TGF-β Family Signaling in Neural and Neuronal Differentiation, Development, and Function

Abstract

The transforming growth factor β (TGF-β) family plays a central role in multiple aspects of nervous system development and function. During embryogenesis, TGF-β family members regulate the initial formation of the nervous system, dorsalization and establishment of rostrocaudal boundaries, patterning of the central nervous system (CNS), stem-cell lineage commitment to neurons and glia, cell migration and axon guidance, synaptogenesis, and cell survival (see Table 1). During adulthood, TGF-β family members modulate inflammatory responses, influence quiescence in neural stem cells of the hippocampus, and have a role in neurodegenerative disease. This review will cover the expression, regulation and function of TGF-β family members in the CNS from early development to adult life.

NEURAL INDUCTION

Formation of the neural plate, the first step in the genesis of the nervous system, is triggered during gastrulation by signals produced by an organizer region on the dorsal blastophore lip (Fig. 1) (Spemann and Mangold 1924). These signals act primarily by inhibiting signaling by TGF-β family members including bone morphogenetic proteins (BMPs), nodal, and activin (Hemmati-Brivanlou and Melton 1992, 1994). Noggin, follistatin, and chordin, the first neural inducers that were identified (Hemmati-Brivanlou et al. 1994; Sasai et al. 1994, 1995), act by suppressing BMP signaling (Piccolo et al. 1996; Zimmerman et al. 1996; Fainsod et al. 1997). Additional BMP antagonists secreted from the organizer include Cerberus, Gremlin, Dan, Drm, Coco, and Ogon/Sizzled (sFRP) (Wagner and Mullins 2002; Yabe et al. 2003).

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Figure 1.

Role of the transforming growth factor-β (TGF-β) family in neural induction. During late gastrulation, the neural tube is specified within ectoderm at the expense of epidermis. Bone morphogenetic proteins (BMPs) and nodal antagonists are secreted by the dorsal mesoderm (organizer) to allow for neural differentiation. BMP, nodal, and their cofactors are secreted from the ventral center to specify prospective epidermis.

Targeted inactivation of any one neural inducer does not prevent formation of the CNS. For example, most mice have a normal CNS after inactivation of the Chordin gene (Bachiller et al. 2000; Anderson et al. 2002), and Noggin^−/− mice also have normal gastrulation and neural plate formation (McMahon et al. 1998). However, Chordin^−/−; Noggin^−/− double-mutant mouse embryos have a severe neural phenotype with a loss of the prosencephalic vesicle and lack of anterior notocord, because of ventralization of the mesoderm (Bachiller et al. 2000). Similarly, antisense oligonucleotides targeting Noggin, Chordin, and Follistatin cause nearly complete loss of the neural plate in Xenopus tropicalis embryos (Khokha et al. 2005). Thus, chordin, noggin and follistatin have redundant functions in the BMP inhibition required for neural formation and patterning of the embryonic axis.

Nodal antagonists secreted from the organizer are also required to induce neural development. Although gastrulation is normal after targeted inactivation of expression of either of the nodal antagonists, Cerberus-like gene or Lefty1, Cerberus^−/−; Lefty1^−/−double-mutant mice have severe developmental impairment of the anterior embryo (Perea-Gomez et al. 2002; Yamamoto et al. 2004). In Xenopus laevis, the asymmetry of nodal signaling is partially regulated by two microRNAs (miRNAs), miR-15 and miR-16. These miRNAs are enriched at the ventral side of the embryo and attenuate the nodal signaling gradient by targeting the nodal receptor activin receptor type 2A (ACVR2A, also known as ActRII) (Martello et al. 2007). Zebrafish use a different miRNA, miR-430, to target nodal signaling (Choi et al. 2007). Taken together, these studies identify a fundamental role for BMP and nodal signaling in neural induction and indicate that these families of antagonists act in a redundant or compensatory manner (Piccolo et al. 1999; Niehrs 2001; Wu and Hill 2009).

Epidermal fate is determined by the expression of the BMP-related Decapentaplegic (Dpp) in Drosophila and BMP-4 in Xenopus explants (Wilson and Hemmati-Brivanlou 1995). Numerous experimental methods have been used to impair BMP signaling, promoting neural induction of the epidermis (Hemmati-Brivanlou and Melton 1994; Hawley et al. 1995; Sasai et al. 1995; Wilson and Hemmati-Brivanlou 1995; Xu et al. 1995). Targeted silencing experiments for BMP-2, BMP-4, and BMP-7 in Xenopus indicate a redundancy in neural tissue specification; however, BMP-4 inhibition has the strongest dorsalized phenotype and is sufficient for neuralization (Reversade et al. 2005). Inactivation of BMP-2, BMP-4, and BMP-7 expression combined with inactivation of the expression of the BMP family member antidorsalizing morphogenetic protein (ADMP), which is expressed in the dorsal gastrula organizing center (Moos et al. 1995), causes complete neuralization of the ectoderm in Xenopus experiments. Restoring signaling by BMP-2, -4, -7 and/or ADMP in embryos with silenced BMP-2, BMP-4, and BMP-7 expression allows for an impressive return of epidermal patterning (Reversade and De Robertis 2005). Further, ADMP competes with nodal for the ACVR2A receptor, which may be a method for self-regulation in the organizer (Inui et al. 2012).

Manipulation of BMP signal transducers and inhibitors of downstream signaling further validates their roles in regulation of epidermal formation. Expression of Smad1 and Smad5, along with the BMP target gene Msx1 prevents neuralization in Xenopus animal caps (Suzuki et al. 1997a,b; Wilson et al. 1997). Interfering with inhibitors of BMP signaling, for example, silencing the expression of the BMP decoy receptor BAMBI, the Smad ubiquitin ligase Smurf1, or the Smad4 E3 ubiquitin ligase ectodermin (also known as TIF1-γ or TRIM33), impair neural induction (Onichtchouk et al. 1999; Zhu et al. 1999b; Dupont et al. 2005). Additionally, BMPs and their antagonists are regulated by extracellular metalloproteinases; specifically Twisted gastrulation (Tsg) binds both BMPs and chordin to modulate BMP signaling toward or against neural induction (Oelgeschläger et al. 2000, 2003; Larraín et al. 2001). This regulation is further complicated by expression of the Tolloid metalloproteinase that cleaves chordin, and Sizzled (sFRP) metalloproteinase, which inhibits Tolloid proteases (Oelgeschläger et al. 2003; Lee et al. 2006; Zakin and De Robertis 2010). Thus, inhibition of BMP signaling during neural induction is tightly regulated at many levels by BMP ligands, signal transducers, target genes, and extracellular metalloproteinases.

Wnt/β-catenin signaling regulates the BMP pathway during early induction by inducing the expression of noggin and chordin in the dorsal organizer region. Activation of β-catenin signaling reduces BMP-4 expression leading to neurulation, which can be inhibited by a constitutively active BMPRIA (BMP type IA receptor, also known as ALK-3), linking both Wnt/β-catenin and BMP signaling (Baker et al. 1999; Kuroda et al. 2004). BMP and Wnt signaling converge later in development when both signaling pathways are integrated through Smad1 phosphorylation of glycogen synthase kinase 3 (GSK3) in gastrulation (Fuentealba et al. 2007). In zebrafish, β-catenin induces a homeobox gene bozozok/dharma that directly represses the expression of BMP2b, which encodes BMP-4 (Leung et al. 2003). Bozozok^−/−; Chordin^−/− zebrafish mutants show synergistic losses of neural tissue and dorsal structures (Gonzalez et al. 2000). Taken together, Wnt/β-catenin signaling acts to inhibit BMP transcripts and increase BMP antagonists to block their function and promote neural induction.

Additional signals regulate BMP activity to influence proper neural induction in the embryo (Lamb and Harland 1995; Fürthauer et al. 1997; Kretzschmar et al. 1997; Wilson et al. 2000; Pera et al. 2001). In particular, fibroblast growth factor (FGF) signaling down-regulates the expression of BMP-4 and BMP-7, and FGF together with insulin-like growth factor (IGF) activate the mitogen-activating protein (MAP) kinase cascade that regulates BMP signaling (Ishimura et al. 2000; Wilson et al. 2000). FGF and IGF obstruct BMP signal transduction by phosphorylating the linker region of Smad1 that blocks translocation of Smad to the nucleus (Pera et al. 2003; Sapkota et al. 2007). The relationship between BMP and FGF is necessary for proper neural development (Khokha et al. 2005); however, FGF signaling has additional roles in development of the embryo that are independent of BMP activity (Linker and Stern 2004; Delaune et al. 2005; Dorey and Amaya 2010).

In conclusion, loss- and gain-of-function experiments in multiple species show that antagonism of BMP signaling is required for proper induction of the nervous system. The importance of this signaling is further represented by the redundancy of BMP ligands and the need of double or triple knockdowns to fully elucidate the role of the TGF-β family.

NEURAL PATTERNING

Following neural induction, organizing centers are responsible for patterning the complex structure of the nervous system (Fig. 2). The roof plate in the dorsal midline and the floor plate and notochord in the ventral midline establish dorsoventral identities. Proper development is induced by the TGF-β family members nodal and BMP, whereas nodal signaling and BMP inhibition form the floor plate and specify ventral patterning. BMP signaling is instrumental in inducing the roof plate and specifying dorsal patterning (Placzek and Briscoe 2005; Lupo et al. 2006; Kiecker and Lumsden 2012).

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Figure 2.

Role of the transforming growth factor-β (TGF-β) family in forming the neural tube and patterning the spinal cord. (A) Antagonism from the notochord inhibits bone morphogenetic proteins (BMPs) signaling to specify the floor plate (FP) (green), whereas the neural plate folds to form the neural tube. The FP and roof plate (RP) (red) form on the ventral and dorsal midline of the neural tube, respectively. Wnt and fibroblast growth factor (FGF) help to regulate BMP signaling to aid in neurulation, and BMP signaling through Lmx1a and Msx forms the RP. (B) BMPs, GDF-5, GDF-6, GDF-7, and Wnts are secreted from the RP to specify neural crest and dorsal interneuron (dI) subtype. Nodal and BMP antagonists are secreted from notochord to specify ventral neuron (vP) subtype. Nodal instructs ventral identity either in parallel or through sonic hedgehog (Shh) signaling.

Nodal signaling is crucial for proper formation and differentiation of the floor plate cells, as impairment of nodal signaling in the zebrafish mutant Cyclops and inhibition of nodal receptors yield severely depleted or absent floor plate cells (Hatta et al. 1991; Feldman et al. 1998; Rebagliati et al. 1998; Dougan et al. 2003). Malformation of the floor plate in the Cyclops mutant could be because of the effects on prechordal mesoderm differentiation that lead to a reduction in other signals, like sonic hedgehog (Shh). In Nodal^−/− mutants, the floor plate is prematurely induced in the anterior epiblast in mouse embryos (Camus et al. 2006), whereas inactivation of the gene encoding the nodal antagonist Lefty increases induction of mesendoderm in formerly ectodermal tissue (Meno et al. 1999; Perea-Gomez et al. 2002). Nodal-deficient mutants show late recovery of midline floor plate cells in the trunk and tail, identifying signaling pathways that ameliorate these effects, specifically Shh and Gli activity (Albert et al. 2003; Norton et al. 2005). This late recovery, however, is not observed in the brain. Variable cycloptic phenotypes are observed in Nodal^−/+ mice, as well as heterozygous or homozygous mutants that inactivate downstream nodal signaling, including Smad2 or Acvr2a/Actr2 (Nomura and Li 1998; Song et al. 1999). Shh is required for floor plate development in many species (Chiang et al. 1996; Wijgerde et al. 2002), and nodal potentiates the ability of Shh to induce floor plate identity in addition to inducing the expression of ventral genes encoding Shh and the forkhead box A2 (FOXA2) transcription factor (Müller et al. 1999, 2000; Rastegar et al. 2002; Patten et al. 2003). Taken together, the role of nodal in ventral patterning may be to (1) sensitize cells to the action of Shh or to enhance Shh signaling (Chen and Schier 2001; Norton et al. 2005), (2) operate in parallel with Shh to trigger the expression of floor-plate-specific genes, or (3) foster expression of floor-plate-specific genes, including Shh, that require Shh for their maintenance (Norton et al. 2005).

In addition to nodal activation, inhibition of BMP signaling is required for proper floor plate induction. Noggin, chordin, and follistatin are expressed in the notochord, prechordal mesoderm, and floor plate that extends from the presumptive anterior diencephalon into the hindbrain (Furuta et al. 1997; Dale et al. 1999; Chapman et al. 2002). Impairments in Shh-expressing ventral midline cells are observed in Noggin^−/− mutant mice (McMahon et al. 1998), whereas the combined actions of Shh and Chordin are able to induce ectopic Shh-expressing floor plate cells in the chick embryo in vivo (Patten and Placzek 2002). Chordin^−/−; Noggin^+/− mice have a dorsalized ventral telencephalon (Anderson et al. 2002), and similar phenotypes are observed in mice lacking Megalin/Lrp2, which encodes a protein responsible for endocytic uptake and lysosomal degradation of BMP-4 (Spoelgen et al. 2005). Further, Bmpr1a^−/−; Bmpr1b^−/− mice fail to form two hemispheres, known as holoprosencephaly, and also fail to form the dorsal midline cell types (Fernandes et al. 2007). Taken together, inhibition of BMP signaling, together with nodal and Shh signaling, is required for proper induction of the floor plate.

Following formation of the floor plate, inhibition of BMP signaling induces normal ventralization of the neural tube (Fig. 2B). This is observed in Noggin^−/− mice, which results in the loss of ventral interneurons and depletion of motor neurons (McMahon et al. 1998; Liem et al. 2000), whereas overexpression of chordin and follistatin expands the motor neuron population at the expense of dorsal populations (Liem et al. 2000; Patten and Placzek 2002). Localized inhibition of BMP signaling confines expression during patterning. The homeodomain protein, Six3, represses BMP-4 expression in the forebrain near the anterior neural plate and is able to restore the size of neural plate in Chordin^−/− mice (Gestri et al. 2005). Meanwhile, Smad7, an inhibitory-Smad, is expressed in the intermediate spinal cord and confines BMP signaling to the dorsal spinal cord (Yan et al. 2009; Hazen et al. 2011). Thus, control of BMP signaling in the spinal cord, through inhibition of signaling or repression of gene expression, is necessary for proper ventral patterning.

The roof plate forms along the dorsal midline of the neural tube and is induced by BMP signaling (Chizhikov and Millen 2004b). BMP-4 and BMP-7 are expressed in the chick epidermal ectoderm at the time of roof plate specification and can induce roof plate cells from cultured chick neural plate, whereas noggin and follistatin inhibit this induction (Liem et al. 1997). Bmp2, Bmp4, and Bmp7 as well as Gdf7 (encoding GDF-7, growth and differentiation factor 7, also known as BMP-12), activin and dorsalin are expressed in the roof plate (Basler et al. 1993; Dickinson et al. 1995; Lee et al. 1998). BMP signaling is necessary and sufficient for chick roof plate development in vivo (Chizhikov and Millen 2004b; Liu et al. 2004) and ectopic activation of BMP signaling expands the roof plate domain to include almost the entire dorsoventral axis of the spinal cord. Bmp5^−/−; Bmp7^−/−double mutants develop an abnormally patterned forebrain, whereas Bmp5^−/−or Bmp7^−/− single mutants do not show developmental defects in the brain (Solloway and Robertson 1999). These studies show the crucial role of the TGF-β family, particularly BMP signaling, in the development of the dorsal organizer.

BMP signaling activates expression of LIM and Msx homeodomain transcription factors to induce the roof plate. Gain- and loss-of-function experiments in chick and mouse models show that expression of Lmx1a is activated by BMP signaling from epidermal ectoderm (Millonig et al. 2000; Chizhikov and Millen 2004a). Interestingly, Lmx1a induces expression of Gdf7/Bmp12 and Bmp4 when ectopically expressed in the developing chick spinal cord or in caudal neural tube explants in vitro (Chizhikov and Millen 2004a). Lmx1a is the main regulator of BMP signaling, as BMP-4 is unable to induce roof plate in explants from Lmx1a^−/− mutants. By itself, Lmx1a can induce roof plate only in the most dorsal region of the neural tube, suggesting that cofactors are necessary for its dorsalizing effects (Chizhikov and Millen 2004a; Liu et al. 2004). These observations indicate a feedback regulation of BMP and Lmx1a signaling. BMP signaling also induces the expression of the Msx family transcription factors (Msx1, Msx2, and Msx3) in the dorsal spinal cord (Timmer et al. 2002; Liu et al. 2004). Single loss-of-function studies have not been informative, likely because of redundancy; however, overexpression of Msx1, but not Msx3, in early chick developing spinal cord is sufficient to induce expression of roof plate markers (Liu et al. 2004). Together, Msx and Lmx1a mediate aspects of BMP signaling effects on roof plate development.

After specifying the roof plate, BMP signaling acts as a morphogen to pattern specific dorsal subtypes in a posterior-caudal to anterorostral direction (for review, see Chizhikov and Millen 2005). BMP signals induce interneuron populations in the dorsal (dI1-dI3), but not more ventral (dI4-dI6) spinal cord (Liem et al. 1995, 1997). Addition of BMP-4, BMP-6, BMP-7, or GDF-7 to naïve caudal neural plate explants mimics roof-plate-mediated induction of dI1 and dI3 interneurons (Liem et al. 1997; Lee et al. 1998; Butler and Dodd 2003), and complete ablation of the roof plate results in failure to form dI1-dI3 interneurons leading to expansion of dI4-dI6 interneurons (Lee et al. 2000). Deletion of both BMPRIA and BMPRIB from the dorsal spinal cord leads to a complete loss of the dorsal-most dI1 interneurons (Wine-Lee et al. 2004), whereas activation of BMPRIA and BMPRIB expands dI1 interneurons at the expense of other interneuron types (Timmer et al. 2002; Chizhikov and Millen 2004a; Liu et al. 2004). Use of small interfering RNA (siRNA) to silence the expression of the downstream BMP signaling mediator, Smad4, has similar effects on dorsal phenotypes (Chesnutt et al. 2004). Thus, BMP signaling is necessary for patterning of dorsal interneurons, and this role is mediated in part by Lmx1a and Msx3 (Millonig et al. 2000; Chizhikov and Millen 2004a; Liu et al. 2004).

Beyond the spinal cord, BMP signaling from the roof plate contributes to the development of the dorsal hindbrain and diencephalon. Hindbrain roof plate cultures express Bmp6, Bmp7, and Gdf7 and these factors are sufficient to induce cerebellar granule neurons (Alder et al. 1999; Machold et al. 2007). BMP-4 and BMP-6 stimulate differentiation and promote survival of granule neurons (Angley et al. 2003; Barneda-Zahonero et al. 2009), whereas targeted inactivation of Bmpr1a and Bmpr1b in mice results in fewer cerebellar granule neurons, causing a smaller cerebellar cortex without foliation (Qin et al. 2006). Application of noggin to the developing midbrain–hindbrain border results in complete loss or dorsal shift of Phox2-positive neurons. The varying response of Phox2-positive neurons indicates that they form in response to a graded BMP signal from either the roof plate or adjacent epidermal ectoderm (Vogel-Höpker and Rohrer 2002). Downstream BMP signaling through Lmx1a plays a unique role in segregating roof plate epithelium lineage from adjacent cerebellar rhombic lip cell lineage. Lmx1a^−/−mouse embryos induce the roof plate normally; however, cells that belong to the roof plate epithelium lineage migrate and adopt fates of neuronal rhombic lip derivatives (Chizhikov et al. 2010). Msx1 signals downstream of BMP in the more rostral roof plate, and targeted inactivation of Msx1 causes a specific loss of the diencephalic roof plate and compromises the dorsal subcommissural organ (Bach et al. 2003). Expression of BMP ligands and receptors both dorsally and ventrally in the diencephalon enhances the complexity of this signaling pathway. Activation of BMPRIA and BMPRIB results in a disruption of proper gene expression patterns in the diencephalon, in addition to an increase in programmed cell death (Lim et al. 2005). However, dominant-negative BMPRIA and BMPRIB did not perturb diencephalic nuclear patterning, suggesting that signaling via BMPRI receptors may not be necessary for patterning in the diencephalon. Thus, BMP signaling functions as a dorsalizing factor in the hindbrain to specify the cerebellum as well as in the spinal cord.

The cerebral hemispheres of the telencephalon surround and dwarf the dorsal midline (roof plate) as it differentiates into the choroid plexus and cortical hem. The dorsomedial telencephalon expresses BMP-2, BMP-4, BMP-6 and BMP-7, as well as the BMP target genes Msx1 and Msx2 (Furuta et al. 1997; Grove et al. 1998). Ablation of the roof plate fails to induce the dorsal telencephalic midline tissue, and gene expression is reduced, including diminished adjacent Lhx2 expression (Monuki et al. 2001; Cheng et al. 2006). Similarly, inactivation of BMP signaling leads to specific loss of the roof-plate-derived choroid plexus, and the cortical hem fails to form (Hébert et al. 2003; Fernandes et al. 2007). Expression of activated BMPRIA in the telencephalon or ectopic expression of BMP-4 can rescue the choroid plexus epithelium without affecting the ventral neural tube (Hébert et al. 2002; Cheng et al. 2006). When BMP signaling is ablated after neural tube closure, holoprosencephaly does not occur, but formation of the dentate gyrus of the hippocampus is affected (Caronia et al. 2010). Indirect evidence suggests a larger role for BMP signaling in the telencephalon. Mutual antagonism exists between BMPs and Wnts in the cortical hem and between BMPs and FGF-8 in the anterior telencephalon. Additionally, there are significant forebrain patterning defects in embryos treated with exogenous BMPs, mice lacking both Bmp5 and Bmp7, and mice lacking Chordin and Noggin (Bachiller et al. 2000). These models are complicated to interpret because they include phenotypes that span multiple regions beyond the roof plate. Thus, understanding the role of BMP signaling in telencephalon patterning (and all patterning along the anterior–posterior axis) must be seen in the context of other signaling cascades and transcriptional networks. However, in general BMP signaling specifies dorsal structures, and its role in ventral instruction is less well known.

CELL FATE SPECIFICATION

Stem and progenitor cells in the proliferative ventricular zone (VZ) and later the subventricular zone (SVZ) generate neurons throughout the CNS, followed by sequential waves of astrocyte and oligodendroglia generation. Remarkably, BMP signaling is critical for progenitor cell fate specification in both neurogenesis and astrogliogenesis, although it inhibits oligodendrogliogenesis at all stages of development.

MIGRATION AND AXON GUIDANCE

Following neurogenesis, the formation of cell layers in the nervous system depends on interactions between neurons, glia, and the extracellular matrix for spatial and temporal migration. The expression patterns of TGF-β ligands and receptors in the cerebral cortex are indicative of their involvement in neuronal migration. TGF-β1 is primarily expressed in the meninges, TGF-β2 and TGF-β3 are expressed by neurons and radial glia, and BMP-6 is expressed by radial glia (Schluesener and Meyermann 1994; Murphy et al. 2004; Ozdamar et al. 2005). TβRI is expressed in the developing murine cortex, and is found postnatally along radial glial fibers, whereas TβRII is primarily expressed by neurons (Tomoda et al. 1996; Miller 2003). Low concentrations of TGF-β1 in culture promote neuronal migration of immature neurons and neuroblastoma cells, whereas high concentrations impair migration. TGF-β1 acts by up-regulating the expression of cell-adhesion molecules, like neural cell-adhesion molecule (N-CAM), and the integrin subunits α₃, α₅, and β₁ (Siegenthaler and Miller 2004). Cell-adhesion molecules assist in cell migration by mediating cell attachments through hemophilic interactions, and integrins act as essential links between the extracellular environment and cytoskeleton. In glioma brain tumors, TGF-β1 and TGF-β2 stimulate migration of glioma cells by inducing the expression of α5β3 integrins, whereas anti-TGF-β neutralizing antibodies reduce glioma cell migration (Paulus et al. 1995; Platten et al. 2000; Wick et al. 2001). TGF-β1, TGF-β2, and TGF-β3 have also been shown to increase the number and length of neurites (Unsicker et al. 1996). Together, these findings indicate that TGF-β signaling is required to induce axon formation and neuronal migration (Yi et al. 2010).

BMP and activin are responsible for migration and axon guidance in the developing brain. BMPs act as inductive signals to promote growth cone guidance, axonal orientation and path finding. During spinal cord development, BMP-6, BMP-7, and GDF-7 are expressed in the rodent roof plate and direct commissural neuron axon extension through the spinal cord toward the floor plate (Lee et al. 1998; Augsburger et al. 1999; Butler and Dodd 2003). Explant cultures treated with BMP-7 mimic the repellent activity of the roof plate for commissural axons and directly act to collapse their growth cones (Augsburger et al. 1999), whereas Bmp7^−/− and Gdf7^−/−explant cultures show very little repellent activity in vitro. Studies show that BMP-7 and GDF-7 form a heterodimer and act cooperatively in vivo for the guidance of commissural axons (Butler and Dodd 2003). Interestingly, BMP-6 and BMP-7 both induce differentiation of commissural neurons, but only BMP-7 is necessary to orient axons (Perron and Dodd 2011, 2012). The fast-acting repellent action of BMP-induced axonal orientation suggests signaling through cytoskeletal actin remodeling and c-Jun amino-terminal kinase (JNK)-mediated microtubule stabilization (Wen et al. 2007; Podkowa et al. 2010; Perron and Dodd 2011). Additional signaling from activin aids in organizing neural circuitry, and the expression of the activin βA subunit at E17 in the telencephalon indicates a possible role in topographical wiring between the neocortex and neostriatum (Andreasson and Worley 1995). In vitro cultures of rat cortical neurons show that activin signaling promotes dendritic complexity through disinhibiting specific signaling factors (Ishikawa et al. 2010). Thus, multiple members of the TGF-β family are highly involved in migration and axon outgrowth and guidance.

SYNAPTOGENESIS AND PLASTICITY

Neurons communicate through synapses that allow for two-way signal transmission from presynaptic and postsynaptic targets. Strengthening synapses is necessary for learning and memory and for regenerating neuronal circuits after injury. BMP signaling regulates neurite outgrowth and dendritic development throughout the CNS, including cortical neurons (Li et al. 1998; Le Roux et al. 1999; Lee-Hoeflich et al. 2004; Podkowa et al. 2010), hippocampal neurons (Withers et al. 2000), and cerebellar neurons (Matsuura et al. 2007). In culture, treatment of sympathetic neurons with BMP-2, BMP-5, BMP-6, or BMP-7 enhances dendritic development (Lein et al. 1995, 1996; Guo et al. 1998; Beck et al. 2001; Horbinski et al. 2002). However, in cerebellar granule neurons, BMP-2 inhibits neurite outgrowth by LIMK-dependent mechanisms (Matsuura et al. 2007). Treatment of cultured rat hippocampal neurons with TGF-β1 or TGF-β2 results in extension of axon-like processes with no effect on dendritogenesis (Ishihara et al. 1994). The postulated role of TGF-β in synapse formation and plasticity in vertebrates is based to a large extent on expression patterns. Inactivation of Tgfb1 in mice results in lower levels of the presynaptic protein synaptophysin in the neocortex and hippocampus, leading to decreased synaptic density (Brionne et al. 2003). TGF-β1 is also necessary for normal expression of the extracellular matrix protein laminin, which has been implicated in learning and memory (Venström and Reichardt 1993; Luckenbill-Edds 1997). Transgenic overexpression of TGF-β1 in astrocytes leads to increased production of laminin, whereas loss of Tgfb1 results in a reduction in laminin expression (Brionne et al. 2003).

Direct evidence for the role of TGF-β family proteins in synaptic differentiation and maturation comes from the study of the glutamatergic neuromuscular junction (NMJ) in Drosophila (for review, Darabid et al. 2014). Evidence of this involvement in synapse development and plasticity has also been provided by studies of the mollusk Aplysia (Schuman 1997; Zhang et al. 1997). During long-term sensitization of the gill- and siphon-withdrawal reflex in this organism, long-term facilitation also results in an increase in TGF-β1 mRNA in sensory neurons (Liu et al. 1997; Zhang et al. 1997). Application of TGF-β1 strengthens the sensory-motor-neuron synaptic connections, whereas a TGF-β receptor antagonist blocks this effect (Zhang et al. 1997; Chin et al. 1999). In the mouse, TGF-β2 treatment in cultured hippocampal neurons alters evoked postsynaptic currents (ePSCs), indicating a role in hippocampal synaptic plasticity (Fukushima et al. 2007). The TGF-β family member activin also affects hippocampal neurons by modifying spine morphology and increasing synaptic contacts (Shoji-Kasai et al. 2007). Thus, the TGF-β family has an important role in both synaptogenesis and in plasticity of the nervous system.

CELL SURVIVAL AND DEATH IN THE NERVOUS SYSTEM

Programmed cell death in the developing vertebrate nervous system is responsible for loss of a large number of neurons. Neuron survival is dependent on competition for a limited supply of survival factors (neurotrophins) provided by target cells. TGF-βs are not independently neurotrophic; however, treatment with TGF-β enhances neurotrophic signaling in cultured neurons grown with low and subthreshold concentrations of neurotrophins. Meanwhile, blocking endogenous TGF-βs lowers the survival-promoting activity of neurotrophin (Krieglstein and Unsicker 1996; Krieglstein et al. 1998). The biological relevance of this synergy is also shown by the increase in TGF-β synthesis and release from neurons in response to neurotrophins (Krieglstein and Unsicker 1996; Krieglstein et al. 1998). Unlike the cotrophic effects of TGF-β signaling, BMP signaling in the developing nervous system triggers apoptosis. BMPs are expressed in the chick embryo in the dorsal regions of the odd-numbered rhombomeres (r3 and r5) and induce segmental apoptosis in these areas (Graham et al. 1994). Loss of BMP antagonists in Chordin^−/−; Noggin^+/− embryos, as well as conditional activation of BMPRIA or BMPRIB expression, increases cell death in the rostral neuroepithelium (Anderson et al. 2002). BMP signaling induces apoptosis of neocortical progenitors, forebrain precursor explants, trigeminal neurons, sympathoadrenal cells, sympathetic neuroblasts, and postmigratory enteric neurons (Chalazonitis et al. 2004). In vitro, treatment of SVZ neurospheres with BMP-4 promotes apoptosis in a dose-dependent manner through Msx2 and the cyclin-dependent kinase inhibitor p21^Cip1 (Israsena and Kessler 2002). BMP signaling can also promote survival through indirect mechanisms such as induction of neurotrophin receptors (Zhang et al. 1998).

The TGF-β family has an essential role in midbrain DN survival. TGF-β1, TGF-β2, and TGF-β3 promote survival of mesencephalic DNs, whereas neutralizing antibodies against TGF-β in the developing chick (E6-10) impairs survival (Krieglstein and Unsicker 1994; Poulsen et al. 1994; Roussa et al. 2004). The effects of TGF-β signaling on DN survival are mediated by HIPK1 (homeodomain interacting protein kinase 1) interacting with Smad3 to regulate target genes (Zhang et al. 2007). In contrast, BMP signaling acts indirectly on dopaminergic neuron survival by stimulating astrocyte differentiation (Jordan et al. 1997; Farkas et al. 2003). BMP-7 induces dopamine neuron differentiation in culture, and promotes survival in the adult nigrostriatal pathway against dopamine toxins in vivo (Lee et al. 2003; Harvey et al. 2004; Chou et al. 2008). Although the direct role of BMP signaling on dopamine neuron induction is unknown, Msx1 and Lmx1a are key determinants of midbrain DNs and are both induced by BMP-Smad activation (Tríbulo et al. 2003; Chizhikov and Millen 2004a; Andersson et al. 2006). Thus, TGF-βs together with BMPs are key regulators in survival and programmed cell death within the developing nervous system.

THE NORMAL AND PATHOLOGICAL ADULT NERVOUS SYSTEM

CONCLUSION

The role of TGF-β family members has been well characterized in the early stages of neural development in several organisms including mice. Spatiotemporal signals mediate effects of TGF-β family members, allowing them to exert a wide range of functions from early stages of neurulation to later stages of development involving processes such as cellular specification, synaptogenesis, and axon guidance. The mechanisms involved later in development are less clear because of the need for multiple TGF-β family gene mutations to overcome their redundant functions. Overall, we conclude that members of the TGF-β family have important roles in virtually every stage of neural development. Continued study in the injured and diseased nervous system will have important clinical implications and could identify novel therapeutic targets.

Footnotes

REFERENCES