Segmentation of the vertebrate skull: neural-crest derivation of adult cartilages in the clawed frog, Xenopus laevis

We utilize a novel, transgenic cell-labeling system to assess the embryonic derivation of cartilages in the post-metamorphic skull of anuran amphibians. Many of these cartilages form de novo at metamorphosis and have no obvious precursors within the larval skeleton. Most adult cartilages are derived from mandibular- or hyoid-stream neural crest, either individually or in combination; branchial-stream neural crest makes a modest contribution. Each stream also contributes to at least one cartilage in the middle ear or external ear. Four cartilages are composite elements; each is derived from at least two distinct cell populations. Many boundaries between adjacent neural-crest territories are cryptic insofar as they do not coincide with anatomical boundaries. The system of adult cranial segmentation revealed by these fate-mapping results differs in important respects from both the segmentation of the ontogenetically earlier larval skull and the cranial segmentation in amniotes. Most striking is the rostral "inversion" of neural-crest-derived cartilages in Xenopus, such that mandibular stream-derived elements are deployed caudal to those derived from the hyoid stream, which predominate anteriorly. This novel pattern of rostral segmentation may be a consequence of the complex, biphasic life history that is characteristic of most species of living amphibians, and especially anurans, in which cranial architecture is significantly reconfigured at metamorphosis. Neural-crest derivation of the vertebrate skull is not invariant; instead, embryonic derivation of individual components of the cranial skeleton may vary widely among species.     

Cranial neural crest cells contribute to connective tissue in cranial muscles in the anuran amphibian, Bombina orientalis.

The contribution of cranial neural crest cells to the development and patterning of cranial muscles in amphibians was investigated in the phylogenetically basal and morphologically generalized frog, Bombina orientalis. Experimental methods included fluorescent marking of premigratory cranial neural crest and extirpation of individual migratory streams. Neural crest cells contributed to the connective tissue component, but not the myofibers, of many larval muscles within the first two branchial arches (mandibular and hyoid), and complex changes in muscle patterning followed neural crest extirpation. Connective tissue components of individual muscles of either arch originate from the particular crest migratory stream that is associated with that arch, and this relationship is maintained regardless of the segmental identity-or embryonic derivation-of associated skeletal components. These developmental relations define a pattern of segmentation in the head of larval anurans that is similar to that previously described in the domestic chicken, the only vertebrate that has been thoroughly investigated in this respect. The fundamental role of the neural crest in patterning skeleton and musculature may represent a primitive feature of cranial development in vertebrates. Moreover, the corresponding developmental processes and cell fates appear to be conserved even when major evolutionary innovations-such as the novel cartilages and muscles of anuran larvae-result in major differences in cranial form.     

Cell migration, pattern formation and cell fate during head development in lungfishes and amphibians

Summary In the evolution of land-living vertebrates, the transition from spending the entire life cycle in the water to first a biphasic (adult on land, eggs and larvae in water) and later a terrestrial life-history mode was achieved by changes in developmental processes and regulatory mechanisms. Lungfishes, salamanders and frogs are studied as examples of species which span this transition. The migration and fate of the embryonic cells that form the head is studied, using experimental embryology (extirpation and transplantation of cells), molecular markers and novel microscopy techniques — such as confocal microscopy. Knowing the migratory routes and fates of the cells that form head structures is important for an elucidation of the changes that took place e.g. when gill arches transformed into head cartilages, and when the specialised larval mouth structures present in today’s frogs and toads arose as an evolutionary innovation. Results so far indicate that the early migration and pattern formation of neural crest cells in the head region is surprisingly conserved. Both the amphibians investigated and the Australian lungfish have the same number of migrating neural crest streams, and the identity of the streams is preserved. The major difference lies in the timing of migration, where there has been a heterochronic shift such that cell migration starts much later in the Australian lungfish than in the amphibians. The molecular mechanisms regulating the formation of streams of cranial neural crest cells seem, at least in part, to be differential expression of ephrins and ephrin receptors, which mediate cell sorting. Our understanding of the behaviour of migrating cells (primarily the more well characterised neural crest cells) could be enhanced by a modelling approach. I present preliminary ideas on how this could be achieved, inspired by recent work on Dictyostelium development and our own previous work on pigment cells and their pattern formation during salamander embryogenesis.     

Evolutionary innovation in the vertebrate jaw: A derived morphology in anuran tadpoles and its possible developmental origin

The mouthparts of anuran tadpoles are highly derived compared to those of caecilians or salamanders. The suprarostral cartilages support the tadpole's upper beak; the infrarostral cartilages support the lower beak. Both supra- and infrarostral cartilages are absent in other vertebrates. These differences reflect the evolutionary origin of a derived feeding mode in anuran tadpoles. We suggest that these unique cartilages stem from the evolution of new articulations within preexisting cartilages, rather than novel cartilage condensations. We propose testing this hypothesis through a search for similarities in the development of the suprarostral and infrarostral cartilage articulations and of the primary jaw joint. In Xenopus, the gene zax is expressed in a region corresponding to the infrarostral cartilage. This gene is related to the bapx1-gene, which regulates jaw joint development. Further investigation of these genes, as well as other genes with joint-related functions, in anuran craniofacial development may provide a connection between the morphological diversity seen in the vertebrate head and the corresponding diversity in genetic regulatory processes. We believe that the evolution of larval jaws in anurans may shed light on the general evolutionary mechanisms of how new articulations, not only in the jaw region, could have arisen in the vertebrate skull.     

Expression patterns of lgr4 and lgr6 during zebrafish development

Leucine-rich repeat (LRR)-containing G protein-coupled receptors (LGRs) belong to the superfamily of G protein-coupled receptors, and are characterized by the presence of seven transmembrane domains and an extracellular domain that contains a series of LRR motifs. Three Lgr proteins – Lgr4, Lgr5, and Lgr6 – were identified as members of the LGR subfamily. Mouse Lgr4 has been implicated in the formation of various organs through regulation of cell proliferation during development, and Lgr5 and Lgr6 are stem cell markers in the intestine or skin. Although the expression of these three genes has already been characterized in adult mice, their expression profiles during the embryonic and larval development of the organism have not yet been defined. We cloned two zebrafish lgr genes using the zebrafish genomic database. Phylogenetic analyses showed that these two genes are orthologs of mammalian Lgr4 and Lgr6. Zebrafish lgr4 is expressed in the neural plate border, Kupffer’s vesicle, neural tube, otic vesicles, midbrain, eyes, forebrain, and brain ventricular zone by 24 h post-fertilization (hpf). From 36 to 96 hpf, lgr4 expression is detected in the midbrain–hindbrain boundary, otic vesicles, pharyngeal arches, cranial cartilages such as Meckel’s cartilages, palatoquadrates, and ceratohyals, cranial cavity, pectoral fin buds, brain ventricular zone, ciliary marginal zone, and digestive organs such as the intestine, liver, and pancreas. In contrast, zebrafish lgr6 is expressed in the notochord, Kupffer’s vesicle, the most anterior region of diencephalon, otic vesicles, and the anterior and posterior lateral line primordia by 24 hpf. From 48 to 72 hpf, lgr6 expression is confined to the anterior and posterior neuromasts, otic vesicles, pharyngeal arches, pectoral fin buds, and cranial cartilages such as Meckel’s cartilages, ceratohyals, and trabeculae. Our results provide a basis for future studies aimed at analyzing the functions of zebrafish Lgr4 and Lgr6 in cell differentiation and proliferation during organ development.     

Cranial neural crest emergence and migration in the Mexican axolotl (Ambystoma mexicanum)

The timing and pattern of cranial neural crest cell emergence and migration in the Mexican axolotl, Ambystoma mexicanum, are assessed using scanning electron microscopy (SEM). Cranial neural crest cells emerge and begin to migrate at the time of neural fold closure and soon form three distinct streams. The most anterior (mandibular) stream emerges first, at the level of the mesencephalon. Cells in this stream migrate rostroventrally around the optic vesicle. The second (hyoid) and third (branchial) streams emerge in close succession at the level of the rhombencephalon and extend ventrolaterally. Cells forming the hyoid stream migrate rostral to the otic vesicle, whereas the branchial stream divides into two parallel streams, which migrate caudal to the otic vesicle. At later stages (stage 26 onwards) the cranial neural crest cells disperse into the adjacent mesoderm and can no longer be followed by dissection and SEM. The pattern of cranial neural crest emergence and migration, and division into migratory streams is similar to that in other amphibians and in the Australian lungfish (Neoceratodus forsteri). Emergence of crest cells from the neural tube, relative to the time of neural tube closure, occurs relatively late in comparison to anurans, but much earlier than in the Australian lungfish. These results establish a morphological foundation for studies in progress on the further development and fate of cranial neural crest cells in the Mexican axolotl, as well as for studies of the role of cranial neural crest in cranial patterning.     

Cranial neural crest contributes to the bony skull vault in adult Xenopus laevis: insights from cell labeling studies

As a step toward resolving the developmental origin of the ossified skull in adult anurans, we performed a series of cell labeling and grafting studies of the cranial neural crest (CNC) in the clawed frog, Xenopus laevis. We employ an indelible, fixative-stable fluorescent dextran as a cell marker to follow migration of the three embryonic streams of cranial neural crest and to directly assess their contributions to the bony skull vault, which forms weeks after hatching. The three streams maintain distinct boundaries in the developing embryo. Their cells proliferate widely through subsequent larval (tadpole) development, albeit in regionally distinct portions of the head. At metamorphosis, each stream contributes to the large frontoparietal bone, which is the primary constituent of the skull vault in adult anurans. The streams give rise to regionally distinct portions of the bone, thereby preserving their earlier relative position anteroposteriorly within the embryonic neural ridge. These data, when combined with comparable experimental observations from other model species, provide insights into the ancestral pattern of cranial development in tetrapod vertebrates as well as the origin of differences reported between birds and mammals.     

Cranial cartilages: Players in the evolution of the cranium during evolution of the chordates in general and of the vertebrates in particular

The present contribution is chiefly a review, augmented by some new results on amphioxus and lamprey anatomy, that draws on paleontological and developmental data to suggest a scenario for cranial cartilage evolution in the phylum chordata. Consideration is given to the cartilage-related tissues of invertebrate chordates (amphioxus and some fossil groups like vetulicolians) as well as in the two major divisions of the subphylum Vertebrata (namely, agnathans, and gnathostomes). In the invertebrate chordates, which can be considered plausible proxy ancestors of the vertebrates, only a viscerocranium is present, whereas a neurocranium is absent. For this situation, we examine how cartilage-related tissues of this head region prefigure the cellular cartilage types in the vertebrates. We then focus on the vertebrate neurocranium, where cyclostomes evidently lack neural-crest derived trabecular cartilage (although this point needs to be established more firmly). In the more complex gnathostome, several neural-crest derived cartilage types are present: namely, the trabecular cartilages of the prechordal region and the parachordal cartilage the chordal region. In sum, we present an evolutionary framework for cranial cartilage evolution in chordates and suggest aspects of the subject that should profit from additional study.     

Evolutionary mechanisms of rib loss in anurans: a comparative developmental approach

ABSTRACT The presence of free ribs is presumed to be a primitive morphological character observed only in a few families of Recent anurans, whereas the absence of ribs has been considered to be a derived condition that is widespread within this order. A comparative study of rib development based on representatives of several anuran lineages (Alytes, Bombina, Bufo, Discoglossus, Hyla, Pelobates, Pelodytes, Rana, and Xenopus) reveals a previously undetected diversity of developmental features in the formation and interaction between neural arches and ribs. The absence of free ribs at premetamorphic or later stages is verified in some groups, but we present for the first time evidence of the existence of larval rib rudiments in others, both in the anterior (Rana, Hyla) and posterior (Bufo, Discoglossus, Pelobates) presacral regions. Heterochrony seems to have played a major role in the processes underlying rib reduction. The intracolumnar differences between anterior (V(2)-V(4)) and posterior (V(5)-V(8)) regions are based on perturbations in the timing of early differentiation. Furthermore, a clear shift in the relative timing of ossification among evolutionary lineages was detected. In this respect Xenopus has a highly derived condition. The use of the morphological character of "rib loss" in phylogenetic analyses must be reconsidered due to the different convergent developmental paths described here. The phylogenetic analysis of a "sequence units" matrix of rib development is compared with current anuran phylogenies. Some evolutionary information appears to be clearly present in the ontogenetic data of this "missing morphology," but its value for evolutionary inferences is rather limited.     

Sequence of chondrocranial development in the oriental fire bellied toad Bombina orientalis

The vertebrate head as a major novelty is directly linked to the evolutionary success of the vertebrates. Sequential information on the embryonic pattern of cartilaginous head development are scarce, but important for the understanding of its evolution. In this study, we use the oriental fire bellied toad, Bombina orientalis, a basal anuran to investigate the sequence and timing of larval cartilaginous development of the head skeleton from the appearance of mesenchymal Anlagen in post-neurulation stages until the premetamorphic larvae. We use different methodological approaches like classic histology, clearing and staining, and antibody staining to examine the larval skeletal morphology. Our results show that in contrast to other vertebrates, the ceratohyals are the first centers of chondrification. They are followed by the palatoquadrate and the basihyal. The latter later fuses to the ceratohyal and the branchial basket. Anterior elements like Meckel's cartilage and the rostralia are delayed in development and alter the ancestral anterior posterior pattern observed in other vertebrates. The ceratobranchials I–IV, components of the branchial basket, follow this strict anterior–posterior pattern of chondrification as reported in other amphibians. Chondrification of different skeletal elements follows a distinct pattern and the larval skeleton is nearly fully developed at Gosner Stage 28. We provide baseline data on the pattern and timing of early cartilage development in a basal anuran species, which may serve as guidance for further experimental studies in this species as well as an important basis for the understanding of the evolutionary changes in head development among amphibians and vertebrates.     

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 splanchnic 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 ventral-ward 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 continuous with the caudal portion of the circumpharyngeal crest. An early contribution to the enteric neuroblasts is apparent in this area.     

Cranial neural crest cell migration in the Australian lungfish, Neoceratodus forsteri

SUMMARY A crucial role for the cranial neural crest in head development has been established for both actinopterygian fishes and tetrapods. It has been claimed, however, that the neural crest is unimportant for head development in the Australian lungfish ( Neoceratodus forsteri   ), a member of the group (Dipnoi) which is commonly considered to be the living sister group of the tetrapods. In the present study, we used scanning electron microscopy to study cranial neural crest development in the Australian lungfish. Our results, contrary to those of Kemp, show that cranial neural crest cells do emerge and migrate in the Australian lungfish in the same way as in other vertebrates, forming mandibular, hyoid, and branchial streams. The major difference is in the timing of the onset of cranial neural crest migration. It is delayed in the Australian lungfish in comparison with their living sister group the Lissamphibia. Furthermore, the delay in timing between the emergence of the hyoid and branchial crest streams is very long, indicating a steeper anterior-posterior gradient than in amphibians. We are now extending our work on lungfish head development to include experimental studies (ablation of selected streams of neural crest cells) and fate mapping (using fluoresent tracer dyes such as DiI) to document the normal fate as well as the role in head patterning of the cranial neural crest in the Australian lungfish.     

Fgf20b is required for the ectomesenchymal fate establishment of cranial neural crest cells in zebrafish

In cranial skeletal development, the establishment of the ectomesenchymal lineage within the cranial neural crest is of great significance. Fgfs are polypeptide growth factors with diverse functions in development and metabolism. Fgf20b knockdown zebrafish embryos showed dysplastic neurocranial and pharyngeal cartilages. Ectomesenchymal cells from cranial neural crest cells were significantly decreased in Fgf20b knockdown embryos, but cranial neural crest cells with a non-ectomesnchymal fate were increased. However, the proliferation and apoptosis of cranial neural crest cells were essentially unchanged. Fgfr1 knockdown embryos also showed dysplastic neurocranial and pharyngeal cartilages. The present findings indicate that Fgf20b is required for ectomesenchymal fate establishment via the activation of Fgfr1 in zebrafish.     

Activity of the RhoU/Wrch1 GTPase is critical for cranial neural crest cell migration

The neural crest (NC) is a stem cell-like population that arises at the border of neural and non-neural ectoderm. During development, NC undergoes an epithelio-mesenchymal transition (EMT), i.e. loss of epithelial junctions and acquisition of pro-migratory properties, invades the entire embryo and differentiates into a wide diversity of terminal tissues. We have studied the implication of Rho pathways in NC development and previously showed that RhoV is required for cranial neural crest (CNC) cell specification. We show here that the non-canonical Wnt response rhoU/wrch1 gene, closely related to rhoV, is also expressed in CNC cells but at later stages. Using both gain- and loss-of-function experiments, we demonstrate that the level of RhoU expression is critical for CNC cell migration and subsequent differentiation into craniofacial cartilages. In in vitro cultures, RhoU activates pathways that cooperate with PAK1 and Rac1 in epithelial adhesion, cell spreading and directional cell migration. These data support the conclusion that RhoU is an essential regulator of CNC cell migration.     

Regulation of neural crest cell populations: occurrence, distribution and underlying mechanisms

Regulation is a significant developmental event because successful cell proliferation and migration are critical to shaping young embryos. Regulation -- the replacement of undifferentiated embryonic cells by other cells in response to signals received from the environment -- is distinct from wound healing and regeneration. Investigations on regulation of neural crest cells span all vertebrates and have revealed that regulative ability varies both among classes (even species), and spatially and temporally within individuals. In general, there is greatest regulation for cranial neural crest cells, less for trunk, and virtually none forcardiac. Regulation also appears to be more complete at early embryonic stages. Fate-mapping studies have demonstrated that large regions of neural crest cells must be removed to generate missing or morphologically reduced structures. Recent studies reveal that less extensive neural crest cell extirpations result in normal morphology of cartilaginous and neuronal elements in the head, and normal development of pigmentation in the trunk. Ablation of cardiac neural crest cells frequently generates abnormalities of the heart, great vessels and parasympathetic nerve innervation. Decreased cell death, increased division, change in fate and altered migration are possible cellular mechanisms of regulation. In mostcases, the specific mechanisms of regulation are unknown, but a major premise underlying regulation is that cell potential is greater than cell fate. This concept was born from studies which demonstrated that some cells were able to express alternative fates if transplanted to a new environment. Among the potential cellular mechanisms for regulation, cell migration has received the most attention. Following ablation of neural crest cells, replacement neural crest cells migrate into gaps, most frequently from anterior/posterior locations. Cells from surrounding epidermal and neural ectoderm may have limited regulative ability, while compensation by cells from the ventral neural tube has been demonstrated to an even lesser extent. Regulation by such non-crest cells would require their transformation into neural crest cells. The potential for regulation of neural crest by placodal cells supports a closer relationship between neural crest and placodal ectoderm than previously recognized. Decreased cell death has been discussed primarily with reference to (1) cranial ganglia that have dual contributions from neural crest and placodal cells and (2) programmed cell death in rhombomeres three and five. Increased cell division in response to neural crest ablation is likely more common than has been reported, but this mechanism is difficult to interpret without a 3-D context for viewing how patterns of division differ from normal. Lastly, changes in cell fate may be the driving factor in regulation of embryonic cells. It has been repeatedly demonstrated thatcell potential is greaterthan cell fate. Once reliable mechanisms for assessing cell potential are established, we may find that fates are commonly altered in response to environmental signals. Regulation is therefore significant both as a basic developmental mechanism and as a mechanism for evolutionary change. The more labile the fate of embryonic cells, the more potential there is for maintaining existing characters and for generating new ones. According to Ettensohn (1992, p. 50), further analysis of such systems might . With regard to the neural crest, studies on regulation of this vital population of cells provide insight to the origin of the neural crest, to embryonic repair, and to the source of many craniofacial malformations, heart and other embryonic defects. (ABSTRACT TRUNCATED)     

Combined intrinsic and extrinsic influences pattern cranial neural crest migration and pharyngeal arch morphogenesis in axolotl

Cranial neural crest cells migrate in a precisely segmented manner to form cranial ganglia, facial skeleton and other derivatives. Here, we investigate the mechanisms underlying this patterning in the axolotl embryo using a combination of tissue culture, molecular markers, scanning electron microscopy and vital dye analysis. In vitro experiments reveal an intrinsic component to segmental migration; neural crest cells from the hindbrain segregate into distinct streams even in the absence of neighboring tissue. In vivo, separation between neural crest streams is further reinforced by tight juxtapositions that arise during early migration between epidermis and neural tube, mesoderm and endoderm. The neural crest streams are dense and compact, with the cells migrating under the epidermis and outside the paraxial and branchial arch mesoderm with which they do not mix. After entering the branchial arches, neural crest cells conduct an "outside-in" movement, which subsequently brings them medially around the arch core such that they gradually ensheath the arch mesoderm in a manner that has been hypothesized but not proven in zebrafish. This study, which represents the most comprehensive analysis of cranial neural crest migratory pathways in any vertebrate, suggests a dual process for patterning the cranial neural crest. Together with an intrinsic tendency to form separate streams, neural crest cells are further constrained into channels by close tissue apposition and sorting out from neighboring tissues.     

The fate of cranial neural crest cells in the Australian lungfish, Neoceratodus forsteri

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.     

Dual origins of the prechordal cranium in the chicken embryo

The prechordal cranium, or the anterior half of the neurocranial base, is a key structure for understanding the development and evolution of the vertebrate cranium, but its embryonic configuration is not well understood. It arises initially as a pair of cartilaginous rods, the trabeculae, which have been thought to fuse later into a single central stem called the trabecula communis (TC). Involvement of another element, the intertrabecula, has also been suggested to occur rostral to the trabecular rods and form the medial region of the prechordal cranium. Here, we examined the origin of the avian prechordal cranium, especially the TC, by observing the craniogenic and precraniogenic stages of chicken embryos using molecular markers, and by focal labeling of the ectomesenchyme forming the prechordal cranium. Subsequent to formation of the paired trabeculae, a cartilaginous mass appeared at the midline to connect their anterior ends. During this midline cartilage formation, we did not observe any progressive medial expansion of the trabeculae. The cartilages consisted of premandibular ectomesenchyme derived from the cranial neural crest. This was further divided anteroposteriorly into two portions, derived from two neural crest cell streams rostral and caudal to the optic vesicle, called preoptic and postoptic neural crest cells, respectively. Fate-mapping analysis elucidated that the postoptic neural crest cells were distributed exclusively in the lateroposterior part of the prechordal cranium corresponding to the trabeculae, whereas the preoptic stream of cells occupied the middle anterior part, differentiating into a cartilage mass corresponding to the intertrabecula. These results suggest that the central stem of the prechordal cranium of gnathostomes is composed of two kinds of distinct cartilaginous modules: a pair of trabeculae and a median intertrabecula, each derived from neural crest cells populating distinct places of the craniofacial primordia through specific migratory pathways.