A histochemical analysis of polyanionic compounds found in the extracellular matrix encountered by migrating cephalic neural crest cells

Neural crest cells destined to form craniofacial primordia initially are “seeded” into and subsequently migrate through the extracellular matrix (ECM) of a cell free space (CFS) between the surface ectoderm and the underlying mesoderm. Utilizing histochemical procedures for polyanionic compounds, we have demonstrated that both sulfated and nonsulfated glycosaminoglycans (GAG) are present in the CFS of the cephalic region of the chick embryo and that their distribution and structural organization vary with the passage of neural crest or mesodermally derived (MD) mesenchymal cells through it. In stages 7 and 8 embryos a predominance of fine filamentous strands composed primarily of nonsulfated, carboxyl-rich GAG is seen spanning intercellular spaces between adjacent tissues and MD mesenchymal cells. In older embryos (stages 9 and 10) much of the filamentous material is replaced by coarse fibrillar strands or amorphous material which coats the surfaces of MD mesenchymal and neural crest cells as they invade the CFS. Using enzymatic digestions (Streptomyces and testicular hyaluronidase) and the critical electrolyte concentration procedure, data suggest that the fine filamentous matrix onto which the neural crest cells migrate consists mainly of hyaluronate with lesser amounts of chondroitin and some sulfated GAG present. The coarse fibrillar matrix that appears after passage of either neural crest or MD mesenchymal cells through the original CFS contains strongly sulfated polyanionic material, predominantly chondroitin sulfates A, C. Since GAG is located ubiquitously within the ECM of embryos at various stages, the role of GAG, if any, in the transfer of developmental information may be of a general nature (ie, stimulus of motility) rather than of specific morphogenetic cues (for specific differentiation into craniofacial primordia).     

The distribution and spatial organization of the extracellular matrix encountered by mesencephalic neural crest cells

Cephalic neural crest (NC) cells enter a cell-free space (CFS) that contains an abundant extracellular matrix (ECM). Numerous in vitro investigations have shown that extracellular matrices can influence cellular activities including NC cell migration. However, little is known about the actual ECM composition of the CFS in vivo, how the components are distributed, or the nature of NC cell interactions with the CFS matrix. Using ultrastructural, autoradiographic, and histochemical techniques we analyzed the composition and spatial organization of the ECM found in the CFS and its interaction with mesencephalic NC cells. We have found that a specific distribution of glycoproteins and sulfated polyanions existed within the CFS prior to the translocation of NC cells and that this ECM was modified in areas occupied by NC. The interaction between the ECM components and the NC cells was not the same for all NC cells in the population. Subpopulations of the NC cell sheet became associated with ECM of the ectoderm (basal lamina) while other NC cells became associated with the ECM of the CFS. Trailing NC cells (NC cells that emerge after the initial appearance of NC cells) encountered a modified ECM due to extensive matrix modifications by the passage of the initial NC cell population.     

Abnormal neural crest cell migration after the in vivo knockdown of tenascin-C expression with morpholino antisense oligonucleotides.

A key feature of vertebrate development is the formation of the neural crest. In the trunk, neural crest cells delaminate from the neural tube shortly after the fusion of the neural folds and migrate ventrally along specific pathways to form the neurons and glia of the peripheral nervous system. As neural crest cells leave the neural tube during the initial stages of their migration, they express the extracellular matrix glycoprotein tenascin-C, which is also found in the stroma of many tumors. We have studied the possible role for tenascin-C during neural crest morphogenesis in vivo by microinjecting tenascin-C morpholino antisense oligonucleotides into the lumen of the avian neural tube in ovo and electroporating the morpholino antisense oligonucleotides into the precursors of the neural crest. After 24 hr, tenascin-C immunostaining is reduced around the dorsal neural tube in the experimental microinjected embryos (12 of 13) but not in embryos microinjected with control morpholino antisense oligonucleotides (n = 3) or subjected to electroporation only (n = 2). In each of the 12 tenascin-C knockdown embryos neural crest cells are seen ectopically in the lumen of the neural tube and in the neuroepithelium; cells that do leave the neural tube after the microinjection fail to disperse laterally from the surface of the neural tube into the somites. The observation that neural crest cells must express tenascin-C to migrate normally is consistent with a role for this glycoprotein in contributing to the invasive behavior of neural crest cells.     

Mapping proteolytic cancer cell-extracellular matrix interfaces

For cancer progression and metastatic dissemination, cancer cells migrate and penetrate through extracellular tissues. Cancer invasion is frequently facilitated by proteolytic processing of components of the extracellular matrix (ECM). The cellular regions mediating proteolysis are diverse and depend upon the physical structure, composition, and dimensionality of the ECM contacted by the cell surface. Cancer cells migrating across 2D substrate contain proteolytic structures such as lamellipodia, invadopodia, and the trailing edge. Likewise, invasive mesenchymal migration through 3D fibrillar ECM, as monitored for HT1080 fibrosarcoma and MDA-MB-231 breast carcinoma cells by submicron-resolved imaging, is mediated by several types of proteolytic structures rich in filamentous actin, ss1 integrin, and MT1-MMP with distinct location and function. These comprise (i) anterior pseudopod bifurcataions and the nucleus corresponding to zones of local cell compression by constraining collagen fibers, (ii) lateral small spikes that protrude into the ECM and cause small spot-like proteolytic foci, and (iii) a strongly proteolytic trailing edge sliding along reorganized ECM fibers. Through their combined action these proteolytic surface structures cleave, remove, and realign ECM barriers, support rear end retraction, generate tube-like matrix defects and laterally widen existing tracks during 3D tissue invasion.     

PNA-positive glycoconjugates are negatively correlated with the access of neural crest cells to the gut in chicken embryos

Neural crest cells give rise to many derivatives, including the neurons and glia of the peripheral nervous system, adrenomedulary cells, and melanocytes, and migrate through precise pathways that differ according to their axial level and/or state of specification. The migratory routes taken by neural crest cells are reported to be regulated by extracellular matrix molecules. We examined the possible influence of glycoconjugates on the establishment of barriers to neural crest access to ventral regions leading to the gut, by labeling stage-16-28 white Leghorn (WL) and Silky (SK) embryos with peanut agglutinin (PNA) at vagal, thoracic, and sacral levels. We observed a transitory expression of glycoconjugates that correlate with a barrier to the entrance of neural crest cells into the gut at the thoracic level, which is not present at vagal and sacral levels. In later stages, neural crest cells of melanocytic lineage were observed entering the gut in embryos of the SK chicken, a mutant with an altered pattern of pigmentation. The ventral regions occupied by melanoblasts in SK embryos were free of PNA labeling, while in WL embryos, in which PNA-positive molecules are strongly expressed, melanoblasts were restricted to peripheral regions. We suggest that PNA-binding glycoconjugates are good molecular marker for barriers that control the access of neural crest cells to the gut.     

Development of the embryonic chick otic placode. II. Electron microscopic analysis

The otic placode takes its origin from surface ectoderm. Prior to the arrival of neural crest cells, surface epithelial cells adjacent to the neural folds are squamous in shape and synthesize primarily interstitial bodies. However, by 26 hours of development, neural crest cells, using the undersurface of the epithelium as a substratum, migrate away from the neural tube. Cells of surface epithelium above the neural crest cells assume a columnar shape, and the amount of intercellular space between adjacent epithelial cells is consequently reduced. Placode cells show extensive interdigitation apically as they pseudostratify and invaginate, while it appears that many of the basal cells contribute components to the underlying extracellular matrix. This extracellular matrix interface between surface epithelium and neural crest cells is distinctly fibrillar and less granular than that found between ordinary head ectoderm and primary mesenchymal cells. Just prior to complete invagination as an otocyst, otic placode cells in a region near the ventrolateral wall of the hindbrain extend cell processes through discontinuities in the basal lamina and leave the otocyst. These are likely to be the cells which contribute to the formation of the acoustico-facialis ganglion. These observations support the hypothesis that the development of the otic placode is the result of a tissue interaction between surface epithelium and neural crest cells.     

Comparative analysis of neural crest cell death, migration, and function during vertebrate embryogenesis.

Cranial neural crest cells are a multipotent, migratory population that generates most of the cartilage, bone, connective tissue and peripheral nervous system in the vertebrate head. Proper neural crest cell patterning is essential for normal craniofacial morphogenesis and is highly conserved among vertebrates. Neural crest cell patterning is intimately connected to the early segmentation of the neural tube, such that neural crest cells migrate in discrete segregated streams. Recent advances in live embryo imaging have begun to reveal the complex behaviour of neural crest cells which involve intricate cell-cell and cell-environment interactions. Despite the overall similarity in neural crest cell migration between distinct vertebrates species there are important mechanistic differences. Apoptosis for example, is important for neural crest cell patterning in chick embryos but not in mouse, frog or fish embryos. In this paper we highlight the potential evolutionary significance of such interspecies differences in jaw development and evolution. Developmental Dynamics 229:14-29, 2004.     

Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells

Neural crest stem cells (NCSCs), a population of multipotent cells that migrate extensively and give rise to diverse derivatives, including peripheral and enteric neurons and glia, craniofacial cartilage and bone, melanocytes and smooth muscle, have great potential for regenerative medicine. Non-human primates provide optimal models for the development of stem cell therapies. Here, we describe the first derivation of NCSCs from cynomolgus monkey embryonic stem cells (CmESCs) at the neural rosette stage. CmESC-derived neurospheres replated on polyornithine/laminin-coated dishes migrated onto the substrate and showed characteristic expression of NCSC markers, including Sox10, AP2α, Slug, Nestin, p75, and HNK1. CmNCSCs were capable of propagating in an undifferentiated state in vitro as adherent or suspension cultures, and could be subsequently induced to differentiate towards peripheral nervous system lineages (peripheral sympathetic neurons, sensory neurons, and Schwann cells) and mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). CmNCSCs transplanted into developing chick embryos or fetal brains of cynomolgus macaques survived, migrated, and differentiated into progeny consistent with a neural crest identity. Our studies demonstrate that CmNCSCs offer a new tool for investigating neural crest development and neural crest-associated human disease and suggest that this non-human primate model may facilitate tissue engineering and regenerative medicine efforts.     

Changes in the extracellular matrix and glycosaminoglycan synthesis during the initiation of regeneration in adult newt forelimbs

The extracellular matrix (ECM) of the distal tissues in a newt limb stump is completely reorganized in the 2-3-week period following amputation. In view of numerous in vitro studies showing that extracellular material influences cellular migration and proliferation, it is likely that the changes in the limb's ECM are important activities in the process leading to regeneration of such limbs. Using biochemical, autoradiographic, and histochemical techniques we studied temporal and spatial differences in the synthesis of glycosaminoglycans (GAGs) during the early, nerve-dependent phase of limb regeneration. Hyaluronic acid synthesis began with the onset of tissue dedifferentiation, became maximal within 1 weeks, and continued throughout the period of active cell proliferation. Chondroitin sulfate synthesis began somewhat later, increased steadily, and reached very high levels during chondrogenesis. During the first 10 days after amputation, distributions of sulfated and nonsulfated GAGs were both uniform throughout dedifferentiating tissues, except for a heavier localization near the bone. Since nerves are necessary to promote the regenerative process, we examined the neural influence on synthesis and accumulation of extracellular GAGs. Denervation decreased GAG production in all parts of the limb stump by approximately 50%. Newt dorsal root ganglia and brain-derived fibroblast growth factor each produced twofold stimulation of GAG synthesis in cultured 7-day regenerates. The latter effect was primarily on synthesis of hyaluronic acid. The results indicate that the trophic action of nerves on amphibian limb regeneration includes a positive influence on synthesis and extracellular accumulation of GAGs. Since the ECM exerts a major influence on cellular proliferation and migration, the effect of nerves on GAG metabolism may have considerable importance for growth and development of the early regenerate.     

Impaired development of the thymic primordium after neural crest ablation

Impaired thymic development as a result of ablation of neural crest has been observed in embryos late in development. The present study was initiated to determine what changes are effected early in thymic development by neural crest ablation. The epithelial primordia of the thymus were studied in chick embryos on the sixth day of incubation. Embryos with neural crest ablations were compared with sham-operated and untreated controls. Neural crest ablation inhibited formation of epithelial thymic primordia. Primordia in experimental embryos were fewer in number and were smaller than in shams and untreated controls. When primordia from shams and controls were transplanted to the chorioallantoic membrane of chick hosts, they were able to develop into organs with the typical features of embryonic thymus. Similar transplantation from neural crest-ablated animals, on the other hand, led to small, predominantly epithelial structures with meager lymphoid development. These findings are consistent with the hypothesis that mesenchyme derived from cranial neural crest is critical in initiating and sustaining the development from pharyngeal pouches of epithelial structures competent to attract and support the proliferation and differentiation of lymphoid stem cells.     

The migration of myogenic cells from the somites into the leg region of avian embryos

The migration of myogenic stem cells into the leg anlagen of chick embryos between stages 16--20 of Hamburger and Hamilton was examined. SEM and TEM studies reveal that cell migration starts at stage 16 from the just-formed somites 26-28. The migrating myogenic cells are elongated and oriented in a medio-lateral direction. The leading ends branch into filopodia which contact a fibrillar network. At first, single cells migrate; later on the cells leaving the ventro-lateral edge of the dermatome migrate in strands and have specialized contacts between them. After reaction with ruthenium red and concanavalin A the migrating cells show a thick surface coat to which ruthenium red-positive particles are attached. The surface coat may be important in the interactions among the migrating cells as well as between the cells and the substrate. The migration of myogenic stem cells was found to take place in a matrix of collagenous fibrils and ruthenium red-positive particles, probably containing glycosaminoglycans. At the onset of migration the fibrillar network exhibits a preferred medio-lateral orientation. Therefore, it may be concluded that this alignment of the fibrils influences the direction of cell migration.     

Structural development of endocardial cushions

Development of chick and rat endocardial cushions (cardiac mesenchyme) was studied histologically (using Nomarski differential interference optics on living and unfixed tissue), ultrastructurally (scanning and transmission electron microscopy), cytochemically (using acidified dialyzed iron as a visual probe for polyanionic material) and autoradiographically (using ³⁵S) to elucidate the origin of the mesenchyme, the morphologic sequences leading to cushion formation and secretion of sulfated glycosaminoglycans, if any, by migrating mesenchymal cells. Cushion formation was similar for both species. Mesenchymal cells appeared initially, in 16- to 18-somite embryos, beneath the endothelium (which lacked a basal lamina) of the future atrioventricular canal and outflow tract. The cytoplasm of cushion mesenchymal cells was structurally similar to the endothelium; probably these cells arose by proliferation of the endothelium. Mitotic figures among the “seeded” cells were also numerous. Cushion cells were initially attached to the endothelium by desmosomes but acquired motile apparatus (pseudopodia and filopodia containing microtubules and microfilamentous bundles). Serial sectioning of successively-aged embryos (20–44 somites) indicated a centrifugal migratory direction. Interaction of the cell processes with extracellular matrix suggested that the latter was used as a migratory substrate. Contact of the advancing wedge of cushion cells with the myocardium produced no alteration in cell structure or mitotic activity. Localization of hyaluronidase-sensitive, dialyzed iron (DI) precipitates in 250-nm Golgi vacuoles and hyaluronidase-sensitive ³⁵S-engendered silver grains over cushion cells indicated that this tissue contributed sulfated macromolecules to the matrix. Localization of hyaluronidase-labile, DI material in coated, endocytic like vesicles and caveolae also suggested potential modification or conditioning of the matrix by migrating mesenchymal cells. Altogether the study established loci in developing cushions where disruption of the developmental sequence could engender valvular or septal defects.     

Bmp2 is required for migration but not for induction of neural crest cells in the mouse.

Bone morphogenetic protein (BMP) signaling is essential for neural crest development in several vertebrates. Genetic experiments in the mouse have shown that Bmp2 is essential for the genesis of migratory neural crest cells. Using several markers and a transgenic reporter approach, we now show that neural crest cells are induced in Bmp2 null mutant embryos, but that these cells fail to migrate out of the neural tube. The absence of migratory neural crest cells in these mutants is not due to their elimination by cell death. The neuroectoderm of Bmp2-/- embryos fail to close and create abnormal folds both along the anterior-posterior and medio-lateral axes, which are associated with an apparent medio-lateral expansion of the neural tube. Finally, our data suggest that the molecular cascade downstream of BMP signaling in early neural crest development may be different in mouse and avian embryos.     

Origin of spinal cord meninges, sheaths of peripheral nerves, and cutaneous receptors including Merkel cells

Summary The origin of cells covering the nervous system and the cutaneous receptors was studied using the quail-chick marking technique and light and electron microscopy. In the first experimental series the brachial neural tube of the quail was grafted in place of a corresponding neural tube segment of the chick embryo at HH-stages 10 to 14. In the second series the leg bud of quail embryos at HH-stages 18–20 was grafted in place of the leg bud of the chick embryos of the same stages and vice versa. It was found that all meningeal layers of the spinal cord, the perineurium and the endoneurium of peripheral nerves, as well as the capsular and inner space cells of Herbst sensory corpuscles, develop from the local mesenchymal cells. Schwann cells and cells of the inner core of sensory corpuscles are of neural crest origin. The precursors of Merkel cells migrate similarly to the Schwann cells into the limb bud where they later differentiate. This means that in addition to the Schwann cells and the melanocytes a further neural crest-derived subpopulation of cells enters the limb.     

Inactivation of TGFbeta signaling in neural crest stem cells leads to multiple defects reminiscent of DiGeorge syndrome

Specific inactivation of TGFbeta signaling in neural crest stem cells (NCSCs) results in cardiovascular defects and thymic, parathyroid, and craniofacial anomalies. All these malformations characterize DiGeorge syndrome, the most common microdeletion syndrome in humans. Consistent with a role of TGFbeta in promoting non-neural lineages in NCSCs, mutant neural crest cells migrate into the pharyngeal apparatus but are unable to acquire non-neural cell fates. Moreover, in neural crest cells, TGFbeta signaling is both sufficient and required for phosphorylation of CrkL, a signal adaptor protein implicated in the development of DiGeorge syndrome. Thus, TGFbeta signal modulation in neural crest differentiation might play a crucial role in the etiology of DiGeorge syndrome.     

Comparative embryology of the carotid body

Vertebrate carotid bodies and related structures (branchial arch oxygen chemoreceptors in fishes, carotid labyrinth in amphibians, chemoreceptors in the wall of the common carotid and its branches in birds) develop in embryos when neural crest cells, blood vessels, and nerve fibers from sympathetic and cranial nerve ganglia invade mesenchymal primordia in the wall of the 3rd branchial arch. This review focuses on literature published since the 1970s investigating similarities and differences in the embryological development of 3rd arch oxygen chemoreceptors, especially between mammals and birds, but also considering reptiles, amphibians and fishes.     

Injectable extracellular matrix derived hydrogel provides a platform for enhanced retention and delivery of a heparin-binding growth factor

Injectable hydrogels derived from the extracellular matrix (ECM) of decellularized tissues have recently emerged as scaffolds for tissue-engineering applications. Here, we introduce the potential for using a decellularized ECM-derived hydrogel for the improved delivery of heparin-binding growth factors. Immobilization of growth factors on a scaffold has been shown to increase their stability and activity. This can be done via chemical crosslinking, covalent bonding, or by incorporating natural or synthetic growth factor-binding domains similar to those found in vivo in sulfated glycosaminoglycans (GAGs). Many decellularized ECM-derived hydrogels retain native sulfated GAGs, and these materials may therefore provide an excellent delivery platform for heparin-binding growth factors. In this study, the sulfated GAG content of an ECM hydrogel derived from decellularized pericardial ECM was confirmed by Fourier transform infrared spectroscopy and its ability to bind basic fibroblast growth factor (bFGF) was established. Delivery in the pericardial matrix hydrogel increased retention of bFGF both in vitro and in vivo in ischemic myocardium compared to delivery in collagen. In a rodent infarct model, intramyocardial injection of bFGF in pericardial matrix enhanced neovascularization by approximately 112% compared to delivery in collagen. Importantly, the newly formed vasculature was anastomosed with existing vasculature. Thus, the sulfated GAG content of the decellularized ECM hydrogel provides a platform for incorporation of heparin-binding growth factors for prolonged retention and delivery.     

Neural crest ablation does not alter pulmonary vein development in the chick embryo

Cranial neural crest, which extends from the mid-diencephalon to somite five, plays an integral role in development of pharyngeal arch derivatives and supplies mesenchyme to the aortic arch arteries. Neural crest cells in pharyngeal arches three, four, and six migrate to the heart and are involved in aorticopulmonary and conotruncal septation. Ablation of the "cardiac" neural crest cells in chick embryos results in a variety of outflow tract anomalies, including persistent truncus arteriosus. Although other studies have shown the importance of the neural crest in the development of the cardiac outflow tract, the role of neural crest in venous development has not been established. This investigation evaluates the effect of cardiac neural crest ablation on the morphological development of the pulmonary vein. The presence of the pulmonary vein was confirmed initially at early stage 15 using histological sections and computer reconstructions of serially sectioned, normal embryos. India ink injections demonstrated a complete, patent pulmonary circuit at stage 18. Cardiac neural crest was ablated at stages 8-10. Operated, sham-operated, and control embryos were sacrificed at incubation day 11, and acrylic plastic casts prepared of the intravascular compartment. In experimental embryos with persistent truncus arteriosus, there were no morphological differences in the pulmonary veins, compared with shams and controls. These data indicate that the lesions of the cardiac neural crest have little morphological impact on pulmonary vein development. It is concluded that alterations in the cardiac neural crest are not involved in venous anomalies such as cor triatriatum and total or partial anomalous pulmonary venous return.     

An in vitro model for characterizing the post-migratory cranial neural crest cells of the first branchial arch.

The cranial neural crest (CNC) is a transient cell population that originates at the crest of the neural fold and gives rise to multiple cell types during craniofacial development. Traditionally, researchers have used tissue explants, such as the neural tube, to obtain primary neural crest cells for their studies. However, this approach has inevitably resulted in simultaneous isolation of neural and non-neural crest cells as both of these cells migrate away from tissue explants. Using the Wnt1-Cre/R26R mouse model, we have obtained a pure population of neural crest cells and established a primary CNC cell culture system in which the cell culture medium best supports the proliferation of E10.5 first branchial arch CNC cells and maintains these cells in their undifferentiated state. Differentiation of CNC cells can be initiated by switching to a differentiation medium. In this model, cultured CNC cells can give rise to neurons, glial cells, osteoblasts, and other cell types, faithfully mimicking the differentiation process of the post-migratory CNC cells in vivo. Taken together, our study shows that the Wnt1-Cre/R26R mouse first branchial arch provides an excellent model for obtaining post-migratory neural crest cells free of any mesodermal contaminants. The cultured neural crest cells are under sustained proliferative, undifferentiated, or lineage-enhanced conditions, hence, serving as a tool for the investigation of the regulatory mechanism of CNC cell fate determination in normal and abnormal craniofacial development.