Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.     

Delta signaling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate

We examined the role of Delta signaling in specification of two derivatives in zebrafish neural plate: Rohon-Beard spinal sensory neurons and neural crest. deltaA-expressing Rohon-Beard neurons are intermingled with premigratory neural crest cells in the trunk lateral neural plate. Embryos homozygous for a point mutation in deltaA, or with experimentally reduced delta signalling, have supernumerary Rohon-Beard neurons, reduced trunk-level expression of neural crest markers and lack trunk neural crest derivatives. Fin mesenchyme, a putative trunk neural crest derivative, is present in deltaA mutants, suggesting it segregates from other neural crest derivatives as early as the neural plate stage. Cranial neural crest derivatives are also present in deltaA mutants, revealing a genetic difference in regulation of trunk and cranial neural crest development.     

Noelin-1 is a secreted glycoprotein involved in generation of the neural crest

The vertebrate neural crest arises at the border of the neural plate during early stages of nervous system development; however, little is known about the molecular mechanisms underlying neural crest formation. Here we identify a secreted protein, Noelin-1, which has the ability to prolong neural crest production. Noelin-1 messenger RNA is expressed in a graded pattern in the closing neural tube. It subsequently becomes restricted to the dorsal neural folds and migrating neural crest. Over expression of Noelin-1 using recombinant retroviruses causes an excess of neural crest emigration and extends the time that the neural tube is competent to generate as well as regenerate neural crest cells. These results support an important role for Noelin-1 in regulating the production of neural crest cells by the neural tube.     

The origins of neural crest cells in the axolotl

We address the question of whether neural crest cells originate from the neural plate, from the epidermis, or from both of these tissues. Our past studies revealed that a neural fold and neural crest cells could arise at any boundary created between epidermis and neural plate. To examine further the formation of neural crest cells at newly created boundaries in embryos of a urodele (Ambystoma mexicanum), we replace a portion of the neural folds of an albino host with either epidermis or neural plate from a normally pigmented donor. We then look for cells that contain pigment granules in the neural crest and its derivatives in intact and sectioned host embryos. By tracing cells in this manner, we find that cells from neural plate transplants give rise to melanocytes and (in one case) become part of a spinal ganglion, and we find that epidermal transplants contribute cells to the spinal and cranial ganglia. Thus neural crest cells arise from both the neural plate and the epidermis. These results also indicate that neural crest induction is (at least partially) governed by local reciprocal interactions between epidermis and neural plate at their common boundary.     

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.     

Molecular mechanisms of neural crest induction

The neural crest is an embryonic cell population that originates at the border between the neural plate and the prospective epidermis. Around the time of neural tube closure, neural crest cells emigrate from the neural tube, migrate along defined paths in the embryo and differentiate into a wealth of derivatives. Most of the craniofacial skeleton, the peripheral nervous system, and the pigment cells of the body originate from neural crest cells. This cell type has important clinical relevance, since many of the most common craniofacial birth defects are a consequence of abnormal neural crest development. Whereas the migration and differentiation of the neural crest have been extensively studied, we are just beginning to understand how this tissue originates. The formation of the neural crest has been described as a classic example of embryonic induction, in which specific tissue interactions and the concerted action of signaling pathways converge to induce a multipotent population of neural crest precursor cells. In this review, we summarize the current status of knowledge on neural crest induction. We place particular emphasis on the signaling molecules and tissue interactions involved, and the relationship between neural crest induction, the formation of the neural plate and neural plate border, and the genes that are upregulated as a consequence of the inductive events.     

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.     

Tumorhead, a Xenopus gene product that inhibits neural differentiation through regulation of proliferation

Tumorhead (TH) is a novel maternal gene product from Xenopus laevis containing several basic domains and a weak coiled-coil. Overexpression of wild-type TH resulted in increased proliferation of neural plate cells, causing expansion of the neural field followed by neural tube and craniofacial abnormalities. Overexpressed TH protein repressed neural differentiation and neural crest markers, but did not inhibit the neural inducers, pan-neural markers or mesodermal markers. Loss of function by injection of anti-TH antibody inhibited cell proliferation. Our data are consistent with a model in which tumorhead functions in regulating differentiation of the neural tissues but not neural induction or determination through its effect on cell proliferation.     

Excess lunatic fringe causes cranial neural crest over-proliferation.

Lunatic fringe is a vertebrate homologue of Drosophila fringe, which plays an important role in modulating Notch signaling. This study examines the distribution of chick lunatic fringe at sites of neural crest formation and explores its possible function by ectopic expression. Shortly after neural tube closure, lunatic fringe is expressed in most of the neural tube, with the exception of the dorsal midline containing presumptive neural crest. Thus, there is a fringe/non-fringe border at the site of neural crest production. Expression of excess lunatic fringe in the cranial neural tube and neural crest by retrovirally mediated gene transfer resulted in a significant increase ( approximately 60%) in the percentage of cranial neural crest cells 1 day after infection. This effect was mediated by an increase in cell division as assayed by BrdU incorporation. Infected embryos had an up-regulation of Delta-1 in the dorsal neural tube and redistribution of Notch-1 to the lumen of the neural tube, confirming that excess fringe modulates Notch signaling. These findings point to a novel role for lunatic fringe in regulating cell division and/or production of neural crest cells by the neural tube.     

Neural fold formation at newly created boundaries between neural plate and epidermis in the axolotl

According to a recent model, the cortical tractor model, neural fold and neural crest formation occurs at the boundary between neural plate and epidermis because random cell movements become organized at this site. If this is correct, then a fold should form at any boundary between epidermis and neural plate. To test that proposition, we created new boundaries in axolotl embryos by juxtaposing pieces of neural plate and epidermis that would not normally participate in fold formation. These boundaries were examined superficially and histologically for the presence of folds, permitting the following observations. Folds form at each newly created boundary, and as many folds form as there are boundaries. When two folds meet they fuse into a hollow "tube" of neural tissue covered by epidermis. Sections reveal that these ectopic folds and "tubes" are morphologically similar to their natural counterparts. Transplanting neural plate into epidermis produces nodules of neural tissue with central lumens and peripheral nerve fibers, and transplanting epidermis into neural plate causes the neural tube and the dorsal fin to bifurcate in the region of the graft. Tissue transplanted homotypically as a control integrates into the host tissue without forming folds. When tissue from a pigmented embryo is transplanted into an albino host, the presence of pigment allows the donor cells to be distinguished from those of the host. Mesenchymal cells and melanocytes originating from neural plate transplants indicate that neural crest cells form at these new boundaries. Thus, any boundary between neural plate and epidermis denotes the site of a neural fold, and the behavior of cells at this boundary appears to help fold the epithelium. Since folds can form in ectopic locations on an embryo, local interactions rather than classical neural induction appear to be responsible for the formation of neural folds and neural crest.     

The neural tube patterns vessels developmentally using the VEGF signaling pathway

Embryonic blood vessels form in a reproducible pattern that interfaces with other embryonic structures and tissues, but the sources and identities of signals that pattern vessels are not well characterized. We hypothesized that the neural tube provides vascular patterning signal(s) that direct formation of the perineural vascular plexus (PNVP) that encompasses the neural tube at mid-gestation. Both surgically placed ectopic neural tubes and ectopic neural tubes engineered genetically were able to recruit a vascular plexus, showing that the neural tube is the source of a vascular patterning signal. In mouse-quail chimeras with the graft separated from the neural tube by a buffer of host cells, graft-derived vascular cells contributed to the PNVP, indicating that the neural tube signal(s) can act at a distance. Murine neural tube vascular endothelial growth factor A (VEGFA) expression was temporally and spatially correlated with PNVP formation, suggesting it is a component of the neural tube signal. A collagen explant model was developed in which presomitic mesoderm explants formed a vascular plexus in the presence of added VEGFA. Co-cultures between presomitic mesoderm and neural tube also supported vascular plexus formation, indicating that the neural tube could replace the requirement for VEGFA. Moreover, a combination of pharmacological and genetic perturbations showed that VEGFA signaling through FLK1 is a required component of the neural tube vascular patterning signal. Thus, the neural tube is the first structure identified as a midline signaling center for embryonic vascular pattern formation in higher vertebrates, and VEGFA is a necessary component of the neural tube vascular patterning signal. These data suggest a model whereby embryonic structures with little or no capacity for angioblast generation act as a nexus for vessel patterning.     

Homeogenetic neural induction in Xenopus

Neural induction is known to involve an interaction of ectoderm with dorsal mesoderm during gastrulation, but several kinds of studies have argued that competent ectoderm can also be neutralized via an interaction with previously neuralized tissue, a process termed homeogenetic neural induction. Although homeogenetic neural induction has been proposed to play an important role in the normal induction of neural tissue, this process has not been subjected to detailed study using tissue recombinants and molecular markers. We have examined the question of homeogenetic neural induction in Xenopus embryos, both in transplant and recombinant experiments, using the expression of two neural antigens to assay the response. When ectoderm that is competent to be neuralized is transplanted to the region adjacent to the neural plate of early neurula embryos, it forms neural tissue, as assayed by staining with antibodies against the neural cell adhesion molecule, N-CAM. Transplants to the ventral region, far from the neural plate, do not express N-CAM, indicating that neuralization is not occurring as a result of the transplantation procedure itself. Because this response might be occurring as a result of interactions of ectoderm with either adjacent neural plate tissue, or with underlying dorsolateral mesoderm, recombinant experiments were performed to determine the source of the neuralizing signal. Ectoderm cultured in combination with neural plate tissue alone expresses neural markers, while ectoderm cultured in combination with dorsolateral mesoderm does not. We conclude that neural tissue can homeogenetically induce competent ectoderm to form neural tissue and argue that this induction occurs via planar signaling within the ectoderm, a mechanism that, in normal development, may be involved in interactions within presumptive neural ectoderm or in specifying structures that lie near the neural plate.     

Plasticity and predetermination of mesencephalic and trunk neural crest transplanted into the region of the cardiac neural crest

The cardiac neural crest contains ectomesenchymal and neural anlagen that are necessary for normal heart development. It is not known whether other regions of the neural crest are capable of supporting normal heart development. In the experiments reported herein, quail donor embryos provided cardiac, trunk, or mesencephalic neural crest to replace or add to the chick host cardiac neural crest. Neither trunk nor mesencephalic neural crest was capable of generating ectomesenchyme competent to effect truncal septation. Addition of mesencephalic neural crest resulted in a high incidence of persistent truncus arteriosus, suggesting that ectomesenchyme derived from the mesencephalic region interferes with ectomesenchyme derived from the cardiac neural crest. Derivatives from the trunk neural crest, on the other hand, did not result in abnormal development of the truncal septum. While mesencephalic neural crest seeded the cardiac ganglia with both neurons and supporting cells, this capability was limited in the trunk neural crest to the more mature regions. These studies indicate a predetermination of the ectomesenchymal derivatives of the cranial neural crest and a possible competition of neural anlagen to form neurons and supporting cells in the cardiac ganglia.     

Differential distribution of competence for panplacodal and neural crest induction to non-neural and neural ectoderm

It is still controversial whether cranial placodes and neural crest cells arise from a common precursor at the neural plate border or whether placodes arise from non-neural ectoderm and neural crest from neural ectoderm. Using tissue grafting in embryos of Xenopus laevis, we show here that the competence for induction of neural plate, neural plate border and neural crest markers is confined to neural ectoderm, whereas competence for induction of panplacodal markers is confined to non-neural ectoderm. This differential distribution of competence is established during gastrulation paralleling the dorsal restriction of neural competence. We further show that Dlx3 and GATA2 are required cell-autonomously for panplacodal and epidermal marker expression in the non-neural ectoderm, while ectopic expression of Dlx3 or GATA2 in the neural plate suppresses neural plate, border and crest markers. Overexpression of Dlx3 (but not GATA2) in the neural plate is sufficient to induce different non-neural markers in a signaling-dependent manner, with epidermal markers being induced in the presence, and panplacodal markers in the absence, of BMP signaling. Taken together, these findings demonstrate a non-neural versus neural origin of placodes and neural crest, respectively, strongly implicate Dlx3 in the regulation of non-neural competence, and show that GATA2 contributes to non-neural competence but is not sufficient to promote it ectopically.     

The universal fuzzy logical framework of neural circuits and its application in modeling primary visual cortex

Analytical study of large-scale nonlinear neural circuits is a difficult task. Here we analyze the function of neural systems by probing the fuzzy logical framework of the neural cells’ dynamical equations. Although there is a close relation between the theories of fuzzy logical systems and neural systems and many papers investigate this subject, most investigations focus on finding new functions of neural systems by hybridizing fuzzy logical and neural system. In this paper, the fuzzy logical framework of neural cells is used to understand the nonlinear dynamic attributes of a common neural system by abstracting the fuzzy logical framework of a neural cell. Our analysis enables the educated design of network models for classes of computation. As an example, a recurrent network model of the primary visual cortex has been built and tested using this approach.