NFIXing Cancer: The Role of NFIX in Oxidative Stress Response and Cell Fate.

NFIX, a member of the nuclear factor I (NFI) family of transcription factors, is known to be involved in muscle and central nervous system embryonic development. However, its expression in adults is limited. Similar to other developmental transcription factors, NFIX has been found to be altered in tumors, often promoting pro-tumorigenic functions, such as leading to proliferation, differentiation, and migration. However, some studies suggest that NFIX can also have a tumor suppressor role, indicating a complex and cancer-type dependent role of NFIX. This complexity may be linked to the multiple processes at play in regulating NFIX, which include transcriptional, post-transcriptional, and post-translational processes. Moreover, other features of NFIX, including its ability to interact with different NFI members to form homodimers or heterodimers, therefore allowing the transcription of different target genes, and its ability to sense oxidative stress, can also modulate its function. In this review, we examine different aspects of NFIX regulation, first in development and then in cancer, highlighting the important role of NFIX in oxidative stress and cell fate regulation in tumors. Moreover, we propose different mechanisms through which oxidative stress regulates NFIX transcription and function, underlining NFIX as a key factor for tumorigenesis.

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture.

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin α2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

Cell-Fibronectin Interactions and Actomyosin Contractility Regulate the Segmentation Clock and Spatio-Temporal Somite Cleft Formation during Chick Embryo Somitogenesis.

Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the unsegmented presomitic mesoderm (PSM). PSM cells undergo cyclic waves of segmentation clock gene expression, followed by Notch-dependent upregulation of meso1 in the rostral PSM which induces somite cleft formation. However, the relevance of the fibronectin matrix for these molecular processes remains unknown. Here, we assessed the role of the PSM fibronectin matrix in the spatio-temporal regulation of chick embryo somitogenesis by perturbing (1) extracellular fibronectin matrix assembly, (2) integrin-fibronectin binding, (3) Rho-associated protein kinase (ROCK) activity and (4) non-muscle myosin II (NM II) function. We found that integrin-fibronectin engagement and NM II activity are required for cell polarization in the nascent somite. All treatments resulted in defective somitic clefts and significantly perturbed meso1 and segmentation clock gene expression in the PSM. Importantly, inhibition of actomyosin-mediated contractility increased the period of hairy1/hes4 oscillations from 90 to 120 min. Together, our work strongly suggests that the fibronectin-integrin-ROCK-NM II axis regulates segmentation clock dynamics and dictates the spatio-temporal localization of somitic clefts.

Linking Oxidative Stress and DNA Damage to Changes in the Expression of Extracellular Matrix Components.

Cells are subjected to endogenous [e.g., reactive oxygen species (ROS), replication stress] and exogenous insults (e.g., UV light, ionizing radiation, and certain chemicals), which can affect the synthesis and/or stability of different macromolecules required for cell and tissue function. Oxidative stress, caused by excess ROS, and DNA damage, triggered in response to different sources, are countered and resolved by specific mechanisms, allowing the normal physiological equilibrium of cells and tissues to be restored. One process that is affected by oxidative stress and DNA damage is extracellular matrix (ECM) remodeling, which is a continuous and highly controlled mechanism that allows tissues to readjust in reaction to different challenges. The crosstalk between oxidative stress/DNA damage and ECM remodeling is not unidirectional. Quite on the contrary, mutations in ECM genes have a strong impact on tissue homeostasis and are characterized by increased oxidative stress and potentially also accumulation of DNA damage. In this review, we will discuss how oxidative stress and DNA damage affect the expression and deposition of ECM molecules and conversely how mutations in genes encoding ECM components trigger accumulation of oxidative stress and DNA damage. Both situations hamper the reestablishment of cell and tissue homeostasis, with negative impacts on tissue and organ function, which can be a driver for severe pathological conditions.

Neonatal Apex Resection Triggers Cardiomyocyte Proliferation, Neovascularization and Functional Recovery Despite Local Fibrosis.

So far, opposing outcomes have been reported following neonatal apex resection in mice, questioning the validity of this injury model to investigate regenerative mechanisms. We performed a systematic evaluation, up to 180 days after surgery, of the pathophysiological events activated upon apex resection. In response to cardiac injury, we observed increased cardiomyocyte proliferation in remote and apex regions, neovascularization, and local fibrosis. In adulthood, resected hearts remain consistently shorter and display permanent fibrotic tissue deposition in the center of the resection plane, indicating limited apex regrowth. However, thickening of the left ventricle wall, explained by an upsurge in cardiomyocyte proliferation during the initial response to injury, compensated cardiomyocyte loss and supported normal systolic function. Thus, apex resection triggers both regenerative and reparative mechanisms, endorsing this injury model for studies aimed at promoting cardiomyocyte proliferation and/or downplaying fibrosis.

Impaired fetal muscle development and JAK-STAT activation mark disease onset and progression in a mouse model for merosin-deficient congenital muscular dystrophy.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a dramatic neuromuscular disease in which crippling muscle weakness is evident from birth. Here, we use the dyW mouse model for human MDC1A to trace the onset of the disease during development in utero. We find that myotomal and primary myogenesis proceed normally in homozygous dyW-/- embryos. Fetal dyW-/- muscles display the same number of myofibers as wildtype (WT) muscles, but by E18.5 dyW-/- muscles are significantly smaller and muscle size is not recovered post-natally. These results suggest that fetal dyW-/- myofibers fail to grow at the same rate as WT myofibers. Consistent with this hypothesis between E17.5 and E18.5 dyW-/- muscles display a dramatic drop in the number of Pax7- and myogenin-positive cells relative to WT muscles, suggesting that dyW-/- muscles fail to generate enough muscle cells to sustain fetal myofiber growth. Gene expression analysis of dyW-/- E17.5 muscles identified a significant increase in the expression of the JAK-STAT target gene Pim1 and muscles from 2-day and 3-week old dyW-/- mice demonstrate a dramatic increase in pSTAT3 relative to WT muscles. Interestingly, myotubes lacking integrin α7ß1, a laminin-receptor, also show a significant increase in pSTAT3 levels compared with WT myotubes, indicating that α7ß1 can act as a negative regulator of STAT3 activity. Our data reveal for the first time that dyW-/- mice exhibit a myogenesis defect already in utero. We propose that overactivation of JAK-STAT signaling is part of the mechanism underlying disease onset and progression in dyW-/- mice.

Axial and limb muscle development: dialogue with the neighbourhood.

Skeletal muscles are part of the musculoskeletal system which also includes nerves, tendons, connective tissue, bones and blood vessels. Here we review the development of axial and limb muscles in amniotes within the context of their surrounding tissues in vivo. We highlight the reciprocal dialogue mediated by signalling factors between cells of these adjacent tissues and developing muscles and also demonstrate its importance from the onset of muscle cell differentiation well into foetal development. Early embryonic tissues secrete factors which are important regulators of myogenesis. However, later muscle development relies on other tissue collaborators, such as developing nerves and connective tissue, which are in turn influenced by the developing muscles themselves. We conclude that skeletal muscle development in vivo is a compelling example of the importance of reciprocal interactions between developing tissues for the complete and coordinated development of a functional system.

Rapid and simple method for in vivo ex utero development of mouse embryo explants.

The in utero development of mammals drastically reduces the accessibility of the mammalian embryo and therefore limits the range of experimental manipulation that can be done to study functions of genes or signaling pathways during embryo development. Over the past decades, tissue and organ-like culture methods have been developed with the intention of reproducing in vivo situations. Developing accessible and simple techniques to study and manipulate embryos is an everlasting challenge. Herein, we describe a reliable and quick technique to culture mid-gestation explanted mouse embryos on top of a floating membrane filter in a defined medium. Viability of the cultured tissues was assessed by apoptosis and proliferation analysis showing that cell proliferation is normal and there is only a slight increase in apoptosis after 12h of culture compared to embryos developing in utero. Moreover, differentiation and morphogenesis proceed normally as assessed by 3D imaging of the transformation of the myotome into deep back muscles. Not only does muscle cell differentiation occur as expected, but so do extracellular matrix organization and the characteristic splitting of the myotome into the three epaxial muscle groups. Our culture method allows for the culture and manipulation of mammalian embryo explants in a very efficient way, and it permits the manipulation of in vivo developmental events in a controlled environment. Explants grown under these ex utero conditions simulate real developmental events that occur in utero.

Fibronectin assembly during early embryo development: A versatile communication system between cells and tissues.

BACKGROUND: Fibronectin extracellular matrix is essential for embryogenesis. Its assembly is a cell-mediated process where secreted fibronectin dimers bind to integrin receptors on receiving cells, which actively assemble fibronectin into a fibrillar matrix. During development, paracrine communication between tissues is crucial for coordinating morphogenesis, typically being mediated by growth factors and their receptors. Recent reports of situations where fibronectin is produced by one tissue and assembled by another, with implications on tissue morphogenesis, suggest that fibronectin assembly may also be a paracrine communication event in certain contexts. RESULTS: Here we addressed which tissues express fibronectin (Fn1) while also localizing assembled fibronectin matrix and determining the mRNA expression and/or protein distribution pattern of integrins α5 and αV, α chains of the major fibronectin assembly receptors, during early chick and mouse development. We found evidence supporting a paracrine system in fibronectin matrix assembly in several tissues, including immature mesenchymal tissues, components of central and peripheral nervous system and developing muscle. CONCLUSIONS: Thus, similarly to growth factor signaling, fibronectin matrix assembly during early development can be both autocrine and paracrine. We therefore propose that it be considered a cell-cell communication event at the same level and significance as growth factor signaling during embryogenesis.

Advantages of the avian model for human ovarian cancer.

Ovarian cancer (OC) is the most lethal gynecological cancer. Early detection of OC is crucial for providing efficient treatment, whereas high mortality rates correlate with late detection of OC, when the tumor has already metastasized to other organs. The most prevalent type of OC is epithelial OC (EOC). Models that have been used to study EOC include the fruit fly, mouse and laying hen, in addition to human EOC cells in 3D culture in vitro. These models have helped in the elucidation of the genetic component of this disease and the development of drug therapies. However, the histological origin of EOC and early markers of the disease remain largely unknown. In this study, we aimed to review the relative value of each of the different models in EOC and their contributions to understanding this disease. It was concluded that the spontaneous occurrence of EOC in the adult hen, the prolific ovulation, the similarity of metastatic progression with that in humans and the advantages of using the chicken embryo for modelling the development of the reproductive system, renders the hen particularly suitable for studying the early development of EOC. Further investigation of this avian model may contribute to a better understanding of EOC, improve clinical insight and ultimately contribute to decreasing its mortality rates among humans.

Molecular cytogenetics of human single pronucleated zygotes.

The aim of the present study was to use fluorescence in situ hybridization to analyze the chromosome status of zygotes with a single pronucleus from in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles. In addition, we performed immunocytochemical detection of nuclear lamins and histone H3 trimethylated at lysine-9, Me(3)H3K9. Zygotes were processed 24 hours after insemination or injection to assure the absence of asynchrony. In opposition to previous results, we observed 2 pronuclei in 16 of 18 IVF zygotes and 40 of 64 ICSI zygotes, suggesting premature pronuclear breakdown. In IVF and ICSI zygotes, the rate of normal diploidy was only 6 of 16 and 27 of 56, respectively, suggesting that monopronucleated zygotes should not be used in assisted reproductive treatments. The possible mechanisms are discussed and compared to previous studies of monopronucleated zygotes.

Dynamics of Akt activation during mouse embryo development: distinct subcellular patterns distinguish proliferating versus differentiating cells.

Akt is a highly conserved serine-threonine protein kinase which has been implicated in a wide variety of cellular functions, from the regulation of growth and metabolism, to activation of pro-survival pathways and cell proliferation, and promotion of differentiation in specific cell types. However, very little is known about the spatial and temporal pattern of Akt activity within cells and whether this pattern changes as cells enter and proceed in their differentiation programs. To address this issue we profiled Akt activation in E8.5-E13.5 mouse embryos and in C2C12 cells. We used a commercial antibody against Akt, phosphorylated on one of its activating residues, Thr-308, and performed high resolution confocal imaging of the immunofluorescence in labeled embryos. We observe strong Akt activity during mitosis in the dermomyotome, the neuroepithelium and some mesenchymal cells. This burst of activity fills the whole cell except for heterochromatin-positive areas in the nucleus. A surge in activity during mitosis is also observed in subconfluent C2C12 cells. Later on in the differentiation programs of skeletal muscle and neural cells, derivatives of the dermomyotome and neuroepithelium, respectively, we find robust, sustained Akt activity in the cytoplasm, but not in the nucleus. Concomitantly with skeletal muscle differentiation, Akt activity becomes concentrated in the sarcomeric Z-disks whereas developing neurons maintain a uniform cytoplasmic pattern of activated Akt. Our findings reveal unprecedented cellular and subcellular details of Akt activity during mouse embryo development, which is spatially and temporally consistent with proposed functions for Akt in mitosis and myogenic and neural differentiation and/or survival. Our results thus demonstrate a subcellular change in the pattern of Akt activation when skeletal muscle and neural progenitor cells cease dividing and progress in their differentiation programs.

Report on the Myomatrix Conference April 22-24, 2012, University of Nevada, Reno, Nevada, USA.

The Myomatrix 2012 conference held April 22-24th, 2012 at the University of Nevada, Reno convened 73 international participants to discuss the dynamic relationship between muscle and its matrix in muscular dystrophy with a specific focus on congenital muscular dystrophy. Seven sessions over 2½ days defined three central themes: (1) the role of extracellular matrix proteins and compartments in development and specifically in congenital muscular dystrophy (CMD) (2) the role of extracellular matrix signaling and adhesion to membrane receptors and (3) the balance and interplay between inflammation and fibrosis as drivers of altered matrix stiffness, impaired regeneration and progressive dystrophy. This report highlights major conference findings and the translational roadmap as defined by conference attendees.

Extracellular matrix assembly and 3D organization during paraxial mesoderm development in the chick embryo.

The extracellular matrix (ECM) is a major player in the microenvironment governing morphogenesis. However, much is yet to be known about how matrix composition and architecture changes as it influences major morphogenetic events. Here we performed a detailed, 3D analysis of the distribution of two ECM components, fibronectin and laminin, during the development of the chick paraxial mesoderm. By resorting to whole mount double immunofluorescence and confocal microscopy, we generated a detailed 3D map of the two ECM components, revealing their supra-cellular architecture in vivo, while simultaneously retaining high resolution cellular detail. We show that fibronectin assembly occurs at the surface of the presomitic mesoderm (PSM), where a gradual increase in the complexity of the fibronectin matrix accompanies PSM maturation. In the rostral PSM, where somites form, fibronectin fibrils are thick and densely packed and some occupy the cleft which comes to separate the newly formed somite from the PSM. Our 3D approach revealed that laminin matrix assembly starts at the PSM surface as small dispersed patches, which are always localized closer to cells than the fibronectin matrix. These patches gradually grow and coalesce with neighboring patches, but do not generate a continuous laminin sheet, not even on epithelial somites and dermomyotome, suggesting that these epithelia develop in contact with a fenestrated laminin matrix. Unexpectedly, as the somite differentiates, its fibronectin and laminin matrices are maintained, thus initially containing both the epithelial dermomyotome and the mesenchymal sclerotome within the somite segment. Our analysis provides unprecedented details of the progressive in vivo assembly and 3D architecture of fibronectin and laminin matrices during paraxial mesoderm development. These data are consistent with the hypothesis that progressive ECM assembly and subsequent 3D organization are active driving and containing forces during tissue development.

Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model.

Myogenesis is a complex process in which committed myogenic cells differentiate and fuse into myotubes that mature into the muscle fibres of adult organisms. This process is initiated by a cascade of myogenic regulatory factors expressed upon entry of the cells into the myogenic differentiation programme. However, external signals such as those provided by the extracellular matrix (ECM) are also important in regulating muscle differentiation and morphogenesis. In the present work, we have addressed the role of various ECM substrata on C2C12 myoblast behaviour in vitro. Cells grown on fibronectin align and fuse earlier than cells on laminin or gelatine. Live imaging of C2C12 myoblasts on fibronectin versus gelatine has revealed that fibronectin promotes a directional collective migratory behaviour favouring cell-cell alignment and fusion. We further demonstrate that this effect of fibronectin is mediated by RGD-binding integrins expressed on myoblasts, that N-cadherin contributes to this behaviour, and that it does not involve enhanced myogenic differentiation. Therefore, we suggest that the collective migration and alignment of cells seen on fibronectin leads to a more predictable movement and a positioning that facilitates subsequent fusion of myoblasts. This study highlights the importance of addressing the role of fibronectin, an abundant component of the interstitial ECM during embryogenesis and tissue repair, in the context of myogenesis and muscle regeneration.

Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse.

BACKGROUND: Skeletal myogenesis is extensively influenced by the surrounding environment. However, how the extracellular matrix (ECM) affects morphogenesis of muscles is not well understood. RESULTS: We mapped the three-dimensional (3D) organization of fibronectin, tenascin, and laminin by immunofluorescence during early epaxial myogenesis in mouse embryos. We define four stages of dermomyotome/myotome development and reveal the 3D organization of myogenic cells within their ECM during those stages. Fibronectin is abundant in all interstitial tissues, while tenascin is restricted to intersegmental borders. Bundles of fibronectin and tenascin also penetrate into the myotome, possibly promoting myocyte alignment. A laminin matrix delineates the dermomyotome and myotome and undergoes dynamic changes, correlating with key developmental events. CONCLUSION: Our observations cast new light on how myotomal cells interact with their environment and suggest that, as the segmented myotomes transform into the epaxial muscle masses, the laminin matrix disassembles and myocytes use the abundant fibronectin matrix to reach their final organization.

The extracellular matrix dimension of skeletal muscle development.

Cells anchor to substrates by binding to extracellular matrix (ECM). In addition to this anchoring function however, cell-ECM binding is a mechanism for cells to sense their surroundings and to communicate and coordinate behaviour amongst themselves. Several ECM molecules and their receptors play essential roles in muscle development and maintenance. Defects in these proteins are responsible for some of the most severe muscle dystrophies at every stage of life from neonates to adults. However, recent studies have also revealed a role of cell-ECM interactions at much earlier stages of development as skeletal muscle forms. Here we review which ECM molecules are present during the early phases of myogenesis, how myogenic cells interact with the ECM that surrounds them and the potential consequences of those interactions. We conclude that cell-ECM interactions play significant roles during all stages of skeletal muscle development in the embryo and suggest that this "extracellular matrix dimension" should be added to our conceptual network of factors contributing to skeletal myogenesis.

A Pax3/Dmrt2/Myf5 regulatory cascade functions at the onset of myogenesis.

All skeletal muscle progenitor cells in the body derive from the dermomyotome, the dorsal epithelial domain of developing somites. These multipotent stem cells express Pax3, and this expression is maintained in the myogenic lineage where Pax3 plays an important role. Identification of Pax3 targets is therefore important for understanding the mechanisms that underlie the onset of myogenesis. In a microarray screen of Pax3-GFP sorted cells, with analysis on Pax3 gain and loss of function genetic backgrounds, we identify Dmrt2, expressed in the dermomyotome, as a Pax3 target. In vitro gel shift analysis and chromatin immunoprecipitation with in vivo extracts show that Pax3 binds to a conserved 286 bp sequence, situated at -18 kb from Dmrt2. This sequence directs reporter transgene expression to the somite, and this is severely affected when the Pax3 site is mutated in the context of the locus. In Dmrt2 mutant embryos, somite maturation is perturbed and the skeletal muscle of the myotome is abnormal. We now report that the onset of myogenesis is also affected. This depends on activation, in the epaxial dermomyotome, of the myogenic determination gene, Myf5, through its early epaxial enhancer. This sequence contains sites that bind Dmrt2, which belongs to the DM class of DNA-binding proteins. Mutation of these sites compromises activity of the enhancer in transgenic embryos where the reporter transgene is under the control of the Myf5 epaxial enhancer. Transactivation of this site by Dmrt2 is demonstrated in vitro, and conditional overexpression of Dmrt2 in Pax3 expressing cells in the somite confirms the role of this factor in the activation of Myf5. These results reveal a novel genetic network, comprising a Pax3/Dmrt2/Myf5 regulatory cascade that operates in stem cells of the epaxial dermomyotome to initiate skeletal muscle formation.

Teaching and research on Developmental Biology in Portugal.

Developmental Biology has established itself as a solid field of teaching and research in Portugal. Its history is recent, generally considered to have started with the pioneering work of Augusto Celestino da Costa at the beginning of the 20th century. However, research groups were very few and, until the early 1990s, teaching beyond morphological and comparative embryology was uncommon. In 1994, the first university course dedicated to Developmental Biology as a separate field from Embryology was created at the Faculty of Sciences of the University of Lisbon and a course on Plant Differentiation and Morphogenesis was also initiated. A Masters programme in Developmental Biology followed at the Lusofona University in 1996. Subsequently, modules of Developmental Biology were included in many Embryology courses and eventually more Developmental Biology courses were created. From 1999 onwards, the number of research groups working in Developmental Biology started to increase, many of which were initiated by researchers who had had the opportunity to pursue their PhD and/or post-doc studies abroad. The Instituto Gulbenkian de Cincia (Gulbenkian Institute of Science) became the first home of most of these groups, but several later spread to other institutions. This increased activity in turn has stimulated teaching of Developmental Biology and more students have been getting interested in the field. This positive feedback loop makes it a nice time to be teaching and working in Developmental Biology in Portugal.