On the origin of cells determined to form skeletal muscle in avian embryos

Summary Pieces of quail embryos from various developmental stages ranging from unincubated blastoderms (before the appearance of a primitive streak) to embryos having formed somites were grafted to the wing buds or into the coelomic cavity of chicken embryos. The grafts, which can be identified on a cellular level by virtue of the prominent nucleolus-associated chromatin, present in the quail and absent in the chicken, were screened after suitable periods of reincubation for the presence or absence of skeletal myotubes containing quail nuclei. Grafts having contributed to such skeletal myotubes were considered as having contained determined myogenic cells at the time of the grafting procedure. Determined myogenic cell appeared first in the primitive streak and in the mesodermal cells formed by the invagination (gastrulation) of epiblastic cells through the primitive streak. This is true for both the head process and the paraxial mesoderm. Epiblastic cells never gave rise to skeletal myotubes. Therefore it can be said, that the onset of myogenic determination coincides with gastrulation. It remains, however, to be established, whether these two events are causally related to one another.     

Derivation of smooth muscle cells with neural crest origin from human induced pluripotent stem cells

The heterogeneity of vascular smooth muscle cells (SMCs) is related to their different developmental origins such as the neural crest and mesoderm. Derivation of SMCs from different origins will provide valuable in vitro models for the investigation of vascular development and diseases. From the perspective of regenerative medicine and tissue engineering, an expandable cell source of SMCs is required for the construction of tissue-engineered blood vessels. In this study, we developed a robust protocol to derive neural crest stem cells (NCSCs) from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). NCSCs derived from ESCs and iPSCs were expandable with similar cell doubling times. NCSCs were capable of differentiating into neural and mesenchymal lineages. TGF-β1 induced the expression of SMC markers calponin-1, SM22α, and smooth muscle myosin heavy chain and resulted in the assembly of smooth muscle α-actin, calponin-1, and SM22α into stress fibers. This work provides a basis for using iPSCs to study SMC biology and deriving vascular cells for tissue engineering.     

Immunophenotypic characterization of enteric neural crest cells in the developing avian colorectum

A chick–chick intraspecies chimera was created by removing the neural tube adjacent to somites 2–6 from a normal chick embryo at E1.5 and replacing it with equivalent tissue from an age‐matched chick‐GFP transgenic embryo. At E10, the colorectum was removed, sectioned, and stained with HNK‐1 antibody (red) to detect neural crest‐derived cells, and with DAPI (blue) to label nuclei. Vagal neural crest‐derived cells are HNK‐1+/GFP+, while sacral neural crest derived‐cells, which comprise the nerve of Remak, are HNK‐1+/GFP?. From Nagy et al., Developmental Dynamics 241:842–851, 2012. © 2012 Wiley Periodicals, Inc.     

Immunophenotypic characterization of enteric neural crest cells in the developing avian colorectum

Background: The enteric nervous system (ENS) develops from neural crest‐derived cells that migrate along the intestine to form two plexuses of neurons and glia. While the major features of ENS development are conserved across species, minor differences exist, especially in the colorectum. Given the embryologic and disease‐related importance of the distal ENS, the aim of this study was to characterize the migration and differentiation of enteric neural crest‐derived cells (ENCCs) in the colorectum of avian embryos. Results: Using normal chick embryos and vagal neural tube transplants from green fluorescent protein (GFP) ‐transgenic chick embryos, we find ENCCs entering the colon at embryonic day (E) 6.5, with colonization complete by E8. Undifferentiated ENCCs at the wavefront express HNK‐1, N‐cadherin, Sox10, p75, and L1CAM. By E7, differentiation begins in the proximal colon, with L1CAM and Sox10 becoming restricted to neuronal and glial lineages, respectively. By E8, multiple markers of differentiation are expressed along the entire colorectum. Conclusions: Our results establish the pattern of ENCC migration and differentiation in the chick colorectum, demonstrate the conservation of marker expression across species, highlight a range of markers, including neuronal cell adhesion molecules, which label cells at the wavefront, and provide a framework for future studies in avian ENS development. Developmental Dynamics 241:842–851, 2012. © 2012 Wiley Periodicals, Inc.     

Cytoskeletal changes during neurogenesis in cultures of avian neural crest cells

Summary Neural crest cells are motile and mitotic, whereas their neuronal derivatives are terminally post-mitotic and consist of stationary cell body from which processes grow. The present study documents changes in the cytoskeleton that occur during neurogenesis in cultures of avian neural crest cells. The undifferentiated neural crest cells contain dense bundles of actin filaments throughout their cytoplasm, and a splayed array of microtubules attached to the centrosome. In newly differentiating neurons, the actin bundles are disrupted and most of the remaining actin filaments are reorganized into a cortical layer underlying the plasma membrane of the cell body and processes. Microtubules are more abundant in newly-differentiating neurons than in the undifferentiated cells, and individual microtubules can be seen dissociated from the centrosome. Neuron-specific -III tubulin appears in some crest cells prior to cessation of motility and cell division, and expression increases with total microtubule levels during neurogenesis. To investigate how these early cytoskeletal changes might contribute to alterations in morphology during neurogenesis, we have disrupted the cytoskeleton with pharmacologic agents. Microfilament disruption by cytochalasin immediately arrests the movement of neural crest cells and causes them to round-up, but does not significantly change the morphology of the immature neurons. Microtubule depolymerization by nocodazole slows the movement of undifferentiated cells and causes retraction of processes extended by the immature neurons. These results suggest that changes in the actin and microtubule arrays within neural crest cells govern distinct aspects of their morphogenesis into neurons.     

Initial migration and distribution of the cardiac neural crest in the avian embryo: An introduction to the concept of the circumpharyngeal crest

The distribution and migration of the cardiac neural crest was studied in chick embryos from stages 11 to 17 that were immunochemically stained in whole-mount and sectioned specimens with a monoclonal antibody, HNK-1. The following results were obtained: (1) The first phase of the migration in the cardiac crest follows the dorsolateral pathway beneath the ectoderm. (2) In the first site of arrest, the cardiac crest forms a longitudinal mass of neural-crest cells, called in the present study, the circumpharyngeal crest; this mass is located dorsolateral to the dorsal edge of the pericardium (pericardial dorsal horn) where splanchic and somatic lateral mesoderm meet. (3) A distinctive strand of neural-crest cells, called the anterior tract, arises from the mid-otic level and ends in the circumpharyngeal crest. (4) By stage 16, after the degeneration of the first somite, another strand of neural-crest cells, called the posterior tract, appears dorsal to the circumpharyngeal crest. It forms an arch-like pathway along the anterior border of the second somite. (5) The seeding of the pharyngeal ectomesenchyme takes place before the formation of pharyngeal arches in the postotic area, i.e., the crest cells are seeded into the lateral body wall ventrally from the circumpharyngeal crest; and, by the ventralward regression of the pericardial dorsal horn, lateral expansion of pharyngeal pouch, and caudal regression of the pericardium, the crest cell population is pushed away by the pharyngeal pouch. Thus the pharyngeal arch ectomesenchyme is segregated. (6) By stage 14, at the occipital somite level, ventrolateral migration of the neural crest is observed within the anterior half of each somite. Some of these crest cells are continuos with the caudal portion of the circumpharyngeal crest. An early contribution to the enteric neuroblasts is apparent in this area.     

Contributions of placodal and neural crest cells to avian cranial peripheral ganglia

The method of embryonic tissue transplantation was used to confirm the dual origin of avian cranial sensory ganglia, to map precise locations of the anlagen of these sensory neurons, and to identify placodal and neural crest-derived neurons within ganglia. Segments of neural crest or strips of presumptive placodal ectoderm were excised from chick embryos and replaced with homologous tissues from quail embryos, whose cells contain a heterochromatin marker. Placode-derived neurons associated with cranial nerves V, VII, IX, and X are located distal to crest-derived neurons. The generally larger, embryonic placodal neurons are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia. Crest-derived neurons are found in the proximal trigeminal ganglion and in the combined proximal ganglion of cranial nerves IX and X. Neurons in the vestibular and acoustic ganglia of cranial nerve VIII derive from placodal ectoderm with the exception of a few neural crest-derived neurons localized to regions within the vestibular ganglion. Schwann sheath cells and satellite cells associated with all these ganglia originate from neural crest. The ganglionic anlagen are arranged in cranial to caudal sequence from the level of the mesencephalon through the third somite. Presumptive placodal ectoderm for the VIIIth, the Vth, and the VIIth, IXth, and Xth ganglia are located in a medial to lateral fashion during early stages of development reflecting, respectively, the dorsolateral, intermediate, and epibranchial positions of these neurogenic placodes.     

Lumbo-sacral neural crest contributes to the avian enteric nervous system independently of vagal neural crest.

Most of the avian enteric nervous system is derived from the vagal neural crest, but a minority of the neural cells in the hindgut, and to an even lesser extent in the midgut, are of lumbo-sacral crest origin. Since the lumbo-sacral contribution was not detected or deemed negligible in the absence of vagal cells, it had been hypothesised that lumbo-sacral neural crest cells require vagal crest cells to contribute to the enteric nervous system. In contrast, zonal aganglionosis, a rare congenital human bowel disease led to the opposite suggestion, that lumbo-sacral cells could compensate for the absence of vagal cells to construct a complete enteric nervous system. To test these notions, we combined E4 chick midgut and hindgut, isolated prior to arrival of neural precursors, with E1. 7 chick vagal and/or E2.7 quail lumbo-sacral neural tube as crest donors, and grafted these to the chorio-allantoic membrane of E9 chick hosts. Double and triple immuno-labelling for quail cells (QCPNA), neural crest cells (HNK-1), neurons and neurites (neurofilament) and glial cells (GFAP) indicated that vagal crest cells produced neurons and glia in large ganglia throughout the entire intestinal tissues. Lumbo-sacral crest contributed small numbers of neurons and glial cells in the presence or absence of vagal cells, chiefly in colorectum, but not in nearby small intestinal tissue. Thus for production of enteric neural cells the avian lumbo-sacral neural crest neither requires the vagal neural crest, nor significantly compensates for its lack. However, enteric neurogenesis of lumbo-sacral cells requires the hindgut microenvironment, whereas that of vagal cells is not restricted to a particular intestinal region.     

Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells.

Fate maps based on quail-chick grafting of avian cephalic neural crest precursors and paraxial mesoderm cells have identified the majority of derivatives from each population but have not unequivocally resolved the precise locations of and population dynamics at the interface between them. The relation between these two mesenchymal tissues is especially critical for the development of skeletal muscles, because crest cells play an essential role in their differentiation and subsequent spatial organization. It is not known whether myogenic mesoderm and skeletogenic neural crest cells establish permanent relations while en route to their final destinations, or later at the sites where musculoskeletal morphogenesis is completed. We applied beta-galactosidase-encoding, replication-incompetent retroviruses to paraxial mesoderm, to crest progenitors, or at the interface between mesodermal and overlying neural crest as both were en route to branchial or periocular regions in chick embryos. With respect to skeletal structures, the results identify the avian neural crest:mesoderm boundary at the junction of the supraorbital and calvarial regions of the frontal bone, lateral to the hypophyseal foramen, and rostral to laryngeal cartilages. Therefore, in the chick embryo, most of the frontal and the entire parietal bone are of mesodermal, not neural crest, origin. Within paraxial mesoderm, the progenitors of each lineage display different behaviors. Chondrogenic cells are relatively stationary and intramembranous osteogenic cells move only in transverse planes around the brain. Angioblasts migrate invasively in all directions. Extraocular muscle precursors form tightly aggregated masses that en masse cross the crest:mesoderm interface to enter periocular territories, while branchial myogenic lineages shift ventrally coincidental with the movements of corresponding neural crest cells. En route to the branchial arches, myogenic mesoderm cells do not maintain constant, nearest-neighbor relations with adjacent, overlying neural crest cells. Thus, progenitors of individual muscles do not establish stable, permanent relations with their connective tissues until both populations reach the sites of their morphogenesis within branchial arches or orbital regions.     

Migratory patterns and developmental potential of trunk neural crest cells in the axolotl embryo.

Using cell markers and grafting, we examined the timing of migration and developmental potential of trunk neural crest cells in axolotl. No obvious differences in pathway choice were noted for DiI-labeling at different lateral or medial positions of the trunk neural folds in neurulae, which contributed not only to neural crest but also to Rohon-Beard neurons. Labeling wild-type dorsal trunks at pre- and early-migratory stages revealed that individual neural crest cells migrate away from the neural tube along two main routes: first, dorsolaterally between the epidermis and somites and, later, ventromedially between the somites and neural tube/notochord. Dorsolaterally migrating crest primarily forms pigment cells, with those from anterior (but not mid or posterior) trunk neural folds also contributing glia and neurons to the lateral line. White mutants have impaired dorsolateral but normal ventromedial migration. At late migratory stages, most labeled cells move along the ventromedial pathway or into the dorsal fin. Contrasting with other anamniotes, axolotl has a minor neural crest contribution to the dorsal fin, most of which arises from the dermomyotome. Taken together, the results reveal stereotypic migration and differentiation of neural crest cells in axolotl that differ from other vertebrates in timing of entry onto the dorsolateral pathway and extent of contribution to some derivatives.     

Coronary artery development in the chick: origin and deployment of smooth muscle cells, and the effects of neural crest ablation.

Previous studies of coronary artery ontogeny have stressed early development and therefore have dwelt mainly upon the origin of the endothelium of the nascent coronary artery stem. This study has analyzed the ontogeny of the vascular smooth muscle cells (VSMC) in the coronary arteries of the domestic chicken, by establishing the timing and deployment of smooth muscle alpha-actin (SMAA). Anti-SMAA was applied to sections of normal embryos, and to sections of experimental embryos that had undergone surgical ablation of the neural crest over somites 1-3. The results show an orderly symmetrical deployment of SMAA in control coronary arteries. SMAA was expressed significantly earlier in the coronary artery VSMC compared with those of the cardiac outflow vessels; this early expression may indicate a unique responsiveness to induction of the smooth muscle phenotype. The normal orderly development of coronary artery VSMC was dependent upon the presence of the neural crest, and therefore was disrupted in the experimental embryos whose neural crest was ablated.     

The role of epithelial coliagen and proteoglycan in the initiation of osteogenesis by avian neural crest cells

Osteogenesis was inhibited when mandibular processes from 3 1/2-day-old embryos were cultured in BUdR, LACA, α, αβ-Dipyridyl, 4-Methylumbelliferone, and 4-Methylumbelliferyl-β-D-glucoside or β-D-xyloside. Mandibular processes were then cultured in the test substances for 3 days, enzymatically separated into their epithelial and ectomesenchymal components, combined with mandibular components from untreated embryos, and either organ-cultured or grafted to chorioallantoic membranes of host embryos. Osteogenesis was inhibited when treated epithelium, but not when treated ectomesenchyme, was present in the tissue recombinations. Analysis of the known action of these inhibitors indicates that proliferation, hydroxylation of collagen, and synthesis of proteoglycans by epithelial cells are all necessary components of this osteogenic epithelial-ectomesenchymal interaction.     

Sclerotomal origin of smooth muscle cells in the wall of the avian dorsal aorta.

The dorsal aorta is the earliest formed intraembryonic blood vessel. It is composed of an inner lining consisting of endothelial cells and an outer wall consisting of smooth muscle cells (SMCs) and fibrocytes. Aortic SMCs have been suggested to arise from several developmental lineages. Cephalic neural crest provides SMCs of the proximal part of the aorta, and SMCs of the distal part are derived from the paraxial mesoderm. Here, we show by using quail-chick chimerization that in the avian embryo, SMCs in the wall of the dorsal aorta at trunk level arise from the sclerotome. Our findings indicate a two-step process of aortic wall formation. First, non-paraxial mesoderm-derived mural cells accumulate at the floor of the aorta. We refer to these cells as primary SMCs. Second, SMCs from the sclerotome are recruited to the roof and sides of the aorta, eventually replacing the primary SMCs in the aortic floor.