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.

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.

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.

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.

A balance between the anti-apoptotic activity of Slug and the apoptotic activity of msx1 is required for the proper development of the neural crest.

We have studied the pattern of programmed cell death in the neural crest and analyzed how it is controlled by the activity of the transcription factors Slug and msx1. Our results indicate that apoptosis is more prevalent in the neural folds than in the rest of the neural ectoderm. Through gain- and loss-of-function experiments with inducible forms of both Slug and msx1 genes, we showed that Slug acts as an anti-apoptotic factor whereas msx1 promotes cell death, either in the neural folds of the whole embryos, in isolated or induced neural crest and in animal cap assays. The protective effect of expressing Slug can be reversed by expressing the apoptotic factor Bax, while the apoptosis promoted by msx1 can be abolished by expressing the Xenopus homologue of Bcl2 (XR11). Furthermore, we show that Slug and msx1 control the transcription of XR11 and several caspases required for programmed cell death. In addition, expression of Bax or Bcl2, produced similar effects on the survival of the neural crest and on the development of its derivatives to those produced by altering the activity of Slug or msx1. Finally, we show that in the neural crest, the region of the neural folds where Slug is expressed, cells undergo less apoptosis, than in the region where the msx1 gene is expressed, which correspond to cells adjacent to the neural crest. We show that the expression of Slug and msx1 controls cell death in certain areas of the neural folds, and we discuss how this equilibrium is necessary to generate sharp boundaries in the neural crest territory, and to precisely control cell number among neural crest derivatives.

Regulation of Msx genes by a Bmp gradient is essential for neural crest specification.

There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, we analyzed how the expression of Bmp targets, the Msx genes, is regulated and the role that Msx genes has in neural crest specification. As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. We propose a model where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds, and that this expression is essential for the early specification of the neural crest.