail–chick chimeras provide a new fate map of the avian scapula

Limbed vertebrates have functionally integrated postcranial axial and appendicular systems derived from two distinct populations of embryonic mesoderm. The axial skeletal elements arise from the paraxial somites, the appendicular skeleton and sternum arise from the somatic lateral plate mesoderm, and all of the muscles for both systems arise from the somites. Recent studies in amniotes demonstrate that the scapula has a mixed mesodermal origin. Here we determine the relative contribution of somitic and lateral plate mesoderm to the avian scapula from quail-chick chimeras. We generate 3D reconstructions of the grafted tissue in the host revealing a very different distribution of somitic cells in the scapula than previously reported. This novel 3D visualization of the cryptic border between somitic and lateral plate populations reveals the dynamics of musculoskeletal morphogenesis and demonstrates the importance of 3D visualization of chimera data. Reconstructions of chimeras make clear three significant contrasts with existing models of scapular development. First, the majority of the avian scapula is lateral plate derived and the somitic contribution to the scapular blade is significantly smaller than in previous models. Second, the segmentation of the somitic component of the blade is partially lost; and third, there are striking differences in growth rates between different tissues derived from the same somites that contribute to the structures of the cervical thoracic transition, including the scapula. These data call for the reassessment of theories on the development, homology, and evolution of the vertebrate scapula.     

Dorso-ventral limb polarity and origin of the ridge: On the fringe of independence?

Molecular and developmental studies of limb pattern formation have recently gained widespread attention. The fact that vertebrate limbs are amenable to both genetic and embryological manipulations has established this model system as a valuable paradigm for studying vertebrate development. Limb buds are polarised along all three major axes and the establishment of the dorso-ventral (DV) polarity is dependent upon cues localised in the trunk, where a DV ectodermal interface is produced by confrontation of dorsal and ventral identities. By analogy to Drosophila imaginal disc development, this interface has been proposed to determine and position an ectodermal organising centre, the Apical Ectodermal Ridge (AER), controlling limb bud outgrowth. Recent fate mapping studies⁽¹⁾ and studies of genes regulating DV limb polarity^(2-6), AER formation^(7,8) and differentiation⁽⁹⁾ suggest, however, that DV patterning and AER induction, though coordinately regulated during limb bud outgrowth, may early on be more dissociated than expected.     

Organization and differentiation of the segmentary plate in common toad embryo Bufo bufo L., following heat shock (author's transl)

A heat shock greatly disturbs the shaping and the growth of embryos with 4 or 5 somites. One part of a segmentary plate with a size including four control somites stays inorganized, whereas posterior segmentation gets on normally. The differentiation of the unsegmented somitic block (fusing myoblasts, myogenesis) is the same as the one of the previous somites; the dermatome and the sclerotome issued from the unsegmented block are not cut up. Ultrastructural observations of treated segmentary plates show that the somitic block undergoes important changes. The non-segmented somitic mesoderm of the control animals obtains a loose aspect; its constitutive cells separated by lacunae are brought into contact by the means of filopods and junctions. 18 hours after the heat shock, the cells of the unsegmented block keep close to each other and are tightened together by many "pseudo-desmosomes", the intercellular spaces are reduced to small meatus. The nuclei of the somitic cells are clear and provided with a restricted amount of chromatine spots; dark granular nuclear like chromatin material is thrown out of the nucleus. In embryos fixed 40 hours after heat shock, several large plurinucleated cells, are formed in the non-segmented somitic block. Inside these plurinucleated cells, there is synthesis of myofilaments and differentiation of myofibrilles. Numerous intercellular lacunae are formed, some yet apparent "pseudo-desmosomes" form "pinchings" between the membranes of muscular cells and therefore confine series of intercellular lacunae.     

Inductive signals from the somatopleure mediated by bone morphogenetic proteins are essential for the formation of the sternal component of avian ribs

The posterior five pairs of avian ribs are composed of vertebral and sternal components, both derived from the somitic mesoderm. For the patterning of the rib cartilage, inductive signals from neighboring tissues on the somitic mesoderm have been suggested to play critical roles. The notochord and surface ectoderm overlying the somitic mesoderm are essentially required for the development of proximal and distal regions of the ribs, respectively. Involvement of the somatopleure in rib development has already been suggested but is less understood than those of the notochord and surface ectoderm. In this study, we reinvestigated the role of the somatopleure during rib development. We first identified the chicken homologue of the mouse Mesenchymal forkhead-1 (cMfh-1) gene based on sequence similarities. cMfh-1 was observed to be expressed in the nonaxial mesoderm, including the somitic mesoderm, and, subsequently, in cartilage forming the ribs, vertebrae, and appendicular skeletal system. In the interlimb region, corresponding to somites 21-25 (or 26), cMfh-1-positive somitic mesoderm was seen penetrating the somatopleure of E4 embryos, and cMfh-1 was used as a molecular marker demarcating prospective rib cartilage. A series of experiments affecting the penetration of the somitic mesoderm into the somatopleure was performed in the present study, resulting in defects in sternal rib formation. The inductive signals emanating from the somatopleure mediated by BMP family proteins were observed to be essentially involved in the ingrowth of the somitic mesoderm. BMP4 alone, however, could not completely replace inductive signals from the somatopleure, suggesting the involvement of additional signals for rib formation.