Frederick S. Jones

The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling.

The determination of animal form depends on the coordination of events that lead to the morphological patterning of cells. This epigenetic view of development suggests that embryonic structures arise as a consequence of environmental influences acting on the properties of cells, rather than an unfolding of a completely genetically specified and preexisting invisible pattern. Specialized cells of developing multicellular organisms are surrounded by a complex extracellular matrix (ECM), comprised largely of different collagens, proteoglycans, and glycoproteins. This ECM is a substrate for tissue morphogenesis, lends support and flexibility to mature tissues, and acts as an epigenetic informational entity in the sense that it transduces and integrates intracellular signals via distinct cell surface receptors. Consequently, ECM‐receptor interactions have a profound influence on major cellular programs including growth, differentiation, migration, and survival. In contrast to many other ECM proteins, the tenascin (TN) family of glycoproteins (TN‐C, TN‐R, TN‐W, TN‐X, and TN‐Y) display highly restricted and dynamic patterns of expression in the embryo, particularly during neural development, skeletogenesis, and vasculogenesis. These molecules are reexpressed in the adult during normal processes such as wound healing, nerve regeneration, and tissue involution, and in pathological states including vascular disease, tumorigenesis, and metastasis. In concert with a multitude of associated ECM proteins and cell surface receptors that include members of the integrin family, TN proteins impart contrary cellular functions, depending on their mode of presentation (i.e., soluble or substrate‐bound) and the cell types and differentiation states of the target tissues. Expression of tenascins is regulated by a variety of growth factors, cytokines, vasoactive peptides, ECM proteins, and biomechanical factors. The signals generated by these factors converge on particular combinations of cis‐regulatory elements within the recently identified TN gene promoters via specific transcriptional activators or repressors. Additional complexity in regulating TN gene expression is achieved through alternative splicing, resulting in variants of TN polypeptides that exhibit different combinations of functional protein domains. In this review, we discuss some of the recent advances in TN biology that provide insights into the complex way in which the ECM is regulated and how it functions to regulate tissue morphogenesis and gene expression. Dev Dyn;218:235–259.

Tenascin-C in development and disease: gene regulation and cell function.

Tenascin-C (TN-C) is a modular and multifunctional extracellular matrix (ECM) glycoprotein that is exquisitely regulated during embryonic development and in adult tissue remodeling. TN-C gene transcription is controlled by intracellular signals that are generated by multiple soluble factors, integrins and mechanical forces. These external cues are interpreted by particular DNA control elements that interact with different classes of transcription factors to activate or repress TN-C expression in a cell type- and differentiation-dependent fashion. Among the transcriptional regulators of the TN-C gene that have been identified, the homeobox family of proteins has emerged as a major player. Downstream from TN-C, intracellular signals that are relayed via specific cell surface receptors often impart contrary cellular functions, even within the same cell type. A key to understanding this behavior may lie in the dual ability of TN-C-enriched extracellular matrices to generate intracellular signals, and to define unique cellular morphologies that modulate these signal transduction pathways. Thus, despite the contention that TN-C null mice appear to develop and act normally, TN-C biology continues to provide a wealth of information regarding the complex nature of the ECM in development and disease.

Gene regulation of cell adhesion: a key step in neural morphogenesis

A mounting body of evidence suggests that cell adhesion molecules (CAMs) play important roles in morphogenetic patterning of the nervous system. The combined factors that control the expression of CAMs during early neural development are, however, largely unknown. We have hypothesized that the coordinate expression of homeobox (Hox) and paired box (Pax) proteins in the neural axis leads to the differential expression of particular CAM genes. Following this hypothesis, we have characterized the promoters and identified cis-regulatory sequences that bind to and respond to Hox and Pax proteins in the genes for three neurally expressed CAMs - the neural cell adhesion molecule, N-CAM, the neuron-glia cell adhesion molecule, Ng-CAM, and L1. Experiments on transgenic mice carrying N-CAM promoter/lacZ reporter gene constructs indicated that mutation of either the HBS or the PBS disrupted patterning of N-CAM expression in the embryonic spinal cord. To examine the factors that restrict the expression of certain CAMs to the nervous system, we identified regulatory elements that block expression of the Ng-CAM and L1 genes in non-neural cells. We characterized a 310 base pair region of the first intron of the Ng-CAM gene containing five neural restrictive silencer elements (NRSEs) and a binding site for the Pax-3 protein. These elements silenced activity of the Ng-CAM promoter in NIH3T3 fibroblasts, but had no effect on its activity in N2A neuroblastoma cells line. Similar analyses of the L1 gene revealed a single NRSE within the second intron that was important for silencing in this cellular transfection system. To analyze the role of the NRSE in vivo, we prepared transgenic mice containing two L1 gene/lacZ constructs, one containing the NRSE and another in which the NRSE was deleted. The wild type L1lacZ transgene showed a neurally restricted pattern of expression, whereas the NRSE-mutated L1 construct showed extensive extraneural expression of the L1 gene. Thus, neural specificity of CAM expression is controlled by the NRSE. The general significance of these observations is that they connect the expression of important families of transcriptional regulators with gene products capable of direct cellular mechanochemistry.

Silencer Elements Modulate the Expression of the Gene for the Neuron-Glia Cell Adhesion Molecule, Ng-CAM

The combined factors that regulate the expression of cell adhesion molecules (CAMs) during development of the nervous system are largely unknown. To identify such factors for Ng-CAM, the neuron-glia CAM, constructs containing portions of the 5′ end of the Ng-CAM gene were examined for activity after transfection into N2A neuroblastoma and NIH3T3 cells. Positive regulatory elements active in both cell types included an Ng-CAM proximal promoter with SP1 and cAMP response element motifs extending 447 base pairs upstream of a single RNA start site and a region within the first exon corresponding to 5′-untranslated sequences. Negative regulatory elements included five neuron-restrictive silencer elements (NRSEs) and a binding site for Pax gene products in a 305-base pair segment of the first intron. Constructs containing the promoter together with the entire first intron were active in N2A cells but were silenced in NIH3T3 cells. This silencer activity was mapped to the NRSEs. In contrast, the Pax motif inhibited activity of Ng-CAM constructs in both cell types. The DNA elements defined in these transfection experiments were examined for their ability to bind nuclear factors. The region within the first exon formed a DNA-protein complex after exposure to nuclear extracts prepared from both NIH3T3 and N2A cells. The NRSE region formed a more prominent complex with proteins prepared from NIH3T3 cells than it did with extracts from N2A cells. A member of the Pax protein family, Pax-3 bound to the Pax motif. Mutations introduced within the Pax motif in its ATTA sequence eliminated this binding whereas mutations in its GTTCC sequence did not, suggesting that paired homeodomain interactions are important for the recognition of Pax-3 by this DNA target sequence. The combined data suggest that negative regulation by NRSEs and Pax proteins may play a key role in the place-dependent expression patterns of Ng-CAM during development.

Drosophila D1 dopamine receptor mediates caffeine-induced arousal

The arousing and motor-activating effects of psychostimulants are mediated by multiple systems. In Drosophila, dopaminergic transmission is involved in mediating the arousing effects of methamphetamine, although the neuronal mechanisms of caffeine (CAFF)-induced wakefulness remain unexplored. Here, we show that in Drosophila, as in mammals, the wake-promoting effect of CAFF involves both the adenosinergic and dopaminergic systems. By measuring behavioral responses in mutant and transgenic flies exposed to different drug-feeding regimens, we show that CAFF-induced wakefulness requires the Drosophila D1 dopamine receptor (dDA1) in the mushroom bodies. In WT flies, CAFF exposure leads to downregulation of dDA1 expression, whereas the transgenic overexpression of dDA1 leads to CAFF resistance. The wake-promoting effects of methamphetamine require a functional dopamine transporter as well as the dDA1, and they engage brain areas in addition to the mushroom bodies.

Knockout of REST/NRSF shows that the protein is a potent repressor of neuronally expressed genes in non‐neural tissues

The protein repressor element 1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF) is a negative regulator of neuronal genes that contain a particular DNA sequence, the neuron restrictive silencer element (NRSE). REST is expressed ubiquitously in non-neural tissues but is down-regulated in neural precursors and turned off in postmitotic neurons, suggesting that it can act both to prevent extraneural expression of certain genes and to delay the differentiation of neuronal subtypes. In a recent paper, Chen et al.(1) describe the production of a null mutant for REST in mice and the mosaic inactivation of REST function in chicken embryos. Knockout of REST led to malformations in several non-neural tissues, as well as apoptosis and embryonic lethality in mice. In addition, the expression of several REST target genes was derepressed in non-neural tissues and in neural progenitors in both mouse and chicken embryos. These studies clearly demonstrate that active repression of tissue-specific genes is required for proper tissue differentiation during embryonic development.

The homeobox transcription factor Barx2 regulates chondrogenesis during limb development

Among the many factors involved in regulation of chondrogenesis, bone morphogenetic proteins (BMPs) and members of the Sox and homeobox transcription factor families have been shown to have crucial roles. Of these regulators, the homeobox transcription factors that function during chondrogenesis have been the least well defined. We show here that the homeobox transcription factor Barx2 is expressed in primary mesenchymal condensations, digital rays, developing joints and articular cartilage of the developing limb, suggesting that it plays a role in chondrogenesis. Using retroviruses and antisense oligonucleotides to manipulate Barx2 expression in limb bud micromass cultures, we determined that Barx2 is necessary for mesenchymal aggregation and chondrogenic differentiation. In accordance with these findings, Barx2 regulates the expression of several genes encoding cell-adhesion molecules and extracellular matrix proteins, including NCAM and collagen II (Col2a1) in the limb bud. Barx2 bound to elements within the cartilage-specific Col2a1 enhancer, and this binding was reduced by addition of Barx2 or Sox9 antibodies, or by mutation of a HMG box adjacent to the Barx2-binding element, suggesting cooperation between Barx2 and Sox proteins. Moreover, both Barx2 and Sox9 occupy Col2a1 enhancer during chondrogenesis in vivo. We also found that two members of the BMP family that are crucial for chondrogenesis, GDF5 and BMP4, regulate the pattern of Barx2 expression in developing limbs. Based on these data, we suggest that Barx2 acts downstream of BMP signaling and in concert with Sox proteins to regulate chondrogenesis.

Cytotactin: a morphoregulatory molecule and a target for regulation by homeo☐ gene products

Two major lines of research in developmental biology should help us to understand the bases of morphogenesis. The first is the analysis of the morphogenetic effects of local expression of various adhesion molecules. The second is the analysis of cascades of regulatory genes that interact during development. Of particular significance are regulatory interactions involving homeobox-containing genes which are expressed in a place-dependent manner in the embryo. Success in connecting these two lines of research would help to resolve the puzzle of how species-specific tissue patterns can arise and be maintained. In this article, we focus on cytotactin, a morphoregulatory molecule of the extracellular matrix that exhibits sharply restricted spatiotemporal patterns of expression during development. Recent experiments indicate the promoter of the cytotactin gene contains target regions that appear to respond to homeodomain proteins. These observations, and those on other morphoregulatory molecules, suggest a possible connection between their effects on cell patterning and control by homeobox-containing genes.

Gene regulation of cell adhesion molecules in neural morphogenesis

A mounting body of evidence suggests that cell‐cell adhesion molecules (CAMs) play critical roles in morphogenetic patterning and in laying down the initial tissue scaffold of the nervous system. Perturbations of CAM binding can lead to altered tissue pattern and interruption of tissue interactions to altered patterns of CAM expression. The combined factors that regulate the expression of CAMs and that drive early neural development are, however, largely unknown. We have hypothesized that the coordinate expression of homeobox (Hox) and paired box (Pax) transcription factors in various axes of the body plan leads to differential expression of particular CAM genes. Following this hypothesis, we have characterized the promoters and other regulatory regions of a number of genes specifying CAMs and have identified cis‐regulatory elements that bind and respond to Hox and Pax proteins. Our recent experiments in vitro indicate, for example, that transcription factors encoded by Hox and Pax genes bind to specific DNA sequences in the N‐CAM promoter and activate expression of the N‐CAM gene. Experiments on transgenic mice carrying either the wild‐type N‐CAM promoter or a variant with mutations in the homeodomain binding sites (HBS) linked to a lac‐Z reporter gene indicate that interactions with these elements are important in establishing and maintaining N‐CAM expression in the spinal cord. We have also examined the regulatory sequences controlling expression of the gene for the neuron‐glia adhesion molecule (Ng‐CAM). Unlike N‐CAM, which is also expressed in many non‐neural sites, Ng‐CAM is restricted to cells of the nervous system. After identifying this promoter for the Ng‐CAM gene, we characterized a silencer region in the first intron of the gene that extinguishes the expression of Ng‐CAM in fibroblasts but not in neuronal cells. Thus, a default mechanism can account for the restriction of Ng‐CAM expression to the nervous system. The silencer region contains five neural‐restrictive silencer elements and a binding site for the Pax3 protein, which also appears to have silencing activity. All of these findings suggest that Hox and Pax transcription factors can have both activating and silencing functions in regulating CAM gene expression. The general significance of these accumulated observations is that they connect the place‐dependent expression of gene products capable of direct morphoregulation to the function of pattern‐forming genes.