Salivary Gland Tissue Recombination Can Modify Cell Fate.

Although mesenchyme is essential for inducing the epithelium of ectodermal organs, its precise role in organ-specific epithelial fate determination remains poorly understood. To elucidate the roles of tissue interactions in cellular differentiation, we performed single-cell RNA sequencing and imaging analyses on recombined tissues, where mesenchyme and epithelium were switched ex vivo between two types of embryonic mouse salivary glands: the parotid gland (a serous gland) and the submandibular gland (a predominantly mucous gland). We found partial induction of molecules that define gland-specific acinar and myoepithelial cells in recombined salivary epithelium. The parotid epithelium recombined with submandibular mesenchyme began to express mucous acinar genes not intrinsic to the parotid gland. While myoepithelial cells do not normally line parotid acini, newly induced myoepithelial cells densely populated recombined parotid acini. However, mucous acinar and myoepithelial markers continued to be expressed in submandibular epithelial cells recombined with parotid mesenchyme. Consequently, some epithelial cells appeared to be plastic, such that their fate could still be modified in response to mesenchymal signaling, whereas other epithelial cells appeared to be already committed to a specific fate. We also discovered evidence for bidirectional induction: transcriptional changes were observed not only in the epithelium but also in the mesenchyme after heterotypic tissue recombination. For example, parotid epithelium induced the expression of muscle-related genes in submandibular fibroblasts that began to mimic parotid fibroblast gene expression. These studies provide the first comprehensive unbiased molecular characterization of tissue recombination approaches exploring the regulation of cell fate.

Extracellular Matrix in Human Craniofacial Development.

The extracellular matrix (ECM) is a highly dynamic amalgamation of structural and signaling molecules whose quantitative and qualitative modifications drive the distinct programmed morphologic changes required for tissues to mature into their functional forms. The craniofacial complex houses a diverse array of tissues, including sensory organs, glands, and components of the musculoskeletal, neural, and vascular systems, alongside several other highly specialized tissues to form the most complex part of the vertebrate body. Through cell-ECM interactions, the ECM coordinates the cell movements, shape changes, differentiation, gene expression changes, and other behaviors that sculpt developing organs. In this review, we focus on several common key roles of the ECM to shape developing craniofacial organs and tissues. We summarize recent advances in our understanding of the ability of the ECM to biochemically and biomechanically orchestrate major events of craniofacial development, and we discuss how dysregulated ECM dynamics contributes to disease and disorders. As we expand our understanding of organ-specific matrix functionality and composition, we will improve our ability to rationally modify matrices to promote regeneration and/or prevent degenerative outcomes in vitro and in vivo.

Human Bone Marrow Cell Extracts Mitigate Radiation Injury to Salivary Gland.

Mitigation of irradiation injury to salivary glands was previously reported using a cell-free extract from mouse bone marrow. However, to bring this potential therapy a step closer to clinical application, a human bone marrow cell extract (BMCE) needs to be tested. Here, we report that irradiation-induced injury of salivary glands in immunocompetent mice treated with human BMCE secreted 50% more saliva than saline-injected mice, and BMCE did not cause additional acute inflammatory reaction. In addition, to identify the cell fraction in BMCE with the most therapeutic activity, we sorted human bone marrow into 3 cell fractions (mononuclear, granulocyte, and red blood cells) and tested their respective cell extracts. We identified that the mononuclear cell extract (MCE) provided the best therapeutic efficacy. It increased salivary flow 50% to 73% for 16 wk, preserved salivary parenchymal and stromal cells, and doubled cell proliferation rates while producing less inflammatory response. In contrast, the cell extract of granulocytes was of shorter efficacy and induced an acute inflammatory response, while that from red blood cells was not therapeutically effective for salivary function. Several proangiogenic (MMP-8, MMP-9, VEGF, uPA) and antiangiogenic factors (TSP-1, PF4, TIMP-1, PAI-1) were identified in MCE. Added advantages of BMCE and MCE for potential clinical use were that cell extracts from both male and female donors were comparably bioactive and that cell extracts could be stored and transported much more conveniently than cells. These findings suggest human BMCE, specifically the MCE fraction, is a promising therapy against irradiation-induced salivary hypofunction.

Integrin transmembrane signaling and cytoskeletal control.

Integrins are remarkably multifunctional: they mediate cell adhesion and migration, orchestrate organization of the actin-based cytoskeleton, and activate signal transduction pathways. Recent studies have identified a variety of steps and hierarchies in these intracellular cytoskeletal and signaling responses, laying the groundwork for future studies on specificity and coordination with responses to growth factors.

Molecular interactions in cell adhesion complexes.

Cell adhesions consist of multimolecular protein complexes of transmembrane adhesion receptors anchoring intracellular cytoskeletal structural proteins and signal transduction molecules. Recent advances reveal that components of cell adhesion complexes display multiple interactions and functions, which cooperate to mediate both cell adhesion and signaling. Cell-matrix and cell-cell adhesions can serve as both recipients and generators of signaling information, using hierarchical and synergistic molecular interactions regulated by aggregation, conformational changes, phosphorylation, and tension.

Cell-to-cell contact and extracellular matrix.

The topics discussed in this overview only scratch the surface of the contents of this issue, and necessarily reflect only some of the highlights that can be gleaned from the articles. The reader will learn a great deal more from this issue, which contains a particularly rich selection of very current topics, in both the traditional and new areas in the field. There continues to be remarkable progress towards understanding the mechanisms by which the different systems of cell-cell and cell-matrix contacts establish and regulate physical adhesive interactions and mediate transmembrane signaling processes in various tissues.

Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts.

Here we use time-lapse microscopy to analyse cell-matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP-paxillin to analyse focal contacts and GFP-tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometers per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain alpha5beta1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometers per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell-matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

VLA-4 mediates CD3-dependent CD4+ T cell activation via the CS1 alternatively spliced domain of fibronectin.

We previously showed that fibronectin (FN) synergized with anti-CD3 in induction of CD4+ T cell proliferation, and that VLA-5 acted as a functional FN receptor in a serum-free culture system. In the present study, we showed that VLA-4 is also involved in this CD3-dependent CD4 cell activation through its interaction with the alternatively spliced CS1 domain of FN. When highly purified CD4 cells were cultured on plates coated with anti-CD3 plus synthetic CS1 peptide-IgG conjugate, significant proliferation could be observed. Neither CS1 alone nor anti-CD3 alone induced this activation. This proliferation was completely blocked by anti-VLA beta 1 (4B4) and anti-VLA-4 (8F2), while anti-VLA-5 (monoclonal antibody [mAb] 16 and 2H6) had no effect. These data indicate that VLA-4 mediates CD3-dependent CD4 cell proliferation via the CS1 domain of FN. Anti-VLA-4 also partially (10-40%) inhibited CD4 cell proliferation induced by native FN plus anti-CD3, implying that the CS1 domain is active in the native plasma FN. However, this native FN-dependent proliferation was entirely abolished by addition of anti-VLA-5 alone. Moreover, when native FN-coated plates were pretreated with anti-FN (mAb 333), which blocks RGDS sites but not CS1 sites, no CD4 cell activation could be observed. These results strongly suggest that CD4 cell activation induced by plasma FN/anti-CD3 may be dependent on both VLA4/CS1 and VLA5/RGDS interactions, although the latter interaction may be required for function of the former.

Activation of CD4 cells by fibronectin and anti-CD3 antibody. A synergistic effect mediated by the VLA-5 fibronectin receptor complex.

In this study, fibronectin synergized with anti-CD3 antibody to promote CD4 cell proliferation in a serum-free culture system. The cell-adhesive domain plus additional regions of the fibronectin molecule are involved in this synergy. Anti4B4(CDw29) antibody blocked the activation of CD4 cells in this system. Furthermore, it is the VLA-5 protein within the set of molecules recognized by anti-4B4 that serves as a fibronectin receptor on the CD4 lymphocytes. The VLA-5 fibronectin receptor was mainly expressed on CD4+ CD45R-CDw29+ cells and may in part contribute to the unique function of these cells.

Single-Cell RNA-seq Identifies Cell Diversity in Embryonic Salivary Glands.

Branching organs, including the salivary and mammary glands, lung, and kidney, arise as epithelial buds that are morphologically very similar. However, the mesenchyme is known to guide epithelial morphogenesis and to help govern cell fate and eventual organ specificity. We performed single-cell transcriptome analyses of 14,441 cells from embryonic day 12 submandibular and parotid salivary glands to characterize their molecular identities during bud initiation. The mesenchymal cells were considerably more heterogeneous by clustering analysis than the epithelial cells. Nonetheless, distinct clusters were evident among even the epithelial cells, where unique molecular markers separated presumptive bud and duct cells. Mesenchymal cells formed separate, well-defined clusters specific to each gland. Neuronal and muscle cells of the 2 glands in particular showed different markers and localization patterns. Several gland-specific genes were characteristic of different rhombomeres. A muscle cluster was prominent in the parotid, which was not myoepithelial or vascular smooth muscle. Instead, the muscle cluster expressed genes that mediate skeletal muscle differentiation and function. Striated muscle was indeed found later in development surrounding the parotid gland. Distinct spatial localization patterns of neuronal and muscle cells in embryonic stages appear to foreshadow later differences in adult organ function. These findings demonstrate that the establishment of transcriptional identities emerges early in development, primarily in the mesenchyme of developing salivary glands. We present the first comprehensive description of molecular signatures that define specific cellular landmarks for the bud initiation stage, when the neural crest-derived ectomesenchyme predominates in the salivary mesenchyme that immediately surrounds the budding epithelium. We also provide the first transcriptome data for the largely understudied embryonic parotid gland as compared with the submandibular gland, focusing on the mesenchymal cell populations.

Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape.

The major cell surface glycoprotein of chick embryo fibroblasts, cellular fibronectin (formerly known as CSP or LETS protein), was purified and used to produce monospecific antisera. After affinity purification, the anti-fibronectin was used to investigate fibronectin's localization, its transfer from intracellular to extracellular pools, its antibody-induced redistribution on the cell surface, and its role in cell shape. Anti-fibronectin localizes to extracellular fibrils located under and between sparse cells, and to a dense matrix that surrounds confluent cells. Cellular fibronectin is also present in granular intracytoplasmic structures containing newly synthesized fibronectin before secretion. This intracellular staining disappears 2 h after treatment with cycloheximide or puromycin, and returns after removal of these protein synthesis inhibitors. In pulse-chase experiments using cycloheximide, fibronectin was sequentially transferred from the intracellular to the fibrillar extracellular forms. Transformation of chick fibroblasts results in decreases in both extracellular and intracellular fibronectin, and in altered cell shape. Treatment of untransformed chick fibroblasts with anti-fibronectin results in rapid (30 min) alteration to a rounder cell shape resembling that of many transformed cells. These rapid shape changes are followed by a slow, antibody-induced redistribution of fibronectin to supranuclear caplike structures. This "capping" is inhibited by metabolic inhibitors. Reconstitution of cell surface fibronectin onto transformed cells restores a more normal fibroblastic phenotype. The reconstituted fibronectin on these cells organizes into fibrillar patterns similar to those of untransformed cells. As with untransformed cells, treatment of these reconstituted cells with anti-fibronectin also results in cell rounding and "capping" of fibronectin.

The zinc-finger protein slug causes desmosome dissociation, an initial and necessary step for growth factor-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an essential morphogenetic process during embryonic development. It can be induced in vitro by hepatocyte growth factor/scatter factor (HGF/SF), or by FGF-1 in our NBT-II cell model for EMT. We tested for a central role in EMT of a zinc-finger protein called Slug. Slug mRNA and protein levels were increased transiently in FGF-1-treated NBT-II cells. Transient or stable transfection of Slug cDNA in NBT-II cells resulted in a striking disappearance of the desmosomal markers desmoplakin and desmoglein from cell-cell contact areas, mimicking the initial steps of FGF-1 or HGF/SF- induced EMT. Stable transfectant cells expressed Slug protein and were less epithelial, with increased cell spreading and cell-cell separation in subconfluent cultures. Interestingly, NBT-II cells transfected with antisense Slug cDNA were able to resist EMT induction by FGF-1 or even HGF/SF. This antisense effect was suppressed by retransfection with Slug sense cDNA. Our results indicate that Slug induces the first phase of growth factor-induced EMT, including desmosome dissociation, cell spreading, and initiation of cell separation. Moreover, the antisense inhibition experiments suggest that Slug is also necessary for EMT.

A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein.

Heat shock proteins of chick embryo fibroblasts were analyzed on SDS polyacrylamide gradient gels and were found to include not only three previously well-characterized proteins of 25,000, 73,000, and 89,000 D, but also a 47,000-D protein. Two-dimensional gel electrophoresis revealed that this protein was unusually basic (pI = 9.0) and corresponded to a recently characterized, major gelatin- and collagen-binding protein. The induction of synthesis of this 47,000-D membrane glycoprotein after heat stress of fibroblasts was particularly apparent in preparations isolated by gelatin-affinity chromatography. Regulation of this 47,000-D phosphoprotein was more sensitive than that of three major heat shock proteins in that a substantial stimulation of synthesis occurred at even 42 degrees C, as well as at higher temperature. Phosphorylation of the 47,000-D protein was not altered after heat shock. These studies establish this phosphorylated membrane glycoprotein as a member of the heat shock/stress protein family, and they add collagen binding to the unexpectedly diverse spectrum of biochemical activities induced by exposure of cells to stress.

Tumor suppressor PTEN inhibits integrin- and growth factor-mediated mitogen-activated protein (MAP) kinase signaling pathways.

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH2-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.

Fibroblast cellular and plasma fibronectins are similar but not identical.

Fibronectins are multimeric, adhesive glycoproteins present on cell surfaces and circulating in blood. Cellular fibronectin produced by fibroblasts in vitro and fibronectin isolated from plasma are known to be very similar immunologically and biochemically. We investigated whether or not they are identifical. Purified chicken and human cell-surface fibronectins are 150-fold more active in hemagglutination of fixed erythrocytes than plasma fibronectins. Cell-surface fibronectin is also 50-fold more active in restoring a more normal morphology to transformed cells originally missing the protein. However, in two other assays that measure cell attachment to collagen and cell spreading, cell-surface and plasma fibronectins have identical specific activities. In sodium dodecyl sulfate polyacrylamide gels, the subunits of human and chicken plasma fibronectins have significantly smaller apparent subunit molecular weights than cellular fibronectins present on cell surfaces or secreted into culture media. These differences are also present in a characteristic large subfragment of both forms of fibronectin after limited proteolysis by trypsin. We conclude that by both biological and biochemical criteria, cellular and plasma fibronectins are similar but not identical.

Quantitation of a transformation-sensitive, adhesive cell surface glycoprotein. Decrease of several untransformed permanent cell lines.

We have quantitated the transformation-sensitive, cell surface LETS glycoprotein on many untransformed cell types. By SDS-polyacrylamide gel electrophoresis, this trypsin-sensitive iodinatable glycoprotein comprises 1-3% of total cellular protein of the seven early passage cell types tested. In contrast, it constitutes less than 0.15% of the protein in four of six continuous cell lines. This decrease is reflected in alterations both in [14C]glucosamine labeling and in the immunofluorescent staining of early passage vs. these four permanent cell lines. These results help to clarify previous experiments in which CSP, a purified LETS protein, partially restored a fibroblastic phenotype to cells transformed by tumor viruses. These findings also indicate that a major decrease in this cell surface glycoprotein can occur in the establishment of a continuous cell line without resulting in cellular transformation.

Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function.

Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5-10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor.

Characterization of a 140-kD avian cell surface antigen as a fibronectin-binding molecule.

A 140,000-D protein cell surface antigen (140k) complex has been implicated in fibronectin-mediated cell-substratum attachment. We have used three different experimental systems to evaluate the hypothesis that this 140k complex can function as a fibronectin receptor. A monoclonal antibody that binds to the 140k complex specifically inhibits the direct binding of 3H-labeled 75,000-D fibronectin cell-binding fragment (f75k) to chicken embryo fibroblasts in suspension. The 140k complex is retarded in its passage through an affinity column consisting of immobilized f75k, and this interaction is specifically inhibited by a synthetic peptide that contains the fibronectin cell-recognition signal sequence. Finally, exogenous purified 140k complex inhibits the attachment and spreading of chicken embryo fibroblasts on fibronectin-coated substrates. Thus, our results indicate that the 140k complex can bind directly to fibronectin and is likely to be a fibronectin receptor for chicken cells.

Regulation of fibronectin receptor distribution.

To determine the role of each intracellular domain of the fibronectin receptor in receptor distribution, chimeric receptors were constructed containing the human interleukin-2 receptor (gp55 subunit) as the extracellular and transmembrane domains, in combination with either the alpha 5 or beta 1 intracellular domain of the fibronectin receptor as the cytoplasmic domain. These chimeric receptors were transiently expressed in normal fibroblasts, and their localization on the cell surface was determined by immunofluorescence using antibodies to the human interleukin-2 receptor. The alpha 5 chimera was expressed diffusely on the plasma membrane. The beta 1 chimera, however, colocalized with the endogenous fibronectin receptor at focal contacts of cells spread on fibronectin. On cells spread in the presence of serum, the beta 1 chimera colocalized both with the fibronectin receptor at sites of extracellular fibronectin fibrils and with the vitronectin receptor at focal contacts. The beta 1 intracellular domain alone, therefore, contains sufficient information to target the chimeric receptor to regions of the cell where ligand-occupied integrin receptors are concentrated. The finding that the beta 1 chimeric protein behaves like a ligand-occupied receptor, even though the beta 1 chimera cannot itself bind extracellular ligand, suggests an intracellular difference between occupied and unoccupied receptors, and predicts that the distribution of integrin receptors can be regulated by ligand occupancy. We tested this prediction by providing a soluble cell-binding fragment of fibronectin to cells spread on laminin. Under conditions preventing further ligand adsorption to the substrate, this treatment nevertheless resulted in the relocation of diffuse fibronectin receptors to focal contacts. Similarly, a redistribution of diffuse vitronectin receptors to focal contacts occurred on cells spread on laminin after the addition of the small soluble peptide GRGDS. We conclude that the propensity for receptor redistribution to focal contacts driven by the beta 1 cytoplasmic domain alone is suppressed in heterodimeric unoccupied fibronectin receptors, and that ligand occupancy can release this constraint. This redistribution of integrin receptors after the binding of a soluble substrate molecule may provide a direct means of assembling adhesion sites.

Dual stimulation of Ras/mitogen-activated protein kinase and RhoA by cell adhesion to fibronectin supports growth factor-stimulated cell cycle progression.

In cellular transformation, activated forms of the small GTPases Ras and RhoA can cooperate to drive cells through the G1 phase of the cell cycle. Here, we show that a similar but substrate-regulated mechanism is involved in the anchorage-dependent proliferation of untransformed NIH-3T3 cells. Among several extracellular matrix components tested, only fibronectin supported growth factor-induced, E2F-dependent S phase entry. Although all substrates supported the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity was specifically enhanced on fibronectin. Moreover, induction of cyclin D1 and suppression of p21(Cip/Waf) occurred specifically, in a Rho-dependent fashion, in cells attached to fibronectin. This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression.