Heterochronic shifts during early cranial neural crest cell migration in two ranid frogs

We describe the development of the cranial neural crest cell streams relative to embryonic events such as neural tube formation and somite appearance in two Eurasian frog species belonging to the Ranidae, Rana temporaria and Sylvirana nigrovittata, and demonstrate developmental heterochronies. The mandibular stream appeared well developed in R. temporaria at a time when the embryo was still spherical, the neural folds were elevated, and the neural plate was wide open, thus earlier than known from any frog species so far. The appearance of the second stream and its division into hyoid and branchial portions was clearly accelerated in R. temporaria relative to other embryonic events when compared to S. nigrovittata. For example, in R. temporaria, the hyoid and branchial portions of the cranial neural crest cell streams were separated before the neural folds had started to fuse, whereas in S. nigrovittata this event took place only after the neural folds had fused completely. Such ostentatious heterochronies related to the characters used herein have formerly only been reported from comparisons between species belonging to different higher taxa. Our results re‐confirm that to understand the full dynamics of the evolution of development, studies need to implement comparative embryological approaches, and include phylogenetically relatively closely related taxa.     

Life, death and Sonic hedgehog

The secreted glycoprotein Sonic hedgehog (SHH), a vertebrate homologue of the Drosophila segment polarity gene Hedgehog, is essential for the development of diverse tissues during embryogenesis. Studies of SHH function during neural tube and somite development have focused on its role in specifying the dorsoventral polarity of these structures, but a recent report by Ahlgren and Bronner-Fraser(1) supports the possibility that SHH has additional functions in cell survival and cell proliferation. Perturbation of SHH signaling after the early dorsoventral specification of the cranial neural tube leads to increased cell death in both the neural tube and the neural crest. This implies that SHH is continually required as a trophic and/or mitogenic factor during brain development, and expands the variety of cellular responses to SHH signaling. BioEssays 22:499-502, 2000.     

The differing effects of occipital and trunk somites on neural development in the chick embryo

In all higher vertebrate embryos the sensory ganglia of the trunk develop adjacent to the neural tube, in the cranial halves of the somite-derived sclerotomes. It has been known for many years that ganglia do not develop in the most cranial (occipital) sclerotomes, caudal to the first somite. Here we have investigated whether this is due to craniocaudal variation in the neural tube or crest, or to an unusual property of the sclerotomes at occipital levels. Using the monoclonal antibody HNK-1 as a marker for neural crest cells in the chick embryo, we find that the crest does enter the cranial halves of the occipital sclerotomes. Furthermore, staining with zinc iodide/osmium tetroxide shows that some of these crest-derived cells sprout axons within these sclerotomes. By stage 23, however, no dorsal root ganglia are present within the five occipital sclerotomes, as assessed both by haematoxylin/eosin and zinc iodide/osmium tetroxide staining. Moreover, despite this loss of sensory cells, motor axons grow out in these segments, many of them later fasciculating to form the hypoglossal nerve. The sclerotomes remain visible until stages 27/28, when they dissociate to form the base of the skull and the atlas and axis vertebrae. After grafting occipital neural tube from quail donor embryos in place of trunk neural tube in host chick embryos, quail-derived ganglia do develop in the trunk sclerotomes. This shows that the failure of occipital ganglion development is not the result of some fixed local property of the neural crest or neural tube at occipital levels. We therefore suggest that in the chick embryo the cranial halves of the five occipital sclerotomes lack factors essential for normal sensory ganglion development, and that these factors are correspondingly present in all the more caudal sclerotomes.     

Critical numbers of neural crest cells are required in the pathways from the neural tube to the foregut to ensure complete enteric nervous system formation

The enteric nervous system (ENS) is mainly derived from vagal neural crest cells (NCC) that arise at the level of somites 1-7. To understand how the size and composition of the NCC progenitor pool affects ENS development, we reduced the number of NCC by ablating the neural tube adjacent to somites 3-6 to produce aganglionic gut. We then back-transplanted various somite lengths of quail neural tube into the ablated region to determine the 'tipping point', whereby sufficient progenitors were available for complete ENS formation. The addition of one somite length of either vagal, sacral or trunk neural tube into embryos that had the neural tube ablated adjacent to somites 3-6, resulted in ENS formation along the entire gut. Although these additional cells contributed to the progenitor pool, the quail NCC from different axial levels retained their intrinsic identities with respect to their ability to form the ENS; vagal NCC formed most of the ENS, sacral NCC contributed a limited number of ENS cells, and trunk NCC did not contribute to the ENS. As one somite length of vagal NCC was found to comprise almost the entire ENS, we ablated all of the vagal neural crest and back-transplanted one somite length of vagal neural tube from the level of somite 1 or somite 3 into the vagal region at the position of somite 3. NCC from somite 3 formed the ENS along the entire gut, whereas NCC from somite 1 did not. Intrinsic differences, such as an increased capacity for proliferation, as demonstrated in vitro and in vivo, appear to underlie the ability of somite 3 NCC to form the entire ENS.     

Cranial neural crest-cell migration in the direct-developing frog, Eleutherodactylus coqui: molecular heterogeneity within and among migratory streams

Direct development is a specialized reproductive mode that has evolved repeatedly in many different lineages of amphibians, especially anurans. A fully formed, albeit miniature adult hatches directly from the egg; there is no free-living larva. In many groups, the evolution of direct development has had profound consequences for cranial development and morphology, including many components that are derived from the embryonic neural crest. Yet, the developmental bases of these effects remain poorly known. In order to more fully characterize these changes, we used three molecular markers to analyze cranial neural crest-cell emergence and migration in the direct-developing frog, Eleutherodactylus coqui: HNK-1 immunoreactivity, Dlx protein expression, and cholinesterase activity. Our study validates and extends earlier results showing that the comprehensive changes in embryonic cranial patterning, differentiation, and developmental timing that are associated with direct development in Eleutherodactylus have not affected gross features of cranial neural crest biology: the relative timing of crest emergence and the number, configuration and identity of the principal migratory streams closely resemble those seen in metamorphic anurans. The three markers are variably expressed within and among neural crest-cell populations. This variation suggests that determination of cranial neural crest-cells may already have begun at or soon after the onset of migration, when the cells emerge from the neural tube. It is not known how or even if this variation correlates with differential cell lineage or fate. Finally, although HNK-1 expression is widely used to study neural crest migration in teleost fishes and amniotes, E. coqui is the only amphibian known in which it effectively labels migrating neural crest-cells. There are not enough comparative data to determine whether this feature is functionally associated with direct development or is instead unrelated to reproductive mode.     

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.     

Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.     

Overlapping origins of pharyngeal arch crest cells on the postotic hind-brain

The developing hind-brain of vertebrates consists of segmental units called rhombomeres. Although crest cells emigrate from the hind-brain, they are subsequently subdivided into several cell populations that are attached to restricted regions of the hind-brain. At the preotic level, only even-numbered rhombomeres are accompanied by crest cells, while the odd-numbered ones are not. At the postotic level, such the birhombomeric repetition becomes obscure. In order to map the origins and distributions of postotic crest cells, focal injections of Dil were made into various axial levels of the postotic neural tube. Cephalic crest cells at the postotic level first form a single cell population deposited by cells along the dorsolateral pathway. They are called the circumpharyngeal crest cells (CP cells) and are secondarily subdivided into each pharyngeal arch ectomesenchyme. The neural tube extending from r5 to the somite 3/4 boundary gave rise to CP cells. The neuraxial origins of each pharyngeal ectomesenchyme extended for more than three somite lengths, most of which overlapped with the other. Unlike in the preotic region, there is no segmental registration between neuraxial levels and pharyngeal arches. Caudal portions of the CP cell population show a characteristic distribution pattern that circumscribes the postotic pharyngeal arches caudally. Heterotopic transplantation of the Dil-labeled neural crest into the somite 3 level resulted in a distribution of labeled cells similar to that of CP cells, suggesting that the pattern of distribution depends upon dynamic modification of the body wall associated with pharyngeal arch formation.     

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.     

Model systems for the study of heart development and disease. Cardiac neural crest and conotruncal malformations

Neural crest cells are multipotential cells that delaminate from the dorsal neural tube and migrate widely throughout the body. A subregion of the cranial neural crest originating between the otocyst and somite 3 has been called "cardiac neural crest" because of the importance of these cells in heart development. Much of what we know about the contribution and function of the cardiac neural crest in cardiovascular development has been learned in the chick embryo using quail-chick chimeras to study neural crest migration and derivatives as well as using ablation of premigratory neural crest cells to study their function. These studies show that cardiac neural crest cells are absolutely required to form the aorticopulmonary septum dividing the cardiac arterial pole into systemic and pulmonary circulations. They support the normal development and patterning of derivatives of the caudal pharyngeal arches and pouches, including the great arteries and the thymus, thyroid and parathyroids. Recently, cardiac neural crest cells have been shown to modulate signaling in the pharynx during the lengthening of the outflow tract by the secondary heart field. Most of the genes associated with cardiac neural crest function have been identified using mouse models. These studies show that the neural crest cells may not be the direct cause of abnormal cardiovascular development but they are a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Since, cardiac neural crest cells span from the caudal pharynx into the outflow tract, they are especially susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations as represented by the DiGeorge syndrome will necessarily require understanding development of the cardiac neural crest.     

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.     

Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp2H) mutant effect

A sub-population of the neural crest is known to play a crucial role in development of the cardiac outflow tract. Studies in avians have mapped the complete migratory pathways taken by 'cardiac' neural crest cells en route from the neural tube to the developing heart. A cardiac neural crest lineage is also known to exist in mammals, although detailed information on its axial level of origin and migratory pattern are lacking. We used focal cell labelling and orthotopic grafting, followed by whole embryo culture, to determine the spatio-temporal migratory pattern of cardiac neural crest in mouse embryos. Axial levels between the post-otic hindbrain and somite 4 contributed neural crest cells to the heart, with the neural tube opposite somite 2 being the most prolific source. Emigration of cardiac neural crest from the neural tube began at the 7-somite stage, with cells migrating in pathways dorsolateral to the somite, medial to the somite, and between somites. Subsequently, cardiac neural crest cells migrated through the peri-aortic mesenchyme, lateral to the pharynx, through pharyngeal arches 3, 4 and 6, and into the aortic sac. Colonisation of the outflow tract mesenchyme was detected at the 32-somite stage. Embryos homozygous for the Sp2H mutation show delayed onset of cardiac neural crest emigration, although the pathways of subsequent migration resembled wild type. The number of neural crest cells along the cardiac migratory pathway was significantly reduced in Sp2H/Sp2H embryos. To resolve current controversy over the cell autonomy of the splotch cardiac neural crest defect, we performed reciprocal grafts of premigratory neural crest between wild type and splotch embryos. Sp2H/Sp2H cells migrated normally in the +/+ environment, and +/+ cells migrated normally in the Sp2H/Sp2H environment. In contrast, retarded migration along the cardiac route occurred when either Sp2H/+ or Sp2H/Sp2H neural crest cells were grafted into the Sp2H/Sp2H environment. We conclude that the retardation of cardiac neural crest migration in splotch mutant embryos requires the genetic defect in both neural crest cells and their migratory environment.     

Cloning and characterization of chicken α5 integrin: Endogenous and experimental expression in early chicken embryos

Key roles for fibronectin and its integrin receptors have been postulated in the multiple cell-matrix interactions essential for chick embryo morphogenesis. However, mechanistic studies of these processes have been hampered by the current absence of sequence data and chicken cDNA clones for the major fibronectin receptor subunit, integrin α5 (ITGA5). We report here the sequence, endogenous expression pattern, and transfection of full-length chicken integrin α5. During early chicken embryonic development, α5 is highly expressed in cranial neural folds and migrating neural crest cells, suggesting potential roles in neural crest formation and migration. In fact, over-expression of this integrin in early neural tube selectively induces BMP5, a growth factor recently implicated in neural crest formation. Availability of these α5 integrin tools should facilitate studies of its functions in early embryonic development.     

In vitro analysis of sympathetic neuron differentiation from chick neural crest cells

Sympathetic neuron differentiation was studied using a fluorescence histochemical assay to detect the appearance of cell-bound catecholamines. Results from in vitro organ cultures indicate that chick neural crest cells must interact with both ventral neural tube (defined throughout as the ventral neural tube plus the notochord) and somitic mesenchyme in order to differentiate into sympathoblasts. Somite, ventral neural tube, and crest were cultured transfilter in various combinations to define these tissue interactions more precisely. Results from these experiments indicate that neural crest cells must be contiguous to somite in order to differentiate into sympathoblasts, but ventral neural tube may act across a Millipore filter membrane (type TH, 25 μm thick) either on somite, crest, or both. To distinguish among these possibilities, somite was cultured transfilter to ventral tube for a short period, after which ventral tube was removed and fresh crest was added to the somite. The results from this and other experiments support the hypothesis that the ventral tube does not act directly on crest cells, but elicits a developmental change in somitic mesenchyme, which then promotes sympathoblast differentiation. To study the relationship of nerve growth factor (NGF) to the differentiation of sympathetic neurons, cultures of somite + crest were temporarily exposed transfilter to ventral tube, in the presence or the absence of exogenous NGF. The results of these and other experiments are consistent with the hypothesis that the continued presence of ventral tube is required to ensure the survival of the differentiating sympathetic neurons. With respect to this second function, ventral tube can be replaced by exogenous NGF.     

Development of the ethmoidal structures of the endocranium in Discoglossus pictus (Anura: Discoglossidae)

We use histological techniques and computer‐aided three‐dimensional reconstructions made from serial histological sections to describe the ontogeny of the ethmoidal endocranium of discoglossid frog Discoglossus pictus. We identify a pattern of development for the suprarostral cartilage that differs from previous findings and probably represents the ancestral anuran pattern. The nasal cartilages, including the inferior prenasal cartilage, are de novo adult structures. The only larva‐derived structures of the adult nasal capsules are the posterior aspects of the solum nasi and septum nasi. We also identify patterns of development for the ethmoid plate and postnasal wall that occur during early in ontogenesis. These patterns are associated with development events during metamorphic climax. The pattern of timing of chondrification of the anterior nasal cartilages more closely coincides with that of the neobatrachian species than that recorded for the pelobatid frog Spea. In addition, this study supports a sister taxon relationship between Discoglossus and Alytes. J. Morphol. 271:1078‐1093, 2010. © 2010 Wiley‐Liss, Inc.     

Notochordal and foregut abnormalities correlate with elevated neural crest apoptosis in Patch embryos

Although Patch mutants show severe abnormalities in many neural crest-derived structures including the face and the heart, there is a paucity of information characterizing the mechanisms underlying these congenital defects. Via manipulating the genetic background to circumvent early embryonic lethality, our results revealed that Patch phenotypes are most likely due to a significant decrease in migratory neural crest lineage due to diminished neural crest survival and elevated apoptosis. Homozygous mutant neural crest precursors can undergo typical expansion within the neural tube, epithelial-to-mesenchymal transformation, and initiate normal neural crest emigration. Moreover, in vitro explant culture demonstrated that when isolated from the surrounding mesenchyme, Patch mutant neural crest cells (NCCs) can migrate appropriately. Additionally, Patch foregut, notochord and somitic morphogenesis, and Sonic hedgehog expression profiles were all perturbed. Significantly, the timing of lethality and extent of apoptosis correlated with the degree of severity of Patch mutant foregut, notochord, and somite dysfunction. Finally, analysis of Balb/c-enriched surviving Patch mutants revealed that not all the neural crest subpopulations are affected and that Patch mutant neural crest-derived sympathetic ganglia and dorsal root ganglia were unaffected. We hypothesize that loss of normal coordinated signaling from the notochord, foregut, and somites underlies the diminished survival of the neural crest lineage within Patch mutants resulting in subsequent neural crest-deficient phenotypes.     

Mapping of neural crest pathways in Xenopus laevis using inter- and intra-specific cell markers

This study examines the pathways of migration followed by neural crest cells in Xenopus embryos using two recently described cell marking techniques. The first is an interspecific chimera created by grafting Xenopus borealis cells into Xenopus laevis hosts. The cells of these closely related species can be distinguished by their nuclear dimorphism. The second type of marker is created by microinjection of lysinated dextrans into fertilized eggs which can then be used for intraspecific grafting. These recently developed fluorescent dyes are fixable and identifiable in both living and fixed embryos. After grafting labeled donor neural tubes into unlabeled host embryos, the distribution of neural crest cells at various stages after grafting was used to define the pathways of neural crest migration. To control for possible grafting artifacts, fluorescent lysinated dextran was injected into a single blastomere which gives rise to a large number of neural crest cells, thereby labeling the neural crest without grafting. By all three techniques, Xenopus neural crest cells were observed along two predominant pathways in the trunk. The majority of neural crest cells were observed along a "ventral" route, between the neural tube and somite, the notochord and somite, and along the dorsal mesentery. A second group of neural crest cells was observed "dorsally" where they populated the dorsal fin. A third minor "lateral" pathway was observed primarily in borealis/laevis chimerae and in blastomere-injected embryos; some neural crest cells were observed underneath the ectoderm lateral to the neural tube. Along the rostrocaudal axis, neural crest cells were not continuously distributed but were primarily located across from the caudal two-thirds of the somite. Fewer than 3% of the neural crest cells were observed across from the rostral third of each somite. When grafted to ventral locations, neural crest cells were not able to migrate dorsally but migrated laterally along the dorsal mesentery. Labeled neural crest cells gave rise to cells of the spinal, sympathetic, and enteric ganglia as well as to adrenal chromaffin cells, Schwann cells, pigment cells, mesenchymal cells of the dorsal fin, and some cells in the integuments and in the region of the pronephros. These results show that the neural crest migratory pathways in Xenopus differ from those in the avian embryo. In avians NC cells migrate as a closely associated sheet of cells while in Xenopus they migrate as individual cells. Both species exhibit a metamerism in the neural crest cell distribution pattern along the rostrocaudal axis.(ABSTRACT TRUNCATED AT 400 WORDS)