17 

 

NEURAL  CREST-DETERMINED  EVOLUTIONARY  NOVELTIES

The proper program of events governing the migration of crest may need first to be established in the hindbrain, to allow migratory crest cells to interpret and respond to environmental signals set up through a series of tissue interactions.

                                                                        P.A. Trainor, D. Sobieszczuk, D. Wilkinson, R. Krumlauf

 

The neural crest is a major evolutionary novelty, unique to vertebrates. It evolved in response to ever-increasing evolutionary pressure for complex structures necessary for the more complex, aggressive and predatory life of the group. Evolution and development of the neural crest is related to the evolution and development of the nervous system and neural tube/CNS. The neural crest consists of multipotent cells that form on/in the neural tube/CNS and during early embryogenesis migrate throughout the animal body to participate in, and direct, the development of the most different organs, tissues and other parts of the body, including the typical vertebrate structures such as vertebrate cranium, jaws, dentition, beak morphology, middle ear ossicles, etc. Before leaving the neural tube/CNS, these cells are provided with epigenetic information on how to find their path through the cytological labyrinth to the migration site and what to transform themselves and the local cells into. Experimental and paleontological evidence suggests that evolution of neural crest-derived structures is not related to, and does not depend on, any changes in genes.

The neural crest is believed to have evolved from an invertebrate neural structure. It evolved to meet the informational requirements related to the ever-increasing complexity of the vertebrate structure and morphology. It is critically involved in the development of numerous, often de novo, organs in vertebrates. To a large extent, neural crest is responsible for the unprecedented rapid rates of vertebrate evolution.

As for its embryonic origin, neural crest cells and nerve cells derive from a common stock of precursor cells and differentiate almost simultaneously in the neural tube. Neural crest cells then delaminate from the neural tube to start an ordered migration throughout the animal body to their target sites where they direct formation of organs other structures.

Empirical evidence shows that before leaving the neural tube, neural crest cells are provided with information not only for finding their way through the maze leading to the target sites, but also on what they have to transform themselves into, as well as for regulating differentiation and proliferation of cells in the target site in the process of the development of particular organs and structures. Evidently, the information that neural crest cells are provided with is epigenetic information (for they share the same genetic information with all the rest of the cells throughout the animal body).

Neural crest cells provided the developmental repertory of vertebrates with a new “do-it-yourself” mechanism, in addition to the “instructionist” mode of control of development accomplished, by communicating developmental information via the brain-hypothalamic-pituitary-peripheral glands axes and local innervation. The neural crest cell-derived structures are among the most malleable structures in vertebrates.

A powerful demonstration of the function of neural crest cells as carriers and providers of the epigenetic information to cells in the sites of their migration is the experimental evidence that interspecific homotopic transplantation of neural crest from a species into another determines formation of donor structures in the body of the recipient organism.

Neural crest cells are essentially involved in the development of almost all vertebrate organs. The omnipresence of neural crest cells in the process of individual development, the accelerated evolution and the malleability of the neural crest -induced organs indicates the important role that they  played in the evolution of vertebrates.

 

Neural Crest-determined Evolution in Vertebrates

 

The rise of vertebrates marks a new stage in metazoan evolution, characterized by accelerated evolutionary rates and increased complexity of structure and function with the morphological diversity as its most visible and amazing manifestation. Transition from invertebrates to vertebrates is characterized not only by accelerated tempo of evolution but also by an unprecedented evolution of de novo structures.

These changes in the tempo and trends of evolution of vertebrates are inextricably related with the advent of the neural crest. All the extant vertebrate species are in possession of neural crest and the gene regulatory networks inducing formation of neural crest are conserved across the vertebrate taxa (Meulemans and Bronner-Fraser, 2004). The neural crest, as a specialized neural tube population of cells, is involved in the individual development both as a contributor of cells to various organs and parts throughout the animal body and as source of inductive signals for morphogenesis and organogenesis at the target sites. In a figure of speech, neural crest cells behave as colonists participating along the local cells in the formation of particular structures and as missionaries, “teaching” local cells what to differentiate into. As a unique developmentally flexible population of cells, neural crest cells represents the main driving force behind the accelerated rates of evolution of vertebrates compared to invertebrates.

 

Origin of the Neural Crest

 

The neural crest is a uniquely vertebrate structure that develops from/on the neural tube after the latter breaks off the ectoderm. It consists of a population of cells arising at the area of contact between the neural tube and ectoderm. This statement does not clarify whether neural crest cells arise from the neural tube or ectoderm and  most investigators do not make it clear.

Some authors believe that neural crest cells detach from the dorsal portion of the neural tube (Sohal et al., 1998). A strong  argument in favor of the idea that the neural crest originates from the neural tube comes from the experimental evidence that, in response to ablation of the cranial neural crest, the neural tube regenerates neural crest cells with all the capacities of the original, species-specific properties. The regeneration of the neural crest from the dorsal neural tube occurs only at the axial level of the ablated neural crest (Scherson et al., 1993).

Evolution of the neural crest and of the ability to use its cellular elements for developing new structures or adaptively modify existing ones for new functions was a result of evolutionary pressures arising from increasing competition under new conditions of living and the resulting predatory life of vertebrates.

Not only developmental evidence but phylogenetic evidence as well suggests that the neural crest is a derivative of the nervous system. According to Gans and Northcutt, the epidermal nerve plexus of “protochordates” is the evolutionary precursor of both the neural crest and neurogenic epidermal placodes. In ascidians, nerve plexus neurons are identified, which, like the vertebrate olfactory placode neurons, migrate to the brain (Stone and Hall, 2004).

It seems plausible that the reason the neural crest took over the functions of mesoderm in the facial cranium has been the failure of the mesoderm to respond to the evolutionary pressure for morphological transformations of the head and jaws that the new predatory style of life of vertebrates required. Vertebrates responded to that pressure by specializing a neural structure, probably an epidermal nerve plexus, which evolved into the neural crest. B.K. Hall also believes that the neural crest could

 

Have existed initially as an epidermal nerve plexus or net controlling ciliary function during movement and filter-feeding. With increasing muscle-based locomotion, the dorsal nerve cord took over innervative control of locomotion, freeing the epidermal nerve cells for other functions. (Hall, 1999b)

 

Based on the tremendous contributions of the neural crest to the morphological diversity of “craniates”, Hall believes it qualifies as a fourth germ layer (Hall, 1999a).

Other authors believe that functionally, the emergence of neural crest cells may be related to the bone-formation throughout the body before the evolution of the bone-forming sclerotome (Gerhart and Kirschner, 1997d). It is noteworthy in this context to remember that the visceral skeleton in vertebrates is of neural crest, not mesodermal, origin (Hall, 1999c).

The neural crest appeared ~450 million years ago, when the earliest vertebrates evolved, i.e. around the time of quadruplication of the Hox gene cluster that occurred in this group. These vertebrates had no axial skeleton but were covered with bony plates. Initially, the neural crest may have been used for developing dentine armor in ostracoderms and only later for producing teeth, gill arches, and jaws (Gerhart and Kirschner, 1997e).

Recent evidence shows that the neural crest may have evolved even earlier. Structures homologue to the neural tube are found in protochordates (Corbo et al., 1997), ascidian urochordates (Baker and Bronner-Fraser, 1997; Jeffery et al., 2004) and amphioxus (Baker and Bronner-Fraser, 1997), all of them presumed to be evolutionary precursors of the vertebrate neural crest. Migratory neural crest-like cells in the ascidian urochordate Ecteinascidia turbinata emerging from the neural tube/central nervous system in an ordered manner migrate into the body wall below the epidermis where they differentiate into pigment cells (Jeffery et al., 2004).

There are several major facts indicating that the neural crest played a crucial role in the evolution of  the morphology of vertebrates.

First, the appearance of the neural crest as a new neural formation coincides with the unprecedented burst of morphological innovations and increased structural complexity characterizing the vertebrate evolution.

Second, neural crest cells migrate to highly specific parts of the animal body to participate in molding local structures.

Third, morphological traits determined and molded by neural crest cells are among the most malleable vertebrate structures.

All the above justify the idea that the neural crest has been “at the centre stage of the vertebrate evolutionary play” (Hall, 1998h).

Genetic and developmental constraints obviously represented a barrier to the evolution of the adaptive structures necessary for the new predominantly predatory life style in vertebrates. The neuroendocrine reprogramming or the “instructive” mode of evolving new adaptations might have been developmentally more costly or inappropriate for developing the extremely complex structures and functions vertebrates had to evolve. This might have given rise to the evolutionary pressure that led to the acquisition by the neural tissue (neural crest) of the unprecedented “do-it-yourself”, executive morphogenetic function in addition to the “instructive” neurohormonal pathways that evolved since the dawn of the metazoan life.

Neural crest cells migrate from the neural tube and colonize particular regions of the  embryonic body for molding local morphologies by participating themselves as cytological building blocks and by instructing local cells to proliferate and differentiate into specific cell types for developing particular structures. This “do-it-yourself” mode of fashioning new morphologies adopted by the neural crest was a developmental “invention” with revolutionary consequences for evolution of vertebrates. It is this novel developmental mode that made possible the extraordinary evolutionary malleability of organs or parts in whose development it is involved. Under appropriate conditions, these structures can dramatically change, sometimes within a small number of generations.

 

Development of the Neural Crest

 

Generally, it is believed that the neural crest develops between the neural tube and the ectoderm after the neural tube breaks off the ectoderm. Its cells delaminate from the neural tube and migrate to specific sites in the animal body to form or contribute to formation of the most different cells, organs, and tissues.  What tremendous role in the evolution of vertebrates the acquisition of the ability of these cells to migrate to their target sites and transform themselves and local cells into cell types characteristic for the developing structures has, is illustrated by the fact that neural crest cells have been essential for the evolution of almost all the new vertebrate morphological features: jaws, pharyngeal jaws in cichlid fish, mammal middle ear ossicles, shell in turtles, feathers in birds, constantly growing incisors in rodents, placenta and viviparity in mammals and reptiles, respectively, lungs in tetrapods, etc. (Hall, 1999g).

Since the middle of the 20^th century, it was believed that formation of the neural crest requires inducing signals from the mesoderm. However, Moury and Jacobson demonstrated that ectopic transplantation of ectoderm to the axolotl (Ambystoma mexicanum) neural tube induces formation of the neural crest (Moury and Jacobson, 1989) and both epidermal and neural plate cells are differentiated into neural crest cells (Moury and Jacobson, 1990). The classical view that neural crest precursors are a distinct population between the epithelium and epidermis

is at variance with its evolutionarily neural origin and recent analyses have demonstrated that neural crest cells and the neural tissue derive from the same cytological precursors. Individual precursor cells within the neural folds can give rise to epidermal-, neural crest-, and neural tube derivatives. The neural plate can induce transformation of the adjacent epidermis into neuroepithelium and, by interacting with the uninduced epidermis, generate neural crest cells (Selleck and  Bronner-Fraser, 1995; Selleck and  Bronner-Fraser, 1996; figure 17.1).

 

                                               

Figure 17.1. Neural plate may induce pluripotent ectoderm cells to become neural, thereby restricting their potential, and concomitantly causing that ectoderm to thicken. Planar interactions between the uninduced prospective epidermis and induced neural plate, may result in the generation of neural crest cells from the latter (From Selleck and Bronner-Fraser, 1995).

Signals from the adjacent tissues also are necessary for induction of the neural crest (figure 17.2). The BMP-4 is sufficient to induce neural crest cells from chick neural explants in vitro. However, in zebrafish neural crest induction takes place in the absence of mesodermal BMP signals.

 

 

Figure 17.2. Neural crest cell induction. Neural crest cells (small circles) are induced to form via planar (double headed arrows) and vertical (arrows) inductive signals at the neural plate border, which is defined as the junction between the dorsal part (black) of the neural tube (big circle) and the adjacent surface ectoderm (light shaded). Three key signaling pathways intersect at the neural plate border. Although a gradient of BMP signaling within the neural plate has been proposed as a requirement for neural crest cell induction, WNT signalling from the ectoderm and FGF signalling from the underlying mesoderm (dark shaded) are also able to induce neural crest cells to form (From Trainor, 2005).

 

After being induced, neural crest cells can differentiate into at least one cell type, the melanophores which migrate to the anterior part of the embryo, a fact that is considered to prove that not only the induction but also  delamination, migration, and differentiation of neural crest cells can occur in the absence of mesoderm-derived signals (Ragland and Raible, 2004)

Tracing back the developmental origins of the signals for neural crest formation, one finally arrives at the maternal factors of neural induction, proneural genes, which stimulate expression of Delta and its receptor, Notch (Itoh et al., 2003) in the neural plate. Notch signals from the neural plate, via induction of the Hairy drive expression of Bmp, Wnt, and Fgf genes, in the underlying mesoderm and adjacent nonneural ectoderm, thus determining lateral inhibition of neurogenesis. These secreted factors, as well as signals from the neural plate (Dlx5) induce expression of “neural plate border specifiers” (Zic factors, Pax3/7, Dlx5, and Msx1/2) (Meulemans and Bronner-Fraser, 2004) (figure 17.3).

Next, the neural plate border specifiers stimulate expression of “neural crest specifiers” (Slug/Snail, AP-2, FoxD3, Sox10, Sox9, and c-Myc), which form a dense network of interacting elements that makes possible expression of the “neural crest effector genes” (Muelemans and Bronner-Fraser, 2004), which in turn determine formation of mature neural crest cells just before delaminating and starting their migration to strictly determined regions all over the animal body.

Experimental evidence shows that the midbrain-hindbrain junction, and the brain inductive signaling center influences the fate of adjacent neural crest (Trainor et al., 2002).

Neural crest cells are transformed from epithelial into mesenchyme cells, which is typical for many neuronal cells.

 

Neural crest cell induction may be an ongoing process, in which an initial induction at the neural plate border is followed by further induction within the dorsal neural tube. (Baker and Bronner-Fraser, 1997)

 

After delaminating from the neural tube/CNS, neural crest cells start migrating to particular regions of the embryo where they direct formation of numerous structures and organs by differentiating themselves into a variety of cell types and by inducing differentiation and proliferation of local cells.

As pointed out, both neurons of the neural tube and neural crest cells derive from common precursor cells. The distinction between neuroepithelial cells of the neural tube and neural crest cells may not be as well defined as is generally believed. After ablation of a part of neural crest in chick embryos, the adjacent neural

tube produces a migratory population of cells that gives rise to neural crest cells:

 

The neural tube cells ventral to the ablation, which normally would not form neural crest cells, regulate to reform the missing regions of the neural tube and the neural crest after ablation of their dorsal neighbors. (Scherson et al., 1993)

 

This view is also supported by the observation that neural crest cells are produced by some spinal cord neurons (Sohal et al., 1998). These experimental facts, and others to be presented later, suggest that the neural tube may be the producer of neural crest cells and the provider of the epigenetic information to the neural crest cells before they delaminate from the neural tube and start migration to specific target sites in the body.

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Evolution of the Mammalian Middle Ear Ossicles

One of the most enigmatic transformations in vertebrates is the evolution of ear ossicles (malleus, incus, stapes, and the tympanic bone), a key morphological innovation that took place in the class of mammals (figure 17.9). Paleontological evidence shows that this has occurred not later than 195 million years ago (Early Jurasic), which is the age of Hadrocodium, the earliest known taxon that lost the mandibular attachment to the middle ear ossicles (Luo,  2001).

Evolution of the middle ear ossicles is result of an evolutionary pressure for hearing under conditions of the new sound-transmission medium (from water to air) related to transition of vertebrates to terrestrial life.

The evolution of mammals from reptiles was characterized by a continuous growth of one of the lower jaw bones, the dentary, at the expense of all six other bones, which their lower jaw consists of. The increase in size of the dentary brought it in contact with the skull and formed the modern squamoso-mandibular joints, thus freeing quadrate and articular bones of their interconnecting function and moving them posteriorly. The evolutionary trend toward freeing reptile lower jaw bones of their articulating function and their posterior displacement to form middle ear ossicles coincided with a heterochronic change in the arrival of neural crest cells in the region where the Meckel’s cartilage forms.

Articulation of jaws in reptiles involves the homologous bones: the articular, the quadrate, and the angular. Human embryos have a primary jaw joint much like fish, amphibian, and reptiles embryos and adults do. The primary jaw joint forms by the assembly of neural crest cells that form the cartilagineous mandibular arch consisting of the quadrate (upper jaw) and Meckel’s cartilage (lower jaw). The mass of neural crest cells between the first and second branchial pouches becomes the hyoid arch. Adult mammals develop a secondary jaw by connecting the dentary and squamosa bones.

 

 

Figure 17.9. Diagrammatic representation of the otic region of a typical mammal. Note the trilaminar structure of the tympanic membrane (eardrum), which is formed by the epithelial layers of the external acoustic meatus and the tubotympanic recess, along with an intervening layer of cells derived from the first and second pharyngeal arches. The tympanic membrane is anchored by the tympanic ring. The three auditory ossicles (malleus, incus and stapes) conduct vibrations from the tympanic membrane across the middle ear cavity, and transmit these vibrations into the inner ear (From Mallo and Gridley, 1996).

Paleontological Evidence of the Evolution of the Middle Ear Ossicles

 

Paleontological record shows that the mandibular precursors of the middle ear ossicles were used for mechanosensation in premammalian groups such as cynodonts and Morganucodon, which continued to conserve these “reptilian” jaw bones but separated by a cartilage mass. Schematized evolution of mandibles and middle ear ossicles from corresponding reptile jaw bones are shown in the figure 17.10 and figure 17.11. An intermediate reduced bone detached from the dentary is found in some mammal fossils of the Early Cretaceous in China (Wang et al., 2001).

Whether the evolutionary transformation of the lower jaw bones into middle ear ossicles in mammals has occurred only once, in their common ancestor (Rowe, 1996), or it has  occurred more than once in different groups, after their divergence from the common ancestor, has been a controversial issue in modern biology. Given the complexity of the structure of the middle ear ossicles and of the process of the transformation of mandibular bones into middle ear ossicles, most biologists believed that this evolution has occurred only once to the common ancestor of all the mammals. This hypothesis of monophyletic origin of middle ear ossicles seems to have been refuted by recent paleontological evidence.

Figure 17.10. (A) Evolution of the mammalian mandible and middle ear (right lateral view), plotted on a phylogeny of selected mammals and their closest extinct relatives. (B) Right lateral view of auditory chain of Didelphis; the stapes is rotated and offset from between the incus and fenestra vestibulae of the inner ear. Crosses signify extinct species.

Abbreviations: FV, fenestra vestibuli; CMJ, craniomandibular joint (From Rowe, 1996).

 

Thomas Rich and coll. (2005) in Australia found a 115 million year old fossil jaw of an Early Cretaceous monotreme, Teinolophos trusleri, considered to be an extinct relative of the modern Australian monotremes, platypus and echidna (Martin and Luo, 2005). The fossil has a trough in which postdentary reduced bones, homologous to the mammalian ear ossicles, were housed, implying that even though the reduced bones might have been used for hearing they were still integral part of the jaw, in the mandibular trough. They represent, thus an important link between the mandibular bones and fully transformed mammalian middle ear ossicles in monotremes (Rich et al., 2005). In view of fact that monotremes, to which Teinolophos trusleri belongs, split off as a separate mammalian group more than 150 million years ago, and modern monotremes have middle ear ossicles, the presence of the bones homologous to the middle ear ossicles still attached to the mandible in T. trusleri, proves that middle ear ossicles in monotremes evolved independently from other groups of mammals.

 

Figure 17.11. (A) Middle ear ossicles (malleus, incus, and stapes) and tympanic ring (ectotympanic) of an adult opossum Didelphis marsupialis in lateral view. (B) Medial view of the lower jaw of a pouch-young opossum, showing that the malleus (formed from the fusion of the ossified posterior end of Meckel's cartilage with a dermal bone, the prearticular) and ectotympanic are components of the lower jaw in early development. The middle portion of Meckel’s cartilage atrophies in early post-hatching stages of monotremes, post-birth stages of marsupials, and late fetal stages of placentals, severing the connection of the malleus and ectotympanic to the dentary. (C) Diagrammatic view of the mandible of the near-mammal (mammaliaform) Morganucodon. Present are both the primitive tetrapod jaw joint, which lies between the articular fossa (ar.f) and the quadrate of the upper jaw, and the neomorphic mammalian jaw joint between the dentary condyle (co) and the squamosal bone of the skull. The angular, which bears a hooklike ventral process, the reflected lamina, is homologous with the ectotympanic of mammals; and the articular and prearticular are homologous with the malleus of mammals. The bone covering the meckelian groove is interpreted as a splenial. The surangular, coronoid, and splenial are absent in living mammals.

Abbreviations: an, angular; ar.f, articular fossa; c, coronoid; co, dentary condyle; d, dentary; e, ectotympanic; i, incus; m, malleus; m.c, Meckel’s cartilage; mg, meckelian groove; p, prearticular; s, stapes; sp, splenial; su, surangular (From Rich et al., 2005).