Evolution of a developmental mechanism: Species-specific regulation of the cell cycle and the timing of events during craniofacial osteogenesis

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.     

Neural crest cells provide species-specific patterning information in the developing branchial skeleton

The skeletal elements of the branchial region are made by neural crest cells, following tissue interactions with the pharyngeal endoderm. Previous transplantation experiments have claimed that the cranial neural crest is morphogenetically prespecified in respect of its branchial skeletal derivatives, that is, that information for the number, size, shape, and position of its individual elements is already determined in these cells when they are still in the neural folds. This positional information would somehow be preserved during delamination from the neural tube and migration into the branchial arches, before being read out as a spatial pattern of chondrogenesis and osteogenesis. However, it now appears that signals from the endoderm are able to specify not only the histogenic differentiation state of neural crest cells but also the identity and orientation of the branchial skeletal elements. It is therefore important to ask whether fine details of branchial skeletal pattern such as those that exist between different species are also governed by extrinsic factors, such as the endoderm, or by the neural crest itself. We have grafted neural crest between duck and quail embryos and show that the shape and size of the resulting skeletal elements is donor derived. The ability to form species-specific patterns of craniofacial skeletal tissue thus appears to be an inherent property of the neural crest, expressed as species-specific responses to endodermal signals.     

Developmental origins of species-specific muscle pattern

Vertebrate jaw muscle anatomy is conspicuously diverse but developmental processes that generate such variation remain relatively obscure. To identify mechanisms that produce species-specific jaw muscle pattern we conducted transplant experiments using Japanese quail and White Pekin duck, which exhibit considerably different jaw morphologies in association with their particular modes of feeding. Previous work indicates that cranial muscle formation requires interactions with adjacent skeletal and muscular connective tissues, which arise from neural crest mesenchyme. We transplanted neural crest mesenchyme from quail to duck embryos, to test if quail donor-derived skeletal and muscular connective tissues could confer species-specific identity to duck host jaw muscles. Our results show that duck host jaw muscles acquire quail-like shape and attachment sites due to the presence of quail donor neural crest-derived skeletal and muscular connective tissues. Further, we find that these species-specific transformations are preceded by spatiotemporal changes in expression of genes within skeletal and muscular connective tissues including Sox9, Runx2, Scx, and Tcf4, but not by alterations to histogenic or molecular programs underlying muscle differentiation or specification. Thus, neural crest mesenchyme plays an essential role in generating species-specific jaw muscle pattern and in promoting structural and functional integration of the musculoskeletal system during evolution.     

The genesis of cartilage size and shape during development and evolution

How do cartilaginous elements attain their characteristic size and shape? Two intimately coupled processes underlie the patterned growth of cartilage. The first is histogenesis, which entails the production of cartilage as a discrete tissue; the second is morphogenesis, which pertains to the origins of three-dimensional form. Histogenesis relies on cues that promote the chondrogenic differentiation of mesenchymal cells, whereas morphogenesis requires information that imbues cartilage with stage-specific (e.g. embryonic versus adult), region-specific (e.g. cranial versus appendicular) and species-specific size and shape. Previous experiments indicate that early programmatic events and subsequent signaling interactions enable chondrogenic mesenchyme to undergo histogenesis and morphogenesis, but precise molecular and cellular mechanisms that generate cartilage size and shape remain unclear. In the face and jaws, neural crest-derived mesenchyme clearly plays an important role, given that this embryonic population serves as the source of chondrocytes and of species-specific patterning information. To elucidate mechanisms through which neural crest-derived mesenchyme affects cartilage size and shape, we made chimeras using quail and duck embryos, which differ markedly in their craniofacial anatomy and rates of maturation. Transplanting neural crest cells from quail to duck demonstrates that mesenchyme imparts both stage-specific and species-specific size and shape to cartilage by controlling the timing of preceding and requisite molecular and histogenic events. In particular, we find that mesenchyme regulates FGF signaling and the expression of downstream effectors such as sox9 and col2a1. The capacity of neural crest-derived mesenchyme to orchestrate spatiotemporal programs for chondrogenesis autonomously, and to implement cartilage size and shape across embryonic stages and between species simultaneously, provides a novel mechanism linking ontogeny and phylogeny.     

Mesenchyme-dependent BMP signaling directs the timing of mandibular osteogenesis

To identify molecular and cellular mechanisms that determine when bone forms, and to elucidate the role played by osteogenic mesenchyme, we employed an avian chimeric system that draws upon the divergent embryonic maturation rates of quail and duck. Pre-migratory neural crest mesenchyme destined to form bone in the mandible was transplanted from quail to duck. In resulting chimeras, quail donor mesenchyme established significantly faster molecular and histological programs for osteogenesis within the relatively slower-progressing duck host environment. To understand this phenotype, we assayed for changes in the timing of epithelial-mesenchymal interactions required for bone formation and found that such interactions were accelerated in chimeras. In situ hybridization analyses uncovered donor-dependent changes in the spatiotemporal expression of genes, including the osteo-inductive growth factor Bmp4. Mesenchymal expression of Bmp4 correlated with an ability of quail donor cells to form bone precociously without duck host epithelium, and also relied upon epithelial interactions until mesenchyme could form bone independently. Treating control mandibles with exogenous BMP4 recapitulated the capacity of chimeras to express molecular mediators of osteogenesis prematurely and led to the early differentiation of bone. Inhibiting BMP signaling delayed bone formation in a stage-dependent manner that was accelerated in chimeras. Thus, mandibular mesenchyme dictates when bone forms by temporally regulating its interactions with epithelium and its own expression of Bmp4. Our findings offer a developmental mechanism to explain how neural crest-derived mesenchyme and BMP signaling underlie the evolution of species-specific skeletal morphology.     

Mesenchymal and mechanical mechanisms of secondary cartilage induction

Secondary cartilage occurs at articulations, sutures, and muscle attachments, and facilitates proper kinetic movement of the skeleton. Secondary cartilage requires mechanical stimulation for its induction and maintenance, and accordingly, its evolutionary presence or absence reflects species-specific variation in functional anatomy. Avians illustrate this point well. In conjunction with their distinct adult mode of feeding via levered straining, duck develop a pronounced secondary cartilage at the insertion (i.e., enthesis) of the mandibular adductor muscles on the lower jaw skeleton. An equivalent cartilage is absent in quail, which peck at their food. We hypothesized that species-specific pattern and a concomitant dissimilarity in the local mechanical environment promote secondary chondrogenesis in the mandibular adductor enthesis of duck versus quail. To test our hypothesis we employed two experimental approaches. First, we transplanted neural crest mesenchyme (NCM) from quail into duck, which produced chimeric “quck” with a jaw complex resembling that of quail, including an absence of enthesis secondary cartilage. Second, we modified the mechanical environment in embryonic duck by paralyzing skeletal muscles, and by blocking the ability of NCM to support mechanotransduction through stretch-activated ion channels. Paralysis inhibited secondary cartilage, as evidenced by changes in histology and expression of genes that affect chondrogenesis, including members of the FGF and BMP pathways. Ion channel inhibition did not alter enthesis secondary cartilage but caused bone to form in place of secondary cartilage at articulations. Thus, our study reveals that enthesis secondary cartilage forms through mechanisms that are distinct from those regulating other secondary cartilage. We conclude that by directing the musculoskeletal patterning and integration of the jaw complex, NCM modulates the mechanical forces and molecular signals necessary to control secondary cartilage formation during development and evolution.     

Do Peanut Agglutinin Receptors on Somites Control the Behavior of Neural Cells?

Peanut agglutinin (PNA) receptors are expressed in the caudal halves of sclerotomes in chick embryos after 3 days of incubation (stages 19–20 of Hamburger & Hamilton). The neural crest cells forming dorsal root ganglia (DRG) and motor nerves appear to avoid PNA positive regions and concentrate into rostral halves of sclerotomes. To investigate the role of PNA receptors in gangliogenesis and nerve growth, we examined PNA binding ability in quail sclerotomes and in chick-quail chimeric embryos made by transplanting quail somites to chick embryos, comparing the development of DRG, motor nerves and sclerotomes. PNA did not bind to any part of the somites of 4.5-day quail embryos, although dorsal root ganglia and motor nerves appeared only in the rostral halves of sclerotomes as in chick embryos. Moreover, in spite of no PNA binding ability of the transplanted quail somite in 4.5-day chick-quail chimeric embryos, DRG and motor nerves derived from chick tissues appeared only in the rostral halves of the sclerotomes derived from these somites. Thus, both quail and chick neural crest cells and motor nerves recognized the difference between the rostral and caudal halves of sclerotomes of quail embryos in the absence of PNA binding ability, indicating that PNA binding site on somite cells does not support the selective neural crest migration and nerve growth.     

Patterning the cranial neural crest: hindbrain segmentation and Hox gene plasticity

Understanding the patterning mechanisms that control head development--particularly the neural crest and its contribution to bones, nerves and connective tissue--is an important problem, as craniofacial anomalies account for one-third of all human congenital defects. Classical models for craniofacial patterning argue that the morphogenic program and Hox gene identity of the neural crest is pre-patterned, carrying positional information acquired in the hindbrain to the peripheral nervous system and the branchial arches. Recently, however, plasticity of Hox gene expression has been observed in the hindbrain and cranial neural crest of chick, mouse and zebrafish embryos. Hence, craniofacial development is not dependent on neural crest prepatterning, but is regulated by a more complex integration of cell and tissue interactions.     

The embryonic origins of avian cephalic and cervical muscles and associated connective tissues

The objective of these experiments was to determine the embryonic origins of craniofacial and cervical voluntary muscles and associated connective tissues in the chick. To accomplish this, suspected primordia, including somitomeres 3-7, somites 1-7, and cephalic neural crest primordia have been transplanted from quail into chick embryos. Quail cells can be detected by the presence of a species-specific nuclear marker. The results are summarized as follows: (table; see text) These results indicate that muscles associated with branchial arch skeletal structures are derived from paraxial mesoderm, as are all other voluntary muscles in the vertebrate embryo. Thus, theories of vertebrate ontogeny and phylogeny based in part on proposed unique features of branchiomeric muscles must be critically reappraised. In addition, many of these cephalic muscles are composites of two separate primordia: the myogenic stem cells of mesodermal origin and the supporting and connective tissues derived from the neural crest or lateral plate mesoderm. Defining these embryonic origins is a necessary prerequisite to understanding how the mesenchymal primordia of cephalic muscles and connective tissues interact to form patterned, species-unique musculoskeletal systems.     

Epithelia-mesenchyme interaction plays an essential role in transdifferentiation of retinal pigment epithelium of silver mutant quail: localization of FGF and related molecules and aberrant migration pattern of neural crest cells during eye rudiment formation

Homozygotes of the quail silver mutation, which have plumage color changes, also display a unique phenotype in the eye: during early embryonic development, the retinal pigment epithelium (RPE) spontaneously transdifferentiates into neural retinal tissue. Mitf is considered to be the responsible gene and to function similarly to the mouse microphthalmia mutation, and tissue interaction between RPE and surrounding mesenchymal tissue in organ culture has been shown to be essential for the initiation of the transdifferentiation process in which fibroblast growth factor (FGF) signaling is involved. The immunohistochemical results of the present study show that laminin and heparan sulfate proteoglycan, both acting as cofactors for FGF binding, are localized in the area of transdifferentiation of silver embryos much more abundantly than in wild-type embryos. More intense immunohistochemical staining with FGF-1 antibody, but not with FGF-2 antibody, is also found in the neural retina, RPE, and choroidal tissue of silver embryos than in wild-type embryos. HNK-1 immunohistochemistry revealed that clusters of HNK-1-positive cells (presumptive migrating neural crest cells) are frequently located around the developing eyes and in the posterior region of the silver embryonic eye. Finally, chick-quail chimerical eyes were made by grafting silver quail optic vesicles to chicken host embryos: in most cases, no transdifferentiation occurs in the silver RPE, but in a few cases, transdifferentiation occurs where silver quail cells predominate in the choroid tissue. These observations together with our previous in vitro study indicate that the silver mutation affects not only RPE cells but also cephalic neural crest cells, which migrate to the eye rudiment, and that these crest cells play an essential role in the transdifferentiation of RPE, possibly by modifying the FGF signaling pathway. The precise molecular mechanism involved in RPE-neural crest cell interaction is still unknown, and the quail silver mutation is considered to be a good experimental model for studying the role of neural crest cells in vertebrate eye development.     

Development of smooth and skeletal muscle cells in the iris of the domestic duck,chick and quail

Summary Embryonic development of the avian iris muscle was studied by light and electron microscopy in order to clarify the origin of the iridial skeletal muscle cells. In normal development of the domestic duck, chick, and quail, the muscle bundles appearing in the iris at stage 35 consisted solely of smooth muscle cells. Undifferentiated cells appeared at stage 36, and finally skeletal muscle cells were observed at stage 37. This sequence suggests that stromal mesenchymal cells migrate into the muscle bundles to become skeletal muscle cells.Tissue culture of whole indes removed from duck embryos at stages 30 through 34 produced skeletal muscle cells while culture of isolated iridial epithelia removed at stages 31 and 32 did not. Removal of the midbrain region of duck embryos at stage 10 frequently produced severe disorganization of the eye concomitant with craniofacial deformities typical of a neural crest mesenchymal defect. These severely disorganized eyes were devoid of iridial skeletal muscle cells. These results also suggest mesenchymal origin of iridial skeletal muscle cells.     

Origins and plasticity of neural crest cells and their roles in jaw and craniofacial evolution

The vertebrate head is a complex assemblage of cranial specializations, including the central and peripheral nervous systems, viscero- and neurocranium, musculature and connective tissue. The primary differences that exist between vertebrates and other chordates relate to their craniofacial organization. Therefore, evolution of the head is considered fundamental to the origins of vertebrates (Gans and Northcutt, 1983). The transition from invertebrate to vertebrate chordates was a multistep process, involving the formation and patterning of many new cell types and tissues. The evolution of early vertebrates, such as jawless fish, was accompanied by the emergence of a specialized set of cells, called neural crest cells which have long held a fascination for developmental and evolutionary biologists due to their considerable influence on the complex development of the vertebrate head. Although it has been classically thought that protochordates lacked neural crest counterparts, the recent identification and characterization of amphioxus and ascidian genes homologous to those involved in vertebrate neural crest development challenges this idea. Instead it suggests thatthe neural crest may not be a novel vertebrate cell population, but could have in fact originated from the protochordate dorsal midline epidermis. Consequently, the evolution of the neural crest cells could be reconsidered in terms of the acquisition of new cell properties such as delamination-migration and also multipotency which were key innovations that contributed to craniofacial development. In this review we discuss recent findings concerning the inductive origins of neural crest cells, as well as new insights into the mechanisms patterning this cell population and the subsequent influence this has had on craniofacial evolution.     

Development of the trigeminal motor neurons in parrots: Implications for the role of nervous tissue in the evolution of jaw muscle morphology

Vertebrates have succeeded to inhabit almost every ecological niche due in large part to the anatomical diversification of their jaw complex. As a component of the feeding apparatus, jaw muscles carry a vital role for determining the mode of feeding. Early patterning of the jaw muscles has been attributed to cranial neural crest‐derived mesenchyme, however, much remains to be understood about the role of nonneural crest tissues in the evolution and diversification of jaw muscle morphology. In this study, we describe the development of trigeminal motor neurons in a parrot species with the uniquely shaped jaw muscles and compare its developmental pattern to that in the quail with the standard jaw muscles to uncover potential roles of nervous tissue in the evolution of vertebrate jaw muscles. In parrot embryogenesis, the motor axon bundles are detectable within the muscular tissue only after the basic shape of the muscular tissue has been established. This supports the view that nervous tissue does not primarily determine the spatial pattern of jaw muscles. In contrast, the trigeminal motor nucleus, which is composed of somata of neurons that innervate major jaw muscles, of parrot is more developed compared to quail, even in embryonic stage where no remarkable interspecific difference in both jaw muscle morphology and motor nerve branching pattern is recognized. Our data suggest that although nervous tissue may not have a large influence on initial patterning of jaw muscles, it may play an important role in subsequent growth and maintenance of muscular tissue and alterations in cranial nervous tissue development may underlie diversification of jaw muscle morphology. J. Morphol. 275:191–205, 2014. © 2013 Wiley Periodicals, Inc.     

Hox genes, neural crest cells and branchial arch patterning

Proper craniofacial development requires the orchestrated integration of multiple specialized tissue interactions. Recent analyses suggest that craniofacial development is not dependent upon neural crest pre-programming as previously thought but is regulated by a more complex integration of cell and tissue interactions. In the absence of neural crest cells it is still possible to obtain normal arch patterning indicating that neural crest is not responsible for patterning all of arch development. The mesoderm, endoderm and surface ectoderm tissues play a role in the patterning of the branchial arches, and there is now strong evidence that Hoxa2 acts as a selector gene for the pathways that govern second arch structures.     

Quail-duck chimeras reveal spatiotemporal plasticity in molecular and histogenic programs of cranial feather development

The avian feather complex represents a vivid example of how a developmental module composed of highly integrated molecular and histogenic programs can become rapidly elaborated during the course of evolution. Mechanisms that facilitate this evolutionary diversification may involve the maintenance of plasticity in developmental processes that underlie feather morphogenesis. Feathers arise as discrete buds of mesenchyme and epithelium, which are two embryonic tissues that respectively form dermis and epidermis of the integument. Epithelial-mesenchymal signaling interactions generate feather buds that are neatly arrayed in space and time. The dermis provides spatiotemporal patterning information to the epidermis but precise cellular and molecular mechanisms for generating species-specific differences in feather pattern remain obscure. In the present study, we exploit the quail-duck chimeric system to test the extent to which the dermis regulates the expression of genes required for feather development. Quail and duck have distinct feather patterns and divergent growth rates, and we exchange pre-migratory neural crest cells destined to form the craniofacial dermis between them. We find that donor dermis induces host epidermis to form feather buds according to the spatial pattern and timetable of the donor species by altering the expression of members and targets of the Bone Morphogenetic Protein, Sonic Hedgehog and Delta/Notch pathways. Overall, we demonstrate that there is a great deal of spatiotemporal plasticity inherent in the molecular and histogenic programs of feather development, a property that may have played a generative and regulatory role throughout the evolution of birds.     

Hox genes, neural crest cells and branchial arch patterning.

Proper craniofacial development requires the orchestrated integration of multiple specialized tissue interactions. Recent analyses suggest that craniofacial development is not dependent upon neural crest pre-programming as previously thought but is regulated by a more complex integration of cell and tissue interactions. In the absence of neural crest cells it is still possible to obtain normal arch patterning indicating that neural crest is not responsible for patterning all of arch development. The mesoderm, endoderm and surface ectoderm tissues play a role in the patterning of the branchial arches, and there is now strong evidence that Hoxa2 acts as a selector gene for the pathways that govern second arch structures.     

Regulation of Cell Number in Formation of the Dorsal Root Ganglion Revealed by Transplantation of Quail Neural Crest Cells into Chick Embryos

Neural crest cells appear transiently in early embryogenesis on the dorsal surface of the neural tube and subsequently migrate along specific pathways. Some migrate to between the neural tube and somites, aggregating to form the rudiments of dorsal root ganglia (DRG). The size of DRG at a given somite level is almost constant in all chick embryos. To determine the mechanisms controlling the size of DRG, we transplanted neural crest cells of 2.5-day-old quail embryos into 2.5-day-old chick embryos between the neural tube and the somites, and examined the size of DRG in these chimeric embryos with extra neural crest cells 2 days after the operation, when natural cell death in DRG had not yet occurred. The DRG on the operated side were composed of both chick and quail cells in various proportions. The cell numbers of these chimeric DRG were almost the same as those of the normal DRG on the opposite side. That is, there were significantly fewer chick cells in chimeric DRG than in DRG composed of only chick cells on the opposite unoperated side. This finding indicates that the size of DRG is not determined in migrating neural crest cells but is regulated by the circumstances.     

Role of morphogens in neural crest cell determination

The neural crest is a transient, migratory cell population found in all vertebrate embryos that generate a diverse range of cell and tissue derivatives including, but not limited, to the neurons and glia of the peripheral nervous system, smooth muscle, connective tissue, melanocytes, craniofacial cartilage, and bone. Over the past few years, many studies have provided tremendous insights into understanding the mechanisms regulating the induction and migration of neural crest cell development. This review highlights the surprising and perhaps unexpected roles for morphogens in these distinct processes. A comparison of studies performed in several different vertebrates emphasizes the requirement for coordination between multiple signaling pathways in the induction and migration of neural crest cells in the developing embryo.     

Insights from a sea lamprey into the evolution of neural crest gene regulatory network

The neural crest is a vertebrate innovation that forms at the embryonic neural plate border, transforms from epithelial to mesenchymal, migrates extensively throughout the embryo along well-defined pathways, and differentiates into a plethora of derivatives that include elements of peripheral nervous system, craniofacial skeleton, melanocytes, etc. The complex process of neural crest formation is guided by multiple regulatory modules that define neural crest gene regulatory network (NC GRN), which allows the neural crest to progressively acquire all of its defining characteristics. The molecular study of neural crest formation in lamprey, a basal extant vertebrate, consisting in identification and functional tests of molecular elements at each regulatory level of this network, has helped address the question of the timing of emergence of NC GRN and define its basal state. The results have revealed striking conservation in deployment of upstream factors and regulatory modules, suggesting that proximal portions of the network arose early in vertebrate evolution and have been tightly conserved for more than 500 million years. In contrast, certain differences were observed in deployment of some neural crest specifier and downstream effector genes expected to confer species-specific migratory and differentiation properties.     

Deconstructing the pharyngeal metamere

A prominent feature of all vertebrate embryos is the presence of a series of bulges on the lateral surface of the head, the pharyngeal arches. These structures constitute a metameric series, with each arch forming a similar set of derivatives. Significantly, the development of the pharyngeal arches is complex as it involves interactions between disparate embryonic cell types: ectoderm, endoderm, mesoderm and neural crest. It is becoming increasingly apparent that the development of the pharyngeal metamere revolves around the pharyngeal endoderm. The segmentation of this tissue is central to the generation of the arches. The pharyngeal endoderm also provides positional cues for the neural crest, and is involved in the induction of a number of components of the pharyngeal metamere. The segmentation of the pharyngeal endoderm has also been key to the evolution of pharyngeal metamerism. It is likely that endodermal segmentation is a deuterostome characteristic and that this basic pattern was sequentially modified and over time the more complex pharyngeal metamere of vertebrates emerged.