Ceramide: a novel inducer for neural tube defects.

BACKGROUND: Circulating plasma ceramides, a class of bioactive sphingolipids, are elevated in metabolic disorders, including obesity. Infants of women with these disorders are at 2- to 3-fold greater risk for developing a neural tube defect (NTD). This study aimed to test the effects of embryonic exposure to C2-ceramides (C2) during neural tube closure. Preliminary data shows an increase in NTDs in chick embryos after C2 exposure, and addresses potential mechanisms. RESULTS: Cell and embryo models were used to examine redox shifts after ceramide exposure. While undifferentiated P19 cells were resistant to ceramide exposure, neuronally differentiated P19 cells exhibited an oxidizing shift. Consistent with these observations, GSH E h curves revealed a shift to a more oxidized state in C2 treated embryos without increasing apoptosis or changing Pax3 expression, however cell proliferation was lower. Neural tube defects were observed in 45% of chick embryos exposed to C2, compared to 12% in control embryos. CONCLUSIONS: C2 exposure during critical developmental stages increased the frequency of NTDs in the avian model. Increased ROS generation in cell culture, along with the more oxidative GSH E h profiles of C2 exposed cells and embryos, support a model wherein ceramide affects neural tube closure via altered tissue redox environments.

Signaling mechanisms controlling cranial placode neurogenesis and delamination.

The neurogenic cranial placodes are a unique transient epithelial niche of neural progenitor cells that give rise to multiple derivatives of the peripheral nervous system, particularly, the sensory neurons. Placode neurogenesis occurs throughout an extended period of time with epithelial cells continually recruited as neural progenitor cells. Sensory neuron development in the trigeminal, epibranchial, otic, and olfactory placodes coincides with detachment of these neuroblasts from the encompassing epithelial sheet, leading to delamination and ingression into the mesenchyme where they continue to differentiate as neurons. Multiple signaling pathways are known to direct placodal development. This review defines the signaling pathways working at the finite spatiotemporal period when neuronal selection within the placodes occurs, and neuroblasts concomitantly delaminate from the epithelium. Examining neurogenesis and delamination after initial placodal patterning and specification has revealed a common trend throughout the neurogenic placodes, which suggests that both activated FGF and attenuated Notch signaling activities are required for neurogenesis and changes in epithelial cell adhesion leading to delamination. We also address the varying roles of other pathways such as the Wnt and BMP signaling families during sensory neurogenesis and neuroblast delamination in the differing placodes.

Vertebrate neurogenic placode development: historical highlights that have shaped our current understanding.

With the flood of published research encountered today, it is important to occasionally reflect upon how we arrived at our current understanding in a particular scientific discipline, thereby positioning new discoveries into proper context with long-established models. This historical review highlights some of the important scientific contributions in the field of neurogenic placode development. By viewing cumulatively the rich historical data, we can more fully appreciate and apply what has been accomplished. Early descriptive work in fish and experimental approaches in amphibians and chick yielded important conceptual models of placode induction and cellular differentiation. Current efforts to discover genes and their molecular functions continue to expand our understanding of the placodes. Carefully considering the body of work may improve current models and help focus modern experimental design.

Pax3 isoforms in sensory neurogenesis: expression and function in the ophthalmic trigeminal placode.

BACKGROUND: In the trigeminal placode, Pax3 is classified as necessary but not sufficient for sensory neuron differentiation. One hypothesis is that different Pax3 isoforms regulate cellular differentiation uniquely. Pax3 is known to sometimes activate and sometimes repress gene transcription, and its activity can be dependent on the isoforms present. Pax3 isoforms had not previously been characterized in chick sensory neurogenesis. RESULTS: Reverse transcriptase-polymerase chain reaction (PCR) analysis revealed three well-expressed Pax3 splice variants: full-length (flPax3), Pax3V1, and Pax3V2. Each was characterized for its effect on neurogenesis by misexpression in placodal ectoderm. The differences observed were more apparent under conditions of enhanced neurogenesis (by means of Notch inhibition), where flPax3 and Pax3V1 caused failed differentiation, while Pax3V2 misexpression resembled the neuronal differentiation seen in controls. Quantitative PCR analysis revealed a progressive increase in Pax3 expression, but no significant change in relative isoform expression. Of interest, Notch inhibition led to a significant increase in Pax3 expression. CONCLUSIONS: We can conclude that: (1) flPax3 and Pax3V1 inhibit neuronal differentiation; (2) Pax3V2 is permissive for neuronal differentiation; (3) while absolute levels change over time, relative splice form expression levels are largely maintained in the trigeminal placode domain; and (4) Pax3 expression generally increases in response to Notch inhibition.

Heterochronic shift in gene expression leads to ontogenetic morphological divergence between two closely related polyploid species.

Heterochrony-alteration to the rate or timing of development-is an important mechanism of trait differentiation associated with speciation. Heterochrony may explain the morphological divergence between two polyploid species, June sucker (Chasmistes liorus) and Utah sucker (Catostomus ardens). The larvae of both species have terminal mouths; however, as adults, June sucker and Utah sucker develop subterminal and ventral mouths, respectively. We document a difference in the timing of shape development and a corresponding change in the timing of gene expression, suggesting the distinctive mouth morphology in June suckers may result from paedomorphosis. Specifically, adult June suckers exhibit an intermediate mouth morphology between the larval (terminal) and ancestral (ventral) states. Endemic and sympatric Chasmistes/Catostomus pairs in two other lakes also are morphologically divergent, but genetically similar. These species pairs could have resulted from the differential expression of genes and corresponding divergence in trait development. Paedomorphosis may lead to adaptive diversification in Catostomids.

Sensory neuron differentiation is regulated by notch signaling in the trigeminal placode.

Trigeminal sensory neurons develop from the neural crest and neurogenic placodes, and have been studied as a principal model of sensory neuron formation. While the Notch pathway has been extensively characterized in central nervous system development and other developmental processes, it has not been well characterized in sensory neurogenesis. Here we studied the functional role of Notch signaling in the trigeminal ophthalmic (opV) placode, a prime model of sensory neurogenesis. To establish a good spatiotemporal description of Notch pathway genes in the chick trigeminal placode, a stage-specific expression analysis was conducted, showing that expression of most Notch pathway genes and effectors are expressed in the placode, with expression primarily being confined to ectodermal cells. Expression was highest at stages of peak neuronal differentiation. To test the function of Notch signaling in opV placode cell differentiation, Notch receptor cleavage was blocked using the gamma-secretase inhibitor, DAPT, or signaling was activated by misexpression of the Notch intracellular domain (NICD). Notch activation resulted in a significant reduction in sensory neurogenesis. Cells remained in the ectoderm and did not differentiate. Expression of the opV specification marker Pax3 was also lost in targeted cells. DAPT exposure resulted in a dramatic increase in neurogenesis without increasing proliferation, where many differentiated cells were found in the mesenchyme and, surprisingly, within the ectoderm. This is the first result clearly showing prolific neuronal differentiation in the ectoderm of the trigeminal placodes after experimental manipulation of a molecular signaling pathway, thus identifying Notch signaling as a primary regulator of the sensory neuron fate in the opV placode.

Activation of Pax3 target genes is necessary but not sufficient for neurogenesis in the ophthalmic trigeminal placode.

Vertebrate cranial neurogenic placodes are relatively simple model systems for investigating the control of sensory neurogenesis. The ophthalmic trigeminal (opV) placode, for which the earliest specific marker is the paired domain homeodomain transcription factor Pax3, forms cutaneous sensory neurons in the ophthalmic lobe of the trigeminal ganglion. We previously showed that Pax3 expression in avian opV placode cells correlates with specification and commitment to a Pax3+, cutaneous sensory neuron fate. Pax3 can act as a transcriptional activator or repressor, depending on the cellular context. We show using mouse Splotch(2H) mutants that Pax3 is necessary for the normal neuronal differentiation of opV placode cells. Using an electroporation construct encoding a Pax3-Engrailed fusion protein, which represses Pax3 target genes, we show that activation of Pax3 target genes is required cell-autonomously within chick opV placode cells for expression of the opV placode markers FGFR4 and Ngn2, maintenance of the preplacodal marker Eya2, expression of Pax3 itself (suggesting that Pax3 autoregulates), neuronal differentiation and delamination. Mis-expression of Pax3 in head ectoderm is sufficient to induce FGFR4 and Ngn2 expression, but neurons do not differentiate, suggesting that additional signals are necessary to enable Pax3+ cells to differentiate as neurons. Mis-expression of Pax3 in the Pax2+ otic and epibranchial placodes also downregulates Pax2 and disrupts otic vesicle closure, suggesting that Pax3 is sufficient to alter the identity of these cells. Overall, our results suggest that activation of Pax3 target genes is necessary but not sufficient for neurogenesis in the opV placode.

The Chicken Embryo as a Model in Developmental Toxicology.

The chicken embryo is a versatile and effective model for studying the effects of teratogenic compounds during early development. Easy access to the embryo allows for exposure and analysis of toxicant effects during embryogenesis. This chapter will provide detailed protocols for embryonic collection and toxicant exposure techniques, including EC culture and Cornish Pasty methods, LysoTracker staining, glutathione redox potential analysis, and 2',7'-dichlorodihydrofluorescein diacetate.

Using a Reconciliation Module Leads to Large Gains in Evolution Acceptance.

Too many students reject the theory of evolution because they view it as incompatible with their religious beliefs. Some have argued that abandoning religious belief is the only way to help religious individuals accept evolution. Conversely, our data support that highlighting faith/evolution compatibility is an effective means to increase student acceptance. We surveyed students enrolled in entry-level biology courses at four religiously affiliated institutions. At each university, teachers gave students a presentation that demonstrated potential compatibility between evolution and faith within the teachings of each university's respective religious affiliation. Students were asked to evaluate their own beliefs about evolution both before and after this instruction. After instruction at each university, students showed significant gains in evolution acceptance without abandoning their religious beliefs. These results demonstrate that giving religious students the opportunity to reconcile their religious beliefs with the theory of evolution under the influence of intentional instruction on the compatibility of belief and evolution can lead to increased evolution acceptance among religious students.

Electroporation strategies for genetic manipulation and cell labeling.

Electroporation has emerged as an effective method for cell labeling and manipulation of gene expression. In the past decade, electroporation applications have expanded to include in vivo chick, mouse, Xenopus, and zebrafish techniques, along with numerous in vitro strategies for cell and tissue culture. We focus on applications relevant to neural stem cell research, providing detailed protocols for in ovo chick electroporation and in vitro targeting of neuroepithelial precursor cells. Electroporation descriptions and related figures identify the tools and reagents needed to carry out targeting of the neuroepithelium. Various applications of the electroporation technique in neural stem cell research are highlighted, along with corresponding publications.

Placental morphology in two sympatric Andean lizards of the genus Liolaemus (Reptilia: Liolaemidae).

Viviparity is a remarkable feature in squamate sauropsids and it has evolved multiple times in parallel with the formation of a placenta. One example of this repeated evolution of viviparity and placentation occurs in the species-rich South American genus Liolaemus with at least six independent origins of viviparity. However, evolutionary studies of placentation in this genus are limited by a lack of data on placental morphology. The aim of this study is to describe and compare the microanatomy and vessel diameter (Dv, a function of blood flow) of the placenta using scanning electron microscopy (SEM) and confocal laser scanning microscopy (cLSM) in two sympatric Andean viviparous but highly divergent species, Liolaemus robustus and Liolaemus walkeri. We found interspecific differences in cell types in the chorion, allantois, and omphalopleure that may be explained by divergent phylogenetic history. Time elapsed since divergence may also explain the pronounced interspecific differences in vessel diameter, and within each species, there are strong differences in Dv between tissue locations. Both species show features to improve gas exchange in the chorioallantoic placenta including absence of eggshell, large Dv in the allantois (L. robustus) or embryonic side of the uterus (L. walkeri), and when present, microvillous cells in the allantois (L. walkeri). Both species also show features that suggest transfer of nutrients or water in the omphaloplacenta, including an almost complete reduction of the eggshell, secretive material (L. robustus), or vesicles (L. walkeri) on cell surface uterus, and when present specialized cells in the omphalopleure (L. walkeri). No statistical differences in Dv were found among stages 32-39 in each species, suggesting that a different mechanism, other than enhanced blood flow, might satisfy the increased oxygen demand of the developing embryos in the hypoxic environments of the high Andes.

Expression of Frizzled genes in the developing chick eye.

Frizzleds are transmembrane receptors that can transduce signals dependent upon binding of Wnts, a large family of secreted glycoproteins homologous to the Drosophila wingless (wg) gene product and critical for a wide variety of normal and pathological developmental processes. In the nervous system, Wnts and Frizzleds play an important role in anterior-posterior patterning, cell fate decisions, proliferation, and synaptogenesis. However, little is known about the role of Frizzled signaling in the developing eye. We isolated cDNAs for ten chick Frizzleds and analyzed the spatial and temporal expression patterns during eye development in the chick embryo. Frizzled-1 to -9 are specifically expressed in the eye at various stages of development and show a complex and partially overlapping pattern of expression.

Expression of green fluorescent protein in the chicken using in vivo transfection of the piggyBac transposon.

The chicken is a well-established model system for studying developmental biology and is recognized as one of the top food production animals in the world. For this reason the chicken is an excellent candidate for transgenic applications, as the technology can be applied to both areas of research. Transgenic technology has not been broadly utilized in the chicken model, however, primarily due to difficulties in targeting germ cells and establishing germ line transmission. Transgenic technologies using non-replicating viral particles have been used in the chick, but are unsuitable for many applications because of size and sequence restraints and low efficiency. To create a more versatile method to target chick germ line stem cells, we utilized the transposable element system piggyBac paired with an in vivo transfection reagent, JetPEI. piggyBac has been previously shown to be highly active in mammalian cells and will transpose into the chicken genome. Here, we show that JetPEI can transfect multiple chick cell types, most notably germline stem cells. We also show that pairing these two reagents is a viable and reproducible method for long-term expression of a transgene in the chicken. Stable expression of the green fluorescent protein (GFP) transgene was seen in multiple tissue types including heart, brain, liver, intestine, kidney and gonad. Combining an in vivo transfection strategy with the PB system provides a simple and flexible method for efficiently producing stable chimeric birds and could be used for production of germ line transgenics.

FGF and Notch signaling in sensory neuron formation: a multifactorial approach to understanding signaling pathway hierarchy.

The ophthalmic trigeminal (opV) placode exclusively gives rise to sensory neurons, making it a good model to study the molecular regulation of sensory neurogenesis. A number of signaling pathways including Wnt, PDGF, FGF, and Notch have been shown to be involved in the process of opV placode cell development. However, the regulatory relationships between these signaling pathways in placode cells are still unknown and have been difficult to study experimentally. Using a novel multifactorial approach in chick embryos that allows for inhibition of FGF throughout the tissue or in individual cells, with simultaneous inactivation of Notch signaling, we investigated the potential interaction between the FGF and Notch signaling pathways in trigeminal sensory neurogenesis. This study builds on prior research describing the individual role of FGF signaling or Notch signaling in opV placode development, where blocking FGF signaling resulted in neurogenesis failure, while blocking Notch signaling resulted in enhanced neurogenesis. Reported here, blocking both pathways simultaneously resulted in a reduction in the number of cells delaminating from the opV placode and undergoing sensory neuron differentiation. Further, Notch inhibition alone did not lead to an increase in the number of cells expressing FGFR4 or in the FGFR4 expression domain, but did result in a highly fragmented basal lamina, which was reversed when blocking FGF signaling. Cumulatively, the results presented here do not support a model of Notch/FGF interdependence, rather that FGF and Notch act in parallel to promote sensory neurogenesis.

Microdistribution of tetrodotoxin in two species of blue-ringed octopuses (Hapalochlaena lunulata and Hapalochlaena fasciata) detected by fluorescent immunolabeling.

Blue-ringed octopuses (genus Hapalochlaena) possess the potent neurotoxin tetrodotoxin (TTX). We examined the microdistribution of TTX in ten tissues of Hapalochlaena lunulata and Hapalochlaena fasciata by immunolabeling for fluorescent light microscopy (FLM). We visualized TTX throughout the posterior salivary gland, but the toxin was concentrated in cells lining the secretory tubules within the gland. Tetrodotoxin was present just beneath the epidermis of the integument (mantle and arms) and also concentrated in channels running through the dermis. This was suggestive of a TTX transport mechanism in the blood of the octopus, which would also explain the presence of the toxin in the blood-rich brachial hearts, gills, nephridia, and highly vascularized Needham's sac (testes contents). We also present the first report of TTX in any cephalopod outside of the genus Hapalochlaena. A specimen of Octopus bocki from French Polynesia contained a small amount of TTX in the digestive gland.

Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells: induction, maintenance, and differentiation into functional schwann cells.

The neural crest (NC) is a transient, multipotent, migratory cell population unique to vertebrates that gives rise to diverse cell lineages. Much of our knowledge of NC development comes from studies of organisms such as chicken and zebrafish because human NC is difficult to obtain because of its transient nature and the limited availability of human fetal cells. Here we examined the process of NC induction from human pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). We showed that NC cells could be efficiently induced from hESCs by a combination of growth factors in medium conditioned on stromal cells and that NC stem cells (NCSCs) could be purified by p75 using fluorescence-activated cell sorting (FACS). FACS-isolated NCSCs could be propagated in vitro in five passages and cryopreserved while maintaining NCSC identity characterized by the expression of a panel of NC markers such as p75, Sox9, Sox10, CD44, and HNK1. In vitro-expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system, as well as mesenchymal derivatives. hESC-derived NCSCs appeared to behave similarly to endogenous embryonic NC cells when injected in chicken embryos. Using a defined medium, we were able to generate and propagate a nearly pure population of Schwann cells that uniformly expressed glial fibrillary acidic protein, S100, and p75. Schwann cells generated by our protocol myelinated rat dorsal root ganglia neurons in vitro. To our knowledge, this is the first report on myelination by hESC- or iPSC-derived Schwann cells.

FGF signaling is essential for ophthalmic trigeminal placode cell delamination and differentiation.

The ophthalmic trigeminal (opV) placode gives rise exclusively to sensory neurons of the peripheral nervous system, providing an advantageous model for understanding neurogenesis. The signaling pathways governing opV placode development have only recently begun to be elucidated. Here, we investigate the fibroblast growth factor receptor-4 (FGFR4), an opV expressed gene, to examine if and how FGF signaling regulates opV placode development. After inhibiting FGFR4, Pax3+ opV placode cells failed to delaminate from the ectoderm and did not contribute to the opV ganglion. Blocking FGF signaling also led to a loss of the early and late neuronal differentiation markers Ngn2, Islet-1, NeuN, and Neurofilament. In addition, without FGF signaling, cells that stalled in the ectoderm lost their opV placode-specific identity by down-regulating Pax3. We conclude that FGF signaling, through FGFR4, is necessary for delamination and differentiation of opV placode cells.

Canonical Wnt signaling is required for ophthalmic trigeminal placode cell fate determination and maintenance.

Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral sensory nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a useful model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the signaling pathways governing opV placode development have not. Here we tested in chick whether the canonical Wnt signaling pathway regulates opV placode development. By introducing a Wnt reporter into embryonic chick head ectoderm, we show that the canonical pathway is active in Pax3+ opV placode cells as, or shortly after, they are induced to express Pax3. Blocking the canonical Wnt pathway resulted in the failure of targeted cells to adopt or maintain an opV fate, as assayed by the expression of various markers including Pax3, FGFR4, Eya2, and the neuronal differentiation markers Islet1, neurofilament, and NeuN, although, surprisingly, it led to upregulation of Neurogenin2, both in the opV placode and elsewhere in the ectoderm. Activating the canonical Wnt signaling pathway, however, was not sufficient to induce Pax3, the earliest specific marker of the opV placode. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells toward an opV placode fate.

Intrinsic signals regulate the initial steps of myogenesis in vertebrates.

In vertebrates, despite the evidence that extrinsic factors induce myogenesis in naive mesoderm, other experiments argue that the initiation of the myogenic program may take place independent of these factors. To resolve this discrepancy, we have re-addressed this issue, using short-term in vivo microsurgery and culture experiments in chick. Our results show that the initial expression of the muscle-specific markers Myf5 and MyoD is regulated in a mesoderm-autonomous fashion. The reception of a Wnt signal is required for MyoD, but not Myf5 expression; however, we show that the source of the Wnt signal is intrinsic to the mesoderm. Gain- and loss-of-function experiments indicate that Wnt5b, which is expressed in the presomitic mesoderm, represents the MyoD-activating cue. Despite Wnt5b expression in the presomitic mesoderm, MyoD is not expressed in this tissue: our experiments demonstrate that this is due to a Bmp inhibitory signal that prevents the premature expression of MyoD before somites form. Our results indicate that myogenesis is a multistep process which is initiated prior to somite formation in a mesoderm-autonomous fashion; as somites form, influences from adjacent tissues are likely to be required for maintenance and patterning of early muscles.