Nicole Dünker

Recruitment and maintenance of tendon progenitors by TGFβ signaling are essential for tendon formation

Tendons and ligaments mediate the attachment of muscle to bone and of bone to bone to provide connectivity and structural integrity in the musculoskeletal system. We show that TGFβ signaling plays a major role in the formation of these tissues. TGFβ signaling is a potent inducer of the tendon progenitor (TNP) marker scleraxis both in organ culture and in cultured cells, and disruption of TGFβ signaling in Tgfb2-/-;Tgfb3-/- double mutant embryos or through inactivation of the type II TGFβ receptor (TGFBR2; also known as TβRII) results in the loss of most tendons and ligaments in the limbs, trunk, tail and head. The induction of scleraxis-expressing TNPs is not affected in mutant embryos and the tendon phenotype is first manifested at E12.5, a developmental stage in which TNPs are positioned between the differentiating muscles and cartilage, and in which Tgfb2 or Tgfb3 is expressed both in TNPs and in the differentiating muscles and cartilage. TGFβ signaling is thus essential for maintenance of TNPs, and we propose that it also mediates the recruitment of new tendon cells by differentiating muscles and cartilage to establish the connections between tendon primordia and their respective musculoskeletal counterparts, leading to the formation of an interconnected and functionally integrated musculoskeletal system.

Reduction of endogenous transforming growth factors |[beta]| prevents ontogeneticneuron death

We show that following immunoneutralization of endogenous transforming growth factors β (TGF-β) in the chick embryo, ontogenetic neuron death of ciliary, dorsal root and spinal motor neurons was largely prevented, and neuron losses following limb bud ablation were greatly reduced. Likewise, preventing TGF-β signaling by treatment with a TβR-II fusion protein during the period of ontogenetic cell death in the ciliary ganglion rescued all neurons that normally die. TUNEL staining revealed decreased numbers of apoptotic cells following antibody treatment. Exogenous TGF-β rescued the TGF-β-deprived phenotype. We conclude that TGF-β is critical in regulating ontogenetic neuron death as well as cell death following neuronal target deprivation.

Tgfß2 –/– Tgfß3 –/– double knockout mice display severe midline fusion defects and early embryonic lethality

Given all known biological activities, it is anticipated that transforming growth factors beta (TGF-ßs) play important roles in many different developmental processes. As all three TGF-ß isoforms display overlapping expression patterns, deletion of one TGF-ß isoform might be compensated for by another. In the present study, targeted disruption of both Tgfß2 and Tgfß3 genes was undertaken to circumvent this problem and determine the essential roles of TGF-ß2 and TGF-ß3 in vivo. Tgfß2 –/– Tgfß3 –/– double knockout mice and their three-allelic Tgfß2 –/– Tgfß3 +/– littermates display a lack of distal parts of the rib, a lack of sternal primordia, and failure in ventral body wall closure, leading to an extrathoracic position of the heart and extrusion of the liver. In addition, abnormalities in connective tissue composition and an early embryonic lethality [around embryonic day (E) 15.5] are seen. In contrast, Tgfß2 +/– Tgfß3 –/– littermates show normal rib and sternum development, normal anterior body wall fusion, and are still alive on E18.5. TGF-ß2 is already known to play a role in skeletal and craniofacial development. The results presented here show that beyond this: (a) TGF-ßs obviously play a fundamental role in midline fusion and (b) the Tgfß2 gene seems to play a more important role in mediating developmental processes than the Tgfß3 gene, since Tgfß2 +/– Tgfß3 –/– mutants – in contrast to their Tgfß2 –/– Tgfß3 + /– littermates – do not display severe malformations.

Transforming Growth Factor β Is Required for Differentiation of Mouse Mesencephalic Progenitors into Dopaminergic Neurons In Vitro and In Vivo: Ectopic Induction in Dorsal Mesencephalon

Tissue engineering is a prerequisite for cell replacement as therapeutic strategy for degenerative diseases, such as Parkinsons disease. In the present study, we investigated regional identity of mesencephalic neural progenitors and characterized their development toward ventral mesencephalic dopaminergic neurons. We show that neural progenitors from ventral and dorsal mouse embryonic day 12 mesencephalon exhibit regional identity in vitro. Treatment of ventral midbrain dissociated neurospheres with transforming growth factor β (TGF‐β) increased the number of Nurr1‐ and tyrosine hydroxylase (TH)‐immunoreactive cells, which can be further increased when the spheres are treated with TGF‐β in combination with sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8). TGF‐β differentiation signaling is TGF‐β receptor‐mediated, involving the Smad pathway, as well as the p38 mitogen‐activated protein kinase pathway. In vivo, TGF‐β2/TGF‐β3 double‐knockout mouse embryos revealed significantly reduced numbers of TH labeled cells in ventral mesencephalon but not in locus coeruleus. TH reduction in Tgfβ2−/−/Tgfβ3+/− was higher than in Tgf‐β2+/−/Tgf‐β3−/−. Most importantly, TGF‐β may ectopically induce TH‐immunopositive cells in dorsal mesencephalon in vitro, in a Shh‐ and FGF8‐independent manner. Together, the results clearly demonstrate that TGF‐β2 and TGF‐β3 are essential signals for differentiation of midbrain progenitors toward neuronal fate and dopaminergic phenotype.

Chick ex ovo culture and ex ovo CAM assay: how it really works.

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci. For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used(1-4), no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay(5,6), (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye(7-9), (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM(10-12), (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice(13) as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM(14). In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.

Reduced programmed cell death in the retina and defects in lens and cornea of Tgfβ2–/–Tgfβ3–/– double-deficient mice

Abstract.We have previously shown that immunoneutralization of transforming growth factor-β (TGF-β) in the chick embryo significantly reduces programmed cell death (PCD) in peripheral neurons, spinal cord, and retina. In order to validate these results we have begun to analyze PCD in mice with targeted ablations of the TGF-β2 and TGF-β3 genes. Recent analyses of mice lacking TGF-β3 had failed to reveal an overt eye phenotype, while retinae of TGF-β2-deficient mice showed retinal hypercellularity. We report now that eyes of Tgfβ2/Tgfβ3 double-deficient mice display severe alterations in the morphology of the retina, lens, and cornea. The inner neural retina—the region where TGF-β receptor (TβR) I and II immunoreactivities are most prominent—is significantly thickened, and numbers of TUNEL-positive cells are significantly reduced compared to wild-type littermates. In Tgfβ2−/−Tgfβ3−/− and Tgfβ2−/−Tgfβ3+/− littermates the retina was consistently detached from the underlying pigment epithelium. Cornea, corneal stroma, and lens epithelium were significantly thinner in these mutants. In contrast, retinal morphology in Tgfβ2+/−Tgfβ3−/−mutant littermates resembles the situation observed in wild-type retinae except for the retinal detachment. Thus, regression in the thickness of cornea and corneal stroma seems to be TGF-β isoform and gene dose dependent. Our results substantiate the notion based on previous analyses of chick embryos with reduced levels of endogenous TGF-β that TGF-β, most notably TGF-β2, is required to mediate PCD in developing retinal cells in vivo. Moreover, our data indicate that TGF-βs play essential roles in cornea and lens development.

Embryonic and larval development in the caecilian Ichthyophis kohtaoensis (Amphibia, gymnophiona): a staging table.

Abstract

TGF-β is required for programmed cell death in interdigital webs of the developing mouse limb

During limb formation massive cell death in the mesenchyme of the interdigital spaces accompanies the formation of free digits. Members of the transforming growth factor beta (TGF-) superfamily were discussed to play a key role in cell-cell interactions, important in the regulation of programmed cell death (PCD). TGF-beta itself is believed to be involved in epithelial-mesenchymal interactions. Here, we demonstrate that PCD is significantly reduced in interdigital spaces of the developing limbs of Tgfbeta2-/-Tgfbeta3-/- double knockouts. The regression of interdigital webs seems to be doses-dependent as interdigital mesenchyme is at least partly reduced in Tgfbeta2-/-Tgfbeta3+/- mutants, whereas interdigital zones of Tgfbeta2-/-Tgfbeta3-/- double knockouts reveal only minimal signs of regression. We conclude that TGF- is a critical extrinsic regulator of PCD.

Immunohistochemical evaluation of matrix molecules associated with wound healing following regenerative periodontal treatment in monkeys.

Abstract. The aim of the study was to investigate immunohistochemically the expression of matrix molecules associated with periodontal tissues reformed after regenerative periodontal treatment. Chronic intrabony defects were treated with guided tissue regeneration, enamel matrix proteins, the combination of both, or access flap surgery. Five months after healing, the animals were killed, and the healed periodontal tissues were evaluated immunohistochemically by means of polyclonal antibodies against osteopontin, collagen I, and collagen III. The intact (nontreated) parts of the periodontium served as controls. As a general observation, the staining for all investigated matrix molecules appeared to be stronger within the regenerated tissues than in the intact ones. The results failed to reveal any differences in terms of staining intensity or distribution pattern of investigated matrix molecules between the four different treatments. Osteopontin expression was most intense at the border near the newly formed cementum and bone. In the regenerated periodontium, collagens I and III were localized throughout the entire periodontal ligament connective tissue. In the regenerated periodontal ligament, collagen III displayed more intense staining than collagen I. The present results suggest that: (1) even after a 5-month period following surgical periodontal therapy, extracellular matrix molecules associated with wound healing and/or remodelling are more strongly expressed in regenerated than in intact tissues and (2) once an environment for periodontal regeneration has been created, the expression of extracellular matrix molecules associated with the healing process seems to display the same pattern, irrespective of treatment modality.

Transforming growth factor‐β and tumor necrosis factor‐α cooperate to induce apoptosis in the oligodendroglial cell line OLI‐neu

As shown previously, transforming growth factor‐β (TGF‐β) plays an important role during the period of developmental cell death in the nervous system. As with neurons, oligodendrocytes are generated in excess and eliminated by apoptosis. The present study was aimed at investigating the possible interaction of TGF‐β with tumor necrosis factor‐α (TNF‐α) in the regulation of cell death in oligodendroglial precursor cells and analyzing the underlying signaling mechanisms. We show that both factors induce apoptosis independently, but cooperate when applied together. The investigation of the signaling events revealed an important role of the JNK pathway during induction of apoptosis. TGF‐β seemed to be more efficient at inducing a release in cytochrome c from mitochondria than TNF‐α. This might be the consequence of decreased Bcl‐xL levels observed in cells treated with TGF‐β but not with TNF‐α. Both factors stimulated caspase‐3 activity, which could be inhibited by caspase‐8 or caspase‐9 inhibitors. Therefore, we conclude that TNF‐α and TGF‐β affect partially common pathways but also regulate different steps in the apoptotic cascade.