Lipid rafts integrity is essential for the prolactin-induced mitogenesis in mouse embryonic stem cells

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constrains and regulation applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using twodimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergentfree preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs are in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

Cortical Interneuron Development: a role for small Rho GTPases

GABAergic interneurons control cortical excitation/inhibition balance and are implicated in severe neurodevelopmental disorders. In contrast to the multiplicity of signals underlying the generation and migration of cortical interneurons, the intracellular proteins mediating the response to these cues are mostly unknown. We have demonstrated the unique and diverse roles of the Rho GTPases Rac1 and 3 in cell cycle and morphology in transgenic animals where Rac1 and Rac1/3 were ablated specifically in cortical interneurons. In the Rac1 mutant, progenitors delay their cell cycle exit probably due to a prolonged G1 phase resulting in a cortex with 50% reductions in interneurons and an imbalance of excitation/inhibition in cortical circuits. This disruption in GABAergic inhibition alters glutamatergic function in the adult cortex that could be reversed by enhancement of GABAergic function during an early postnatal period. Furthermore, this disruption disturbs the neuronal synchronization in the adult cortex. In the double mutant, additional severe cytoskeletal defects result in an 80% interneuron decrease. Both lines die from epileptic seizures postnatally. We have made progress towards characterizing the cell cycle defect in Rac1 mutant interneuron progenitors, determining the morphological and synaptic characteristics of single and double mutant interneurons and identifying some of the molecular players by which Racs exert their actions by proteomic analysis. In our present work, we review these studies and discuss open questions and future perspectives. We expect that our data will contribute to the understanding of the function of cortical interneurons, especially since preclinical models of interneuron-based cell therapies are being established.

Repression of differentiation genes by Hes transcription factors fuels neural tumour growth in Drosophila

Background: Neural stem cells (NSC) in divide asymmetrically to generate a cell that retains stem cell identity and another that is routed to differentiation. Prolonged mitotic activity of the NSCs gives rise to the plethora of neurons and glial cells that wire the brain and nerve cord. Genetic insults, such as excess of Notch signaling, perturb the normal NSC proliferation programs and trigger the formation of NSC hyperplasias, that can later progress to malignancies. Hes proteins are crucial mediators of Notch signaling and in the NSC context they act by repressing a cohort of early pro-differentiation transcription factors. Downregulation of these pro-differentiation factors makes NSC progeny cells susceptible to adopting an aberrant stem cell program. We have recently shown that Hes overexpression in Drosophila leads to NSC hyperplasias that progress to malignant tumours after allografting to adult hosts. Methods: We have combined genetic analysis, tissue allografting and transcriptomic approaches to address the role of Hes genes in NSC malignant transformation. Results: We show that the E(spl) genes are important mediators in the progression of Notch hyperplasias to malignancy, since allografts lacking the E(spl) genes grow much slower. We further present RNA profiling of Hes-induced tumours at two different stages after allografting. We find that the same cohort of differentiation-promoting transcription factors that are repressed in the primary hyperplasias continue to be downregulated after transplantation. This is accompanied by an upregulation of stress-response genes and metabolic reprogramming. Conclusions: The combination of dedifferentiation and cell physiology changes most likely drive tumour growth.

SV40T reprograms Schwann cells into stem-like cells that can re-differentiate into terminal nerve cells

Background: The specific effect of SV40T on neurocytes has been rarely investigated by the researchers. We transfected Schwann cells (SCs) that did not have differentiation ability with MPH 86 plasmid containing SV40T in order to explore the effects of SV40T on Schwann cells.Methods: SCs were transfected with MPH 86 plasmid carrying the SV40T gene and cultured in different media, as well as co-cultured with neural stem cells (NSCs). In our study, SCs overexpressing SV40T were defined as SV40T-SCs. The proliferation of these cells was detected by WST-1, and the expression of different biomarkers was analyzed by qPCR and immunohistochemistry. Results: SV40T induced the characteristics of NSCs, such as the ability to grow in suspension, form spheroid colonies and proliferate rapidly, in the SCs, which were reversed by knocking out SV40T by the Flip-adenovirus. In addition, SV40T upregulated the expressions of neural crest-associated markers Nestin, Pax3 and Slug, and down-regulated S100b as well as the markers of mature SCs MBP, GFAP and Olig1/2. These cells also expressed NSC markers like Nestin, Sox2, CD133 and SSEA-1, as well as early development markers of embryonic stem cells (ESCs) like BMP4, c-Myc, OCT4 and Gbx2. Co-culturing with NSCs induced differentiation of the SV40T-SCs into neuronal and glial cells. Conclusions: SV40T reprograms Schwann cells to stem-like cells at the stage of neural crest cells (NCCs) that can differentiate to neurocytes.

Development of growth factor-incorporating liposomes for integration into scaffolds as a method to improve tissue regeneration

This review is an update about the efforts to develop liposomal carriers for growth factor delivery. It is well known that growth factors have the potential to enhance/accelerate tissue regeneration, however their poor stability which results in rapid loss of their activity, together with their rapid clearance from defected tissues (when applied as free molecules) is a serious drawback for their use; their highly hydrophilic nature and low capability to permeate through biological barriers (cell membranes) are additional factors that limit their applicability. In the last years, the advantages of liposomal drug delivery systems have motivated efforts to deliver growth factors (GFs) in liposomal form. Herein, after briefly introducing the basic structural characteristics of liposome types and their advantages when used as drug carriers, as well as the basic problems encountered when GFs are applied for tissue regeneration, we focus on recent reports about development and potential regenerative effects of liposomal GFs, towards defects of various tissues. The methodologies used for incorporation, attachment or immobilization of liposomal GFs in order to sustain their retention at the defected tissues, are highlighted as well.

On the role of pleiotrophin and its receptors in development and angiogenesis

The secreted growth factor pleiotrophin (PTN) is expressed in all species and is evolutionary highly conserved, suggesting that it is significant for the regulation of important processes. The observation that it is highly expressed at early stages during development and at embryonic progenitor cells, highlights a potential important contribution to development. There is ample evidence for the role of PTN in the development of the nervous system and hematopoiesis, some but still not conclusive evidence for its role in the skeletomuscular system and limited evidence for its role in the development of other organs. Studies on its role in the cardiovascular system and angiogenesis suggest that PTN has a significant regulatory effect by acting on endothelial cells, while its role in smooth or cardiac muscle cells’ functions has not been studied. This review highlights what is known up to date on the role of PTN in the development of various organs and in angiogenesis. Wherever known, evidence on the crosstalk among the receptors that mediate PTN’s functions is also quoted, highlighting the complex regulatory pathways that affect development and angiogenesis.

Zebrafish research in Greece: swimming against the current

Zebrafish is a vertebrate model extensively used in Developmental Biology and Human Disease modeling as it shares high genetic and physiological similarities with humans. It has become the second most popular animal model, following mice, with several advantages: zebrafish are easily housed and cared for, the cost of installing and maintaining a zebrafish facility is significantly lower than mice, they reproduce often and develop quickly. Using zebrafish complies with the 3Rs principles of laboratory animal use. Zebrafish embryos develop externally and are transparent, allowing for in vivo non-invasive imaging. There are many transgenic and mutant lines available that mimic most Human Diseases, including reporter lines for most signaling pathways. There are also several reverse genetic tools to functionally verify genes or variants of unknown significance, identified in Genome-Wide Association Studies (GWAS) or using Next Generation Sequencing (NGS) approaches. In addition, the model emerges as an invaluable whole animal platform for various stages of drug discovery efforts by exploring the ability to do high-throughput phenotypic-driven screens. These include phenotypic screenings, determinations of general and/or specific toxicity (cardiac, renal, hepatotoxicity etc.), and mechanism of action studies. Finally, zebrafish are able to retain their capacity to regenerate most organs during their entire life span, making them a well-established model to study organ regeneration. The European Zebrafish Society consists of more than 180 research labs throughout Europe. In Greece however, zebrafish use remains rather limited. I present here a brief historical overview of zebrafish research in Greece.

Canonical Wnt signaling regulates branching morphogenesis of submandibular gland by modulating levels of lama5

Background: Branching morphogenesis is a crucial developmental mechanism for the formation of a typical bush-like structure of the submandibular gland (SMG). However, the detailed mechanism underlying this process remains to be fully understood. Here, we investigate whether a cross-talking may exist between Wnt/beta-catenin signaling pathway and lama5 during the branching process in SMG development. Methods: Embryonic mouse SMG organ culture model was established, and the validity of this model was confirmed. The roles of Wnt/beta-catenin signaling pathway, FGF signaling, and Lama5 in the branching process were investigated by morphogenesis assays. And the interactions between these signaling were also investigated and demonstrated by morphogenesis assays and gene expression patterns. Results: We demonstrated that E12 or E13 SMG organ culture model can be used as an ideal approach to study the process of branching morphogenesis. And branching morphogenesis assay revealed that the epithelial branching process would be promoted when the canonical Wnt pathway was inhibited and be significantly suppressed when wnt pathway is over activated. Further experiments indicated that FGF signaling acts most likely acts upstream as a negative regulator of the canonical Wnt pathway during the branching process, whose effect could be partially reversed by Wnt3a. And we further demonstrated that Wnt/beta-catenin signaling regulates the branching morphogenesis through Lama5. Conclusion: Our present work demonstrated that Wnt/beta-catenin signaling pathway acting downstream of FGF signaling may serve as a negative regulatory mechanism in the process of SMG branching morphogenesis through Lama5.

Keratinocyte differentiation and proteolytic pathways in skin (patho)physiology

Epidermis is a stratified epithelium that forms the barrier between the organism and its environment. It is mainly composed of keratinocytes at different stages of differentiation. Stratum corneum is the outermost layer of the epidermis and is formed of multiple layers of anucleated keratinocytes called corneocytes. We aim to highlight the roles of epidermal differentiation and proteolysis in skin diseases. Skin biopsies isolated from Spink5-/- mice, the established model of Netherton syndrome (NS), and from patients with NS, seborrheic dermatitis (SD) and psoriasis, as well as healthy controls, were analyzed by histology and immunohistochemistry. Our results showed that NS, SD, and psoriasis are all characterized by abnormal epidermal differentiation, manifested by hyperplasia, hyperkeratosis, and parakeratosis. At the molecular level, abnormal differentiation is accompanied by increased expression of involucrin and decreased expression of loricrin in NS and psoriasis. Increased epidermal proteolysis associated with increased kallikrein-related peptidases (KLK) expression is also observed in both NS and psoriatic epidermis. Further, reduced expression of desmosomal proteins is observed in NS but increased in psoriasis. Since desmosomal protein are proteolytic substrates and control keratinocyte differentiation, their altered expression directly links epidermal proteolysis to differentiation. In conclusion, abnormal cellular differentiation and proteolysis are interconnected and underlie the pathology of NS, SD and psoriasis.

Evidence of Swim secretion and association with extracellular matrix in the Drosophila embryo

Secreted wingless-interacting protein (Swim) is the Drosophila ortholog gene of the mammalian Tubulointerstitial Nephritis Antigen Like 1 (TINAGL1), known also as lipocalin-7 (LCN7), or adrenocortical zonation factor 1 (AZ-1). Swim and TINAGL1 proteins share a significant homology, including the somatomedin B and the predictive inactive C1 cysteine peptidase domains. In mammals, both TINAGL1 and its closely related homolog TINAG have been identified in basement membranes, where they may function as modulators of integrin-mediated adhesion. In Drosophila, Swim was initially identified in the eggshell matrix and subsequently was detected in the culture medium of S2 cells. Further biochemical analysis indicated that Swim binds to wingless (wg) in a lipid-dependent manner. This observation together with RNAiknockdown studies suggested that Swim is an essential cofactor of wg-signalling. However, recent elegant genetic studies ruled out the possibility that Swim is required alone to facilitate wgsignalling in Drosophila, because flies without Swim are viable and fertile. Here, we use the UAS/Gal4 expression system together with confocal imaging to analyze the in vivo localization of a chimeric Swim-GFP in the developing Drosophila embryo. Our data fully support the notion that Swim is an extracellular matrix component that upon ectopic expression is secreted and preferentially associates with the basement membranes of various organs and with the specialized tendon matrix at the muscle attachment sites (MAS). Interestingly, the accumulation of Swim at the MAS does not require integrins. In conclusion, Swim is an extracellular matrix component, and it is possible that Swim exhibits overlapping functions in concert with other undefined components.