Verónica Palma

Calcium mediates dorsoventral patterning of mesoderm in Xenopus.

Calcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that alpha1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity.

Functional analysis reveals angiogenic potential of human mesenchymal stem cells from Wharton’s jelly in dermal regeneration

Abstract Disorders in skin wound healing are a major health problem that requires the development of innovative treatments. The use of biomaterials as an alternative of skin replacement has become relevant, but its use is still limited due to poor vascularization inside the scaffolds, resulting in insufficient oxygen and growth factors at the wound site. In this study, we have developed a cell-based wound therapy consisting of the application of collagen-based dermal scaffolds containing mesenchymal stem cells from Wharton’s jelly (WJ-MSC) in an immunocompetent mouse model of angiogenesis. From our comparative study on the secretion profile between WJ-MSC and adipose tissue-derived MSC, we found a stronger expression of several well-characterized growth factors, such as VEGF-A, angiopoietin-1 and aFGF, which are directly linked to angiogenesis, in the culture supernatant of WJ-MSC, both on monolayer and 3D culture conditions. WJ-MSC proved to be angiogenic both in vitro and in vivo, through tubule formation and CAM assays, respectively. Moreover, WJ-MSC consistently improved the healing response in vivo in a mouse model of human-like dermal repair, by triggering angiogenesis and further providing a suitable matrix for wound repair, without altering the inflammatory response in the animals. Since these cells can be easily isolated, cultured with high expansion rates and cryopreserved, they represent an attractive stem cell source for their use in allogeneic cell transplant and tissue engineering.

Sonic Hedgehog in cancer stem cells: a novel link with autophagy

The Sonic Hegdehog/GLI (SHH/GLI) pathway has been extensively studied for its role in developmental and cancer biology. During early embryonic development the SHH pathway is involved mainly in pattern formation, while in latter stages its function in stem cell and progenitor proliferation becomes increasingly relevant. During postnatal development and in adult tissues, SHH/GLI promotes cell homeostasis by actively regulating gene transcription, recapitulating the function observed during normal tissue growth. In this review, we will briefly discuss the fundamental importance of SHH/GLI in tumor growth and cancer evolution and we will then provide insights into a possible novel mechanism of SHH action in cancer through autophagy modulation in cancer stem cells. Autophagy is a homeostatic mechanism that when disrupted can promote and accelerate tumor progression in both cancer cells and the stroma that harbors tumorigenesis. Understanding possible new targets for SHH signaling and its contribution to cancer through modulation of autophagy might provide better strategies in order to design combined treatments and perform clinical trials.

Sonic hedgehog (Shh)-Gli signaling controls neural progenitor cell division in the developing tectum in zebrafish

Despite considerable progress, the mechanisms that control neural progenitor differentiation and behavior, as well as their functional integration into adult neural circuitry, are far from being understood. Given the complexity of the mammalian brain, non‐mammalian models provide an excellent model to study neurogenesis, including both the cellular composition of the neurogenic microenvironment, and the factors required for precursor growth and maintenance. In particular, we chose to address the question of the control of progenitor proliferation by Sonic hedgehog (Shh) using the zebrafish dorsal mesencephalon, known as the optic tectum (OT), as a model system. Here we show that either inhibiting pharmacologically or eliminating hedgehog (Hh) signaling by using mutants that lack essential components of the Hh pathway reduces neural progenitor cell proliferation affecting neurogenesis in the OT. On the contrary, pharmacological gain‐of‐function experiments result in significant increase in proliferation. Importantly, Shh‐dependent function controls neural progenitor cell behavior as sox2‐positive cell populations were lost in the OT in the absence of Hh signaling, as evidenced in slow‐muscle‐omitted (smu) mutants and with timed cyclopamine inhibition. Expressions of essential components of the Hh pathway reveal for the first time a late dorsal expression in the embryonic OT. Our observations argue strongly for a role of Shh in neural progenitor biology in the OT and provide comparative data to our current understanding of progenitor/stem cell mechanisms that place Shh as a key niche factor in the dorsal brain.

Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel

Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant alpha-subunits with the beta1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant α-subunits with the β1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.

Sonic Hedgehog Stimulates Proliferation of Human Periodontal Ligament Stem Cells

Regulation of cell renewal in the periodontium is a critical cell function that has not been clarified. Sonic hedgehog (Shh) is a secreted signaling molecule that plays a key role during development and adult tissue homeostasis. In the present study, we have analyzed the role played by Shh in human periodontal ligament stem cell (HPLSC) proliferation. HPLSC were isolated with anti-STRO-1 antibodies. Shh increased the expression of GLI1 and PTC-1 and selectively stimulated cell proliferation in STRO-1(+) derived from adult periodontal ligament. Shh components were localized to primary cilia in STRO-1(+) cells after Shh stimulation. STRO-1(+) also expressed Shh, suggesting an autocrine-regulated phenomenon. Thus, we propose that Shh plays a critical role in the regulation of cell proliferation in STRO-1(+)/HPLSC.

Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation

Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors.

Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development

BackgroundThe increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh) pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1) and novel Hh-regulated genes in zebrafish embryos.ResultsThe combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo.ConclusionA recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in different contexts of vertebrate embryonic development.

Skeletal plasticity in response to embryonic muscular activity underlies the development and evolution of the perching digit of birds

Most birds have an opposable digit 1 (hallux) allowing the foot to grasp, which evolved from the non-opposable hallux of early theropod dinosaurs. An important morphological difference with early theropods is the twisting of the long axis of its metatarsal. Here, we show how embryonic musculature and the onset of its activity are required for twisting of metatarsal 1 (Mt1) and retroversion of the hallux. Pharmacologically paralyzed embryos do not fully retrovert the hallux and have a straight Mt1 shaft, phenocopying the morphology of early tetanuran dinosaurs. Molecular markers of cartilage maturation and ossification show that differentiation of Mt1 is significantly delayed compared to Mt2-4. We hypothesize on how delayed maturation may have increased plasticity, facilitating muscular twisting. Our experimental results emphasize the importance of embryonic muscular activity in the evolutionary origin of a crucial adaptation.

Neogenin1 is a sonic hedgehog target in medulloblastoma and is necessary for cell cycle progression

The canonical Sonic Hedgehog (Shh)/Gli pathway plays multiples roles during central nervous system (CNS) development. To elucidate the molecular repertoire of Shh mediators, we have recently described novel transcriptional targets in response to Shh pathway modulation. Among them, we were able to identify Neogenin1 (Neo1), a death dependence receptor, as a new direct Shh downstream regulator in neural precursor proliferation. As appropriate Shh signaling is required for cerebellar growth and alterations cause Shh‐driven medulloblastoma (MB), here we have addressed the role of the Shh/Neogenin1 interaction in the context of cerebellar development and cancer. We demonstrate that the Shh pathway regulates Neogenin1 expression in mouse models that recapitulate the Shh MB subtype. We show that the canonical Shh pathway directly regulates the Neo1 gene acting through an upstream sequence in its promoter both in vitro and in vivo in granule neuron precursor cells. We also identified and characterized a functional Gli‐binding site in the first intron of the human NEO1 gene. Gene expression profiling of more than 300 MB shows that NEO1 is indeed upregulated in SHH tumors compared to the other MB subgroups. Finally, we provide evidence that NEO1 is necessary for cell cycle progression in a human MB cell line, because a loss of function of NEO1 arrests cells in the G2/M phase. Taken together, these results highlight Neogenin1 as a novel downstream effector of the Shh pathway in MB and a possible therapeutic target.