A monoclonal antibody against a laminin-heparan sulfate proteoglycan complex perturbs cranial neural crest migration in vivo

INO (inhibitor of neurite outgrowth) is a monoclonal antibody that blocks axon outgrowth, presumably by functionally blocking a laminin-heparan sulfate proteoglycan complex (Chiu, A. Y., W. D. Matthew, and P. H. Patterson. 1986. J. Cell Biol. 103: 1382-1398). Here the effect of this antibody on avian neural crest cells was examined by microinjecting INO onto the pathways of cranial neural crest migration. After injection lateral to the mesencephalic neural tube, the antibody had a primarily unilateral distribution. INO binding was observed in the basal laminae surrounding the neural tube, ectoderm, and endoderm, as well as within the cranial mesenchyme on the injected side of the embryo. This staining pattern was indistinguishable from those observed with antibodies against laminin or heparan sulfate proteoglycan. The injected antibody remained detectable for 18 h after injection, with the intensity of immuno-reactivity decreasing with time. Embryos ranging from the neural fold stage to the 9-somite stage were injected with INO and subsequently allowed to survive for up to 1 d after injection. These embryos demonstrated severe abnormalities in cranial neural crest migration. The predominant defects were ectopic neural crest cells external to the neural tube, neural crest cells within the lumen of the neural tube, and neural tube deformities. In contrast, embryos injected with antibodies against laminin or heparan sulfate proteoglycan were unaffected. When embryos with ten or more somites were injected with INO, no effects were noted, suggesting that embryos are sensitive for only a limited time during their development. Immunoprecipitation of the INO antigen from 2-d chicken embryos revealed a 200-kD band characteristic of laminin and two broad smears between 180 and 85 kD, which were resolved into several bands at lower molecular mass after heparinase digestion. These results indicate that INO precipitates both laminin and proteoglycans bearing heparan sulfate residues. Thus, microinjection of INO causes functional blockage of a laminin-heparan sulfate proteoglycan complex, resulting in abnormal cranial neural crest migration. This is the first evidence that a laminin-heparan sulfate proteoglycan complex is involved in aspects of neural crest migration in vivo.     

Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of laminin and collagens during avian neural crest development

The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscle. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues. The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and lamin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development. These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny. (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration. (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis. (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.     

In vivo evidence for short- and long-range cell communication in cranial neural crest cells

The proper assembly of craniofacial structures and the peripheral nervous system requires neural crest cells to emerge from the neural tube and navigate over long distances to the branchial arches. Cell and molecular studies have shed light on potential intrinsic and extrinsic cues, which, in combination, are thought to ensure the induction and specification of cranial neural crest cells. However, much less is known about how migrating neural crest cells interpret and integrate signals from the microenvironment and other neural crest cells to sort into and maintain the stereotypical pattern of three spatially segregated streams. Here, we explore the extent to which cranial neural crest cells use cell-to-cell and cell-environment interactions to pathfind. The cell membrane and cytoskeletal elements in chick premigratory neural crest cells were labeled in vivo. Three-dimensional reconstructions of migrating neural crest cells were then obtained using confocal static and time-lapse imaging. It was found that neural crest cells maintained nearly constant contact with other migrating neural crest cells, in addition to the microenvironment. Cells used lamellipodia or short, thin filopodia (1-2 microm wide) for local contacts (<20 microm). Non-local, long distance contact (up to 100 microm) was initiated by filopodia that extended and retracted, extended and tracked, or tethered two non-neighboring cells. Intriguingly, the cell-to-cell contacts often stimulated a cell to change direction in favor of a neighboring cell's trajectory. In summary, our results present in vivo evidence for local and long-range neural crest cell interactions, suggesting a possible role for these contacts in directional guidance.     

BMP signaling is essential for development of skeletogenic and neurogenic cranial neural crest

BMP signaling is essential for a wide variety of developmental processes. To evaluate the role of Bmp2/4 in cranial neural crest (CNC) formation or differentiation after its migration into the branchial arches, we used Xnoggin to block their activities in specific areas of the CNC in transgenic mice. This resulted in depletion of CNC cells from the targeted areas. As a consequence, the branchial arches normally populated by the affected neural crest cells were hypomorphic and their skeletal and neural derivatives failed to develop. In further analyses, we have identified Bmp2 as the factor required for production of migratory cranial neural crest. Its spatial and temporal expression patterns mirror CNC emergence and Bmp2 mutant embryos lack both branchial arches and detectable migratory CNC cells. Our results provide functional evidence for an essential role of BMP signaling in CNC development.     

An assay system to study migratory behavior of cranial neural crest cells in Xenopus

An increasing number of genes are known to show expression in the cranial neural crest area. So far it is very difficult to analyze their effect on neural crest cell migration because of the lack of transplantation techniques. This paper presents a simple method to study the migratory behavior of cranial neural crest cells by homo- and heterotopic transplantations: Green fluorescent protein (GFP) RNA was injected into one blastomere of Xenopus laevis embryos at the 2-cell stage. The cranial neural crest area of stage 14 embryos was transplanted into the head or trunk region of an uninjected host embryo, and the migration was monitored by GFP fluorescence. The transplants were further examined by double immunostaining and confocal microscopy to trace migratory routes inside the embryo, and to exclude contaminations of grafts with foreign tissues. Our results demonstrate that we developed a highly efficient and reproducible technique to study the migratory ability of cranial neural crest cells. It offers the possibility to analyze genes involved in neural crest cell migration by coinjecting their RNA with that of GFP. Received: 28 September 1999 / Accepted: 17 November 1999     

In vivo analysis reveals a critical role for neuropilin-1 in cranial neural crest cell migration in chick

The neural crest provides an excellent model system to study invasive cell migration, however it is still unclear how molecular mechanisms direct cells to precise targets in a programmed manner. We investigate the role of a potential guidance factor, neuropilin-1, and use functional knockdown assays, tissue transplantation and in vivo confocal time-lapse imaging to analyze changes in chick cranial neural crest cell migratory patterns. When neuropilin-1 function is knocked down in ovo, neural crest cells fail to fully invade the branchial arches, especially the 2nd branchial arch. Time-lapse imaging shows that neuropilin-1 siRNA transfected neural crest cells stop and collapse filopodia at the 2nd branchial arch entrances, but do not die. This phenotype is cell autonomous. To test the influence of population pressure and local environmental cues in driving neural crest cells to the branchial arches, we isochronically transplanted small subpopulations of DiI-labeled neural crest cells into host embryos ablated of neighboring, premigratory neural crest cells. Time-lapse confocal analysis reveals that the transplanted cells migrate in narrow, directed streams. Interestingly, with the reduction of neuropilin-1 function, neural crest cells still form segmental migratory streams, suggesting that initial neural crest cell migration and invasion of the branchial arches are separable processes.     

Isotretinoin embryopathy and the cranial neural crest: an in vivo and in vitro study

Severe congenital malformations have been associated with the inadvertant use in early pregnancy of a new dermatological drug, isotretinoin. We present proposals for the pathogenesis of this embryopathy based on the study of animal models. The characteristic malformations of the face, thymus, and great vessels were induced in mice by prenatal exposure to the drug during the early somite stages of development. From histological examination of mouse embryos it was shown that the drug directly interferes with the development of cranial neural crest cells. Subsequent deficiency of crest cell-derived mesenchyme adequately explains most of the observed malformations. Rat embryo culture studies showed that, when used at concentrations of 500 ng/ml, both isotretinoin and its main metabolite in the human, 4-oxo-isotretinoin, induce malformations similar to those seen in vivo. Since during normal repetitive dosing in the human the mean trough blood concentration of isotretinoin ranges from 132 to 196 ng/ml, while 4-oxo-isotretinoin ranges from 610 to 791 ng/ml, it is likely that the metabolite plays a major role in the induction of the isotretinoin embryopathy.     

The development and distribution of the cranial neural crest in the rat embryo

Summary The head region of rat embryos was investigated by scanning electron microscopy after removal of the surface ectoderm with adhesive tape. Observations were made in embryos from 6-somite to 11-somite stages of development, in order to determine: (1) the sequence of emigration of neural crest cells from the different regions of the future brain; (2) the appearance of crest cells before, during, and after their conversion from an epithelial to a mesenchymal form; (3) the migration pathways.Emigration occurs first from the midbrain, and next from the rostral hindbrain; crest cells from these two regions migrate into the first visceral arch. Subsequently cells emigrate from the caudal hindbrain, but not in a rostrocaudal sequence. At the time of crest cell emigration, the neural fold morphology varies from a slightly convex, widely open plate (midbrain) to a closed tube (caudal hindbrain). Thus the timing of emigration is related neither to age (as reflected in rostrocaudal levels) nor to morphology of the neural epithelium.     

Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.

Cranial neural crest (CNC) cells migrate extensively, typically in a pattern of cell streams. In Xenopus, these cells express the adhesion molecule Xcadherin-11 (Xcad-11) as they begin to emigrate from the neural fold. In order to study the function of this molecule, we have overexpressed wild-type Xcad-11 as well as Xcad-11 mutants with cytoplasmic (deltacXcad-11) or extracellular (deltaeXcad-11) deletions. Green fluorescent protein (GFP) was used to mark injected cells. We then transplanted parts of the fluorescent CNC at the premigratory stage into non-injected host embryos. This altered not only migration, but also the expression of neural crest markers. Migration of transplanted cranial neural crest cells was blocked when full-length Xcad-11 or its mutant lacking the beta-catenin-binding site (deltacXcad-11) was overexpressed. In addition, the expression of neural crest markers (AP-2, Snail and twist) diminished within the first four hours after grafting, and disappeared completely after 18 hours. Instead, these grafts expressed neural markers (2G9, nrp-I and N-Tubulin). Beta-catenin co-expression, heterotopic transplantation of CNC cells into the pharyngeal pouch area or both in combination failed to prevent neural differentiation of the grafts. By contrast, deltaeXcad-11 overexpression resulted in premature emigration of cells from the transplants. The AP-2 and Snail patterns remained unaffected in these migrating grafts, while twist expression was strongly reduced. Co-expression of deltaeXcad-11 and beta-catenin was able to rescue the loss of twist expression, indicating that Wnt/beta-catenin signalling is required to maintain twist expression during migration. These results show that migration is a prerequisite for neural crest differentiation. Endogenous Xcad-11 delays CNC migration. Xcad-11 expression must, however, be balanced, as overexpression prevents migration and leads to neural marker expression. Although Wnt/beta-catenin signalling is required to sustain twist expression during migration, it is not sufficient to block neural differentiation in non-migrating grafts.     

Fate of cranial neural crest cells during craniofacial development in endothelin-A receptor-deficient mice

Most of the bone, cartilage and connective tissue of the lower jaw is derived from cranial neural crest cells (NCCs) arising from the posterior midbrain and hindbrain. Multiple factors direct the patterning of these NCCs, including endothelin-1-mediated endothelin A receptor (Edn1/Ednra) signaling. Loss of Ednra signaling results in multiple defects in lower jaw and neck structures, including homeotic transformation of lower jaw structures into upper jaw-like structures. However, since the Ednra gene is expressed by both migrating and post-migrating NCCs, the actual function of Ednra in cranial NCC development is not clear. Ednra signaling could be required for normal migration or guidance of NCCs to the pharyngeal arches or in subsequent events in post-migratory NCCs, including proliferation and survival. To address this question, we performed a fate analysis of cranial NCCs in Ednra-/- embryos using the R26R;Wnt1-Cre reporter system, in which Cre expression within NCCs results in permanent beta-galactosidase activity in NCCs and their derivatives. We find that loss of Ednra does not detectably alter either migration of most cranial NCCs into the mandibular first arch and second arch or their subsequent proliferation. However, mesenchymal cell apoptosis is increased two fold in both E9.5 and E10.5 Ednra-/- embryos, with apoptotic cells being present in and just proximal to the pharyngeal arches. Based on these studies, Ednra signaling appears to be required by most cranial NCCs after they reach the pharyngeal arches. However, a subset of NCCs appear to require Ednra signaling earlier, with loss of Ednra signaling likely leading to premature cessation of migration into or within the arches and subsequent cell death.     

Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling

1-Phenyl 2-thiourea (PTU) is a tyrosinase inhibitor commonly used to block pigmentation and aid visualization of zebrafish development. At the standard concentration of 0.003% (200 μM), PTU inhibits melanogenesis and reportedly has minimal other effects on zebrafish embryogenesis. We found that 0.003% PTU altered retinoic acid and insulin-like growth factor (IGF) regulation of neural crest and mesodermal components of craniofacial development. Reduction of retinoic acid synthesis by the pan-aldehyde dehydrogenase inhibitor diethylbenzaldehyde, only when combined with 0.003% PTU, resulted in extraocular muscle disorganization. PTU also decreased retinoic acid-induced teratogenic effects on pharyngeal arch and jaw cartilage despite morphologically normal appearing PTU-treated controls. Furthermore, 0.003% PTU in combination with inhibition of IGF signaling through either morpholino knockdown or pharmacologic inhibition of tyrosine kinase receptor phosphorylation, disrupted jaw development and extraocular muscle organization. PTU in and of itself inhibited neural crest development at higher concentrations (0.03%) and had the greatest inhibitory effect when added prior to 22 hours post fertilization (hpf). Addition of 0.003% PTU between 4 and 20 hpf decreased thyroxine (T4) in thyroid follicles in the nasopharynx of 96 hpf embryos. Treatment with exogenous triiodothyronine (T3) and T4 improved, but did not completely rescue, PTU-induced neural crest defects. Thus, PTU should be used with caution when studying zebrafish embryogenesis as it alters the threshold of different signaling pathways important during craniofacial development. The effects of PTU on neural crest development are partially caused by thyroid hormone signaling.     

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers (foxb1.2/mariposa, krox20, pax2.1 and pax6.1), including anteriorly expressed Hox genes (hoxb1a, hoxa2 and hoxb2), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1, but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development.     

Eif3ba regulates cranial neural crest development by modulating p53 in zebrafish

Congenital diseases caused by abnormal development of the cranial neural crest usually present craniofacial malformations and heart defects while the precise mechanism is not fully understood. Here, we show that the zebrafish eif3ba mutant caused by pseudo-typed retrovirus insertion exhibited a similar phenotype due to the hypogenesis of cranial neural crest cells (NCCs). The derivatives of cranial NCCs, including the NCC-derived cell population of pharyngeal arches, craniofacial cartilage, pigment cells and the myocardium derived from cardiac NCCs, were affected in this mutant. The expression of several neural crest marker genes, including crestin, dlx2a and nrp2b, was specifically reduced in the cranial regions of the eif3ba mutant. Through fluorescence-tracing of the cranial NCC migration marker nrp2b, we observed reduced intensity of NCC-derived cells in the heart. In addition, p53 was markedly up-regulated in the eif3ba mutant embryos, which correlated with pronounced apoptosis in the cranial area as shown by TUNEL staining. These findings suggest a novel function of eif3ba during embryonic development and a novel level of regulation in the process of cranial NCC development, in addition to providing a potential animal model to mimic congenital diseases due to cranial NCC defects. Furthermore, we report the identification of a novel transgenic fish line Et(gata2a:EGFP)^pku418 to trace the migration of cranial NCCs (including cardiac NCCs); this may serve as an invaluable tool for investigating the development and dynamics of cranial NCCs during zebrafish embryogenesis.     

Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus

Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca(2+)-calmodulin-binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration.     

Contributions of placodal and neural crest cells to avian cranial peripheral ganglia

The method of embryonic tissue transplantation was used to confirm the dual origin of avian cranial sensory ganglia, to map precise locations of the anlagen of these sensory neurons, and to identify placodal and neural crest-derived neurons within ganglia. Segments of neural crest or strips of presumptive placodal ectoderm were excised from chick embryos and replaced with homologous tissues from quail embryos, whose cells contain a heterochromatin marker. Placode-derived neurons associated with cranial nerves V, VII, IX, and X are located distal to crest-derived neurons. The generally larger, embryonic placodal neurons are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia. Crest-derived neurons are found in the proximal trigeminal ganglion and in the combined proximal ganglion of cranial nerves IX and X. Neurons in the vestibular and acoustic ganglia of cranial nerve VIII derive from placodal ectoderm with the exception of a few neural crest-derived neurons localized to regions within the vestibular ganglion. Schwann sheath cells and satellite cells associated with all these ganglia originate from neural crest. The ganglionic anlagen are arranged in cranial to caudal sequence from the level of the mesencephalon through the third somite. Presumptive placodal ectoderm for the VIIIth, the Vth, and the VIIth, IXth, and Xth ganglia are located in a medial to lateral fashion during early stages of development reflecting, respectively, the dorsolateral, intermediate, and epibranchial positions of these neurogenic placodes.     

TGFbeta-mediated FGF signaling is crucial for regulating cranial neural crest cell proliferation during frontal bone development

The murine frontal bone derives entirely from the cranial neural crest (CNC) and consists of the calvarial (lateral) aspect that covers the frontal lobe of brain and the orbital aspect that forms the roof of bony orbit. TGFbeta and FGF signaling have important regulatory roles in postnatal calvarial development. Our previous study has demonstrated that conditional inactivation of Tgfbr2 in the neural crest results in severe defects in calvarial development, although the cellular and molecular mechanisms by which TGFbeta signaling regulates the fate of CNC cells during frontal bone development remain unknown. Here, we show that TGFbeta IIR is required for proliferation of osteoprogenitor cells in the CNC-derived frontal bone anlagen. FGF acts downstream of TGFbeta signaling in regulating CNC cell proliferation, and exogenous FGF2 rescues the cell proliferation defect in the frontal primordium of Tgfbr2 mutant. Furthermore, the CNC-derived frontal primordium requires TGFbeta IIR to undergo terminal differentiation. However, this requirement is restricted to the developing calvarial aspect of the frontal bone, whereas the orbital aspect forms despite the ablation of Tgfbr2 gene, implying a differential requirement for TGFbeta signaling during the development of various regions of the frontal bone. This study demonstrates the biological significance of TGFbeta-mediated FGF signaling cascade in regulating frontal bone development, suggests that TGFbeta functions as a morphogen in regulating the fate of the CNC-derived osteoblast and provides a model for investigating abnormal craniofacial development.