Anthony Graham

Pharyngeal arch patterning in the absence of neural crest

Pharyngeal arches are a prominent and critical feature of the developing vertebrate head. They constitute a series of bulges within which musculature and skeletal elements form; importantly, these tissues derive from different embryonic cell types [1]. Numerous studies have emphasised the role of the cranial neural crest, from which the skeletal components derive, in patterning the pharyngeal arches [2-4]. It has never been clear, however, whether all arch patterning is completely dependent on this cell type. Here, we show that pharyngeal arch formation is not coupled to the process of crest migration and, furthermore, that pharyngeal arches form, are regionalized and have a sense of identity even in the absence of the neural crest. Thus, vertebrate head morphogenesis can now be seen to be a more complex process than was previously believed and must result from an integration of both neural-crest-dependent and -independent patterning mechanisms. Our results also reflect the fact that the evolutionary origin of pharyngeal segmentation predates that of the neural crest, which is an exclusively vertebrate characteristic.

Patterning the pharyngeal arches.

The presence of a muscularised pharynx with skeletal support is a fundamental vertebrate characteristic. Developmentally, the pharynx arises from the pharyngeal arches on either side of the head of vertebrate embryos. The development of the pharyngeal arches is complex involving a number of disparate embryonic populations, ectoderm, endoderm, neural crest and mesoderm, which must be co‐ordinated to generate the components and overall identity of each of the arches. Previous studies suggested that it is the neural crest that plays a pivotal role in patterning the pharyngeal arches. It is now also becoming clear, however, that there are crest‐independent patterning mechanisms. Therefore, pharyngeal arch patterning is more complex than was previously believed and there must be an integration of crest‐dependent and ‐independent patterning mechanisms. BioEssays 23:54–61, 2001.

Trunk neural crest has skeletogenic potential.

During early vertebrate development, neural crest cells emerge from the dorsal neural tube, migrate into the periphery, and form a wide range of derivatives. There is, however, a significant difference between the cranial and trunk neural crest with respect to the diversity of cell types that each normally produces. Thus, while crest cells from all axial levels form neurons, glia, and melanocytes, the cranial crest additionally generates skeletal derivatives such as bone and cartilage; trunk crest cells are generally thought to lack skeletogenic potential. Here, we show, however, that if avian trunk neural crest cells are cultured in appropriate media, they form both bone and cartilage cells, and if placed into the developing head, they contribute to cranial skeletal components. Thus, the neural crest from all axial levels can generate the full repertoire of crest derivatives. The skeletogenic potential of the trunk neural crest is significant, as it was likely realized in early vertebrates, which had extensive postcranial exoskeletal coverings.

Early steps in the production of sensory neurons by the neurogenic placodes.

Neurogenic placodes are specialized regions of the embryonic ectoderm that generate the majority of the neurons of the cranial sensory ganglia. Here we have accurately determined the onset of neurogenesis in each of the placodes in the chick, and we have also analyzed the expression profiles of genes that are believed to be involved in determining the types of sensory neurons produced by each placode. Interestingly, we find that there is a major difference in the expression domains of neurogenin-1 and neurogenin-2 in the chick, when compared with those reported for the mouse. We do find, however, that Brn-3a and Phox-2a and Phox-2b which are also associated with the specification of neuronal type are expressed in the same domains in the chick as they are in the mouse. These results suggest that neurogenin-1 and neurogenin-2 are functionally interchangeable in neurogenic placodes. We have also found major differences between the ophthalmic and maxillomandibular trigeminal placodes, and while all of the other placodes generate mitotically active cells the ophthalmic trigeminal placode seems to throw off postmitotic neuronal cells.

Significance of the cranial neural crest

The cranial neural crest has long been viewed as being of particular significance. First, it has been held that the cranial neural crest has a morphogenetic role, acting to coordinate the development of the pharyngeal arches. By contrast, the trunk crest seems to play a more subservient role in terms of embryonic patterning. Second, the cranial crest not only generates neurons, glia, and melanocytes, but additionally forms skeletal derivatives (bones, cartilage, and teeth, as well as smooth muscle and connective tissue), and this potential was thought to be a unique feature of the cranial crest. Recently, however, several studies have suggested that the cranial neural crest may not be so influential in terms of patterning, nor so exceptional in the derivatives that it makes. It is now becoming clear that the morphogenesis of the pharyngeal arches is largely driven by the pharyngeal endoderm. Furthermore, it is now apparent that trunk neural crest cells have skeletal potential. However, it has now been demonstrated that a key role for the cranial neural crest streams is to organise the innervation of the hindbrain by the cranial sensory ganglia. Thus, in the past few years, our views of the significance of the cranial neural crest for head development have been altered. Developmental Dynamics 229:5–13, 2004.

The origin and evolution of the neural crest

Many of the features that distinguish the vertebrates from other chordates are derived from the neural crest, and it has long been argued that the emergence of this multipotent embryonic population was a key innovation underpinning vertebrate evolution. More recently, however, a number of studies have suggested that the evolution of the neural crest was less sudden than previously believed. This has exposed the fact that neural crest, as evidenced by its repertoire of derivative cell types, has evolved through vertebrate evolution. In this light, attempts to derive a typological definition of neural crest, in terms of molecular signatures or networks, are unfounded. We propose a less restrictive, embryological definition of this cell type that facilitates, rather than precludes, investigating the evolution of neural crest. While the evolutionary origin of neural crest has attracted much attention, its subsequent evolution has received almost no attention and yet it is more readily open to experimental investigation and has greater relevance to understanding vertebrate evolution. Finally, we provide a brief outline of how the evolutionary emergence of neural crest potentiality may have proceeded, and how it may be investigated. BioEssays 30:530–541, 2008.

Development of the pharyngeal arches

The oro‐pharyngeal apparatus has its origin in a series of bulges that is found on the lateral surface of the embryonic head, the pharyngeal arches. The development of the pharyngeal arches is complex involving a number of disparate embryonic cell types: ectoderm, endoderm, neural crest and mesoderm, whose development must be co‐ordinated to generate the functional adult apparatus. In the past, most studies have emphasised the role played by the neural crest, which generates the skeletal elements of the arches, in directing pharyngeal arch development, but it has also become apparent that the other tissues of the arches, most notably the endoderm, also plays a prominent role in directing arch development. Thus pharyngeal arch development is more complex, and more consensual, than was previously believed.

Conserved deployment of genes during odontogenesis across osteichthyans

Odontogenesis has only been closely scrutinized at the molecular level in the mouse, an animal with an extremely restricted dentition of only two types and one set. However, within osteichthyans many species display complex and extensive dentitions, which questions the extent to which information from the mouse is applicable to all osteichthyans. We present novel comparative molecular and morphological data in the rainbow trout (Oncorhynchus mykiss) that show that three genes, essential for murine odontogenesis, follow identical spatial-temporal expression. Thus, at all tooth bud sites, epithelial genes Pitx–2 and Shh initiate the odontogenic cascade, resulting in dental mesenchymal Bmp–4 expression, importantly, including the previously unknown formation of replacement teeth. Significantly, this spatial–temporal sequence is the same for marginal and lingual dentitions, but we find notable differences regarding the deployment of Pitx–2 in the developing pharyngeal dentition. This difference may be highly significant in relation to the theory that dentitions may have evolved from pharyngeal tooth sets in jawless fishes. We have provided the first data on operational genes in tooth development to show that the same signalling genes choreograph this evolutionary stable event in fishes since the osteichthyan divergence 420 Myr ago, with the identical spatial–temporal expression as in mammals.

Wnt6 marks sites of epithelial transformations in the chick embryo

In a screen for Wnt genes executing the patterning function of the vertebrate surface ectoderm, we have isolated a novel chick Wnt gene, chick Wnt6. This gene encodes the first pan-epidermal Wnt signalling molecule. Further sites of expression are the boundary of the early neural plate and surface ectoderm, the roof of mesencephalon, pretectum and dorsal thalamus, the differentiating heart, and the otic vesicle. The precise sites of Wnt6 expression coincide with crucial changes in tissue architecture, namely epithelial remodelling and epithelial-mesenchymal transformation (EMT). Moreover, the expression of Wnt6 is closely associated with areas of Bmp signalling.

The role of the endoderm in the development and evolution of the pharyngeal arches

The oro‐pharyngeal apparatus has its origin in a series of bulges found on the lateral surface of the embryonic head, the pharyngeal arches. Significantly, the development of these structures is extremely complex, involving interactions between a number of disparate embryonic cell types: ectoderm, endoderm, mesoderm and neural crest, each of which generates particular components of the arches, and whose development must be co‐ordinated to generate the functional adult oro‐pharyngeal apparatus. In the past most studies have emphasized the role played by the neural crest, which generates the skeletal elements of the arches, in directing pharyngeal arch development. However, it is now apparent that the pharyngeal endoderm plays an important role in directing arch development. Here we discuss the role of the pharyngeal endoderm in organizing the development of the pharyngeal arches, and the mechanisms that act to pattern the endoderm itself and those which direct its morphogenesis. Finally, we discuss the importance of modification to the pharyngeal endoderm during vertebrate evolution. In particular, we focus on the emergence of the parathyroid gland, which we have recently shown to be the result of the internalization of the gills.