Drew M. Noden

The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues

The morphology of skeletal tissues formed in each of the branchial arches of higher vertebrates is unique. In addition to these structures, which are derived from the neural crest, the crest-derived connective tissues and mesodermal muscles also form different patterns in each of the branchial arches. The objective of this study was to examine how these patterns arise during avian embryonic development. Presumptive second or third arch neural crest cells were excised from chick hosts and replaced with presumptive first arch crest cells. Both quail and chick embryos were used as donors; orthotopic crest grafts were performed as controls. Following heterotopic transplantation, the hosts developed several unexpected anomalies. Externally they were characterized by the appearance of ectopic, beak-like projections from the ventrolateral surface of the neck and also by the formation of supernumerary external auditory depressions located immediately caudal to the normal external ear. Internally, the grafted cells migrated in accordance with normal, second arch pathways but then formed a complete, duplicate first arch skeletal system in their new location. Squamosal, quadrate, pterygoid, Meckels, and angular elements were present in most cases. In addition, anomalous first arch-type muscles were found associated with the ectopic skeletal tissues in the second arch. These results indicate that the basis for patterning of branchial arch skeletal and connective tissues resides within the neural crest population prior to its emigration from the neural epithelium, and not within the pharynx or pharyngeal pouches as had previously been suggested. Furthermore, the patterns of myogenesis by mesenchymal populations derived from paraxial mesoderm is dependent upon properties inherent to the neural crest.

Relations and interactions between cranial mesoderm and neural crest populations

The embryonic head is populated by two robust mesenchymal populations, paraxial mesoderm and neural crest cells. Although the developmental histories of each are distinct and separate, they quickly establish intimate relations that are variably important for the histogenesis and morphogenesis of musculoskeletal components of the calvaria, midface and branchial regions. This review will focus first on the genesis and organization within nascent mesodermal and crest populations, emphasizing interactions that probably initiate or augment the establishment of lineages within each. The principal goal is an analysis of the interactions between crest and mesoderm populations, from their first contacts through their concerted movements into peripheral domains, particularly the branchial arches, and continuing to stages at which both the differentiation and the integrated three‐dimensional assembly of vascular, connective and muscular tissues is evident. Current views on unresolved or contentious issues, including the relevance of head somitomeres, the processes by which crest cells change locations and constancy of cell–cell relations at the crest–mesoderm interface, are addressed.

The Differentiation and Morphogenesis of Craniofacial Muscles

Unraveling the complex tissue interactions necessary to generate the structural and functional diversity present among craniofacial muscles is challenging. These muscles initiate their development within a mesenchymal population bounded by the brain, pharyngeal endoderm, surface ectoderm, and neural crest cells. This set of spatial relations, and in particular the segmental properties of these adjacent tissues, are unique to the head. Additionally, the lack of early epithelialization in head mesoderm necessitates strategies for generating discrete myogenic foci that may differ from those operating in the trunk. Molecular data indeed indicate dissimilar methods of regulation, yet transplantation studies suggest that some head and trunk myogenic populations are interchangeable. The first goal of this review is to present key features of these diversities, identifying and comparing tissue and molecular interactions regulating myogenesis in the head and trunk. Our second focus is on the diverse morphogenetic movements exhibited by craniofacial muscles. Precursors of tongue muscles partly mimic migrations of appendicular myoblasts, whereas myoblasts destined to form extraocular muscles condense within paraxial mesoderm, then as large cohorts they cross the mesoderm:neural crest interface en route to periocular regions. Branchial muscle precursors exhibit yet another strategy, establishing contacts with neural crest populations before branchial arch formation and maintaining these relations through subsequent stages of morphogenesis. With many of the prerequisite stepping‐stones in our knowledge of craniofacial myogenesis now in place, discovering the cellular and molecular interactions necessary to initiate and sustain the differentiation and morphogenesis of these neglected craniofacial muscles is now an attainable goal. Developmental Dynamics 235:1194–1218, 2006.

Vertebrate Craniofacial Development: The Relation between Ontogenetic Process and Morphological Outcome; pp. 190–207

Many structures that are present, often transiently, in the head of extant vertebrate embryos appear to be segmentally organized. These include the brain, particularly the hindbrain (e.g., rhombomeres), and adjacent axial structures such as paraxial mesoderm (e.g., somites, somitomeres) and neural crest cells. Also present in the head are additional sets of serially arranged structures that develop in more ventral and lateral locations. Examples of these are epibranchial placodes, aortic arches, and pharyngeal pouches. All these embryonic structures are frequently used both individually and collectively as characters to assist in defining homologies. New cell labeling and identification methods are providing detailed accounts of cell movements and tissue lineages that reveal a range of disparate behaviors not previously appreciated. The well-known migrations of neural crest cells bring all but the neurogenic members of this mesenchymal population form dorsal, axial locations into ventral and rostral locations where they largely surround the pharynx, stomdeum, and prosencephalon. Equally dramatic movements of neural plate cells, myoblasts, angioblasts, and placode-derived cells have recently been documented. These movements may occur in concert with those of other nearby tissues (e.g., branchiomeric myoblasts, neural crest cells, and surface ectoderm) or may be independent (e.g., placodal neuroblasts). Migrating cells may be clustered and follow definable pathways towards their destination (e.g., neural crest cells), or they may be solitary and wander invasively without a prespecified destination (e.g., angioblasts). These extensive morphogenetic movements bring cells into contact with a greater variety of other tissues and matrix environments than has heretofore been recognized. Moreover, because of these rearrangements, the cells present in a particular location, such as a branchial arch, may trace their ancestry to many axial levels, which complicates the analyses of segmental relations. Comparative morphological studies of craniofacial development have recently been augmented by descriptions of the sites and times of expression of many matrix components, growth factors and their receptors, and regulatory genes. Particularly important has been the discovery of a network of genes called the homeobox family. These genes are similar in their sequence and their organization along a chromosome to genes that establish the spatial identity of prospective body parts in drosophila. The combination of cellular and molecular descriptive studies of vertebrate craniofacial development provide exciting opportunities to catalogue patterns of gene expression and morphogenesis during the gastrula, neurula, and early organogenesis stages. Moreover, such data form the basis for proposing and then testing hypotheses about the mechanisms controlling cell movements, tissue formation, and the assembly of functionally integrated sets of structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.

Differentiation of Avian Craniofacial Muscles: I. Patterns of Early Regulatory Gene Expression and Myosin Heavy Chain Synthesis

Myogenic populations of the avian head arise within both epithelial (somitic) and mesenchymal (unsegmented) mesodermal populations. The former, which gives rise to neck, tongue, laryngeal, and diaphragmatic muscles, show many similarities to trunk axial, body wall, and appendicular muscles. However, muscle progenitors originating within unsegmented head mesoderm exhibit several distinct features, including multiple ancestries, the absence of several somite lineage‐determining regulatory gene products, diverse locations relative to neuraxial and pharyngeal tissues, and a prolonged and necessary interaction with neural crest cells. The object of this study has been to characterize the spatial and temporal patterns of early muscle regulatory gene expression and subsequent myosin heavy chain isoform appearance in avian mesenchyme‐derived extraocular and branchial muscles, and compare these with expression patterns in myotome‐derived neck and tongue muscles. Myf5 and myoD transcripts are detected in the dorsomedial (epaxial) region of the occipital somites before stage 12, but are not evident in the ventrolateral domain until stage 14. Within unsegmented head mesoderm, myf5 expression begins at stage 13.5 in the second branchial arch, followed within a few hours in the lateral rectus and first branchial arch myoblasts, then other eye and branchial arch muscles. Expression of myoD is detected initially in the first branchial arch beginning at stage 14.5, followed quickly by its appearance in other arches and eye muscles. Multiple foci of myoblasts expressing these transcripts are evident during the early stages of myogenesis in the first and third branchial arches and the lateral rectus‐pyramidalis/quadratus complex, suggesting an early patterned segregation of muscle precursors within head mesoderm. Myf5‐positive myoblasts forming the hypoglossal cord emerge from the lateral borders of somites 4 and 5 by stage 15 and move ventrally as a cohort. Myosin heavy chain (MyHC) is first immunologically detectable in several eye and branchial arch myofibers between stages 21 and 22, although many tongue and laryngeal muscles do not initiate myosin production until stage 24 or later. Detectable synthesis of the MyHC‐S3 isoform, which characterizes myofibers as having “slow” contraction properties, occurs within 1–2 stages of the onset of MyHC synthesis in most head muscles, with tongue and laryngeal muscles being substantially delayed. Such a prolonged, 2‐ to 3‐day period of regulatory gene expression preceding the onset of myosin production contrasts with the interval seen in muscles developing in axial (approximately 18 hr) and wing (approximately 1–1.5 days) locations, and is unique to head muscles. This finding suggests that ongoing interactions between head myoblasts and their surroundings, most likely neural crest cells, delay myoblast withdrawal from the mitotic pool. These descriptions define a spatiotemporal pattern of muscle regulatory gene and myosin heavy chain expression unique to head muscles. This pattern is independent of origin (somitic vs. unsegmented paraxial vs. prechordal mesoderm), position (extraocular vs. branchial vs. subpharyngeal), and fiber type (fast vs. slow) and is shared among all muscles whose precursors interact with cephalic neural crest populations. Dev Dyn 1999;216:96–112. ©1999 Wiley‐Liss, Inc.

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 β‐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. Developmental Dynamics 235:1310–1325, 2006.

Origins and assembly of avian embryonic blood vessels.

Two processes by which embryonic blood vessels develop are well-known: angiogenesis (growth by budding and branching of existing vessels) and local formation of endothelial vesicles that coalesce with elongating vessels. The former process appears to be more prevalent, with the latter restricted to vessels that form near the endoderm-mesoderm interface. The contributions of endothelial cells formed by each of these processes to specific blood vessels has not been defined, however, nor have the origins of precursors (angioblasts) of intraembryonic endothelial populations been established. To identify the origins of endothelial cells, precursor populations from quail embryos were transplanted into chick embryos. Antibodies that recognize quail endothelial cells were applied to sections from chimeric embryos fixed 2-5 days after surgery. These experiments reveal that all intraembryonic mesodermal tissues, except the notochord and prechordal plate, contain angiogenic precursors. Many angioblasts emigrate from the grafted tissue, invading surrounding mesenchyme and contributing to the formation of arteries, veins, and capillaries in a wide area. The invasive behavior of these angioblasts is unlike that of any other embryonic mesenchymal cell type and represents a third process operating during embryonic blood vessel formation. Transplanted angioblasts, even those excised from quail trunk regions, form normal craniofacial vascular channels, including the cardiac outflow tract. These results demonstrate that the control over blood vessel assembly resides within the connective tissue-forming mesenchyme of the embryo, not within endothelial precursors.

A new heart for a new head in vertebrate cardiopharyngeal evolution

It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts — both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates.

Cell origins and tissue boundaries during outflow tract development

Morphogenesis of the cardiac outflow tract and aortic sac regions requires the progressive immigration and integrated differentiation of cells having very divergent embryonic histories. Mesodermal cells originating both within and beside the developing head contribute to endocardium and myocardium. These cells, together with later arriving neural crest cells, participate in the formation of the aorticopulmonary septum, truncal cushions, and semilunar valves, although there is uncertainty regarding the precise contributions of each. In addition, precursors of the enveloping epicardium and coronary arteries move into the outflow tract. Defining the spatial and temporal contributions of these disparate populations and the boundaries between them as the outflow tract shifts caudally is an essential prerequisite to understanding normal heart morphogenesis as well as the etiology of outflow tract dysmorphologies.