In juxtacrine interactions, proteins from the inducing cell interact with receptor proteins of adjacent responding cells. The inducer does not diffuse from the cell producing it. There are three types of juxtacrine interactions. In the first type, a protein on one cell binds to its receptor on the adjacent cell. We saw this type of juxtacrine interaction when we discussed the interaction between the Bride of sevenless protein and its receptor, Sevenless. In the second type, a receptor on one cell binds to its ligand on the extracellular matrix secreted by another cell. In the third type, the signal is transmitted directly from the cytoplasm of one cell through small conduits into the cytoplasm of an adjacent cell.
Figure 6.29
Mechanism of Notch activity. (A) Model for the activation of Notch. A ligand (Delta, Jagged, or Serrate protein) on one cell binds to the extracellular domain of the Notch protein on an adjacent cell. This binding causes a shape change in the intracellular domain of Notch, which activates a protease. The protease cleaves Notch and allows the intracellular region of the Notch protein to enter the nucleus and activate a transcription factor of the CSL family (such as Suppressor of hairless or CBF1). The activated CSL can then transcribe its target genes. (B) Inhibition of neurogenesis in the mouse. Delta family gene Delta-1 (Dll1) signals Notch family genes Notch-1 (and probably Notch-3). These proteins activate a CSL transcription factor (RBP), which transcribes the Hes5 gene. Hes5 protein inhibits the Mash1 gene, which would otherwise activate the neural determining gene NeuroD. Thus, neural determination is blocked. Hes5 also inhibits
another gene (probably for neurogenin) that would otherwise activate the production of Delta-1. (A after Schroeder et al. 1998; B after Blaschuk and ffrench-Constant 1998.
Notch proteins are extremely important receptors in the nervous system. In both the vertebrate and Drosophila nervous system, the binding of Delta to Notch tells the receiving cell not to become neural (Figure 6.29B; Chitnis et al. 1995; Wang et al. 1998). In the vertebrate eye, the interactions between Notch and its ligands seem to regulate which cells become optic neurons and which become glial cells (Dorsky et al. 1997; Wang et al. 1998).
Figure 6.32
Fibronectin in the developing frog embryo. (A) Fluorescent antibodies to fibronectin show fibronectin deposition as a green band in the Xenopus embryo during gastrulation. The fibronectin will orient the mesoderm movements of the cells.
(B) Structure and binding domains of fibronectin. The rectangles represent protease-resistant domains. The fibroblastbinding domain consists of two units, the RGD site and the high-affinity site, both of which are essential for cell binding. Avian neural crest cells have another site that is necessary for them to migrate on a fibronectin substrate. Other regions of fibronectin enable it to bind to collagen, heparin, and other molecules of the extracellular matrix. (A courtesy of M. Marsden and D. W. DeSimone; B after Dufour et al. 1988.)
Laminin and type IV collagen are major components of a type of extracellular matrix called the basal lamina (Figure 6.33). This basal lamina is characteristic of the closely knit sheets that surround epithelial tissue. The adhesion of epithelial cells to laminin (upon which they sit) is much greater than the affinity of mesenchymal cells for fibronectin (to which they must bind and release if they are to migrate). Like fibronectin, laminin plays a role in assembling the extracellular matrix, promoting cell adhesion and growth, changing cell shape, and permitting cell migration (Hakamori et al. 1984).
Figure 6.33
Location and formation of extracellular matrices in the chick embryo. The scanning electron micrograph shows the extracellular matrix at the junction of the epithelial cells (above) and mesenchymal cells (below). The epithelial cells synthesize a tight, glycoprotein-based basal lamina, while the mesenchymal cells secrete a loose reticular lamina made primarily of collagen. (Courtesy of R. L. Trelsted.)
Figure 6.34
Speculative diagram relating the binding of cytoskeleton to the extracellular matrix through the integrin molecule
Bissell and her colleagues (1982; Martins-Green and Bissell 1995) have proposed that the extracellular matrix is capable of inducing specific gene expression in developing tissues, especially those of the liver, testis, and mammary gland, in which the induction of specific transcription factors depends on cell-substrate binding (Figure 6.35; Liu et al. 1991; Streuli et al. 1991; Notenboom et al. 1996). Often, the presence of bound integrin prevents the activation of genes that specify apoptosis (Montgomery et al. 1994; Frisch and Ruoslahti 1997). The chondrocytes that produce the cartilage of our vertebrae and limbs can survive and differentiate only if they are surrounded by an extracellular matrix and are joined to that matrix through their integrins (Hirsch et al. 1997). If chondrocytes from the developing chick sternum are incubated with antibodies that block the binding of integrins to the extracellular matrix, they shrivel up and die. While the mechanisms by which bound integrins inhibit apoptosis remain controversial (see Howe et al. 1998), the extracellular matrix is obviously an important source of signals that can be transduced into the nucleus to produce specific gene expression.
Figure 6.35
Role of the extracellular matrix in cell differentiation. Light micrographs of rat Sertoli testis cells grown for two weeks (A) on tissue culture plastic dishes and (B) on dishes coated with basal lamina. The two photographs were taken at the same magnification, 1200. (From Hadley et al. 1985; photographs courtesy of M. Dym.)
Some of the genes induced by matrix attachment are being identified. When plated onto tissue culture plastic, mouse mammary gland cells will divide (Figure 6.36). Indeed, the genes for cell division (c- myc, cyclinD1) are expressed, while the genes for the differentiated products of the mammary gland (casein, lactoferrin, whey acidic protein) are not expressed. If the same cells are plated onto plastic coated with a laminin-containing basement membrane, the cells stop dividing and the differentiated genes of the mammary gland are expressed. This happens only after the integrins of the mammary gland cells bind to the laminin of the extracellular basement membrane. Then the gene for lactoferrin is expressed, as is the gene for p21, a cell division inhibitor. The c-myc and cyclinD1 genes become silent. Eventually, all the genes for the developmental products of the mammary gland are expressed and the cell division genes remain turned off. At this time, the mammary cells will have enveloped themselves in the basement membrane, forming a secretory epithelium reminiscent of the mammary gland tissue. The binding of integrins to laminin is essential for the transcription of the casein gene, and the integrins act in concert with prolactin (see Figure 6.21) to activate that gene's expression (Roskelley et al. 1994; Muschler et al. 1999).
Figure 6.36
Basement membrane-directed gene expression in mammary gland tissue. (A) Mouse mammary gland tissue divides when placed on tissue culture plastic. Cell division genes are on, and the genes capable of synthesizing the differentiated products of the mammary gland (lactoferrin, casein, whey acidic protein) are off. (B) When presented with basement membrane that contains laminin, the genes for cell division proteins are turned off, while the gene inhibiting cell division (p21) and the gene for lactoferrin are turned on. (C, D) Mammary gland cells wrap the basement membrane about them, forming a secretory epithelium. The genes for casein and whey protein are sequentially activated. (After Bissell, personal communication.)
Recent studies have shown that the binding of integrins to the extracellular matrix can stimulate the RTKRas pathway. When an integrin on the cell membrane of one cell binds to the fibronectin or collagen secreted by a neighboring cell, the integrin can activate the tyrosine kinase cascade through an adaptor protein-like complex that connects the integrin to the Ras G protein (Figure 6.37A; Wary et al. 1998). Cadherins and other cell adhesion molecules can also transmit signals by hijacking the FGF receptors (Williams et al. 1994b; Clark and Brugge 1995). Cadherins, for example, are able to bind to the intramembrane region of FGF receptors and thereby dimerize these receptors just like the normal FGF ligands (Figure 6.37B; Williams et al. 1994a; Doherty et al. 1995).
Figure 6.37
Two types of activation by cell adhesion molecules. (A) Cell-substrate adhesion molecules such as integrins may transmit a signal from the cytoplasmic portion of the integrin protein to the Ras G protein through a cascade involving caveolin and Fyn proteins. (B) The FGF receptors may be hijacked by cell adhesion molecules and dimerized. They may be brought together by the interaction of opposite cell adhesion molecules, or the crosslinking of FGF receptors by the apposing cell membrane may activate their kinase domains. (A after Wary et al.1998.)
Figure 6.38
Gap junctions. (A) Electron micrograph of a row of gap junctions connecting two apposed cells. (B) Fluorescence micrograph of gap junctions in a 17-day embryonic mouse kidney tubule. (C) Compartments formed by gap junction proteins between cells that communicate with one another. This compartment in the mouse gastrula can be seen by injecting Lucifer Yellow dye into one cell and seeing it transferred to a small group of cells. (D) Subunit structure of the gap junction. (A from Peracchia and Dulhunty 1976, courtesy of C. Peracchia; B from Sainio et al. 1992, courtesy of K. Sainio; C from Kalimi and Lo 1988, courtesy of C. Lo; D after Darnell et al. 1986.)
The gap junction channels are made of connexin proteins. In each cell, six identical connexins in the membrane group together to form a transmembrane channel containing a central pore. The channel complex of one cell connects to the channel complex of another cell, enabling the cytoplasms of both cells to be joined (Figure 6.38D). Different types of connexin proteins have separate, but overlapping, roles in normal development. For example, connexin-43 is found throughout the developing mouse embryo, in nearly every tissue. However, if the connexin-43 genes are knocked out by gene targeting, the mouse embryo will still develop. It appears that most of the functions of the connexin-43 protein can be taken over by other related connexin proteinsbut not all. Shortly after birth, connexin-43-deficient mice take gasping breaths, turn bluish, and die. Autopsies of these mice show that the right ventriclethe chamber that pumps blood through the pulmonary artery to the lungsis filled with tissue that occludes the chamber and obstructs blood flow (Reaume et al. 1995; Huang et al. 1998). Although loss of the connexin-43 protein can be compensated for in many tissues, it appears to be critical for normal heart development. The importance of gap junctions in development has been demonstrated in amphibian and mammalian embryos (Warner et al. 1984). When antibodies to connexins were microinjected into one specific cell of an 8-cell Xenopus blastula, the progeny of that cell, which are usually coupled through gap junctions, could no longer pass ions or small molecules from cell to cell. Moreover, the tadpoles that resulted from such treated blastulae showed defects specifically relating to the developmental fate of the injected cell (Figure 6.39). The progeny of the injected cell did not die, but they were unable to develop normally (Warner et al. 1984). In the mouse embryo, the first eight blastomeres are also connected to one another by gap junctions. Although
loosely associated with one another, these eight cells move together to form a compacted embryo. If compaction is inhibited by antibodies against connexins, the treated blastomeres continue to divide, but further development ceases (Lo and Gilula 1979; Lee et al. 1987). If antisense RNA to connexin messages is injected into one of the blastomeres of a normal mouse embryo, that cell will not form gap junctions and will not be included in the embryo (Bevilacqua et al. 1989).