Signalling between the hindbrain and paraxial tissues dictates neural crest migration pathways.

Cranial neural crest cells are a pluripotent population of cells derived from the neural tube that migrate into the branchial arches to generate the distinctive bone, connective tissue and peripheral nervous system components characteristic of the vertebrate head. The highly conserved segmental organisation of the vertebrate hindbrain plays an important role in patterning the pathways of neural crest cell migration and in generating the distinct or separate streams of crest cells that form unique structures in each arch. We have used focal injections of DiI into the developing mouse hindbrain in combination with in vitro whole embryo culture to map the patterns of cranial neural crest cell migration into the developing branchial arches. Our results show that mouse hindbrain-derived neural crest cells migrate in three segregated streams adjacent to the even-numbered rhombomeres into the branchial arches, and each stream contains contributions of cells from three rhombomeres in a pattern very similar to that observed in the chick embryo. There are clear neural crest-free zones adjacent to r3 and r5. Furthermore, using grafting and lineage-tracing techniques in cultured mouse embryos to investigate the differential ability of odd and even-numbered segments to generate neural crest cells, we find that odd and even segments have an intrinsic ability to produce equivalent numbers of neural crest cells. This implies that inter-rhombomeric signalling is less important than combinatorial interactions between the hindbrain and the adjacent arch environment in specific regions, in the process of restricting the generation and migration of neural crest cells. This creates crest-free territories and suggests that tissue interactions established during development and patterning of the branchial arches may set up signals that the neural plate is primed to interpret during the progressive events leading to the delamination and migration of neural crest cells. Using interspecies grafting experiments between mouse and chick embryos, we have shown that this process forms part of a conserved mechanism for generating neural crest-free zones and contributing to the separation of migrating crest populations with distinct Hox expression during vertebrate head development.     

In ovo time-lapse analysis of chick hindbrain neural crest cell migration shows cell interactions during migration to the branchial arches

Hindbrain neural crest cells were labeled with DiI and followed in ovo using a new approach for long-term time-lapse confocal microscopy. In ovo imaging allowed us to visualize neural crest cell migration 2-3 times longer than in whole embryo explant cultures, providing a more complete picture of the dynamics of cell migration from emergence at the dorsal midline to entry into the branchial arches. There were aspects of the in ovo neural crest cell migration patterning which were new and different. Surprisingly, there was contact between neural crest cell migration streams bound for different branchial arches. This cell-cell contact occurred in the region lateral to the otic vesicle, where neural crest cells within the distinct streams diverted from their migration pathways into the branchial arches and instead migrated around the otic vesicle to establish a contact between streams. Some individual neural crest cells did appear to cross between the streams, but there was no widespread mixing. Analysis of individual cell trajectories showed that neural crest cells emerge from all rhombomeres (r) and sort into distinct exiting streams adjacent to the even-numbered rhombomeres. Neural crest cell migration behaviors resembled the wide diversity seen in whole embryo chick explants, including chain-like cell arrangements; however, average in ovo cell speeds are as much as 70% faster. To test to what extent neural crest cells from adjoining rhombomeres mix along migration routes and within the branchial arches, separate groups of premigratory neural crest cells were labeled with DiI or DiD. Results showed that r6 and r7 neural crest cells migrated to the same spatial location within the fourth branchial arch. The diversity of migration behaviors suggests that no single mechanism guides in ovo hindbrain neural crest cell migration into the branchial arches. The cell-cell contact between migration streams and the co-localization of neural crest cells from adjoining rhombomeres within a single branchial arch support the notion that the pattern of hindbrain neural crest cell migration emerges dynamically with cell-cell communication playing an important guidance role.     

Temporal restriction of migratory and lineage potential in rhombomere 1 and 2 neural crest

Migratory cranial neural crest cells differentiate into a wide range of cell types, such as ectomesenchymal tissue (bone and connective tissues) ventrally in the branchial arches and neural tissue (neurons and glia) dorsally. We investigated spatial and temporal changes of migration and differentiation potential in neural crest populations derived from caudal midbrain and rhombomeres 1 and 2 by back-transplanting cells destined for the first branchial arch and trigeminal ganglion from HH8-HH19 quail into HH7-HH11 chicks. Branchial arch cells differentiated down ectomesenchymal lineages but largely lost both the ability to localize to the trigeminal position and neurogenic differentiation capacity by HH12-HH13, even before the arch is visible, and lost long distance migratory ability around HH17. In contrast, neural crest-derived cells from trigeminal ganglia lost ectomesechymal differentiation potential by HH17. Despite this, they retain the ability to migrate into the branchial arches until at least HH19. However, many of the neural crest-derived trigeminal ganglia cells in the branchial arch localized to the non-neural crest core of the arch from HH13 and older donors. These results suggest that long distance migration ability, finer scale localization, and lineage restriction may not be coordinately regulated in the cranial neural crest population.     

The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues

The morphology of skeletal tissues formed in each of the branchial arches of higher vertebrates is unique. In addition to these structures, which are derived from the neural crest, the crest-derived connective tissues and mesodermal muscles also form different patterns in each of the branchial arches. The objective of this study was to examine how these patterns arise during avian embryonic development. Presumptive second or third arch neural crest cells were excised from chick hosts and replaced with presumptive first arch crest cells. Both quail and chick embryos were used as donors; orthotopic crest grafts were performed as controls. Following heterotopic transplantation, the hosts developed several unexpected anomalies. Externally they were characterized by the appearance of ectopic, beak-like projections from the ventrolateral surface of the neck and also by the formation of supernumerary external auditory depressions located immediately caudal to the normal external ear. Internally, the grafted cells migrated in accordance with normal, second arch pathways but then formed a complete, duplicate first arch skeletal system in their new location. Squamosal, quadrate, pterygoid, Meckel's, and angular elements were present in most cases. In addition, anomalous first arch-type muscles were found associated with the ectopic skeletal tissues in the second arch. These results indicate that the basis for patterning of branchial arch skeletal and connective tissues resides within the neural crest population prior to its emigration from the neural epithelium, and not within the pharynx or pharyngeal pouches as had previously been suggested. Furthermore, the patterns of myogenesis by mesenchymal populations derived from paraxial mesoderm is dependent upon properties inherent to the neural crest.     

Pitx2c patterns anterior myocardium and aortic arch vessels and is required for local cell movement into atrioventricular cushions

Inactivation of the left-right asymmetry gene Pitx2 has been shown, in mice, to result in right isomerism with associated defects that are similar to that found in humans. We show that the Pitx2c isoform is expressed asymmetrically in a presumptive secondary heart field within the branchial arch and splanchnic mesoderm that contributes to the aortic sac and conotruncal myocardium. Pitx2c was expressed in left aortic sac mesothelium and in left splanchnic and branchial arch mesoderm near the junction of the aortic sac and branchial arch arteries. Mice with an isoform-specific deletion of Pitx2c had defects in asymmetric remodeling of the aortic arch vessels. Fatemapping studies using a Pitx2 cre recombinase knock-in allele showed that daughters of Pitx2-expressing cells populated the right and left ventricles, atrioventricular cushions and valves and pulmonary veins. In Pitx2 mutant embryos, descendents of Pitx2-expressing cells failed to contribute to the atrioventricular cushions and valves and the pulmonary vein, resulting in abnormal morphogenesis of these structures. Our data provide functional evidence that the presumptive secondary heart field, derived from branchial arch and splanchnic mesoderm, patterns the forming outflow tract and reveal a role for Pitx2c in aortic arch remodeling. Moreover, our findings suggest that a major function of the Pitx2-mediated left right asymmetry pathway is to pattern the aortic arches, outflow tract and atrioventricular valves and cushions.     

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.     

Patterning the vertebrate head: murine Hox 2 genes mark distinct subpopulations of premigratory and migrating cranial neural crest.

The structures of the face in vertebrates are largely derived from neural crest. There is some evidence to suggest that the form of the facial pattern is determined by the crest, and that it is specified before migration as to the structures that is is able to form. The neural crest is able to control the form of surrounding, non-neural crest tissues by an instructive interaction. Some of this cranial crest is derived from a region of the hindbrain that expresses Hox 2 homeobox genes in an overlapping and segment-restricted pattern. We have found that neurogenic and mesenchymal neural crest expresses Hox 2 genes from its point of origin beside the neural plate, during migration and after migration has ceased and that rhombomeres 3 and 5 do not have any expressing neural crest beside them. Each branchial arch expresses a different combination or code of Hox genes in a segment-restricted way. The surface ectoderm over the arches initially does not express Hox genes, and later adopts an expression pattern that reflects that of neural crest that has come to underlie it. We suggest that initially the neural plate and neural crest are spatially specified, while the surface ectoderm is unpatterned. Subsequently some positional information could be transferred to the surface ectoderm as a result of an interaction with the neural crest. Given that the role of the homologous genes in insects is position specification, and that neural crest is imprinted before migration, we suggest that Hox 2 genes are providing part of this positional information to the neural crest and hence are involved in patterning the structures of the branchial arches.     

Neural tube derived signals and Fgf8 act antagonistically to specify eye versus mandibular arch muscles

Recent knockout experiments in the mouse generated amazing craniofacial skeletal muscle phenotypes. Yet none of the genes could be placed into a molecular network, because the programme to control the development of muscles in the head is not known. Here we show that antagonistic signals from the neural tube and the branchial arches specify extraocular versus branchiomeric muscles. Moreover, we identified Fgf8 as the branchial arch derived signal. However, this molecule has an additional function in supporting the proliferative state of myoblasts, suppressing their differentiation, while a further branchial arch derived signal, namely Bmp7, is an overall negative regulator of head myogenesis.     

Preferential expression of Mdm2 oncogene during the development of neural crest and its derivatives in mouse early embryogenesis

The Mdm2 oncoprotein acts as the principal negative regulator of p53 activities and is essential for its control during mouse early development, at least before implantation. We analyzed Mdm2 expression between 7.5 and 9 days post-coitum (dpc) by whole-mount in situ hybridization and report here a novel expression pattern during neural crest development. At 7.5 dpc Mdm2 becomes preferentially expressed at the top of the neural folds. Between 8 and 9 dpc, this preferential expression is also observed in neural crest cells migrating from the closing brain towards craniofacial regions and the first three branchial arches. It persists in the craniofacial mesenchyme and the first branchial arch in 9 dpc embryos. Migrating neural crest cells in the tail region are also preferentially labeled at this stage. At day 9.5 Mdm2 becomes more ubiquitously expressed throughout the embryo as reported before.     

Spatial integration among cells forming the cranial peripheral nervous system

Neural crest cells represent a unique link between axial and peripheral regions of the developing vertebrate head. Although their fates are well catalogued, the issue of their role in spatial organization is less certain. Recent data, particularly on patterns of expression of Hox genes in the hindbrain and crest cells, have raised anew the debate whether a segmental arrangement is the basis for positional specification of craniofacial epithelial and mesenchymal tissues or is but one manifestation of underlying spatial programming processes. The mechanisms of positional specification of sensory neurons derived from the neural crest and placodes are unknown. This review examines the spatial organization of cells and tissues that develop in proximity to sensory neurons; some of these tissues share a common ancestry, others are targets of cranial sensory and motor nerves. All share the necessity of acquiring and expressing site-specific properties in a functionally integrated manner. This integration occurs in part by coordinating patterns of cell migration, as occurs between migrating crest cells and branchial arch myoblasts. Constant rostro-caudal relations are maintained among these precursors as they move dorsoventrally from the hindbrain–paraxial regions to establish branchial arches. During this period the interactions among these and other mesenchymal cells are hierarchical; each cell population differentially integrates its past with cues emanating from new microenvironments. Analyses of tissue interactions indicate that neural crest cells play a dominant role in this scenario. © 1993 John Wiley & Sons, Inc.     

Differential growth and tissue shifts in the developing neck of the chick embryo. An experimental study

The differential growth of the neck was studied by means of linear marks in the chick embryo. The marks were inserted into 3 different zones: Zone I, between the level of 1st and 2nd branchial cleft, zone II, between the level of the 2nd and 3rd branchial cleft, and zone III, between the level of the 3rd branchial cleft and the Cuvier duct. The deformation and dispersion of marks after further incubation of labelled embryos indicate: 1. A caudocranially oriented growth of axial and paraaxial structures causing the displacement of branchial to axial and paraxial structures, 2. an extent growth in the region of the 3rd branchial arch in contrast to other branchial arches with 2 exceptions: the extent craniocaudal growth of the operculum (2nd branchial arch) and the extent caudocranially oriented growth of the hypobranchial region. On the base of our findings the differences in the topography of some neck organs of birds and mammals could be explained.     

Domains of cellular retinoic acid-binding protein I (CRABP I) expression in the hindbrain and neural crest of the mouse embryo.

We describe here the distribution of cellular retinoic acid-binding protein I (CRABP I) in the head of the early mouse embryo from day 8 to day 13 of gestation, using both in situ hybridisation to localise mRNA and immunocytochemistry to localise protein. The distribution of mRNA and protein was found to be identical. CRABP I first appeared in part of the presumptive hindbrain of the presomite embryo and then became localised to rhombomeres 2, 4, 5 and 6. The only other area of expression in the cephalic neuroepithelium was in a part of the midbrain roof. The neural crest and its mesenchymal derivatives, the branchial arches, expressed CRABP I and crest could be seen streaming from the neuroepithelium of individual rhombomeres into particular branchial arches. This suggested a fate map could be constructed describing the rhombomeric origin of branchial arch mesenchyme. Later in development, axons throughout the hindbrain expressed CRABP I. The results are considered in terms of the role of retinoic acid in the specification of neuronal phenotype in the hindbrain and in axon outgrowth.     

Retinoic acid-induced developmental defects are mediated by RARbeta/RXR heterodimers in the pharyngeal endoderm

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor beta (RARbeta) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, as they are similar to those generated by a selective pan-RAR agonist, are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarb-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarb and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARbeta/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARbeta. Endodermal cells lining the first two branchial arches respond to treatment with the RARbeta agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, we provide the first in vivo evidence that the synthetic RARbeta agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes.     

Fibroblast growth factor signalling and regional specification of the pharyngeal ectoderm

Branchial arch development involves dynamic interactions between neural crest cells as well as ectodermal, endodermal and mesodermal cell populations. Despite their importance and evolutionary conservation, the intercellular interactions guiding the early development of the branchial arches are still poorly understood. We have here studied fibroblast growth factor (FGF) signalling in early pharyngeal development. In mice homozygous for a hypomorphic allele of Fgfr1, neural crest cells migrating from the hindbrain mostly fail to enter the second branchial arch. This defect is non-cell-autonomous suggesting that Fgfr1 provides a permissive environment for neural crest cell migration. Here we demonstrate localized down-regulation of the expression of the FGF responsive gene, Sprouty1 in the epithelium covering the presumptive second branchial arch of hypomorphic Fgfr1 mutants. This appears to result in a failure to establish an ectodermal signalling center expressing Fgf3 and Fgf15. We also studied differentiation of the ectoderm in the second branchial arch region. Development of the geniculate placode as well as the VIIth cranial ganglion is affected in Fgfr1 hypomorphs. Our results suggest that Fgfr1 is important for localized signalling in the pharyngeal ectoderm and consequently for normal tissue interactions in the developing second branchial arch.