Deltex/Dtx mediates NOTCH signaling in regulation of Bmp4 expression in cranial neural crest formation during avian development

NOTCH signaling plays a key role in cell fate determination in both vertebrates and invertebrates. It is well known that Su(H)/RBP-J is a major mediator of NOTCH signaling. In a previous study, it was shown that NOTCH signaling was involved in cranial neural crest formation in avian embryos. However, Su(H)/RBP-J activity did not appear to be required in this process. In this study, the Deltex/Dtx gene was focussed on as a potential mediator of NOTCH signaling in neural crest formation. At the time of neural crest formation, quail Deltex2 was expressed throughout the ectoderm. Misexpression of a dominant-negative form of Deltex in the ectoderm caused reduced expression of Slug, a neural crest marker. Dominant-negative Deltex expression reduced the expression of Bmp4, a neural crest inducer, whereas co-transfection of Bmp4 with dominant-negative Deltex rescued Slug expression. In parallel, Hairy2 expression in the epidermis was regulated by a Su(H)-dependent pathway. These results indicate that NOTCH signaling has dual functions mediated by either Su(H) or Deltex in the avian embryonic ectoderm.     

Both inhibition and activation of Notch signaling rely on a conserved Neuralized-binding motif in Bearded proteins and the Notch ligand Delta

Lateral inhibition is one of the key functions of Notch signaling during animal development. In the proneural clusters that give rise to Drosophila mechanosensory bristles, Delta (Dl) ligand in the sensory organ precursor (SOP) cell is targeted for ubiquitination by the E3 ligase Neuralized (Neur), resulting in activation of Dl's capacity to signal to the Notch receptor on neighboring cells. The cells that receive this signal activate a genetic program that suppresses their SOP fate potential, insuring that only a single SOP develops within each cluster. Using multiple lines of investigation, we provide evidence that members of the Bearded family of proteins (BFMs) inhibit Dl activation in non-SOP cells by binding to Neur and preventing it from interacting with Dl. We show that this activity of BFMs is dependent on the conserved NXXN motif, and report the unexpected finding that several BFMs include multiple functional copies of this motif. We find that a conserved NXXN motif in the intracellular domain of Dl is responsible for its interaction with Neur, indicating direct competition between Dl and BFMs for binding to Neur, and we show that Neur-dependent endocytosis of Dl requires the integrity of its NXXN motif. Our results illuminate the mechanism of an important regulatory event in Notch signaling that appears to be conserved between insects and crustaceans.     

Different downstream pathways for Notch signaling are required for gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells

We examined the roles of Notch signaling and fibroblast growth factors (FGFs) in the gliogenesis of mouse mesencephalic neural crest cells. The present study demonstrated that Notch activation or FGF treatment promotes the differentiation of glia expressing glial fibrillary acidic protein. Notch activation or FGF2 exposure during the first 48 h in culture was critical for glial differentiation. The promotion of gliogenesis by FGF2 was significantly suppressed by the inhibition of Notch signaling using Notch-1 siRNA. These data suggest that FGFs activate Notch signaling and that this activation promotes the gliogenic specification of mouse mesencephalic neural crest cells. Notch activation and FGF treatment have been shown to participate in the chondrogenic specification of these cells [Nakanishi, K., Chan, Y.S., Ito, K., 2007. Notch signaling is required for the chondrogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 124, 190–203]. Therefore, we analyzed whether or not there were differences between gliogenic and chondrogenic specifications in the downstream pathway of the Notch receptor. Whereas the activation of only the Deltex-mediated pathway was sufficient to promote glial specification, the activation of both RBP-J- and Deltex-dependent pathways was required for chondrogenic specification. These results suggest that the different downstream pathways of the Notch receptor participate in the gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells.     

Melanophore sublineage-specific requirement for zebrafish touchtone during neural crest development

The specification, differentiation and maintenance of diverse cell types are of central importance to the development of multicellular organisms. The neural crest of vertebrate animals gives rise to many derivatives, including pigment cells, peripheral neurons, glia and elements of the craniofacial skeleton. The development of neural crest-derived pigment cells has been studied extensively to elucidate mechanisms involved in cell fate specification, differentiation, migration and survival. This analysis has been advanced considerably by the availability of large numbers of mouse and, more recently, zebrafish mutants with defects in pigment cell development. We have identified the zebrafish mutant touchtone (tct), which is characterized by the selective absence of most neural crest-derived melanophores. We find that although wild-type numbers of melanophore precursors are generated in the first day of development and migrate normally in tct mutants, most differentiated melanophores subsequently fail to appear. We demonstrate that the failure in melanophore differentiation in tct mutant embryos is due at least in part to the death of melanoblasts and that tct function is required cell autonomously by melanoblasts. The tct locus is located on chromosome 18 in a genomic region apparently devoid of genes known to be involved in melanophore development. Thus, zebrafish tct may represent a novel as well as selective regulator of melanoblast development within the neural crest lineage. Further, our results suggest that, like other neural crest-derived sublineages, melanogenic precursors constitute a heterogeneous population with respect to genetic requirements for development.     

Induction of the neural crest and the opportunities of life on the edge

The neural crest is a multipotent population of migratory cells unique to the vertebrate embryo. Neural crest arises at the lateral edge of the neural plate and migrates throughout the embryo to give rise to a wide variety of cell types including peripheral and enteric neurons and glia, craniofacial cartilage and bone, smooth muscle, and pigment cells. Here we review recent studies that have addressed the role of several signaling pathways in the induction of the neural crest. Work in the mouse, chick, Xenopus, and zebrafish have shown that a complex network of genes is activated at the neural plate border in response to neural crest-inducing signals. We also summarize some of these findings and discuss how the differential activation of these genes may contribute to the establishment of neural crest diversity.     

Ephrin-B2 forward signaling regulates somite patterning and neural crest cell development

Genetic studies in the mouse have implicated ephrin-B2 (encoded by the gene Efnb2) in blood vessel formation, cardiac development and remodeling of the lymphatic vasculature. Here we report that loss of ephrin-B2 leads to defects in populations of cranial and trunk neural crest cells (NCC) and to defective somite development. In addition, we show that Efnb1/Efnb2 double heterozygous embryos exhibit phenotypes in a number of NCC derivatives. Expression of one copy of a mutant version of Efnb2 that lacks tyrosine phosphorylation sites was sufficient to rescue the embryonic phenotypes associated with loss of Efnb2. Our results uncover an important role for ephrin-B2 in NCC and somites during embryogenesis and suggest that ephrin-B2 exerts many of its embryonic function via activation of forward signaling.     

Cardiac neural crest ablation alters Id2 gene expression in the developing heart

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.     

Morphology and behaviour of neural crest cells of chick embryo in vitro

Summary Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamellae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm or ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.     

Multiple roles of Notch signaling in cochlear development

Notch signaling inhibits hair cell differentiation, based on studies on mice deficient in Notch signaling-related genes and its downstream genes. However, the precise mechanisms of this inhibition are unknown because it is difficult to control the timing and duration of the suppression of Notch signaling. Here, we developed a novel in vitro culture and analysis method for mouse fetal cochleae and examined the roles of Notch signaling by its reversible inhibition through the use of Notch signaling inhibitors of gamma-secretase and TNF-alpha-converting enzyme. Notch inhibition with Notch signaling inhibitor treatment increases the number of cochlear hair cells, as observed in gene deletion experiments. We elucidated that this increase is regulated by the dichotomy between hair cells and supporting cells from common progenitors. We also propose other roles of Notch signaling in cochlear development. First, Notch signaling arrests the cell cycle of the cochlear epithelium containing putative hair cells and supporting cell progenitors because Notch inhibition with inhibitor treatment increases the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells that can differentiate into hair cells or supporting cells. Second, Notch signaling is required for the induction of Prox1-positive supporting cells. Third, Notch signaling is required for the maintenance of supporting cells.     

Quantitative distribution of chick neural crest cells during gangliogenesis

Summary We have quantitated the distribution of chick neural crest cells after they have completed early migration and are aggregating into ganglia. Variables tested for an influence on the distribution of cells include stage, level of somites, position in each of the primary body axes, and individual embryo. The 11th–15th cervical somites of embryos at stages 30, 35, and 40 somites (s) incubated for 2.5, 3.0, and 3.5 days were labeled with antibody to HNK-1 to detect neural crest cells, and doubly labeled with antibody to HNK-1 and to the 150 kD neurofilament subunit to detect neural crest-derived neurons. Significantly more neural crest cells appear at older stages, but cells are uniformly distributed among the 11th–15th somites at any given stage. Significant differences in the total number of neural crest cells among three embryos sampled at the same stage indicate that the number of cells is independent of the staging series used. As early as the 35 s stage about one-third of the neural crest cells throughout the somite exhibit NF staining. At the 40 s stage, doubly labeled NF cells, as well as HNK-1 labeled cells, aggregate in a circumscribed portion of the mediolateral axis to form presumptive sensory ganglia in the dorsal region of the somites. Also at 40 s a wave of cell aggregation into sympathetic ganglia proceeds anteroposteriorly along the ventral border of the somitic mesenchyme. The results show a sequence of phenotypic expression beginning with neurofilament antigen, then ganglionic aggregation, and finally, in the case of sympathetic neurons, catecholamine transmitter.     

Tfap2a and Foxd3 regulate early steps in the development of the neural crest progenitor population

The neural crest is a stem cell-like population exclusive to vertebrates that gives rise to many different cell types including chondrocytes, neurons and melanocytes. Arising from the neural plate border at the intersection of Wnt and Bmp signaling pathways, the complexity of neural crest gene regulatory networks has made the earliest steps of induction difficult to elucidate. Here, we report that tfap2a and foxd3 participate in neural crest induction and are necessary and sufficient for this process to proceed. Double mutant tfap2a (mont blanc, mob) and foxd3 (mother superior, mos) mob;mos zebrafish embryos completely lack all neural crest-derived tissues. Moreover, tfap2a and foxd3 are expressed during gastrulation prior to neural crest induction in distinct, complementary, domains; tfap2a is expressed in the ventral non-neural ectoderm and foxd3 in the dorsal mesendoderm and ectoderm. We further show that Bmp signaling is expanded in mob;mos embryos while expression of dkk1, a Wnt signaling inhibitor, is increased and canonical Wnt targets are suppressed. These changes in Bmp and Wnt signaling result in specific perturbations of neural crest induction rather than general defects in neural plate border or dorso-ventral patterning. foxd3 overexpression, on the other hand, enhances the ability of tfap2a to ectopically induce neural crest around the neural plate, overriding the normal neural plate border limit of the early neural crest territory. Although loss of either Tfap2a or Foxd3 alters Bmp and Wnt signaling patterns, only their combined inactivation sufficiently alters these signaling gradients to abort neural crest induction. Collectively, our results indicate that tfap2a and foxd3, in addition to their respective roles in the differentiation of neural crest derivatives, also jointly maintain the balance of Bmp and Wnt signaling in order to delineate the neural crest induction domain.     

Quail neural crest cells cannot read positional values in the dorsal trunk feathers of the chicken embryo

Summary If quail neural crest cells are grafted to the chick, they migrate into the feathers of the host and produce melanin pigment. In one study, the dorsal trunk feathers of the chimaera were found to have quail-like pigment patterns. This was interpreted in terms of a positional information model. By contrast, in another study it was found that pigment patterns in the wing plumage of the chimaera bore little or no resemblance to the quail, showing instead a rather uniform, dark pigmentation. This was interpreted in terms of a prepattern in the ectoderm. This striking difference in results could be because the wing and trunk plumages have their pigment patterns specified in different ways. We have examined this possibility by making detailed maps of the dorsal trunk plumage of the normal quail and the quail-chick chimaera. Using this novel technique, we can accurately record the secondary pigment patterns in the embryonic down plumage. In the quail there are well-defined, longitudinal stripes running down the back, whereas the chimaera shows rather uniform, dark pigment in this area. There is little or no indication of stripes and some chimaerae develop asymmetric, mottled patterns. Grafts to the cephalic region also produce uniform pigmentation with no quail-like patterning. These findings indicate that neural crest cells cannot read positional values in the feathers of another species.     

Morphogenesis of sclerotome and neural crest in avian embryos

Summary The distribution of sclerotome and neural crest cells of avian embryos was studied by light and electron microscopy. Sclerotome cells radiated from the somites towards the notochord, to occupy the perichordal space. Neural crest cells, at least initially, also entered cell-free spaces. At the cranial somitic levels they moved chiefly dorsal to the somites, favouring the rostral part of each somite. These cells did not approach the perichordal space. More caudally (i.e. trunk levels), neural crest cells initially moved ventrally between the somites and neural tube. Adjacent to the caudal half of each somite, these cells penetrated no further than the myosclerotomal border, but opposite the rostral somite half, they were found next to the sclerotome almost as far ventrally as the notochord. However, they did not appear to enter the perichordal space, in contrast to sclerotome cells.When tested in vitro, sclerotome cells migrated towards notochords co-cultured on fibronectin-rich extracellular material, and on collagen gels. In contrast, neural crest cells avoided co-cultured notochords. This avoidance was abolished by inclusion of testicular hyaluronidase and chondroitinase ABC in the culture medium, but not by hyaluronidase from Streptomyces hyalurolyticus. The results suggest that sclerotome and neural crest mesenchyme cells have a different distribution with respect to the notochord, and that differential responses to notochordal extracellular material, possibly chondroitin sulphate proteoglycan, may be responsible for this.     

Visualization of spatiotemporal activation of Notch signaling: live monitoring and significance in neural development

Notch signaling plays various key roles in cell fate determination during CNS development in a context-dependent fashion. However, its precise physiological role and the localization of its target cells remain unclear. To address this issue, we developed a new reporter system for assessing the RBP-J-mediated activation of Notch signaling target genes in living cells and tissues using a fluorescent protein Venus. Our reporter system revealed that Notch signaling is selectively activated in neurosphere-initiating multipotent neural stem cells in vitro and in radial glia in the embryonic forebrain in vivo. Furthermore, the activation of Notch signaling occurs during gliogenesis and is required in the early stage of astroglial development. Consistent with these findings, the persistent activation of Notch signaling inhibits the differentiation of GFAP-positive astrocytes. Thus, the development of our RBP-J-dependent live reporter system, which is activated upon Notch activation, together with a stage-dependent gain-of-function analysis allowed us to gain further insight into the complexity of Notch signaling in mammalian CNS development.