Juan A. Montero

Transforming Growth Factors β Coordinate Cartilage and Tendon Differentiation in the Developing Limb Mesenchyme

Transforming growth factor β (TGFβ) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGFβ signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGFβs shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved down-regulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGFβ signaling up-regulates Sox9 in the in vivo experimental model system in which TGFβ treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGFβ signaling, we found that TGFβs appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGFβ signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGFβ signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGFβ treatments.

Anatomy, Biomechanics, Imaging, and Management of Ligamentum Teres Injuries

The ligamentum teres has traditionally been viewed as an embryonic remnant with no role in the biomechanics or vascularity of adult hips. However, the ligamentum teres is a strong intraarticular ligament that is anatomically and biochemically similar to the anterior cruciate ligament of the knee. It is composed of two bands that originate from the acetabular transverse ligament and the pubic and ischial margins of the acetabular notch. Among other functions, the ligamentum teres is an important stabilizer of the hip, particularly in adduction, flexion, and external rotation. Abnormalities of the ligamentum teres account for 4%-15% of sports-related injuries and should be considered in the differential diagnosis of patients with hip pain. Lesions of the ligamentum teres include partial or complete traumatic tears, degenerative tears, avulsion fractures of the ligament at its insertion into the fovea capitis femoris, and a congenital absence of the ligament. Magnetic resonance arthrography and computed tomographic arthrography are the preferred modalities for precise preoperative diagnosis of ligamentum teres injuries and may be used to rule out other associated intraarticular injuries. Treatment of these lesions is still evolving; at present, treatment of most injuries is limited to arthroscopic débridement.

Reducing GABAA α5 Receptor-Mediated Inhibition Rescues Functional and Neuromorphological Deficits in a Mouse Model of Down Syndrome

Down syndrome (DS) is associated with neurological complications, including cognitive deficits that lead to impairment in intellectual functioning. Increased GABA-mediated inhibition has been proposed as a mechanism underlying deficient cognition in the Ts65Dn (TS) mouse model of DS. We show that chronic treatment of these mice with RO4938581 (3-bromo-10-(difluoromethyl)-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]diazepine), a selective GABAA α5 negative allosteric modulator (NAM), rescued their deficits in spatial learning and memory, hippocampal synaptic plasticity, and adult neurogenesis. We also show that RO4938581 normalized the high density of GABAergic synapse markers in the molecular layer of the hippocampus of TS mice. In addition, RO4938581 treatment suppressed the hyperactivity observed in TS mice without inducing anxiety or altering their motor abilities. These data demonstrate that reducing GABAergic inhibition with RO4938581 can reverse functional and neuromorphological deficits of TS mice by facilitating brain plasticity and support the potential therapeutic use of selective GABAA α5 NAMs to treat cognitive dysfunction in DS.

Activin/TGFβ and BMP crosstalk determines digit chondrogenesis

The progress zone (PZ) is a specialized area at the distal margin of the developing limb where mesodermal cells are kept in proliferation and undifferentiated, allowing limb outgrowth. At stages of digit morphogenesis the PZ cells can undergo two possible fates, either aggregate initiating chondrogenic differentiation to configure the digit blastemas, or to die by apoptosis if they are incorporated in the interdigital mesenchyme. While both processes are controlled by bone morphogenetic proteins (BMPs) the molecular basis for such contrasting differential behavior of the autopodial mesoderm remains unknown. Here we show that a well-defined crescent domain of high BMP activity located at the tip of the forming digits, which we termed the digit crescent (DC), directs incorporation and differentiation of the PZ mesenchymal cells into the digit aggregates. The presence of this domain does not correlate with an exclusive expression domain of BMP receptors and its abrogation by surgical approaches or by local application of BMP antagonists is followed by digit truncation and cell death. We further show that establishment of the DC is directed by Activin/TGFbeta signaling, which inhibits Smad 6 and Bambi, two specific BMP antagonists expressed in the interdigits and progress zone mesoderm. The interaction between Activin/TGFbeta and BMP pathways at the level of DC promotes the expression of the chondrogenic factor SOX9 accompanied by a local decrease in cell proliferation. Characteristically, the DC domain is asymmetric, it being extended towards the posterior interdigit. The presence of the DC is transitorily dependent of the adjacent posterior interdigit and its maintenance requires also the integrity of the AER.

Sculpturing digit shape by cell death

Physiological cell death is a key mechanism that ensures appropriate development and maintenance of tissues and organs in multicellular organisms. Most structures in the vertebrate embryo exhibit defined areas of cell death at precise stages of development. In this regard the areas of interdigital cell death during limb development provide a paradigmatic model of massive cell death with an evident morphogenetic role in digit morphogenesis. Physiological cell death has been proposed to occur by apoptosis, cellular phenomena genetically controlled to orchestrate cell suicide following two main pathways, cytochrome C liberation from the mitochondria or activation of death receptors. Such pathways converge in the activation of cysteine proteases known as caspases, which execute the cell death program, leading to typical morphologic changes within the cell, termed apoptosis. According to these findings it would be expected that caspases loss of function experiments could cause inhibition of interdigital cell death promoting syndactyly phenotypes. A syndactyly phenotype is characterized by absence of digit freeing during development that, when caused by absence of interdigital cell death, is accompanied by the persistence of an interdigital membrane. However this situation has not been reported in any of the KO mice or chicken loss of function experiments ever performed. Moreover histological analysis of dying cells within the interdigit reveals the synchronic occurrence of different types of cell death. All these findings are indicative of caspase alternative and/or complementary mechanisms responsible for physiological interdigital cell death. Characterization of alternative cell death pathways is required to explain vertebrate morphogenesis. Today there is great interest in cell death via autophagy, which could substitute or act synergistically to the apoptotic pathway. Here we discuss what is known about physiological cell death in the developing interdigital tissue of vertebrate embryos, paying special attention to the avian species.

Expression of Sox8, Sox9 and Sox10 in the developing valves and autonomic nerves of the embryonic heart.

We describe the expression pattern of Sox8, Sox9 and Sox10 during the development of the chick embryo heart. These Sox genes constitute the group E of the large Sox family of transcription factors. We show that the expression of Sox8, Sox9 and Sox10 in the developing heart correlates with heart septation and with the differentiation of the connective tissue of the valve leaflets. Sox10 appears also as a specific marker of developing heart nerves. These findings fit with the occurrence of morphological and functional anomalies of the heart reported in humans deficient for Sox9 and Sox10.

Defining the earliest transcriptional steps of chondrogenic progenitor specification during the formation of the digits in the embryonic limb.

The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The “pre-condensation stage”, occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the “condensation stage” is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the “pre-cartilage period”, precedes the formation of molecularly identifiable cartilage by 2–3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo.

Cellular Plasticity Confers Migratory and Invasive Advantages to a Population of Glioblastoma‐Initiating Cells that Infiltrate Peritumoral Tissue

Glioblastoma (GBM) is associated with infiltration of peritumoral (PT) parenchyma by isolated tumor cells that leads to tumor regrowth. Recently, GBM stem‐like or initiating cells (GICs) have been identified in the PT area, but whether these GICs have enhanced migratory and invasive capabilities compared with GICs from the tumor mass (TM) is presently unknown. We isolated GICs from the infiltrated PT tissue and the TM of three patients and found that PT cells have an advantage over TM cells in two‐dimensional and three‐dimensional migration and invasion assays. Interestingly, PT cells display a high plasticity in protrusion formation and cell shape and their migration is insensitive to substrate stiffness, which represent advantages to infiltrate microenvironments of different rigidity. Furthermore, mouse and chicken embryo xenografts revealed that only PT cells showed a dispersed distribution pattern, closely associated to blood vessels. Consistent with cellular plasticity, simultaneous Rac and RhoA activation are required for the enhanced invasive capacity of PT cells. Moreover, Rho GTPase signaling modulators αVβ3 and p27 play key roles in GIC invasiveness. Of note, p27 is upregulated in TM cells and inhibits RhoA activity. Gene silencing of p27 increased the invasive capacity of TM GICs. Additionally, β3 integrin is upregulated in PT cells. Blockade of dimeric integrin αVβ3, a Rac activator, reduced the invasive capacity of PT GICs in vitro and abrogated the spreading of PT cells into chicken embryos. Thus, our results describe the invasive features acquired by a unique subpopulation of GICs that infiltrate neighboring tissue. STEM Cells 2013;31:1075–1085

Tgfβ2 and 3 are coexpressed with their extracellular regulator Ltbp1 in the early limb bud and modulate mesodermal outgrowth and BMP signaling in chicken embryos

BackgroundTransforming growth factor β proteins (Tgfβs) are secreted cytokines with well-defined functions in the differentiation of the musculoskeletal system of the developing limb. Here we have studied in chicken embryos, whether these cytokines are implicated in the development of the embryonic limb bud at stages preceding tissue differentiation.ResultsImmunohistochemical detection of phosphorylated Smad2 and Smad3 indicates that signaling by this pathway is active in the undifferentiated mesoderm and AER. Gene expression analysis shows that transcripts of tgfβ2 and tgfβ3 but not tgfβ1 are abundant in the growing undifferentiated limb mesoderm. Transcripts of tgfβ2 are also found in the AER, which is the signaling center responsible for limb outgrowth. Furthermore, we show that Latent Tgfβ Binding protein 1 (LTBP1), which is a key extracellular modulator of Tgfβ ligand bioavailability, is coexpressed with Tgfβs in the early limb bud. Administration of exogenous Tgfβs to limb buds growing in explant cultures provides evidence of these cytokines playing a role in the regulation of mesodermal limb proliferation. In addition, analysis of gene regulation in these experiments revealed that Tgfβ signaling has no effect on the expression of master genes of musculoskeletal tissue differentiation but negatively regulates the expression of the BMP-antagonist Gremlin.ConclusionWe propose the occurrence of an interplay between Tgfβ and BMP signaling functionally associated with the regulation of early limb outgrowth by modulating limb mesenchymal cell proliferation.

Divergent Differentiation of Skeletal Progenitors into Cartilage and Tendon: Lessons from the Embryonic Limb

Repairing damaged cartilage and tendons is a major challenge of regenerative medicine. There has been great progress in the past decade toward obtaining stem cells for regenerative purposes from a variety of sources. However, the development of procedures to direct and maintain the differentiation of progenitors into cartilage or tendon is still a hurdle to overcome in regenerative medicine of the musculoskeletal system. This is because connective tissues often lack stable phenotypes and retain plasticity to return to the initial stages of differentiation or to transdifferentiate into another connective tissue cell lineage. This makes it necessary to unravel the molecular basis that is responsible for the differentiation of connective tissue cell lineages. In this review, we summarize the investigations performed in the past two decades to unravel the signals that regulate the differentiation of skeletal cell progenitors into cartilage and tendons during embryonic limb development. The data obtained in those studies demonstrate that Tgfβ, BMP, FGF, and Wnt establish a complex signaling network that directs the differentiation of skeletal cell progenitors. Remarkably, in the embryonic digit model, the divergent differentiation of progenitors depends on the temporal coordination of those signals, rather than being specified by an individual signaling pathway. Due to its potential medical relevance, we highlight the importance of the coordinate influence of the Tgfβ and BMP pathways in the differentiation of cell progenitors into tendon or cartilage.