Widely Conserved Signaling Pathways in the Establishment of Cell Polarity

How are the asymmetric distributions of proteins, lipids, and RNAs established and maintained in various cell types? Studies from diverse organisms show that Par proteins, GTPases, kinases, and phosphoinositides participate in conserved signaling pathways to establish and maintain cell polarity.The asymmetric distribution of proteins, lipids, and RNAs is necessary for cell fate determination, differentiation, and specialized cell functions that underlie morphogenesis (St Johnston 2005; Gonczy 2008; Knoblich 2008; Macara and Mili 2008; Martin-Belmonte and Mostov 2008). A fundamental question is how this asymmetric distribution is established and maintained in different types of cells and tissues. The formation of a specialized apical surface on an epithelial cell seems quite different from the specification of axons versus dendrites in a neuron, or the asymmetric division of a nematode zygote. Yet, remarkably, a conserved molecular toolbox is used throughout the metazoa to establish and maintain cell polarity in these and many other contexts. This toolbox consists of proteins that are components of signal transduction pathways (Goldstein and Macara 2007; Assemat et al. 2008; Yamanaka and Ohno 2008). However, our understanding of these pathways, and their intersection with other signaling networks, remains incomplete. Moreover, the regulation and cross talk between the polarity proteins and other signaling components varies from one context to another, which complicates the task of dissecting polarity protein function. Nonetheless, rapid progress is being made in our understanding of polarity signaling, which is outlined in this article, with an emphasis on the Par proteins, because these proteins play major roles integrating diverse signals that regulate cell polarity (Fig. 1) (see Munro and Bowerman 2009; Prehoda 2009; Nelson 2009).Open in a separate windowFigure 1.An overview of Par complex signaling, showing inputs (bottom) and outputs (top) with cellular functions that are targeted by these pathways (italics).     

Fibronectins,Their Fibrillogenesis,and In Vivo Functions

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN–FN and cell–FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.As a ubiquitous component of the extracellular matrix (ECM), fibronectin (FN) provides essential connections to cells through integrins and other receptors and regulates cell adhesion, migration, and differentiation. FN is secreted as a large dimeric glycoprotein with subunits that range in size from 230 kDa to 270 kDa (Mosher 1989; Hynes 1990). Variation in subunit size depends primarily on alternative splicing. FN was first isolated from blood more than 60 years ago (Edsall 1978), and this form is called plasma FN. The other major form, called cellular FN, is abundant in the fibrillar matrices of most tissues. Although FN is probably best known for promoting attachment of cells to surfaces, this multidomain protein has many interesting structural features and functional roles beyond cell adhesion.FN is composed of three different types of modules termed type I, II, and III repeats (Fig. 1) (Petersen et al. 1983; Hynes 1990). These repeats have distinct structures. Although the conformations of type I and type II repeats are maintained by pairs of intramodule disulfide bonds, the type III repeat is a 7-stranded β-barrel structure that lacks disulfide bonds (Main et al. 1992; Leahy et al. 1996, 1992) and, therefore, can undergo conformational changes. FN type III repeats are widely distributed among animal, bacterial, and plant proteins and are found in both extracellular and intracellular proteins (Bork and Doolittle 1992; Tsyguelnaia and Doolittle 1998).Open in a separate windowFigure 1.FN domain organization and isoforms. Each FN monomer has a modular structure consisting of 12 type I repeats (cylinders), 2 type II repeats (diamonds), and 15 constitutive type III repeats (hexagons). Two additional type III repeats (EIIIA and EIIIB, green) are included or omitted by alternative splicing. The third region of alternative splicing, the V region (green box), is included (V120), excluded (V0), or partially included (V95, V64, V89). Sets of modules comprise domains for binding to other extracellular molecules as indicated. Domains required for fibrillogenesis are in red: the assembly domain (repeats I_1-5) binds FN, III_9-10 contains the RGD and synergy sequences for integrin binding, and the carboxy-terminal cysteines form the disulfide-bonded FN dimer (‖). The III_1-2 domain (light red) has two FN binding sites that are important for fibrillogenesis. The amino-terminal 70-kDa fragment contains assembly and gelatin-binding domains and is routinely used in FN binding and matrix assembly studies.Sets of adjacent modules form binding domains for a variety of proteins and carbohydrates (Fig. 1). ECM proteins, including FN, bind to cells via integrin receptors, αβ heterodimers with two transmembrane subunits (Hynes 2002). FN-binding integrins have specificity for one of the two cell-binding sites within FN, either the RGD-dependent cell-binding domain in III₁₀ (Pierschbacher and Ruoslahti 1984) or the CS1 segment of the alternatively spliced V region (IIICS) (Wayner et al. 1989; Guan and Hynes 1990). Some integrins require a synergy sequence in repeat III₉ for maximal interactions with FN (Aota et al. 1994; Bowditch et al. 1994). Another family of cell surface receptors is the syndecans, single-chain transmembrane proteoglycans (Couchman 2010). Syndecans use their glycosaminoglycan (GAG) chains to interact with FN at its carboxy-terminal heparin-binding (HepII) domain (Fig. 1) (Saunders and Bernfield 1988; Woods et al. 2000), which binds to heparin, heparan sulfate, and chondroitin sulfate GAGs (Hynes 1990; Barkalow and Schwarzbauer 1994). Syndecan binding to the HepII domain enhances integrin-mediated cell spreading and intracellular signaling, suggesting that syndecans act as coreceptors with integrins in cell–FN binding (Woods and Couchman 1998; Morgan et al. 2007).A major site for FN self-association is within the amino-terminal assembly domain spanning the first five type I repeats (I_1-5) (Fig. 1) (McKeown-Longo and Mosher 1985; McDonald et al. 1987; Schwarzbauer 1991b; Sottile et al. 1991). This domain plays an essential role in FN fibrillogenesis. As a major blood protein, FN interacts with fibrin during blood coagulation, also using the I_1-5 domain (Mosher 1989; Hynes 1990). As fibrin polymerizes, factor XIII transglutaminase covalently cross-links glutamine residues near the amino terminus of FN to fibrin α chains (Mosher 1975; Corbett et al. 1997). The amino-terminal domain has multiple binding partners in addition to FN and fibrin; these include heparin, S. aureus, and other bacteria, thrombospondin-1, and tenascin-C (Hynes 1990; Ingham et al. 2004; Schwarz-Linek et al. 2006). Adjacent to this domain is the gelatin/collagen-binding domain composed of type I and type II modules (Ingham et al. 1988). This domain also binds to tissue transglutaminase (Radek et al. 1993) and fibrillin-1 (Sabatier et al. 2009). Within the 15 type III repeats reside several FN binding sites that interact with the amino-terminal assembly domain as well as three sites of alternative splicing that generate multiple isoforms. At the carboxyl terminus is a pair of cysteine residues that form the FN dimer through antiparallel disulfide bonds (Hynes 1990). This dimerization may be facilitated by disulfide isomerase activity located in the last set of type I repeats (Langenbach and Sottile 1999).The diverse set of binding domains provides FN with the ability to interact simultaneously with other FN molecules, other ECM components (e.g., collagens and proteoglycans), cell surface receptors, and extracellular enzymes (Pankov and Yamada 2002; Fogelgren et al. 2005; Hynes 2009; Singh et al. 2010). Multitasking by FN probably underlies its essential role during embryogenesis (George et al. 1993). Furthermore, FN''s interactions can be modulated by exposure or sequestration of its binding sites within matrix fibrils, through the presence of ECM proteins that bind to FN, or through variation in structure by alternative splicing.     

The Desmosome

Desmosomes are intercellular junctions that tether intermediate filaments to the plasma membrane. Desmogleins and desmocollins, members of the cadherin superfamily, mediate adhesion at desmosomes. Cytoplasmic components of the desmosome associate with the desmosomal cadherin tails through a series of protein interactions, which serve to recruit intermediate filaments to sites of desmosome assembly. These desmosomal plaque components include plakoglobin and the plakophilins, members of the armadillo gene family. Linkage to the cytoskeleton is mediated by the intermediate filament binding protein, desmoplakin, which associates with both plakoglobin and plakophilins. Although desmosomes are critical for maintaining stable cell–cell adhesion, emerging evidence indicates that they are also dynamic structures that contribute to cellular processes beyond that of cell adhesion. This article outlines the structure and function of the major desmosomal proteins, and explores the contributions of this protein complex to tissue architecture and morphogenesis.The desmosome is an adhesive intercellular junction that is crucial to tissues that experience mechanical stress, such as the myocardium, bladder, gastrointestinal mucosa, and skin (Getsios et al. 2004b; Holthofer et al. 2007). The desmosome was first observed in the spinous layer of epidermis by the Italian pathologist Giulio Bizzozero (1846–1901). Bizzozero''s observations of these small dense nodules, subsequently named “nodes of Bizzozero,” led him to the insightful interpretation of these structures as adhesive cell–cell contact points. The term desmosome was later coined by Josef Schaffer in 1920 and is derived from the Greek words “desmo,” meaning bond or fastening, and “soma,” meaning body (Wells 2005; Calkins and Setzer 2007). The introduction of electron microscopy yielded a series of advances by Porter, Odland, and Kelly in the 1950s and 1960s, which revealed desmosome organization at the ultrastructural level. These studies and others indicated that the desmosome can be divided into three morphologically identifiable zones: the extracellular core region (desmoglea), the outer dense plaque (ODP), and the inner dense plaque (IDP) (Fig. 1A) (Kowalczyk et al. 1994; Schmidt et al. 1994; Green and Jones 1996; North et al. 1999; Garrod and Chidgey 2008).Open in a separate windowFigure 1.A model for the structure of desmosomes. (A) Electron micrograph of a desmosome. (B) Schematic of desmosomal proteins and relative distance from the plasma membrane (PM). The desmosomal cadherins, the desmogleins and desmocollins, extend into extracellular core and outer dense plaque (ODP) to establish contact and adhere to neighboring cells in a Ca²⁺-dependent manner. The cadherin cytoplasmic tails associate linker proteins, plakoglobin (PG), the plakophilins (PKP), and desmoplakin (DP). DP binds to keratin intermediate filaments (KIF) within the inner dense plaque (IDP), serving to tether the intermediate filaments to the plasma membrane. (Adapted with permission from Kottke et al. 2006.)In the mid 1970s, Skerrow and Matoltsy (Skerrow and Matoltsy 1974a; Skerrow and Matoltsy 1974b) advanced the field by isolating desmosomes using biochemical approaches (Bass-Zubek and Green 2007).These landmark studies provided a foundation for the Franke and Steinberg laboratories to characterize the transmembrane glycoproteins and cytoplasmic plaque proteins that linked the structure to the intermediate filament cytoskeleton, and to develop immunological tools for localizing specific components (Franke et al. 1981; Kapprell et al. 1985; Steinberg et al. 1987). Collectively, these and other studies shaped our current view of how desmosomal components are organized.The transmembrane glycoproteins, termed desmogleins and desmocollins (Garrod and Chidgey 2008), represent separate subfamilies of the cadherin superfamily of calcium dependent adhesion molecules. The extracellular domains of the desmogleins and desmocollins mediate adhesion, whereas the cytoplasmic tails of these cadherins associate with the desmosomal plaque proteins. The outer dense plaque consists of the cytoplasmic tails of the desmosomal cadherins, which bind to members of the armadillo and plakin family of linker proteins (Kowalczyk et al. 1994; Getsios et al. 2004b; Garrod and Chidgey 2008). Plakoglobin, a member of the armadillo family, binds directly to the cytoplasmic tails of both the desmogleins and the desmocollins (Wahl et al. 1996; Witcher et al. 1996). Desmoplakin, a member of the plakin family, interacts with both plakoglobin and another subgroup of armadillo family proteins, the plakophilins (Cowin and Burke 1996). Finally, the interaction between desmoplakin and the keratin filaments forms the inner dense plaque, tethering the cytoskeletal network to the adhesion complex (Fig. 1B) (Kowalczyk et al. 1994; Getsios et al. 2004b; Garrod and Chidgey 2008).The following sections of this article describe the structural and functional characteristics of the major desmosomal proteins. In addition, we discuss differences in tissue expression patterns of desmosomal proteins and the role of desmosomes in human disease. A comprehensive review of additional proteins found to regulate or associate with desmosomes is provided elsewhere (Holthofer et al. 2007) and discussion of desmosome dynamics is provided in Green et al. 2009.     

mGluR1/TRPC3-mediated Synaptic Transmission and Calcium Signaling in Mammalian Central Neurons

Metabotropic glutamate receptors type 1 (mGluR1s) are required for a normal function of the mammalian brain. They are particularly important for synaptic signaling and plasticity in the cerebellum. Unlike ionotropic glutamate receptors that mediate rapid synaptic transmission, mGluR1s produce in cerebellar Purkinje cells a complex postsynaptic response consisting of two distinct signal components, namely a local dendritic calcium signal and a slow excitatory postsynaptic potential. The basic mechanisms underlying these synaptic responses were clarified in recent years. First, the work of several groups established that the dendritic calcium signal results from IP₃ receptor-mediated calcium release from internal stores. Second, it was recently found that mGluR1-mediated slow excitatory postsynaptic potentials are mediated by the transient receptor potential channel TRPC3. This surprising finding established TRPC3 as a novel postsynaptic channel for glutamatergic synaptic transmission.Glutamate is the predominant neurotransmitter used by excitatory synapses in the mammalian brain (Hayashi 1952; Curtis et al. 1959). At postsynaptic sites, glutamate binds to two different classes of receptors, namely the ionotropic glutamate receptors (iGluRs) and the metabotropic glutamate receptors (mGluRs) (Sladeczek et al. 1985; Nicoletti et al. 1986; Sugiyama et al. 1987). The iGluRs represent ligand-gated nonselective cation channels that underlie excitatory postsynaptic currents (EPSCs). Based on their subunit composition, gating, and permeability properties, they are subdivided into three groups named after specific agonists: AMPA- (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), NMDA receptors (N-methyl D-aspartate receptors) and kainate receptors (Alexander et al. 2009). The other class of glutamate receptors, the mGluRs, consists of receptors that are coupled to G proteins and act through distinct downstream signaling cascades. They are structurally different from iGluRs and characterized by the presence of seven transmembrane domains (Houamed et al. 1991; Masu et al. 1991). The mGluRs exist as homodimers that do not by themselves form an ion-permeable pore in the membrane (Ozawa et al. 1998). To date, eight different genes (and more splice variants) encoding mGluRs have been identified and form the mGluR1 through mGluR8 subtypes (Alexander et al. 2009). Based on the amino acid sequence homology, downstream signal transduction pathways, and pharmacological properties, each of the subtypes was assigned to one of three groups. Group I receptors consist of mGluR1 and mGluR5 that positively couple to the phospholipase C (PLC). The receptors mGluR2 and mGluR3 constitute group II, whereas the remaining mGluRs, namely mGluR4, mGluR6, mGluR7, and mGluR8, belong to group III. Both groups II and III inhibit the adenylyl cyclase and thereby reduce the concentration of cAMP in the cytosol.Of all different subtypes, mGluR1 is the most abundantly expressed mGluR in the mammalian central nervous system. In the brain, mGluR1 is highly expressed in the olfactory bulb, dentate gyrus, and cerebellum (Lein et al. 2007). The highest expression level of mGluR1 in the brain is found in Purkinje cells, the principal neurons of the cerebellar cortex (Shigemoto et al. 1992; Lein et al. 2007). Together with the AMPA receptors, mGluR1s are part of the excitatory synapses formed between parallel fibers and Purkinje cells (Fig. 1A). Each Purkinje cell is innervated by 100,000–200,000 parallel fibers (Ito 2006) that are axons of the cerebellar granule cells, the most abundant type of neuron in the brain. A second type of excitatory input to Purkinje cells is represented by the climbing fibers that originate in the inferior olive in the brain stem (Ito 2006). The two excitatory synaptic inputs to Purkinje cells are important determinants for the main functions of the cerebellum, including the real-time control of movement precision, error-correction, and control of posture as well as the procedural learning of complex movement sequences and conditioned responses.Open in a separate windowFigure 1.Parallel fiber-evoked mGluR1-dependent signals. (A) Diagram showing the parallel fiber synaptic input to Purkinje cell dendrites. (B) Microelectrode recording of glutamatergic postsynaptic potentials from a Purkinje cell in an acute slice of adult rat cerebellum. Short trains of stimuli to the parallel fibers (5–6 at 50 Hz) caused summation of the early AMPA receptor-dependent EPSPs (leading to spike firing) and a slow, delayed, depolarizing potential (slow EPSP), which was reversibly inhibited by antagonist of mGluRs (+)-MCPG (1mM). (C) Confocal image of a patch-clamped Purkinje cell in a cerebellar slice of an adult mouse. The patch-clamp pipette and the glass capillary used for electrical stimulation of parallel fibers are depicted schematically. The site of stimulation is shown at higher magnification in D. (D) Left: Parallel fiber-evoked (five pulses at 200 Hz, in 10 mM CNQX) synaptic responses consisting of a dendritic mGluR1-dependent Ca²⁺ transient (ΔF/F, top) and an early rapid and a slow excitatory postsynaptic current (EPSC, bottom). Block of the mGluR1-dependent components by the group I-specific mGluR-antagonist CPCCOEt (200 µM) is shown as indicated. Right: Pseudocolor image of the synaptic Ca²⁺ signal. (B, Reprinted with modifications, with permission, from Batchelor and Gaithwaite 1997 [Nature Publishing Group].)It is expected that mGluR1 is involved in many of these cerebellar functions. This view is supported by the observation that mGluR1-deficient knockout mice show severe impairments in motor coordination. In particular, the gait of these mice is strongly affected as well as their ability for motor learning and general coordination (Aiba et al. 1994). The phenotype of the general mGluR1-knockout mice is rescued by the insertion of the gene encoding mGluR1 exclusively into cerebellar Purkinje cells (Ichise et al. 2000) and blockade of mGluR1 expression only in Purkinje cells of adult mice leads to impaired motor coordination (Nakao et al. 2007). These findings established mGluR1 in Purkinje cell as synaptic receptors that are indispensable for a normal cerebellar function.Synaptic transmission involving mGluR1s is found at both parallel fiber-Purkinje cell synapses (Batchelor and Garthwaite 1993; Batchelor et al. 1994) as well as at climbing fiber-Purkinje cell synapses (Dzubay and Otis 2002). Most of our knowledge on the mGluR1 was gained from the analysis of the parallel fiber synapses. The parallel fiber synapse is quite unique in the central nervous system regarding its endowment with neurotransmitter receptors. In contrast to most other glutamatergic synapses in the mammalian brain, it lacks functional NMDA receptors (Shin and Linden 2005). The entire synaptic transmission at these synapses relies on AMPA receptors and on mGluR1 (Takechi et al. 1998). Although AMPA receptors are effectively activated even with single shock stimuli (Konnerth et al. 1990; Llano et al. 1991b), activation of mGluRs requires repetitive stimulation (Batchelor and Garthwaite 1993; Batchelor et al. 1994; Batchelor and Garthwaite 1997; Takechi et al. 1998). A possible explanation for the need of repetitive stimulation may relate to the observation that mGluR1s are found mostly at the periphery of the subsynaptic region (Nusser et al. 1994). At these sites outside the synaptic cleft, glutamate levels that are sufficiently high for receptor activation may be reached only with repetitive stimulation.At parallel fiber-Purkinje cell synapses, repetitive stimulation produces an initial AMPA receptor postsynaptic signal component, followed by a more prolonged mGluR1 component (Fig. 1). Figure 1B shows a current clamp recording of this response consisting of an early burst of action potentials, followed by a prolonged depolarization known as a “slow excitatory postsynaptic potential” (slow EPSP) (Batchelor and Garthwaite 1993; Batchelor et al. 1994; Batchelor and Garthwaite 1997). Voltage-clamp recordings allow a clear separation of the initial rapid, AMPA receptor mediated excitatory postsynaptic current (EPSC) and the mGluR1-mediated slow EPSC (Fig. 1D) (Takechi et al. 1998; Hartmann et al. 2008). In addition of inducing the slow EPSPs, mGluR1s mediate a large and highly localized dendritic calcium transient in cerebellar Purkinje cells (Fig. 1D) (Llano et al. 1991a; Finch and Augustine 1998; Takechi et al. 1998).     

Biology of the TAM Receptors

The TAM receptors—Tyro3, Axl, and Mer—comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands—Gas6 and Protein S—are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.The name of the TAM family is derived from the first letter of its three constituents—Tyro3, Axl, and Mer (Prasad et al. 2006). As detailed in Figure 1, members of this receptor tyrosine kinase (RTK) family were independently identified by several different groups and appear in the early literature under multiple alternative names. However, Tyro3, Axl, and Mer (officially c-Mer or MerTK for the protein, Mertk for the gene) have now been adopted as the NCBI designations. The TAMs were first grouped into a distinct RTK family (the Tyro3/7/12 cluster) in 1991, through PCR cloning of their kinase domains (Lai and Lemke 1991). The isolation of full-length cDNAs for Axl (O''Bryan et al. 1991), Mer (Graham et al. 1994), and Tyro3 (Lai et al. 1994) confirmed their segregation into a structurally distinctive family of orphan RTKs (Manning et al. 2002b). The two ligands that bind and activate the TAMs—Gas6 and Protein S (Pros1)—were identified shortly thereafter (Ohashi et al. 1995; Stitt et al. 1995; Mark et al. 1996; Nagata et al. 1996).Open in a separate windowFigure 1.TAM receptors and ligands. The TAM receptors (red) are Tyro3 (Lai and Lemke 1991; Lai et al. 1994)—also designated Brt (Fujimoto and Yamamoto 1994), Dtk (Crosier et al. 1994), Rse (Mark et al. 1994), Sky (Ohashi et al. 1994), and Tif (Dai et al. 1994); Axl (O''Bryan et al. 1991)—also designated Ark (Rescigno et al. 1991), Tyro7 (Lai and Lemke 1991), and Ufo (Janssen et al. 1991); and Mer (Graham et al. 1994)—also designated Eyk (Jia and Hanafusa 1994), Nyk (Ling and Kung 1995), and Tyro12 (Lai and Lemke 1991). The TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The TAM ligands (blue) are Gas6 and Protein S (Pros1). The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When γ-carboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. See text for details. (From Lemke and Burstyn-Cohen 2010; adapted, with permission, from the authors.)Subsequent progress on elucidating the biological roles of the TAM receptors was considerably slower and ultimately required the derivation of mouse loss-of-function mutants (Camenisch et al. 1999; Lu et al. 1999). The fact that Tyro3^−/−, Axl^−/−, and Mer^−/− mice are all viable and fertile permitted the generation of a complete TAM mutant series that included all possible double mutants and even triple mutants that lack all three receptors (Lu et al. 1999). Remarkably, these Tyro3^−/−Axl^−/−Mer^−/− triple knockouts (TAM TKOs) are viable, and for the first 2–3 wk after birth, superficially indistinguishable from their wild-type counterparts (Lu et al. 1999). Because many RTKs play essential roles in embryonic development, even single loss-of-function mutations in RTK genes often result in an embryonic-lethal phenotype (Gassmann et al. 1995; Lee et al. 1995; Soriano 1997; Arman et al. 1998). The postnatal viability of mice in which an entire RTK family is ablated completely—the TAM TKOs can survive for more than a year (Lu et al. 1999)—is therefore highly unusual. Their viability notwithstanding, the TAM mutants go on to develop a plethora of phenotypes, some of them debilitating (Camenisch et al. 1999; Lu et al. 1999; Lu and Lemke 2001; Scott et al. 2001; Duncan et al. 2003; Prasad et al. 2006). Almost without exception, these phenotypes are degenerative in nature and reflect the loss of TAM signaling activities in adult tissues that are subject to regular challenge, renewal, and remodeling. These activities are the subject of this review.     

Diversity of Neural Precursors in the Adult Mammalian Brain

Aided by advances in technology, recent studies of neural precursor identity and regulation have revealed various cell types as contributors to ongoing cell genesis in the adult mammalian brain. Here, we use stem-cell biology as a framework to highlight the diversity of adult neural precursor populations and emphasize their hierarchy, organization, and plasticity under physiological and pathological conditions.The adult mammalian brain displays remarkable structural plasticity by generating and incorporating new neural cell types into an already formed brain (Kempermann and Gage 1999). Largely restricted within the subventricular zone (SVZ) along the lateral ventricle and the subgranular zone (SGZ) in the dentate gyrus (DG), neural genesis is thought to arise from neural stem cells (NSCs) (Ming and Song 2011). Stem cells are defined by hallmark functions: capacity to self-renew, maintenance of an immature state over a long duration, and ability to generate specialized cell types (Fig. 1). These features distinguish stem cells from committed progenitor cells that more readily differentiate into specialized cell types (Fig. 1). Stem and progenitor cells (collectively called precursors) are additionally characterized by their lineage capacity. For example, multipotential neural precursors generate neurons and glia, whereas unipotential cells produce only one cell type, such as neurons (Gage 2000; Ma et al. 2009). The classical NSC definition is based on cell culture experiments in which a single cell can self-renew and generate neurons, astrocytes, and oligodendrocytes (Gage 2000; Ma et al. 2009). Yet, reprogramming studies have raised the question of whether cultured lineage-restricted neural progenitors acquire additional potential not evident in vivo (Palmer et al. 1999; Kondo and Raff 2000; Gabay et al. 2003). As a result, various lineage models have been proposed to explain cell generation in the adult brain (Fig. 1) (Ming and Song 2011). In one model, bona fide adult stem cells generate multiple lineages at the individual cell level. In another, cell genesis represents a collective property from a mixed population of unipotent progenitors. Importantly, these models are not mutually exclusive as evidence for the coexistence of multiple precursors has been observed in several adult somatic tissues, in which one population preferentially maintains homeostasis and another serves as a cellular reserve (Li and Clevers 2010; Mascre et al. 2012). Recent technical advances, including single-cell lineage tracing (Kretzschmar and Watt 2012), have made it possible to dissect basic cellular and behavioral processes of neural precursors in vivo (Fig. 4) (Bonaguidi et al. 2012). In this work, we review our current knowledge of precursor cell identity, hierarchical organization, and regulation to examine the diverse origins of cell genesis in the adult mammalian brain.Open in a separate windowFigure 1.Models of generating cell diversity in the adult tissues. (A,B) Definitions of stem and progenitor cells. In A, quiescent stem cells (S_q) become active stem cells (S_a) that proliferate to generate different types of specialized cells (C₁, C₂, C₃) and new stem cells (S). The active stem cell can return to quiescence and remain quiescent over long periods of time. In B, lineage-restricted progenitor cells lacking self-renewal capacity (P₁, P₂, P₃) each give rise to distinct populations of specialized cells (C₁, C₂, C₃). (C) Generation of specialized cells in a tissue could be explained by three models. (1) The stem-cell model, in which multipotent stem cells give rise to all the specialized cells in the tissue. (2) The progenitor cell model, in which diverse, lineage-restricted progenitor cells give rise to different cell types in the tissue. (3) A hybrid model, in which a mixture of stem cells and lineage-restricted progenitor cells generate specialized cells of the adult tissue.

Table 1.

Comparison of different methods used to study the generation of new cells in the adult mammalian nervous system

Toll-Like Receptor Signaling

Toll-like receptors sense pathogen-associated molecular patterns (e.g., lipopolysaccharides) and trigger gene-expression changes that ultimately eradicate the invading microbes.Toll-like receptors (TLRs) are protective immune sentries that sense pathogen-associated molecular patterns (PAMPs) such as unmethylated double-stranded DNA (CpG), single-stranded RNA (ssRNA), lipoproteins, lipopolysaccharide (LPS), and flagellin. In innate immune myeloid cells, TLRs induce the secretion of inflammatory cytokines (Newton and Dixit 2012), thereby engaging lymphocytes to mount an adaptive, antigen-specific immune response (see Fig. 1) that ultimately eradicates the invading microbes (Kawai and Akira 2010).Open in a separate windowFigure 1.TLR signaling (simplified view).Identification of TLR innate immune function began with the discovery that Drosophila mutants in the Toll gene are highly susceptible to fungal infection (Lemaitre et al. 1996). This was soon followed by identification of a human Toll homolog, now known as TLR4 (Medzhitov et al. 1997). To date, 10 TLR family members have been identified in humans, and at least 13 are present in mice. All TLRs consist of an amino-terminal domain, characterized by multiple leucine-rich repeats, and a carboxy-terminal TIR domain that interacts with TIR-containing adaptors. Nucleic acid–sensing TLRs (TLR3, TLR7, TLR8, and TLR9) are localized within endosomal compartments, whereas the other TLRs reside at the plasma membrane (Blasius and Beutler 2010; McGettrick and O’Neill 2010). Trafficking of most TLRs from the endoplasmic reticulum (ER) to either the plasma membrane or endolysosomes is orchestrated by ER-resident proteins such as UNC93B (for TLR3, TLR7, TLR8, and TLR9) and PRAT4A (for TLR1, TLR2, TLR4, TLR7, and TLR9) (Blasius and Beutler 2010). Once in the endolysosomes, TLR3, TLR7, and TLR9 are subject to stepwise proteolytic cleavage, which is required for ligand binding and signaling (Barton and Kagan 2009). For some TLRs, ligand binding is facilitated by coreceptors, including CD14 and MD2.Following ligand engagement, the cytoplasmic TIR domains of the TLRs recruit the signaling adaptors MyD88, TIRAP, TRAM, and/or TRIF (see Fig. 2). Depending on the nature of the adaptor that is used, various kinases (IRAK4, IRAK1, IRAK2, TBK1, and IKKε) and ubiquitin ligases (TRAF6 and pellino 1) are recruited and activated, culminating in the engagement of the NF-κB, type I interferon, p38 MAP kinase (MAPK), and JNK MAPK pathways (Kawai and Akira 2010; Morrison 2012). TRAF6 is modified by K63-linked autoubiquitylation, which enables the recruitment of IκB kinase (IKK) through a ubiquitin-binding domain of the IKKγ (also known as NEMO) subunit. In addition, a ubiquitin-binding domain of TAB2 recognizes ubiquitylated TRAF6, causing activation of the associated TAK1 kinase, which then phosphorylates the IKKβ subunit. Pellino 1 can modify IRAK1 with K63-linked ubiquitin, allowing IRAK1 to recruit IKK directly. TLR4 signaling via the TRIF adaptor protein leads to K63-linked polyubiquitylation of TRAF3, thereby promoting the type I interferon response via interferon regulatory factor (IRFs) (Hacker et al. 2011). Alternatively, TLR4 signaling via MyD88 leads to the activation of TRAF6, which modifies cIAP1 or cIAP2 with K63-linked polyubiquitin (Hacker et al. 2011). The cIAPs are thereby activated to modify TRAF3 with K48-linked polyubiquitin, causing its proteasomal degradation. This allows a TRAF6–TAK1 complex to activate the p38 MAPK pathway and promote inflammatory cytokine production (Hacker et al. 2011). TLR signaling is turned off by various negative regulators: IRAK-M and MyD88 short (MyD88s), which antagonize IRAK1 activation; FADD, which antagonizes MyD88 or IRAKs; SHP1 and SHP2, which dephosphorylate IRAK1 and TBK1, respectively; and A20, which deubiquitylates TRAF6 and IKK (Flannery and Bowie 2010; Kawai and Akira 2010).Open in a separate windowFigure 2.TLR signaling. (Adapted with kind permission of Cell Signaling Technology [http://www.cellsignal.com].)Deregulation of the TLR signaling cascade causes several human diseases. Patients with inherited deficiencies of MyD88, IRAK4, UNC93B1, or TLR3 are susceptible to recurrent bacterial or viral infections (Casanova et al. 2011). Chronic TLR7 and/or TLR9 activation in autoreactive B cells, in contrast, underlies systemic autoimmune diseases (Green and Marshak-Rothstein 2011). Furthermore, oncogenic activating mutations of MyD88 occur frequently in the activated B-cell-like subtype of diffuse large B-cell lymphoma and in other B-cell malignancies (Ngo et al. 2011). Inhibitors of various TLRs or their associated kinases are currently being developed for autoimmune or inflammatory diseases and also hold promise for the treatment of B-cell malignancies with oncogenic MyD88 mutations. Many TLR7 and TLR9 agonists are currently in clinical trials as adjuvants to boost host antitumor responses in cancer patients (Hennessy et al. 2010).     

The Role of Functional Prion-Like Proteins in the Persistence of Memory

Prions are a self-templating amyloidogenic state of normal cellular proteins, such as prion protein (PrP). They have been identified as the pathogenic agents, contributing to a number of diseases of the nervous system. However, the discovery that the neuronal RNA-binding protein, cytoplasmic polyadenylation element-binding protein (CPEB), has a prion-like state that is involved in the stabilization of memory raised the possibility that prion-like proteins can serve normal physiological functions in the nervous system. Here, we review recent experimental evidence of prion-like properties of neuronal CPEB in various organisms and propose a model of how the prion-like state may stabilize memory.Prions are proteinaceous infectious agents that were discovered in the 1980s by Stanley Prusiner while studying Creutzfeldt–Jakob disease (Prusiner 1982). Prusiner and colleagues showed them to be an amyloidogenic, self-perpetuating, forms of a normal cellular protein, termed prion protein or PrP. Prp in its self-perpetuating state kills cells. Prusiner and colleagues found that PrPs exist in at least two conformations: monomeric and aggregated (Fig. 1). The transition among these forms occurs spontaneously and only the aggregated conformation is pathogenic. Soon, PrPs were found to contribute to other neurodegenerative disorders in people, including kuru, transmissible spongiform encephalopathies, as well as bovine spongiform encephalopathy in cows (Prusiner 1994; Aguzzi and Weissmann 1998).Open in a separate windowFigure 1.Pathogenic prions exist in two states (soluble and aggregated and self-perpetuating). The conversion from the soluble to the aggregated form is spontaneous and the aggregated, self-perpetuating form is often toxic and kills the cell.There is now a growing consensus that similar prion-like, self-templating mechanisms underlie a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Polymenidou and Cleveland 2012).Not all prions, however, appear to be disease causing. Fungal prions, for instance, are nontoxic, and some may even be beneficial to the cells that harbor them (Wickner 1994; Shorter and Lindquist 2005; Crow and Li 2011). In 2003, Si and Kandel serendipitously discovered a prion-like protein in multicellular eukaryotes—the nervous system of the marine snail Aplysia—whose aggregated and self-perpetuating form contributes to the maintenance of long-term changes in synaptic efficacy. This functional prion-like protein differs from pathogenic prions in two important ways: (1) The conversion to the prion-like state is regulated by a physiological signal, and (2) the aggregated form has an identified physiological function (Fig. 2). Recent identification of new functional prion-like proteins in various organisms, including human, supports the idea that nonpathogenic prions may perform a wide range of biologically meaningful roles (Coustou et al. 1997; Eaglestone et al. 1999; True and Lindquist 2000; Ishimaru et al. 2003; True et al. 2004; Hou et al. 2011; Jarosz et al. 2014).Open in a separate windowFigure 2.“Functional” prion: memory. “Functional” prions differ from conventional prions in two ways. First, the conversion is triggered by a physiological signal, and second, the aggregated, self-perpetrating forms have a physiological function. 5-HT, Serotonin; DA, dopamine.In this review, we focus on functional prion-like proteins in the brain and specifically on the prion-like properties of the cytoplasmic polyadenylation element-binding protein (CPEB), and examine how the prion-like state can control protein synthesis at the synapse and, thereby, synaptic plasticity and long-lasting memory. We anticipate the studies of CPEB would also provide some generalizable concepts as to how prion-based protein switches in multicellular eukaryotes may work.     

Gap Junctions

Gap junctions are aggregates of intercellular channels that permit direct cell–cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hexamers of medium-sized families of integral proteins: connexins in chordates and innexins in precordates. The functions of gap junctions have been explored by studying mutations in flies, worms, and humans, and targeted gene disruption in mice. These studies have revealed a wide diversity of function in tissue and organ biology.Gap junctions are clusters of intercellular channels that allow direct diffusion of ions and small molecules between adjacent cells. The intercellular channels are formed by head-to-head docking of hexameric assemblies (connexons) of tetraspan integral membrane proteins, the connexins (Cx) (Goodenough et al. 1996). These channels cluster into polymorphic maculae or plaques containing a few to thousands of units (Fig. 1). The close membrane apposition required to allow the docking between connexons sterically excludes most other membrane proteins, leaving a narrow ∼2 nm extracellular “gap” for which the junction is named (Fig. 2). Gap junctions in prechordates are composed of innexins (Phelan et al. 1998; Phelan 2005). In chordates, connexins arose by convergent evolution (Alexopoulos et al. 2004), to expand by gene duplication (Cruciani and Mikalsen 2007) into a 21-member gene family. Three innexin-related proteins, called pannexins, have persisted in vertebrates, although it is not clear if they form intercellular channels (Panchin et al. 2000; Bruzzone et al. 2003). 7Å-resolution electron crystallographic structures of intercellular channels composed of either a carboxy-terminal truncation of Cx43 (Unger et al. 1999; Yeager and Harris 2007) or an M34A mutant of Cx26 (Oshima et al. 2007) are available. The overall pore morphologies are similar with the exception of a “plug” in the Cx26 channel pore. The density of this plug is substantively decreased by deletion of amino acids 2–7, suggesting that the amino-terminus contributes to this structure (Oshima et al. 2008). A 3.5-Å X-ray crystallographic structure has visualized the amino-terminus of Cx26 folded into the mouth of the channel without forming a plug, thought to be an image of the open channel conformation (Maeda et al. 2009). The amino-terminus has been physiologically implicated in voltage-gating of the Cx26 and Cx32 channels (Purnick et al. 2000; Oh et al. 2004), lending support to a role for the amino-terminus as a gating structure. However, Cx43 also shows voltage-gating, and its lack of any structure resembling a plug remains unresolved. A comparison of a 1985 intercellular channel structure (Makowski 1985) with the 2009 3.5Å structure (Maeda et al. 2009) summarizes a quarter-century of X-ray progress (Fig. 3).Open in a separate windowFigure 1.A diagram showing the multiple levels of gap junction structure. Individual connexins assemble intracellularly into hexamers, called connexons, which then traffic to the cell surface. There, they dock with connexons in an adjacent cell, assembling an axial channel spanning two plasma membranes and a narrow extracellular “gap.”Open in a separate windowFigure 2.Electron microscopy of gap junctions joining adjacent hepatocytes in the mouse. The gap junction (GJ) is seen as an area of close plasma membrane apposition, clearly distinct from the tight junction (TJ) joining these cells. (Inset A) A high magnification view of the gap junction revealing the 2–3 nm “gap” (white arrows) separating the plasma membranes. (Inset B) A freeze-fracture replica of a gap junction showing the characteristic particles on the protoplasmic (P) fracture face and pits on the ectoplasmic (E) fracture face. The particles and pits show considerable disorder in their packing with an average 9-nm center-to-center spacing.Open in a separate windowFigure 3.A comparison of axial sections through gap-junction structures deduced from X-ray diffraction. The 1985 data (Makowski 1985) were acquired from gap junctions isolated biochemically from mouse liver containing mixtures of Cx32 and Cx26. The intercellular channel (CHANNEL) is blocked at the two cytoplasmic surfaces by electron density at the channel mouths along the sixfold symmetry axis. The 2009 data (Maeda et al. 2009), acquired from three-dimensional crystals of recombinant Cx26, resolve this density at the channel opening as the amino-termini of the connexin proteins, the 2009 model possibly showing an open channel structure.Most cells express multiple connexins. These may co-oligomerize into the same (homomeric) or mixed (heteromeric) connexons, although only certain combinations are permitted (Falk et al. 1997; Segretain and Falk 2004). A connexon may dock with an identical connexon to form a homotypic intercellular channel or with a connexon containing different connexins to form a heterotypic channel (Dedek et al. 2006). Although only some assembly combinations are permitted (White et al. 1994), the number of possible different intercellular channels formed by this 21-member family is astonishingly large. This diversity has significance because intercellular channels composed of different connexins have different physiological properties, including single-channel conductances and multiple conductance states (Takens-Kwak and Jongsma 1992), as well as permeabilities to experimental tracers (Elfgang et al. 1995) and to biologically relevant permeants (Gaunt and Subak-Sharpe 1979; Veenstra et al. 1995; Bevans et al. 1998; Gong and Nicholson 2001; Goldberg et al. 2002; Ayad et al. 2006; Harris 2007).Opening of extrajunctional connexons in the plasma membrane, described as “hemichannel” activity, can be experimentally induced in a variety of cell types. Because first observations of hemichannel activity were in an oocyte expression system (Paul et al. 1991) and dissociated retinal horizontal cells (DeVries and Schwartz 1992), the possible functions of hemichannels composed of connexins and pannexins has enjoyed vigorous investigation (Goodenough and Paul 2003; Bennett et al. 2003; Locovei et al. 2006; Evans et al. 2006; Srinivas et al. 2007; Schenk et al. 2008; Thompson and MacVicar 2008; Anselmi et al. 2008; Goodenough and Paul 2003). Hemichannels have been implicated in various forms of paracrine signaling, for example in providing a pathway for extracellular release of ATP (Cotrina et al. 1998; Kang et al. 2008), glutamate (Ye et al. 2003), NAD⁺ (Bruzzone et al. 2000), and prostaglandins (Jiang and Cherian 2003).     

DNA Replication Origins

The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.Timely duplication of the genome is an essential step in the reproduction of any cell, and it is not surprising that chromosomal DNA synthesis is tightly regulated by mechanisms that determine precisely where and when new replication forks are assembled. The first model for a DNA synthesis regulatory circuit was described about 50 years ago (Jacob et al. 1963), based on the idea that an early, key step in building new replication forks was the binding of a chromosomally encoded initiator protein to specialized DNA regions, termed replication origins (Fig. 1). The number of replication origins in a genome is, for the most part, dependent on chromosome size. Bacterial and archaeal genomes, which usually consist of a small circular chromosome, frequently have a single replication origin (Barry and Bell 2006; Gao and Zhang 2007). In contrast, eukaryotic genomes contain significantly more origins, ranging from 400 in yeast to 30,000–50,000 in humans (Cvetic and Walter 2005; Méchali 2010), because timely duplication of their larger linear chromosomes requires establishment of replication forks at multiple locations. The interaction of origin DNA and initiator proteins (Fig. 1) ultimately results in the assembly of prereplicative complexes (pre-RCs), whose role is to load and activate the DNA helicases necessary to unwind DNA before replication (Remus and Diffley 2009; Kawakami and Katayama 2010). Following helicase-catalyzed DNA unwinding, replisomal proteins become associated with the single-stranded DNA, and new replication forks proceed bidirectionally along the genome until every region is duplicated (for review, see O’Donnell 2006; Masai et al. 2010).Open in a separate windowFigure 1.Revised versions of the replicon model for all domains of life. For cells of each domain type, trans-acting initiators recognize replication origins to assemble prereplicative complexes required to unwind the DNA and load DNA helicase. Eukaryotic initiators are preassembled into hexameric origin recognition complexes (ORCs) before interacting with DNA. In prokaryotes, single initiators (archaeal Orc1/Cdc6 or bacterial DnaA) bind to recognition sites and assemble into complexes on DNA. In all cases, the DNA helicases (MCMs or DnaB) are recruited to the origin and loaded onto single DNA strands. In bacteria, DNA-bending proteins, such as Fis or IHF, may modulate the assembly of pre-RC by bending the origin DNA. Two activities of DnaA are described in the figure. The larger version binds to recognition sites, and the smaller version represents DnaA required to assist DnaC in loading DnaB helicase on single-stranded DNA.Initiator proteins from all forms of life share structural similarities, including membership in the AAA⁺ family of proteins (ATPases associated with various cellular activities) (Duderstadt and Berger 2008; Wigley 2009) that are activated by ATP binding and inactivated by ATP hydrolysis (Duderstadt and Berger 2008; Duncker et al. 2009; Kawakami and Katayama 2010). Despite these similarities, initiators assemble into prereplicative complexes in two fundamentally different ways (Fig. 2). In prokaryotes, initiator monomers interact with the origin at multiple repeated DNA sequence motifs, and the arrangement of these motifs (see below) can direct assembly of oligomers that mediate strand separation (Erzberger et al. 2006; Rozgaja et al. 2011). In eukaryotes, a hexameric origin recognition complex (ORC) binds to replication origins and then recruit additional factors (as Cdc6 and Cdt1) that will themselves recruit the hexameric MCM2-7 DNA helicase to form a prereplicative complex (for review, see Diffley 2011). This process occurs during mitosis and along G₁ and is called “DNA replication licensing,” a crucial regulation of eukaryotic DNA replication (for review, see Blow and Gillespie 2008). Importantly, this complex is still inactive, and only a subset of these preassembled origins will be activated in S phase. This process is, therefore, fundamentally different from initiation of replication in bacteria. Moreover, because sequence specificity appears more relaxed in large eukaryotic genomes, prokaryotic mechanisms that regulate initiator–DNA site occupation must be replaced by alternative mechanisms, such as structural elements or the use of epigenetic factors.Open in a separate windowFigure 2.Functional elements in some well-studied prokaryotic replication origins. (A) Bacterial oriCs. The DNA elements described in the text are (arrows) DnaA recognition boxes or (boxes) DNA unwinding elements (DUEs). When recognition site affinities are known, colored arrows designate high- (K_d > 100 nm) and low- (K_d < 100 nm) affinity sites. (B) Archaeal oriCs. Arrows and boxes designate DNA elements as in A, but the initiator protein is Orc1/Cdc6 rather than DnaA. (Thick arrows) Long origin recognition boxes (ORBs); (thin arrows) shorter versions (miniORBs). Both ORBs and miniORBs are identified in Pyrococcus. DUEs are not yet well defined for Helicobacter or Sulfolobus genera and are not labeled in this figure.Here, we describe replication origins on prokaryotic and eukaryotic genomes below, with a particular focus on the attributes responsible for orderly initiator interactions and origin selection specificity, as well as on the shift from origin sequence-dependent regulation to epigenetic regulation. You are also referred to other related articles in this collection and several recent reviews covering the topics of DNA replication initiation in more detail (Méchali 2010; Beattie and Bell 2011; Blow et al. 2011; Bryant and Aves 2011; Ding and MacAlpine 2011; Dorn and Cook 2011; Kaguni 2011; Leonard and Grimwade 2011; Sequeira-Mendes and Gomez 2012).     

Wnt Signaling in Vertebrate Axis Specification

The Wnt pathway is a major embryonic signaling pathway that controls cell proliferation, cell fate, and body-axis determination in vertebrate embryos. Soon after egg fertilization, Wnt pathway components play a role in microtubule-dependent dorsoventral axis specification. Later in embryogenesis, another conserved function of the pathway is to specify the anteroposterior axis. The dual role of Wnt signaling in Xenopus and zebrafish embryos is regulated at different developmental stages by distinct sets of Wnt target genes. This review highlights recent progress in the discrimination of different signaling branches and the identification of specific pathway targets during vertebrate axial development.Wnt pathways play major roles in cell-fate specification, proliferation and differentiation, cell polarity, and morphogenesis (Clevers 2006; van Amerongen and Nusse 2009). Signaling is initiated in the responding cell by the interaction of Wnt ligands with different receptors and coreceptors, including Frizzled, LRP5/6, ROR1/2, RYK, PTK7, and proteoglycans (Angers and Moon 2009; Kikuchi et al. 2009; MacDonald et al. 2009). Receptor activation is accompanied by the phosphorylation of Dishev-elled (Yanagawa et al. 1995), which appears to transduce the signal to both the cell membrane and the nucleus (Cliffe et al. 2003; Itoh et al. 2005; Bilic et al. 2007). Another common pathway component is β-catenin, an abundant component of adherens junctions (Nelson and Nusse 2004; Grigoryan et al. 2008). In response to signaling, β-catenin associates with T-cell factors (TCFs) and translocates to the nucleus to stimulate Wnt target gene expression (Behrens et al. 1996; Huber et al. 1996; Molenaar et al. 1996).This β-catenin-dependent activation of specific genes is often referred to as the “canonical” pathway. In the absence of Wnt signaling, β-catenin is destroyed by the protein complex that includes Axin, GSK3, and the tumor suppressor APC (Clevers 2006; MacDonald et al. 2009). Wnt proteins, such as Wnt1, Wnt3, and Wnt8, stimulate Frizzled and LRP5/6 receptors to inactivate this β-catenin destruction complex, and, at the same time, trigger the phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2 (HIPK2) (Hikasa et al. 2010; Hikasa and Sokol 2011). Both β-catenin stabilization and the regulation of TCF protein function by phosphorylation appear to represent general strategies that are conserved in multiple systems (Sokol 2011). Thus, the signaling pathway consists of two branches that together regulate target gene expression (Fig. 1).Open in a separate windowFigure 1.Conserved Wnt pathway branches and components. In the absence of Wnt signals, glycogen synthase kinase 3 (GSK3) binds Axin and APC to form the β-catenin destruction complex. Some Wnt proteins, such as Wnt8 and Wnt3a, stimulate Frizzled and LRP5/6 receptors to inhibit GSK3 activity and stabilize β-catenin (β-cat). Stabilized β-cat forms a complex with T-cell factors (e.g., TCF1/LEF1) to activate target genes. Moreover, GSK3 inhibition leads to target gene derepression by promoting TCF3 phosphorylation by homeodomain-interacting protein kinase 2 (HIPK2) through an unknown mechanism, for which β-catenin is required as a scaffold. This phosphorylation results in TCF3 removal from target promoters and gene activation. Other Wnt proteins, such as Wnt5a and Wnt11, use distinct receptors such as ROR2 and RYK, in addition to Frizzled, to control the the cytoskeletal organization through core planar cell polarity (PCP) proteins, small GTPases (Rho/Rac/Cdc42), and c-Jun amino-terminal kinase (JNK).Other Wnt proteins, such as Wnt5a or Wnt11, strongly affect the cytoskeletal organization and morphogenesis without stabilizing β-catenin (Torres et al. 1996; Angers and Moon 2009; Wu and Mlodzik 2009). These “noncanonical” ligands do not influence TCF3 phosphorylation (Hikasa and Sokol 2011), but may use distinct receptors such as ROR1/2 and RYK instead of or in addition to Frizzled (Hikasa et al. 2002; Lu et al. 2004; Mikels and Nusse 2006; Nishita et al. 2006, 2010; Schambony and Wedlich 2007; Grumolato et al. 2010; Lin et al. 2010; Gao et al. 2011). In such cases, signaling mechanisms are likely to include planar cell polarity (PCP) components, such as Vangl2, Flamingo, Prickle, Diversin, Rho GTPases, and c-Jun amino-terminal kinases (JNKs), which do not directly affect β-catenin stability (Fig. 1) (Sokol 2000; Schwarz-Romond et al. 2002; Schambony and Wedlich 2007; Komiya and Habas 2008; Axelrod 2009; Itoh et al. 2009; Tada and Kai 2009; Sato et al. 2010; Gao et al. 2011). This simplistic dichotomy of the Wnt pathway does not preclude some Wnt ligands from using both β-catenin-dependent and -independent routes in a context-specific manner.Despite the existence of many pathway branches, only the β-catenin-dependent branch has been implicated in body-axis specification. Recent experiments in lower vertebrates have identified additional pathway components and targets and provided new insights into the underlying mechanisms.     

Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity.

The evolution of modern cells is arguably the most challenging and important problem the field of biology has ever faced …—Carl R. Woese(Woese 2002)

Current models may accept that all modern eukaryotic cells arose from a single common ancestor (the cenancestral eukaryote), the nature of which is—owing to the lack of direct living or fossil descendants—still highly under debate (de Duve 2007). The chimeric nature of eukaryotic genomes with eubacterial and archaebacterial shares led to a discussion about the origin of this first “proto-eukaryote.” Several models exist (see Fig. 1), which either place the evolution of the nucleus before or after the emergence of the mitochondrion (outlined in Koonin 2010; Martijn and Ettema 2013). According to the different postulated scenarios (summarized in Embley and Martin 2006), eukaryotes in the latter case might have evolved by endosymbiosis between a hydrogen-producing, oxygen-producing, or sulfur-dependent α-proteobacterium and an archaebacterial host (Fig. 1C). The resulting mitochondriate prokaryote would have evolved the nucleus subsequently. In other scenarios (Fig. 1B), the cenancestral eukaryote emerged by cellular fusion or endosymbiosis of a Gram-negative, maybe hydrogen-producing, eubacterium and a methanogenic archaebacterium or eocyte, leading to a primitive but nucleated amitochondrial (archezoan) cell (Embley and Martin 2006, and references therein). As a third alternative, Cavalier-Smith (2002) suggested a common eubacterial ancestor for eukaryotes and archaebacteria (the Neomuran hypothesis) (Fig. 1A).Open in a separate windowFigure 1.Evolution of the last common ancestor of all eukaryotic cells. A schematic depiction of the early eukaryogenesis. Because of the lack of living and fossil descendants, several opposing models are discussed (A–C). The anticipated order of events is shown as a flow chart. For details, see text. (Derived from Embley and Martin 2006; Koonin 2010.)     

Neurogenesis in the Adult Avian Song-Control System

New neurons are added throughout the forebrain of adult birds. The song-control system is a model to investigate the addition of new long-projection neurons to a cortical circuit that regulates song, a learned sensorimotor behavior. Neuroblasts destined for the song nucleus HVC arise in the walls of the lateral ventricle, and wander through the pallium to reach HVC. The survival of new HVC neurons is supported by gonadally secreted testosterone and its downstream effectors including neurotrophins, vascularization, and electrical activity of postsynaptic neurons in nucleus RA (robust nucleus of the arcopallium). In seasonal species, the HVC→RA circuit degenerates in nonbreeding birds, and is reconstructed by the incorporation of new projection neurons in breeding birds. There is a functional linkage between the death of mature HVC neurons and the birth of new neurons. Various hypotheses for the function of adult neurogenesis in the song system can be proposed, but this remains an open question.Song behavior in oscine birds is regulated by a network of pallial and striatal nuclei. The song-control system shows extensive plasticity in adults, including ongoing neurogenesis in several nuclei (Brenowitz 2008). The addition of new neurons to the adult brain of higher vertebrates was first suggested by the pioneering studies of Altman and Das (1965) and Kaplan and Hinds (1977). They reported that labeled cells were present in the dentate gyrus (DG) of rats following the injection of ³H-thymidine. Their claims, however, met with skepticism and the neuronal identity of the new cells that they observed was called into question (Gross 2000). In an influential study, Rakic (1985) injected adult rhesus monkeys with ³H-thymidine and reported that, “all neurons of the rhesus monkey brain are generated during prenatal and early postnatal life.” The study of neuronal addition to the adult brain, was subsequently dropped for ∼20 years in the face of the dogma that neurogenesis was largely completed by birth (Gross 2000). This prevailing view only started to be overturned when Nottebohm and colleagues published a series of studies showing that new cells are added to the cortical-like song nucleus HVC (Fig. 1) of adult canaries (Serinus canarius) (Goldman and Nottebohm 1983). These new cells have neuronal morphology, some of these cells fire action potentials in response to sound (Paton and Nottebohm 1984), receive synaptic input (Burd and Nottebohm 1985), may synapse on neurons in the efferent robust nucleus of the arcopallium (RA) (Alvarez-Buylla et al. 1990), and express neuron-specific proteins (Barami et al. 1995). Together, these studies in songbirds showed that new neurons are born and incorporated into functional circuits in the brains of adults of higher vertebrates (Nottebohm 2004). This research on adult neurogenesis in songbirds stimulated investigators to re-examine this topic in mammals. It soon became clear that new neurons are added throughout life to the DG and olfactory bulb of mammals including humans (Cameron and Gould 1994; Gould et al. 1997, 1999a; Lim et al. 1997; Eriksson et al. 1998). Because of these initial confirmatory reports, there has been explosive growth in study of the mechanisms and functions of adult neurogenesis in the mammalian DG and olfactory bulb.Open in a separate windowFigure 1.A schematic of the neurogenic regions in the avian brain overlaid on the avian song circuits. Neurogenic regions are shown in red. Note the proximity of HVC (and hippocampus [HC]) to the ventricular zone (VZ). A schematic version of the motor pathway for song production is shown in blue. A schematic of the ascending auditory pathway is shown in green. The dotted line indicates an indirect route through many nuclei of the ascending auditory pathway leading to field L in the telencephalon. The anterior forebrain circuit for song learning and plasticity is shown in yellow. NCM, Caudomedial nidopallium; RA, arcopallium; LMAN, lateral magnocellular nucleus of the anterior neostriatum; OB, olfactory bulb; DLM, dorsolateral medial; PAm, parambigualis; RAm, retroambigualisBirds continue to be a productive model for the study of neurogenesis in the adult brain, as discussed below. In this article, we will focus on neurogenesis in the song-control system as this is the most intensively studied model in birds. (For a review of neurogenesis in the avian hippocampus [HC], see Barnea and Pravosudov 2011.) We will discuss the mechanisms of neurogenesis in the song system, intrinsic and extrinsic factors that influence neuronal addition, a linkage between cell death and neurogenesis, seasonal plasticity, and consider potential functions of adult neurogenesis.