The crucial role of model systems in understanding the complexity of cell signaling in human neurocristopathies.

Animal models are useful to study the molecular, cellular, and morphogenetic mechanisms underlying normal and pathological development. Cell-based study models have emerged as an alternative approach to study many aspects of human embryonic development and disease. The neural crest (NC) is a transient, multipotent, and migratory embryonic cell population that generates a diverse group of cell types that arises during vertebrate development. The abnormal formation or development of the NC results in neurocristopathies (NCPs), which are characterized by a broad spectrum of functional and morphological alterations. The impaired molecular mechanisms that give rise to these multiphenotypic diseases are not entirely clear yet. This fact, added to the high incidence of these disorders in the newborn population, has led to the development of systematic approaches for their understanding. In this article, we have systematically reviewed the ways in which experimentation with different animal and cell model systems has improved our knowledge of NCPs, and how these advances might contribute to the development of better diagnostic and therapeutic tools for the treatment of these pathologies. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics Congenital Diseases > Stem Cells and Development Congenital Diseases > Molecular and Cellular Physiology Neurological Diseases > Genetics/Genomics/Epigenetics.

Rhodotorula glutinis T13 as a potential source of microbial lipids for biodiesel generation.

Single cell oils (SCO) are a promising source of oils that could be exploited in different industrial areas. SCO for biodiesel production circumvents the controversy food vs. fuel, does not require large land areas for culture, and is independent of climate and seasonal variations, among other advantages in comparison to vegetable oils. In this study, a red yeast isolated from a mountain water source, identified as Rhodotorula glutinis T13, showed high potential for lipid production (40% w/w) with suitable growth parameters, yields, and fatty acids profile. Yeast lipids showed a high content of unsaturated fatty acids (56.44%; C18:1, C18:2), and the fuel properties (cetane number, iodine value, density, kinematic viscosity, etc.) of yeast oil analysed were in good agreement with international biodiesel standards. The results show that R. glutinis T13 can be used in the future as a promising microorganism for the commercial production of biodiesel.

Geoffroea decorticans fruit extracts inhibit the wnt/ß-catenin pathway, a therapeutic target in cancer.

Geoffroea decorticans (chañar) is commonly used for culinary and medicinal purposes in rural communities. The aim of this work was to chemically characterize three Geoffroea decorticans extracts and determine their capacity to modulate the wnt/ß-catenin pathway. This signaling pathway plays a key role in embryonic development but its overactivation leads to cancer cell growth. Phytochemical analysis of extracts showed presence of major classes of phytochemicals. Gas chromatography-mass spectrometry results revealed the presence of acids, esters and furanic compounds. Using Xenopus embryos as in vivo model organisms, we found that the extracts modulated dorso-ventral axis formation and rescued hyperdorsalized phenotypes produced by LiCl treatment. In agreement with these findings, Geoffroea decorticans extracts decreased ß-catenin levels and suppressed the expression of wnt target genes such as xnr3 and chordin, thus demonstrating an inhibitory regulation of the wnt/ß-catenin signaling pathway. All these results support a new role for Geoffroea decorticans fruit derivatives with possible anti-carcinogenic actions.

Neurogenesis From Neural Crest Cells: Molecular Mechanisms in the Formation of Cranial Nerves and Ganglia.

The neural crest (NC) is a transient multipotent cell population that originates in the dorsal neural tube. Cells of the NC are highly migratory, as they travel considerable distances through the body to reach their final sites. Derivatives of the NC are neurons and glia of the peripheral nervous system (PNS) and the enteric nervous system as well as non-neural cells. Different signaling pathways triggered by Bone Morphogenetic Proteins (BMPs), Fibroblast Growth Factors (FGFs), Wnt proteins, Notch ligands, retinoic acid (RA), and Receptor Tyrosine Kinases (RTKs) participate in the processes of induction, specification, cell migration and neural differentiation of the NC. A specific set of signaling pathways and transcription factors are initially expressed in the neural plate border and then in the NC cell precursors to the formation of cranial nerves. The molecular mechanisms of control during embryonic development have been gradually elucidated, pointing to an important role of transcriptional regulators when neural differentiation occurs. However, some of these proteins have an important participation in malformations of the cranial portion and their mutation results in aberrant neurogenesis. This review aims to give an overview of the role of cell signaling and of the function of transcription factors involved in the specification of ganglia precursors and neurogenesis to form the NC-derived cranial nerves during organogenesis.

Growth and lipid production of Rhodotorula glutinis R4, in comparison to other oleaginous yeasts.

Some Rhodotorula spp. have been characterized as oleaginous yeasts. Under certain culture conditions they can accumulate neutral lipids, which are mainly triglycerides (TAG). Microbial TAG that can be used as raw material for biodiesel synthesis are attractive for the biofuel industry. In this study, the ability to synthesize lipids of Rhodotorula glutinis R4, isolated in Antarctica, was compared with eight strains belonging to the genera Rhodotorula and Yarrowia with the aim of proposing a novel source of oils for biodiesel synthesis. All strains were cultured under nitrogen (N) limiting conditions and an excess of carbon (C) in the culture medium. We found that yeasts accumulated between 9-48.9 % (w/w) of lipids. Among them, R. glutinis R4 showed the highest growth (14 g L-1, µmax 0,092 h-1) and lipid production (7 g L-1; 47 % w/w). Microbial oils produced by R. glutinis R4 are similar to vegetable oils, with 61 % of oleic acid, indicating that it is adequate for biodiesel synthesis. Our results demonstrate that biodiesel derived from R. glutinis R4 complies with international fuel standards ASTM D6751 and EN 14214. Therefore, this work demonstrates that Rhodotorula glutinis R4 is a novel and valuable source of microbial oils for biodiesel synthesis.

The role of teratogens in neural crest development.

The neural crest (NC), discovered by Wilhelm His 150 years ago, gives rise to a multipotent migratory embryonic cell population that generates a remarkably diverse and important array of cell types during the development of the vertebrate embryo. These cells originate in the neural plate border (NPB), which is the ectoderm between the neural plate and the epidermis. They give rise to the neurons and glia of the peripheral nervous system, melanocytes, chondrocytes, smooth muscle cells, odontoblasts and neuroendocrine cells, among others. Neurocristopathies are a class of congenital diseases resulting from the abnormal induction, specification, migration, differentiation or death of NC cells (NCCs) during embryonic development and have an important medical and societal impact. In general, congenital defects affect an appreciable percentage of newborns worldwide. Some of these defects are caused by teratogens, which are agents that negatively impact the formation of tissues and organs during development. In this review, we will discuss the teratogens linked to the development of many birth defects, with a strong focus on those that specifically affect the development of the NC, thereby producing neurocristopathies. Although increasing attention is being paid to the effect of teratogens on embryonic development in general, there is a strong need to critically evaluate the specific role of these agents in NC development. Therefore, increased understanding of the role of these factors in NC development will contribute to the planning of strategies aimed at the prevention and treatment of human neurocristopathies, whose etiology was previously not considered.

Activation of Hes1 and Msx1 in Transgenic Mouse Embryonic Stem Cells Increases Differentiation into Neural Crest Derivatives.

The neural crest (NC) comprises a multipotent cell population that produces peripheral neurons, cartilage, and smooth muscle cells, among other phenotypes. The participation of Hes1 and Msx1 when expressed in mouse embryonic stem cells (mESCs) undergoing NC differentiation is unexplored. In this work, we generated stable mESCs transfected with constructs encoding chimeric proteins in which the ligand binding domain of glucocorticoid receptor (GR), which is translocated to the nucleus by dexamethasone addition, is fused to either Hes1 (HGR) or Msx1 (MGR), as well as double-transgenic cells (HGR+MGR). These lines continued to express pluripotency markers. Upon NC differentiation, all lines exhibited significantly decreased Sox2 expression and upregulated Sox9, Snai1, and Msx1 expression, indicating NC commitment. Dexamethasone was added to induce nuclear translocation of the chimeric proteins. We found that Collagen IIa transcripts were increased in MGR cells, whereas coactivation of HGR+MGR caused a significant increase in Smooth muscle actin (α-Sma) transcripts. Immunostaining showed that activation in HGR+MGR cells induced higher proportions of ß-TUBULIN III⁺, α-SMA⁺ and COL2A1⁺ cells. These findings indicate that nuclear translocation of MSX-1, alone or in combination with HES-1, produce chondrocyte-like cells, and simultaneous activation of HES-1 and MSX-1 increases the generation of smooth muscle and neuronal cells.

Molecular characterization of wdr68 gene in embryonic development of Xenopus laevis.

WDR68, also known as DCAF7, is a WD40 repeated domain protein highly conserved in eukaryotic organisms in both plants and animals. This protein participates in numerous cellular processes and exerts its function through interaction with other proteins. In the present work, we isolated, sequenced and characterized cDNA corresponding to the wdr68 gene in embryos of the amphibian Xenopus laevis. Syntenic analysis revealed high conservation of the genomic region containing the WDR68 locus in amniotes. Nevertheless, in fishes and amphibians, we observed that the tandem genes surrounding wdr68 undergoes certain rearrangements with respect to the organization found in amniotes. We also defined the temporal and spatial expression pattern of the wdr68 gene in the development of Xenopus laevis through whole mount in situ hybridization and RT-PCR techniques. We observed that wdr68 is ubiquitously expressed during early embryonic development but, during the neurula stage, it undergoes a strong expression in the neural tube and in the migratory cephalic streams of the neural crest. At the tailbud stages, it is strongly expressed in the cephalic region, particularly in otic and optic vesicles, in addition to branchial arches. In contrast, wdr68 transcripts are localized in the somitic mesoderm in the trunk. The expression area that includes the migratory neural crest of the head and the branchial arches suggest that this gene would be involved in jaws formation, probably through a hierarchical relationship with the component genes of the endothelin-1/endothelin receptor type A cell signaling pathway.

Gli2 is required for the induction and migration of Xenopus laevis neural crest.

The neural crest (NC) is a multipotent migratory embryonic population that is formed during late gastrulation and gives rise to a wide array of derivatives, including cells from the peripheral nervous system (PNS), the craniofacial bones and cartilages, peripheral glial cells, and melanocyte cells, among others. In this work we analyzed the role of the Hedgehog signaling pathway effector gli2 in Xenopus NC. We provide evidence that the gli2 gene is expressed in the prospective, premigratory and migratory NC. The use of a specific morpholino against gli2 and the pharmacological specific inhibitor GANT61 in different experimental approaches allowed us to determine that gli2 is required for the induction and specification of NC cells as a transcriptional activator. Moreover, gli2 also acts by reducing apoptosis in the NC without affecting its cell proliferation status. We also demonstrated that gli2 is required cell-autonomously for NC migration, and for the formation of NC derivatives such as the craniofacial cartilages, melanocytes and the cranial ganglia. Altogether, our results showed that gli2 is a key transcriptional activator to accomplish the proper specification and development of Xenopus NC cells.

Neurocristopathies: New insights 150 years after the neural crest discovery.

The neural crest (NC) is a transient, multipotent and migratory cell population that generates an astonishingly diverse array of cell types during vertebrate development. These cells, which originate from the ectoderm in a region lateral to the neural plate in the neural fold, give rise to neurons, glia, melanocytes, chondrocytes, smooth muscle cells, odontoblasts and neuroendocrine cells, among others. Neurocristopathies (NCP) are a class of pathologies occurring in vertebrates, especially in humans that result from the abnormal specification, migration, differentiation or death of neural crest cells during embryonic development. Various pigment, skin, thyroid and hearing disorders, craniofacial and heart abnormalities, malfunctions of the digestive tract and tumors can also be considered as neurocristopathies. In this review we revisit the current classification and propose a new way to classify NCP based on the embryonic origin of the affected tissues, on recent findings regarding the molecular mechanisms that drive NC formation, and on the increased complexity of current molecular embryology techniques.

Trunk neural crest cells: formation, migration and beyond.

Neural crest cells (NCCs) are a multipotent, migratory cell population that generates an astonishingly diverse array of cell types during vertebrate development. The trunk neural crest has long been considered of particular significance. First, it has been held that the trunk neural crest has a morphogenetic role, acting to coordinate the development of the peripheral nervous system, secretory cells of the endocrine system and pigment cells of the skin. Second, the trunk neural crest additionally has skeletal potential. However, it has been demonstrated that a key role of the trunk neural crest streams is to organize the innervation of the intestine. Although trunk NCCs have a limited capacity for self-renewal, sometimes they become neural-crest-derived tumor cells and reveal the fact that that NCCs and tumor cells share the same molecular machinery. In this review we describe the routes taken by trunk NCCs and consider the signals and cues that pattern these trajectories. We also discuss recent advances in the characterization of the properties of trunk NCCs for various model organisms in order to highlight common themes. Finally, looking to the future, we discuss the need to translate the wealth of data from animal studies to the clinical area in order to develop treatments for neural crest-related human diseases.

Oleaginous yeasts from Antarctica: Screening and preliminary approach on lipid accumulation.

The capability of 17 Rhodotorula spp. isolated from Antarctica to accumulate intracellular lipids in nitrogen-limited medium was investigated. As results, 10 isolates were selected by Nile red staining, while 12 isolates were selected as oleaginous by analysis of total lipid content (20.4-73%, w/w of dry biomass). The higher lipid production and accumulation was exhibited for six strains belonging to three species of Rhodotorula (Rhodotorula glutinis, Rhodotorula glacialis, and Rhodotorula laryngis). This is the first report where R. laryngis have been identified within oleaginous specie. Lipid accumulation was evaluated comparatively in two nitrogen-limited glucose-based media (MI and MII). MI (low C/N ratio) was more suitable for biomass and lipid production while in MII (high C/N ratio) total lipid content was improved. R. glutinis R4, R. glacialis R15, and R. glutinis R48 showed high lipid concentrations (4.65-6.93 g L-1 ) and they were able to accumulate large amounts of lipids per gram of biomass (47-77%, w/w). A similar profile in fatty acids composition and content of neutral lipids to vegetable oils was observed, indicating that lipids produced by oleaginous Antarctic yeasts can be considered an alternative feedstock for biodiesel production. Antarctica represents an important source of oleaginous yeasts with adaptive capabilities to accumulate considerable amounts of lipids with biotechnological interest at 15 °C and 25 °C.

Functional analysis of Hairy genes in Xenopus neural crest initial specification and cell migration.

BACKGROUND: Neural crest formation is one of the fundamental processes in the early stages of embryonic development in vertebrates. This transient and multipotent embryonic cell population is able to generate a variety of tissues and cell types in the adult body. hairy genes are transcription factors that contain a basic helix-loop-helix domain which binds to DNA. In Xenopus three hairy genes are known: hairy1, hairy2a, and hairy2b. The requirement of hairy genes was explored in early neural crest development although the late requirements of these genes during neural crest maintenance, migration and derivatives formation are still unknown. RESULTS: In this work, we extended the analysis of Xenopus hairy genes expression patterns and described new domains of expression. Functional analysis showed that hairy genes are required for the induction and migration of the neural crest and for the control of apoptosis. Moreover, we showed that hairy genes function as transcriptional repressors and that they are down-regulated by bone morphogenetic protein-Smad signaling and positively regulated by the Notch/Delta-Su(h) pathway. CONCLUSIONS: Our results indicate that hairy genes have a functional equivalence between them and that they are required for multiple processes during neural crest development.

Two different vestigial like 4 genes are differentially expressed during Xenopus laevis development.

The vestigial gene (vg) was first characterized in Drosophila and several homologues were identified in vertebrates and called vestigial like 1-4 (vgll1-4). Vgll proteins interact with the transcription factors TEF-1 and MEF-2 through a conserved region called TONDU (TDU). Vgll4s are characterized by two tandem TDU domains which differentiate them from other members of the vestigial family. In Xenopus two genes were identified as vgll4. Our bioinformatic analysis demonstrated that these two genes are paralogues and must be named differently. We designated them as vgll4 and vgll4l. In situ hybridization analysis revealed that the expression of these two genes is rather different. At gastrula stage, both were expressed in the animal pole. However, at neurula stage, vgll4 was mainly expressed in the neural plate and neural folds, while vgll4l prevailed in the neural folds and epidermis. From the advanced neurula stage onward, expression of both genes was strongly enhanced in neural tissues, anterior neural plate, migrating neural crest, optic and otic vesicles. Nevertheless, there were some differences: vgll4 presented somite expression and vgll4l was localized at the skin and notochord. Our results demonstrate that Xenopus has two orthologues of the vgll4 gene, vgll4 and vgll4l with differential expression in Xenopus embryos and they may well have different roles during development.

Developmental expression and role of Kinesin Eg5 during Xenopus laevis embryogenesis.

BACKGROUND: The neural crest is a transient multipotent migratory cell population unique to vertebrates. These cells undergo an epithelial-to-mesenchymal transition and migrate extensively through the embryo. They differentiate into numerous diverse derivatives including the peripheral nervous system, melanocytes,and craniofacial cartilages. The development of the neural crest is mediated by complex interactions of multiple signals and transcription factors. The kinesin Eg5 is a plus end-directed microtubule-based motor protein that is essential for bipolar spindle formation during mitosis and meiosis, axon growth, and mammal embryonic development. RESULTS: We analyzed in detail the expression pattern of eg5 and established that it is expressed at the prospective neural fold, in the premigratory and migratory neural crest. Functional analysis revealed that in Xenopus, early embryogenesis eg5 function is required during neural crest induction, specification, and maintenance. eg5 is also required during neural crest migration and for derivatives formation. Moreover, we demonstrated a hierarchical relationship with the Indian Hedgehog signaling pathway. CONCLUSIONS: Our results show that eg5 is essential for the specification and maintenance of neural crest progenitors during Xenopus early embryogenesis rather than cell proliferation and survival.

Indian hedgehog signaling is required for proper formation, maintenance and migration of Xenopus neural crest.

Neural crest induction is the result of the combined action at the neural plate border of FGF, BMP, and Wnt signals from the neural plate, mesoderm and nonneural ectoderm. In this work we show that the expression of Indian hedgehog (Ihh, formerly named Banded hedgehog) and members of the Hedgehog pathway occurs at the prospective neural fold, in the premigratory and migratory neural crest. We performed a functional analysis that revealed the requirement of Ihh signaling in neural crest development. During the early steps of neural crest induction loss of function experiments with antisense morpholino or locally grafted cyclopamine-loaded beads suppressed the expression of early neural crest markers concomitant with the increase in neural and epidermal markers. We showed that changes in Ihh activity produced no alterations in either cell proliferation or apoptosis, suggesting that this signal involves cell fate decisions. A temporal analysis showed that Hedgehog is continuously required not only in the early and late specification but also during the migration of the neural crest. We also established that the mesodermal source of Ihh is important to maintain specification and also to support the migratory process. By a combination of embryological and molecular approaches our results demonstrated that Ihh signaling drives in the migration of neural crest cells by autocrine or paracrine mechanisms. Finally, the abrogation of Ihh signaling strongly affected only the formation of cartilages derived from the neural crest, while no effects were observed on melanocytes. Taken together, our results provide insights into the role of the Ihh cell signaling pathway during the early steps of neural crest development.

ΔNp63 is regulated by BMP4 signaling and is required for early epidermal development in Xenopus.

BACKGROUND: It has been established in several models that the p63 gene has an important role in the development of the epidermis and its derivatives. In Xenopus, only the ΔNp63 isoform of this gene has been cloned and its role during epidermal development remains unknown. RESULTS: In this work, we showed that ΔNp63 is expressed in the nonneural ectoderm since the gastrula stage and that it is regulated by the bone morphogenetic protein 4 (BMP4) signaling pathway. Our in vivo and in vitro experiments demonstrated that ΔNp63 is required in the earliest inductive steps of epidermal development. The overexpression of ΔNp63 caused an increase in epidermal markers with a suppression of neural induction while the blocking of ΔNp63 led to the opposite results. Finally, we found that ΔNp63 acts as an anti-apoptotic gene, regulating the transcription of some apoptotic and anti-apoptotic factors. CONCLUSION: The results suggest that ΔNp63 is an essential gene in early epidermal specification under the control of BMP4.

A new role for the Endothelin-1/Endothelin-A receptor signaling during early neural crest specification.

The neural crest is induced at the border of the neural plate in a multistep process by signals emanated from the epidermis, neural plate and mesoderm. In this work we show for the first time the existence of a neural crest maintenance step which is dependent on signals released from the mesoderm. We identified Endothelin-1 (Edn1) and its receptor (Ednra) as key players of this signal and we show that Edn1/Ednra signaling is required for maintenance of the neural crest by a dual mechanism of cell specification and cell survival. We show that: (i) Ednra is expressed in prospective neural crest; (ii) loss-of-function experiments with antisense morpholino or with specific chemical inhibitor suppress the expression of early neural crest markers; (iii) gain-of-function experiments expand the neural crest territory; (iv) epistatic experiments show that Ednra/Edn1 is downstream of the early neural crest gene Msx1 and upstream of the late genes Sox9 and Sox10; and (v) Edn1/Ednra signaling inhibits apoptosis and controls cell specification of the neural crest. Together, our results provide insight on a new role of Edn1/Ednra cell signaling pathway during early neural crest development.

Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains.

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.

Cloning and functional characterization of two key enzymes of glycosphingolipid biosynthesis in the amphibian Xenopus laevis.

Gangliosides are a subfamily of complex glycosphingolipids (GSLs) with important roles in many biological processes. In this study, we report the cDNA cloning, functional characterization, and the spatial and temporal expression of Xlcgt and Xlgd3 synthase during Xenopus laevis development. Xlcgt was expressed both maternally and zigotically persisting at least until stage 35. Maternal Xlgd3 synthase mRNA could not be detected and showed a steady-state expression from gastrula to late tailbud stage. Xlcgt is mainly present in involuted paraxial mesoderm, neural folds, and their derivatives. Xlgd3 synthase transcripts were detected in the dorsal blastoporal lip, in the presumptive neuroectoderm, and later in the head region, branchial arches, otic and optic primordia. We determined the effect of glycosphingolipid depletion with 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP) in mesodermal layer. PPMP-injected embryos showed altered expression domains in the mesodermal markers. Our results suggest that GSL are involved in convergent-extension movements during early development in Xenopus.