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.     

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.     

Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development

Background

In vertebrates, the skeletal elements of the jaw, together with the connective tissues and tendons, originate from neural crest cells, while the associated muscles derive mainly from cranial mesoderm. Previous studies have shown that neural crest cells migrate in close association with cranial mesoderm and then circumscribe but do not penetrate the core of muscle precursor cells of the branchial arches at early stages of development, thus defining a sharp boundary between neural crest cells and mesodermal muscle progenitor cells. Tendons constitute one of the neural crest derivatives likely to interact with muscle formation. However, head tendon formation has not been studied, nor have tendon and muscle interactions in the head.

Methodology/Principal Findings

Reinvestigation of the relationship between cranial neural crest cells and muscle precursor cells during development of the first branchial arch, using quail/chick chimeras and molecular markers revealed several novel features concerning the interface between neural crest cells and mesoderm. We observed that neural crest cells migrate into the cephalic mesoderm containing myogenic precursor cells, leading to the presence of neural crest cells inside the mesodermal core of the first branchial arch. We have also established that all the forming tendons associated with branchiomeric and eye muscles are of neural crest origin and express the Scleraxis marker in chick and mouse embryos. Moreover, analysis of Scleraxis expression in the absence of branchiomeric muscles in Tbx1^−/− mutant mice, showed that muscles are not necessary for the initiation of tendon formation but are required for further tendon development.

Conclusions/Significance

This results show that neural crest cells and muscle progenitor cells are more extensively mixed than previously believed during arch development. In addition, our results show that interactions between muscles and tendons during craniofacial development are similar to those observed in the limb, despite the distinct embryological origin of these cell types in the head.     

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.     

Patterning of avian craniofacial muscles

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

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.     

Twist function is required for the morphogenesis of the cephalic neural tube and the differentiation of the cranial neural crest cells in the mouse embryo

Loss of Twist function in the cranial mesenchyme of the mouse embryo causes failure of closure of the cephalic neural tube and malformation of the branchial arches. In the Twist(-/-) embryo, the expression of molecular markers that signify dorsal forebrain tissues is either absent or reduced, but those associated with ventral tissues display expanded domains of expression. Dorsoventral organization of the mid- and hindbrain and the anterior-posterior pattern of the neural tube are not affected. In the Twist(-/-) embryo, neural crest cells stray from the subectodermal migratory path and the late-migrating subpopulation invades the cell-free zone separating streams of cells going to the first and second branchial arches. Cell transplantation studies reveal that Twist activity is required in the cranial mesenchyme for directing the migration of the neural crest cells, as well as in the neural crest cells within the first branchial arch to achieve correct localization. Twist is also required for the proper differentiation of the first arch tissues into bone, muscle, and teeth.     

The location and distribution of neural crest-derived Schwann cells in developing peripheral nerves in the chick forelimb.

The location and distribution of neural crest-derived Schwann cells during development of the peripheral nerves of chick forelimbs were examined using chick-quail chimeras. Neural crest cells were labeled by transplantation of the dorsal part of the neural tube from a quail donor to a chick host at levels of the neural tube destined to give rise to brachial innervation. The ventral roots, spinal nerves, and peripheral nerves innervating the chick forelimb were examined for the presence of quail-derived neural crest cells at several stages of embryonic development. These quail cells are likely to be Schwann cells or their precursors. Quail-derived Schwann cells were present in ventral roots and spinal nerves, and were distributed along previously described neural crest migratory pathways or along the peripheral nerve fibers at all stages of development examined. During early stages of wing innervation, quail-derived Schwann cells were not evenly distributed, but were concentrated in the ventral root and at the brachial plexus. The density of neural crest-derived Schwann cells decreased distal to the plexus, and no Schwann cells were ever seen in advance of the growing nerve front. When the characteristic peripheral nerve branching pattern was first formed, Schwann cells were clustered where muscle nerves diverged from common nerve trunks. In still older embryos, neural crest-derived Schwann cells were evenly distributed along the length of the peripheral nerves from the ventral root to the distal nerve terminations within the musculature of the forelimb. These observations indicate that Schwann cells accompany axons into the developing limb, but they do not appear to lead or direct axons to their targets. The transient clustering of neural crest-derived Schwann cells in the ventral root and at places where axon trajectories diverge from one another may reflect a response to some environmental feature within these regions.     

Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

Consequences of somite manipulation on the pattern of dorsal root ganglion development

We have investigated dorsal root ganglion formation, in the avian embryo, as a function of the composition of the paraxial somitic mesoderm. Three or four contiguous young somites were unilaterally removed from chick embryos and replaced by multiple cranial or caudal half-somites from quail embryos. Migration of neural crest cells and formation of DRG were subsequently visualized both by the HNK-1 antibody and the Feulgen nuclear stain. At advanced migratory stages (as defined by Teillet et al. Devl Biol. 120, 329-347 1987), neural crest cells apposed to the dorsolateral faces of the neural tube were distributed in a continuous, nonsegmented pattern that was indistinguishable on unoperated sides and on sides into which either half of the somites had been grafted. In contrast, ventrolaterally, neural crest cells were distributed segmentally close to the neural tube and within the cranial part of each normal sclerotome, whereas they displayed a nonsegmental distribution when the graft involved multiple cranial half-somites or were virtually absent when multiple caudal half-somites had been implanted. In spite of the identical dorsal distribution of neural crest cells in all embryos, profound differences in the size and segmentation of DRG were observed during gangliogenesis (E4-9) according to the type of graft that had been performed. Thus when the implant consisted of compound cranial half-somites, giant, coalesced ganglia developed, encompassing the entire length of the graft. On the other hand, very small, dorsally located ganglia with irregular segmentation were seen at the level corresponding to the graft of multiple caudal half-somites. We conclude that normal morphogenesis of dorsal root ganglia depends upon the craniocaudal integrity of the somites.     

Development of cephalic neural crest cells in embryos of Lampetra japonica, with special reference to the evolution of the jaw

Neural crest cells contribute extensively to vertebrate head morphogenesis and their origin is an important question to address in understanding the evolution of the craniate head. The distribution pattern of cephalic crest cells was examined in embryos of one of the living agnathan vertebrates, Lampetra japonica. The initial appearance of putative crest cells was observed on the dorsal aspect of the neural rod at stage 20.5 and ventral expansion of these cells was first seen at the level of rostral somites. As in gnathostomes, cephalic crest cells migrate beneath the surface ectoderm and form three major cell populations, each being separated at the levels of rhombomeres (r) 3 and r5. The neural crest seems initially to be produced at all neuraxial levels except for the rostral-most area, and cephalic crest cells are secondarily excluded from levels r3 and r5. Such a pattern of crest cell distribution prefigures the morphology of the cranial nerve anlage. The second or middle crest cell population passes medial to the otocyst, implying that the otocyst does not serve as a barrier to separate the crest cell populations. The three cephalic crest cell populations fill the pharyngeal arch ventrally, covering the pharyngeal mesoderm laterally with the rostral-most population covering the premandibular region and mandibular arch. The third cell population is equivalent to the circumpharyngeal crest cells in the chick, and its influx into the pharyngeal region precedes the formation of postotic pharyngeal arches. Focal injection of DiI revealed the existence of an anteroposterior organization in the neural crest at the neurular stage, destined for each pharyngeal region. The crest cells derived from the posterior midbrain that express the LjOtxA gene, the Otx2 cognate, were shown to migrate into the mandibular arch, a pattern which is identical to gnathostome embryos. It was concluded that the head region of the lamprey embryo shares a common set of morphological characters with gnathostome embryos and that the morphological deviation of the mandibular arch between the gnathostomes and the lamprey is not based on the early embryonic patterning.     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

Embryonic origin of avian corneal sensory nerves.

Sensory nerves play a vital role in maintaining corneal transparency. They originate in the trigeminal ganglion, which is derived from two embryonic cell populations (cranial neural crest and ectodermal placode). Nonetheless, it is unclear whether corneal nerves arise from neural crest, from placode, or from both. Quail-chick chimeras and species-specific antibodies allowed tracing quail-derived neural crest or placode cells during trigeminal ganglion and corneal development, and after ablation of either neural crest or placode. Neural crest chimeras showed quail nuclei in the proximal part of the trigeminal ganglion, and quail nerves in the pericorneal nerve ring and in the cornea. In sharp contrast, placode chimeras showed quail nuclei in the distal part of the trigeminal ganglion, but no quail nerves in the cornea or in the pericorneal nerve ring. Quail placode-derived nerves were present, however, in the eyelids. Neural crest ablation between stages 8 and 9 resulted in diminished trigeminal ganglia and absence of corneal innervation. Ablation of placode after stage 11 resulted in loss of the ophthalmic branch of the trigeminal ganglion and reduced corneal innervation. Noninnervated corneas still became transparent. These results indicate for the first time that although both neural crest and placode contribute to the trigeminal ganglion, corneal innervation is entirely neural crest-derived. Nonetheless, proper corneal innervation requires presence of both cell types in the embryonic trigeminal ganglion. Also, complete lack of innervation has no discernible effect on development of corneal transparency or cell densities.     

Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite

The ventro-medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso-lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri-potency of somite cell differentiation, apparent pluri-potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4-day chimera embryos and to form muscle and dermal tissue in 9-day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.     

Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras.

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.     

The distribution of neural crest-derived Schwann cells from subsets of brachial spinal segments into the peripheral nerves innervating the chick forelimb.

Neural crest cells from brachial levels of the neural tube populate the ventral roots, spinal nerves, and peripheral nerves of the chick forelimb where they give rise to Schwann cells. The distribution of neural crest cells in the developing forelimb was examined using homotopic and heterotopic chick-quail chimeras to label neural crest cells from subsets of the brachial spinal segments. Neural crest cells from particular regions of the spinal cord populated ventral roots and spinal nerves adjacent to or immediately posterior to the graft. Crest cells also populated the brachial plexus in accord with their segmental origins. In the forelimb, neural crest cells populated muscle nerves with anterior brachial spinal segments populating nerves to anterior musculature of the forelimb and posterior brachial spinal segments populating nerves to posterior musculature. Similar patterns were seen following both homotopic and heterotopic transplantation. In both types of grafts, the distribution of neural crest cells largely matched the sensory and motor projection pattern from the same spinal segmental level. This suggests that neural crest-derived Schwann cells from a particular spinal segment may use sensory and motor fibers emerging from the same segmental level as substrates to guide their migration into the periphery.     

A novel role for retinoids in patterning the avian forebrain during presomite stages

Retinoids, and in particular retinoic acid (RA), are known to induce posterior fates in neural tissue. However, alterations in retinoid signalling dramatically affect anterior development. Previous reports have demonstrated a late role for retinoids in patterning craniofacial and forebrain structures, but an earlier role in anterior patterning is not well understood. We show that enzymes involved in synthesizing retinoids are expressed in the avian hypoblast and in tissues directly involved in head patterning, such as anterior definitive endoderm and prechordal mesendoderm. We found that in the vitamin A-deficient (VAD) quail model, which lacks biologically active RA from the first stages of development, anterior endodermal markers such as Bmp2, Bmp7, Hex and the Wnt antagonist crescent are affected during early gastrulation. Furthermore, prechordal mesendodermal and prospective ventral telencephalic markers are expanded posteriorly, Shh expression in the axial mesoderm is reduced, and Bmp2 and Bmp7 are abnormally expressed in the ventral midline of the neural tube. At early somite stages, VAD embryos have increased cell death in ventral neuroectoderm and foregut endoderm, but normal cranial neural crest production, whereas at later stages extensive apoptosis occurs in head mesenchyme and ventral neuroectoderm. As a result, VAD embryos end up with a single and reduced telencephalic vesicle and an abnormally patterned diencephalon. Therefore, we propose that retinoids have a dual role in patterning the anterior forebrain during development. During early gastrulation, RA acts in anterior endodermal cells to modulate the anteroposterior (AP) positional identity of prechordal mesendodermal inductive signals to the overlying neuroectoderm. Later on, at neural pore closure, RA is required for patterning of the mesenchyme of the frontonasal process and the forebrain by modulating signalling molecules involved in craniofacial morphogenesis.     

Effect of hyaluronidase treatment on the distribution of cranial neural crest cells in the chick embryo

The cranial paraxial mesoblast is patterned into segmental units termed somitomeres. Recently we demonstrated the morphological relationship between the migratory pathways of cranial neural crest cells and the patterned primary mesenchyme of chick embryos (Anderson and Meier, '81). Since extracellular matrix, particularly hyaluronate, is also distributed in cranial crest pathways, embryos were given sub-blastodisc injections of hyaluronidase just prior to neural tube fusion and neural crest migration to remove matrix. Histological sections of enzyme-treated embryos showed that Alcian blue staining of hyaluronate was significantly reduced. Surface ectoderm appeared collapsed on the subjacent mesoderm as well. Examination of embryos with the scanning electron microscope (SEM) revealed that paraxial mesoderm remained segmentally patterned even though it appeared more condensed because of a reduction in intercellular space between mesenchymal cells. In enzyme-treated embryos, the rostral crest cells spread over the dorsal surfaces of the first four somitomeres, as they would do normally. This distribution of neural crest cells occurs even when enzyme treatment interferes with neural tube fusion at that level. We conclude that 1) neural tube fusion is not a prerequisite for the timely release of cranial crest in the chick embryo and 2) that much of the organized hyaluronate-rich matrix that lies in the path of cranial crest is not essential for crest emigration or patterned distribution.     

CEPU-1 expression in the early embryonic chick brain

We describe the expression pattern of CEPU-1, a cell adhesion molecule of the immunoglobulin superfamily, in the early chick embryo brain. An initially broad domain of expression, encompassing forebrain, midbrain and anterior hindbrain, is subsequently narrowed down to a ring-shaped domain at the midbrain-hindbrain boundary, co-localizing precisely with the expression of Wnt1 at the isthmus. In addition, CEPU-1 is expressed in the dorsal aspect of rhombomere 4 and its emigrating neural crest cells. Later in development, we also find CEPU-1 expression in other parts of the developing nervous system such as sensory ganglia and in the ventral aspect of forebrain, midbrain and hindbrain.