**2. The role of Smads during neural/non-neuronal development**

#### **2.1. Already early development requires Smad expression**

co-operating Smad(Co-Smad)4, which is required for the nuclear translocation of activated R-Smads to subsequently control Smad sensitive promoter activity. Moreover, I-Smads support recruitment of HECT-type E3-ubiquitin ligases Smurf1/2, which allow type I receptor ubiqui‐ tination and its degradation. Members of HECT-type and RING-type E3 ubiquitin ligases have also been implicated in Smad degradation [13]. Regulating Smad activity allows the control of highly complex developmental networks, e.g. the patterning of ventro-lateral mesoderm, the decisive development of epidermal/neural cell lineages including the induction and estab‐ lishment of neural plate border, the dorso-ventral patterning of the neural tube, or the

**Figure 1. Synopsis of canonical Smad signalling**. The upper panel shows the binding of ligands belonging to the TGFβ superfamily to their serine/threonine kinase receptors types I and II, leading to intracellular activation of R-Smads by receptor type I induced phosphorylation of the C-terminal Smad motif SSXS. The left and right receptor complexes represent the TGFβ/activin/nodal linked Smad2/3 signalling and the BMP/GDF linked Smad1/5/8 signal‐ ling, respectiviely. The middle panel also displays a set of relevant binding partners of Smad proteins and shows the generation of heterotrimer or heterodimer Smad complexes consisting of C-terminally phosphorylated R-Smads and Co-Smad4, which are transported into the nucleus to excert their transcriptional activity by binding to susceptible pro‐ moter sequences (a). In (b) and (c) examples of the agonist-induced Smad linker phosphorylation mediated by cyclindependent kinases are shown, which increases Smad transcriptional activity prior to signal termination and Smad degradation [14, 17]. In contrast, the MAPK triggered linker phosphorylation of Smads in cytoplasm diminishes the receptor type I responsible Smad phosphorylation and favours the cytoplasmic retention and degradation of Smad. More information is provided in the main text.*Note*: Numbers in square brackets indicate relevant references included in the reference list provided at the chapters end.*Abbreviations*: CDK, cyclin-dependent kinase; dynein, dynein light chain km23-1 or km23-2; MAPK, mitogen activated protein kinase; MT, microtubuli; Nedd4L, neuronal precursor cell expressed developmentally down-regulated 4-like ubiquitin ligase; Nuc214, nucleoporin 214; SARA, Smad achor for

receptor activation; Smurf1, HECT-domain ubiquitin ligase Smurf1.

migration of neural crest cells [18-21].

76 Trends in Cell Signaling Pathways in Neuronal Fate Decision

During early ontogenesis, development and aging as well as prior to individual cell death Smad proteins differentially affect cellular function, depending on time and local partners. Already at very early stages of postnatal rat development Smads1-7 were detectable in the ovary [39]. At all stages of follicular development (e.g. in primordial, primary and secondary follicles) Smad proteins are present in oocytes as well as in granulosa cells and theca cells, though with different concentrations [39-41]. While Smad3,6 and 8 content did not change, the expression of Smad1,2,4,5,6 and 7 seems to be regulated in ovary [39]. In mice oocytes a clear and persistent nuclear localization of phosphorylated Smad1,5,9 and Smad2,3 demonstrates activated activin/GDF9/nodal/TGFβ and BMP/MIS pathways, respectively, and confirms oocytes as a major target for Smad signalling pathways [42]. Smad proteins play also important roles already during maturation of human oocytes [43]. In unfertilized oocytes, Smad2 and 3 and TGFβ receptors I and II are present [44]. After fertilization at the 4-cell and 8-cell stages Smad2 and 3 are also present, while neither of the TGFβ receptors is detectable. Later on in the blastocyte stage Smad2 and 3 kept present and TGFβ receptor I again appears [44]. Human granulosa cells express Smad1-7 and 9, but Smad2,3, and 4 with the highest expression levels [45]. A mutual interaction between oocytes and granulosa cells is necessary for normal folliculogenesis. However, if Smad4 is deleted in granulosa cells infertility results, while deletion of Smad4 in oocytes only marginally effects the primordial follicle stage [46]. How‐ ever, Smad2 reduction in zebrafish oocytes completely blocks activin A-induced oocyte maturation and a Smad2 knockdown decreases basal and hCG-induced oocyte maturation demonstrating a key role of Smads during oocyte maturation [47].

Remarkably, conversely, it has also been suggested from very early studies on Smad2+/-; Smad3-/- mutants in zebrafish showing anterior truncations [49], that Smad2 and Smad3, which are mainly effectors of TGFβ/nodal pathways, are also positively involved in neural develop‐ ment [61]. Dominant negative Smad3 mutants inhibit the expression of early neural markers sox2 and sox3 at the onset of gastrulation and lead to reduction of anterior and posterior neuroectodermal markers otx2 and hoxb1b, respectively, during late gastrulation. According‐ ly, elevated Smad2 and Smad3 activities increased sox2 and sox3 expression, probably at least partly due to its positive impact on chordin expression, which is a BMP antagonist [61]. In mouse embryo BMP signalling also inhibits premature neural differentiation, but in contrast to Xenopus [62] and chick [63,64] FGF signalling seems additionally required to induce neural

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In summary, the data indicate that simultaneous suppression of both Smad1,5,8 and Smad2,3 pathways, e.g. suppression of mesoderm- and ectoderm-inducing Smad signals, respectively, are required for neural induction. In principle, the `dual Smad inhibition` method is adopted for induction of efficient neuralization of human (-induced) pluripotent stem cells (hiPSCs,

To establish a precise borderline between neuroectoderm (containing low BMP2/4 levels) and non-neuroectoderm (containing high BMP2/4 levels) regions during early neurulation, specific strategies are required and examined in urchin embryos, where Fez, a zinc finger protein, drops down the BMP controlled pSmad1,5,8 levels [68]. It is probable that Fez acts as a transcriptional repressor of Smad or interferes with genes attenuating intracellular BMP signalling e.g. SIP1,

Once the neuroectoderm forming is initiated a flat neural plate rolls up, the process of neural plate folding proceeds and is finished by neural tube closure [69]. The closure of neural tube is a complex process involving hinge points (HP), representing specialized neural plate cells. The cells of ventral midline/median hinge point (MHP) as well as both dorso-lateral hinge points (DLHPs) are affected by dynamic BMP signalling [70-73]. Folding of neural plate is connected with a two-dimentionally spatiotemporal gradient of phosphorylated Smad1,5,8 (pSmad1,5,8) [71,73]. While initially a mosaic labelling of pSmad1,5,8 was observed at the apical surface of the presumptive neuroectoderm later two intersecting Smad gradients were exerted: a lateromedial gradient with the lowest pSmad1,5,8 expression at the MHP and a second gradient along the apicobasal axis of the presumptive midbrain plate with mosaic expression in apical nuclei [71]. Some of the apical pSmad1,5,8 positive cells proliferate and thus support the neural fold elevation [71]. Disturbances of the phosphorylation state of Smad1,5,8 alter midbrain shape by multiple hinge-like invaginations but do not affect the ventral cell-fate specification [71]. However, the extent of Smad1,5,8 phosphorylation seems to control MHP formation during neurulation in vertebrates. The correct MHF formation is obviously directly linked to the pSmad1,5,8 controlled regulation of apicobasal polarity of neural plates cells. After phosphorylation, Smad1,5,8 interact with the PAR3-PAR6-aPKC complex and stabilizes linked tight junctions, while inversely low pSmad1,5,8 phosphorylation

differentiation [65].

hPSCs) [66,67].

*2.2.2. Neural tube closure*

Smurf1, Ski or SnoN.

#### **2.2. Gastrulation and neurulation**

#### *2.2.1. Suppression of Smad signalling specifies neuroectoderm generation*

Initially, the ectoderm cells in Xenopus are pluripotent until gastrulation [48]. Early gastrula‐ tion steps are characterized by the formation of mesenchymal cells and the induction of mesoderm in the marginal-zone by an epithelial-to-mesenchymal transition (EMT) process, which involves activation of Smad2,3,4 signalling by TGFβ/nodal ligands after binding to corresponding receptors [49-53].

Mesoderm induction requires a subtle coordination of the canonical TGFβ/nodal signalling, with Smad4 as a key player. However, initially it was shown, that a simple ectopic Smad4 expression in the Xenopus animal cap was inefficient to induce mesoderm [54], due to its binding to ectodermin, a RING-type ubiquitin ligase, which is highly expressed in the animal cap [53]. Ectodermin limits Smad4 function via antagonizing nuclear accumulation of Smad4 and supporting Smad4 degradation by ubiquitin-proteosome pathway [53]. By this, ectoder‐ min ensures that ectoderm cells do only develop to a mesodermal lineage, but also supports neural differentiation of ectoderm towards neuroectodermal fates by interfering with BMP signalling via limiting Smad4 function [53]. However, the stable presence of soluble BMP which prevents the execution of an intrinsic program of ectodermal cells to form neural tissue can also be undermined by the release of BMP antagonists as noggin, chordin, follistatin or cerberus, which however, allows creation and development of neural cells. This interrelation is basically reflected in the ´default model´ of neural induction [55,56]. Confirming this model it was shown, that the simultaneous depletion of BMP2, BMP4 and BMP7 induces massive brain formation [57]. Accordingly, during early gastrulation-stage of chick embryo Smad1,5,8 signalling is undetectable [21] but is activated at neurula stage. So, phosphorylated Smad1 becomes detectable only at late gastrulation stages in the posterior territory of the embryo [21].

The default model has been further modified due to experiments showing that inhibition of BMP/Smad1 signalling, e.g. by Smad6, was insufficient to induce neural markers efficiently [58], while the additional suppression of Smad2 was sufficient [59]. Moreover, stimulation of Smad2 signalling blocks neural induction even at gastrula stages and indicate prospective neural cells to further undergo mesodermal and non-neural fates [59]. These data are sup‐ ported by experiments in sea urchin embryos showing that Smad2 and 3 suppress neural differentiation in the oral ectoderm [60].

Remarkably, conversely, it has also been suggested from very early studies on Smad2+/-; Smad3-/- mutants in zebrafish showing anterior truncations [49], that Smad2 and Smad3, which are mainly effectors of TGFβ/nodal pathways, are also positively involved in neural develop‐ ment [61]. Dominant negative Smad3 mutants inhibit the expression of early neural markers sox2 and sox3 at the onset of gastrulation and lead to reduction of anterior and posterior neuroectodermal markers otx2 and hoxb1b, respectively, during late gastrulation. According‐ ly, elevated Smad2 and Smad3 activities increased sox2 and sox3 expression, probably at least partly due to its positive impact on chordin expression, which is a BMP antagonist [61]. In mouse embryo BMP signalling also inhibits premature neural differentiation, but in contrast to Xenopus [62] and chick [63,64] FGF signalling seems additionally required to induce neural differentiation [65].

In summary, the data indicate that simultaneous suppression of both Smad1,5,8 and Smad2,3 pathways, e.g. suppression of mesoderm- and ectoderm-inducing Smad signals, respectively, are required for neural induction. In principle, the `dual Smad inhibition` method is adopted for induction of efficient neuralization of human (-induced) pluripotent stem cells (hiPSCs, hPSCs) [66,67].

#### *2.2.2. Neural tube closure*

the blastocyte stage Smad2 and 3 kept present and TGFβ receptor I again appears [44]. Human granulosa cells express Smad1-7 and 9, but Smad2,3, and 4 with the highest expression levels [45]. A mutual interaction between oocytes and granulosa cells is necessary for normal folliculogenesis. However, if Smad4 is deleted in granulosa cells infertility results, while deletion of Smad4 in oocytes only marginally effects the primordial follicle stage [46]. How‐ ever, Smad2 reduction in zebrafish oocytes completely blocks activin A-induced oocyte maturation and a Smad2 knockdown decreases basal and hCG-induced oocyte maturation

Initially, the ectoderm cells in Xenopus are pluripotent until gastrulation [48]. Early gastrula‐ tion steps are characterized by the formation of mesenchymal cells and the induction of mesoderm in the marginal-zone by an epithelial-to-mesenchymal transition (EMT) process, which involves activation of Smad2,3,4 signalling by TGFβ/nodal ligands after binding to

Mesoderm induction requires a subtle coordination of the canonical TGFβ/nodal signalling, with Smad4 as a key player. However, initially it was shown, that a simple ectopic Smad4 expression in the Xenopus animal cap was inefficient to induce mesoderm [54], due to its binding to ectodermin, a RING-type ubiquitin ligase, which is highly expressed in the animal cap [53]. Ectodermin limits Smad4 function via antagonizing nuclear accumulation of Smad4 and supporting Smad4 degradation by ubiquitin-proteosome pathway [53]. By this, ectoder‐ min ensures that ectoderm cells do only develop to a mesodermal lineage, but also supports neural differentiation of ectoderm towards neuroectodermal fates by interfering with BMP signalling via limiting Smad4 function [53]. However, the stable presence of soluble BMP which prevents the execution of an intrinsic program of ectodermal cells to form neural tissue can also be undermined by the release of BMP antagonists as noggin, chordin, follistatin or cerberus, which however, allows creation and development of neural cells. This interrelation is basically reflected in the ´default model´ of neural induction [55,56]. Confirming this model it was shown, that the simultaneous depletion of BMP2, BMP4 and BMP7 induces massive brain formation [57]. Accordingly, during early gastrulation-stage of chick embryo Smad1,5,8 signalling is undetectable [21] but is activated at neurula stage. So, phosphorylated Smad1 becomes detectable only at late gastrulation stages in the posterior territory of the embryo [21].

The default model has been further modified due to experiments showing that inhibition of BMP/Smad1 signalling, e.g. by Smad6, was insufficient to induce neural markers efficiently [58], while the additional suppression of Smad2 was sufficient [59]. Moreover, stimulation of Smad2 signalling blocks neural induction even at gastrula stages and indicate prospective neural cells to further undergo mesodermal and non-neural fates [59]. These data are sup‐ ported by experiments in sea urchin embryos showing that Smad2 and 3 suppress neural

demonstrating a key role of Smads during oocyte maturation [47].

*2.2.1. Suppression of Smad signalling specifies neuroectoderm generation*

**2.2. Gastrulation and neurulation**

78 Trends in Cell Signaling Pathways in Neuronal Fate Decision

corresponding receptors [49-53].

differentiation in the oral ectoderm [60].

To establish a precise borderline between neuroectoderm (containing low BMP2/4 levels) and non-neuroectoderm (containing high BMP2/4 levels) regions during early neurulation, specific strategies are required and examined in urchin embryos, where Fez, a zinc finger protein, drops down the BMP controlled pSmad1,5,8 levels [68]. It is probable that Fez acts as a transcriptional repressor of Smad or interferes with genes attenuating intracellular BMP signalling e.g. SIP1, Smurf1, Ski or SnoN.

Once the neuroectoderm forming is initiated a flat neural plate rolls up, the process of neural plate folding proceeds and is finished by neural tube closure [69]. The closure of neural tube is a complex process involving hinge points (HP), representing specialized neural plate cells. The cells of ventral midline/median hinge point (MHP) as well as both dorso-lateral hinge points (DLHPs) are affected by dynamic BMP signalling [70-73]. Folding of neural plate is connected with a two-dimentionally spatiotemporal gradient of phosphorylated Smad1,5,8 (pSmad1,5,8) [71,73]. While initially a mosaic labelling of pSmad1,5,8 was observed at the apical surface of the presumptive neuroectoderm later two intersecting Smad gradients were exerted: a lateromedial gradient with the lowest pSmad1,5,8 expression at the MHP and a second gradient along the apicobasal axis of the presumptive midbrain plate with mosaic expression in apical nuclei [71]. Some of the apical pSmad1,5,8 positive cells proliferate and thus support the neural fold elevation [71]. Disturbances of the phosphorylation state of Smad1,5,8 alter midbrain shape by multiple hinge-like invaginations but do not affect the ventral cell-fate specification [71]. However, the extent of Smad1,5,8 phosphorylation seems to control MHP formation during neurulation in vertebrates. The correct MHF formation is obviously directly linked to the pSmad1,5,8 controlled regulation of apicobasal polarity of neural plates cells. After phosphorylation, Smad1,5,8 interact with the PAR3-PAR6-aPKC complex and stabilizes linked tight junctions, while inversely low pSmad1,5,8 phosphorylation supports re-arrangement of neural plate cells, a prerequisite for MHP formation and neural fold elevation [72].

signalling pathways is required [84] for a timely and locally well tuned progression. Besides Wnt signalling a major role in NC induction plays canonical BMP signalling [85-90] combined with FGF signalling [21,91]. The inductive step of NPB development during gastrulation requires a concerted action of activated Wnt signals and inhibition of BMP signalling repre‐ sented by low Smad1,5,8 activity. Later, when neurulation proceeds and NC develops from NPB, activated Wnt signals cooperate with a robust Smad1,5,8 activation in NPB. While during the inductive step of NC gastrulation FGF downregulates Smad1,5,8 activity by triggering MAPK, the catalysed phosphorylation of Smad1,5,8 linker region is nearly completely absent in NPB during neurulation. In consequence, a strongly elevated Smad1,5,8 signalling is observed suggesting a ´two step model of NC development´ with respect to activated

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The further outcome of NC cells including the generation of the PNS also strongly depends on Smad signalling. Interestingly, Smad expression regulates both the formation of neurons

Precursors that emigrate from the neural crest to the bowel generates the enteric nervous system (ENS) belonging to PNS. If fetal enteral neural crest-derived cells (ENCDC) of gut are exposed to BMP2 or BMP4 phosphorylated Smad1 translocates to the nucleus and the cells develop processes, indicating an essential role of Smad phosphorylation for neuron induction in the gut [92]. However, Smad phosphorylation alone is not sufficient to direct development of ENCDC towards ENS neurons or glial cells but requires further factors e.g. glial growth factor 2 (GGF2) [93]. Moreover, besides generation, also maturation of enteric neurons (as well as regulation of gliogenesis) during postnatal development is Smad-dependent. This function was identified because mice lacking in the homeodomain interacting protein kinase 2 (HIPK2), which can interact with Smad1, Smad2 and Smad3 [94] and therefore control transcription of subsequent Smad-dependent promoters, are characterized by a progressive loss of enteric neurons and an arrest in synaptic maturation postnatally. Additionally, in the HIPK2-/- mice the remaining enteric neurons exhibit an increased number of cells with nuclear Smad1,5,8 phosphorylation [95]. Interestingly, both in the enteric nervous system and in the midbrain [96], HIPK2 reduction severely reduces survival of dopaminergic neurons through interfer‐ ence with Smad signalling pathways, regulated by BMP2/4 or TGFβ, respectively. Whether altered Smad signalling, affected by HIPK2, is also responsible for Parkinson´s disease and would allow to develop a therapeutic intervention has to be investigated in the future.

Recent data demonstrate, that Smad1 signalling by BMP2 induction is implicated in differen‐ tiation of primary enteric neurons to catecholaminergic (TH expression) but not to cholinergic

Peripheral glia precursors derived from neural-crest and exhibiting characteristic expression of immature glia markers e.g. nestin, are critically influenced by BMP2/Smad signalling. Using Smad1 activation, neural-crest precursor cells, that begin to develop along glial pathway are kept in an undifferentiated immature multipotent state, thus allowing migration to their postmigratory locations [98] where they later acquire myelinating properties. Smad-linked

Smad1,5,8 signalling [21].

**2.3. Peripheral nervous system (PNS)**

(ChAT expression) neuronal subtypes [97].

of PNS as well as early stages of peripheral glial development.

#### *2.2.3. Patterning and developing of the spinal cord*

After closure, the neural tube starts to develop the spinal cord pattern along its dorso-ventral axis which is essentially established by the expression of Wnt and BMPs, the main roof plate morphogens, and sonic hedgehog (SHH) and TGFβs, the morphogens released from the floor plate [74-76]. On the basis of provided intricated morphogenic gradients functionally distinct neurons are generated.

Both roof plate derived BMP6 and BMP7 are evoking Smad1,5,8 phosphorylation and subsequent induction of distinct dorsal interneurons (dI) via BMP receptor I activation [77-79]. While the BMP7/pSmad1,5 activated induction of dI1, dI3 and dI5 is independent of the patterned expression of progenitor proteins, e.g. Pax7, Gsh1/2 or Olig3, a Smad regulated expression of specific proneural proteins, e.g. cAth, and cAscl1, is required [79]. The patterning of dorsal progenitor proteins could be associated with other BMPs, e.g. BMP4 [80]. Smad1 and Smad5 activity seems to be important for the maintenance of neural cells as committed progenitors, because the loss of Smad1,5 reduced the total number of newly generated neurons and forces cell cycle exit and premature neurogenesis of neural progenitors [79]. The inhibitory Smad6 also promotes the neuronal differentiation in the spinal cord by interfering with Wntsignalling [81]. Hazen and colleagues demonstrated, that the inhibitory Smad6 and Smad7 function to restrict the action of roof plate released BMPs to distinct dorsal interneurons and participate in the determination of dI4-dI6 spinal neuron number [82].

However, the subsequent orientation of spinal neurons is also controlled by BMP7 but not via activation of Smad signalling instead using phosphoinosite-3-kinase (PI3K) activation by the BMP receptor type II [77]. Nevertheless, Smad6 most potently allows blocking dI1 axon outgrowth [82].

While Smad3 was also expressed in the developing spinal cord, Smad2 was not detected [83]. Contrasting to Smad1,5,8, the expression of Smad3 in the developing spinal cord depends on progenitor proteins, e.g. Nkx6.1, Olig2, Nkx2.2, which directs the Smad3 expression mainly to discrete ventral progenitor domains [83]. Smad3 activity in turn reduces expression of progenitor proteins and promotes activation of neuronal differentiation, e.g. by supporting cell cycle exit via activating of p27Kip1 expression [83]. The Smad3 expression is sufficient to promote differentiation of ventral and various dorsal interneurons, while differentiation of motor neurons is impaired. Therefore Smad3 expression is excluded in the prospective motor neuron progenitor cells [83].

#### *2.2.4. Induction of neural crest (NC)*

Multipotent embryonic cells of the dorsal region of the neural tube, an area existing immedi‐ ately before forming the neural plate border (NPB), are the origin of neural crest cells (NC) that migrate to initiate a panel of diverse derivatives including various non-neural but also neural cells e.g. sensory neurons of the peripheral nervous system. The combination of several signalling pathways is required [84] for a timely and locally well tuned progression. Besides Wnt signalling a major role in NC induction plays canonical BMP signalling [85-90] combined with FGF signalling [21,91]. The inductive step of NPB development during gastrulation requires a concerted action of activated Wnt signals and inhibition of BMP signalling repre‐ sented by low Smad1,5,8 activity. Later, when neurulation proceeds and NC develops from NPB, activated Wnt signals cooperate with a robust Smad1,5,8 activation in NPB. While during the inductive step of NC gastrulation FGF downregulates Smad1,5,8 activity by triggering MAPK, the catalysed phosphorylation of Smad1,5,8 linker region is nearly completely absent in NPB during neurulation. In consequence, a strongly elevated Smad1,5,8 signalling is observed suggesting a ´two step model of NC development´ with respect to activated Smad1,5,8 signalling [21].

#### **2.3. Peripheral nervous system (PNS)**

supports re-arrangement of neural plate cells, a prerequisite for MHP formation and neural

After closure, the neural tube starts to develop the spinal cord pattern along its dorso-ventral axis which is essentially established by the expression of Wnt and BMPs, the main roof plate morphogens, and sonic hedgehog (SHH) and TGFβs, the morphogens released from the floor plate [74-76]. On the basis of provided intricated morphogenic gradients functionally distinct

Both roof plate derived BMP6 and BMP7 are evoking Smad1,5,8 phosphorylation and subsequent induction of distinct dorsal interneurons (dI) via BMP receptor I activation [77-79]. While the BMP7/pSmad1,5 activated induction of dI1, dI3 and dI5 is independent of the patterned expression of progenitor proteins, e.g. Pax7, Gsh1/2 or Olig3, a Smad regulated expression of specific proneural proteins, e.g. cAth, and cAscl1, is required [79]. The patterning of dorsal progenitor proteins could be associated with other BMPs, e.g. BMP4 [80]. Smad1 and Smad5 activity seems to be important for the maintenance of neural cells as committed progenitors, because the loss of Smad1,5 reduced the total number of newly generated neurons and forces cell cycle exit and premature neurogenesis of neural progenitors [79]. The inhibitory Smad6 also promotes the neuronal differentiation in the spinal cord by interfering with Wntsignalling [81]. Hazen and colleagues demonstrated, that the inhibitory Smad6 and Smad7 function to restrict the action of roof plate released BMPs to distinct dorsal interneurons and

However, the subsequent orientation of spinal neurons is also controlled by BMP7 but not via activation of Smad signalling instead using phosphoinosite-3-kinase (PI3K) activation by the BMP receptor type II [77]. Nevertheless, Smad6 most potently allows blocking dI1 axon

While Smad3 was also expressed in the developing spinal cord, Smad2 was not detected [83]. Contrasting to Smad1,5,8, the expression of Smad3 in the developing spinal cord depends on progenitor proteins, e.g. Nkx6.1, Olig2, Nkx2.2, which directs the Smad3 expression mainly to discrete ventral progenitor domains [83]. Smad3 activity in turn reduces expression of progenitor proteins and promotes activation of neuronal differentiation, e.g. by supporting cell cycle exit via activating of p27Kip1 expression [83]. The Smad3 expression is sufficient to promote differentiation of ventral and various dorsal interneurons, while differentiation of motor neurons is impaired. Therefore Smad3 expression is excluded in the prospective motor

Multipotent embryonic cells of the dorsal region of the neural tube, an area existing immedi‐ ately before forming the neural plate border (NPB), are the origin of neural crest cells (NC) that migrate to initiate a panel of diverse derivatives including various non-neural but also neural cells e.g. sensory neurons of the peripheral nervous system. The combination of several

participate in the determination of dI4-dI6 spinal neuron number [82].

fold elevation [72].

neurons are generated.

outgrowth [82].

neuron progenitor cells [83].

*2.2.4. Induction of neural crest (NC)*

*2.2.3. Patterning and developing of the spinal cord*

80 Trends in Cell Signaling Pathways in Neuronal Fate Decision

The further outcome of NC cells including the generation of the PNS also strongly depends on Smad signalling. Interestingly, Smad expression regulates both the formation of neurons of PNS as well as early stages of peripheral glial development.

Precursors that emigrate from the neural crest to the bowel generates the enteric nervous system (ENS) belonging to PNS. If fetal enteral neural crest-derived cells (ENCDC) of gut are exposed to BMP2 or BMP4 phosphorylated Smad1 translocates to the nucleus and the cells develop processes, indicating an essential role of Smad phosphorylation for neuron induction in the gut [92]. However, Smad phosphorylation alone is not sufficient to direct development of ENCDC towards ENS neurons or glial cells but requires further factors e.g. glial growth factor 2 (GGF2) [93]. Moreover, besides generation, also maturation of enteric neurons (as well as regulation of gliogenesis) during postnatal development is Smad-dependent. This function was identified because mice lacking in the homeodomain interacting protein kinase 2 (HIPK2), which can interact with Smad1, Smad2 and Smad3 [94] and therefore control transcription of subsequent Smad-dependent promoters, are characterized by a progressive loss of enteric neurons and an arrest in synaptic maturation postnatally. Additionally, in the HIPK2-/- mice the remaining enteric neurons exhibit an increased number of cells with nuclear Smad1,5,8 phosphorylation [95]. Interestingly, both in the enteric nervous system and in the midbrain [96], HIPK2 reduction severely reduces survival of dopaminergic neurons through interfer‐ ence with Smad signalling pathways, regulated by BMP2/4 or TGFβ, respectively. Whether altered Smad signalling, affected by HIPK2, is also responsible for Parkinson´s disease and would allow to develop a therapeutic intervention has to be investigated in the future.

Recent data demonstrate, that Smad1 signalling by BMP2 induction is implicated in differen‐ tiation of primary enteric neurons to catecholaminergic (TH expression) but not to cholinergic (ChAT expression) neuronal subtypes [97].

Peripheral glia precursors derived from neural-crest and exhibiting characteristic expression of immature glia markers e.g. nestin, are critically influenced by BMP2/Smad signalling. Using Smad1 activation, neural-crest precursor cells, that begin to develop along glial pathway are kept in an undifferentiated immature multipotent state, thus allowing migration to their postmigratory locations [98] where they later acquire myelinating properties. Smad-linked activation of GFAP promoter is part of this process [99]. Generally, oligodendroglial differen‐ tiation of precursor cells is actively suppressed by BMPs concomitant with the stimulation of astrocytic differentiation [100]. Accordingly, BMP4-activation directs progenitor cells in vivo to commit to the astrocytic rather than the oligodendroglial lineage [101].

*3.1.2. Neurons*

[120].

[121] or in SVZ after stroke [122].

The commitment of cell fates in the nervous system is strongly dependent on Smad2,3 signalling cascade. However, there have been contradictory results on the role of Smad3 for neuronal precursor proliferation and their differentiation, indicating a complex Smad signal‐

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Some reports show, that TGFβ, which activates Smad3 signalling, diminishes neurogenesis in hippocampus after its chronical increase [116], and in SVZ and DG after intracerebroventric‐ ular TGFβ1 infusion [117] and promotes neuronal differentiation from hippocampal and cortical progenitors [118]. An clear inhibitory function of Smad3 on neural precursor prolif‐ eration was shown in the developing spinal cord [119], where Smad3 also promotes differen‐ tiation and influences the fate of selected neurons. The antiproliferative role of Smad3 and Smad4 plays a role during early-postnatal differentiation of cerebellar neurons into postmitotic neurons, where TGFβ stimulation induces nuclear translocation of phosphorylated Smads and induction of cyclin-dependent kinase inhibitors p21, p27 and markers of neuronal maturity

However, other reports show that TGFβ increased neurogenesis in DG after adrenalectomy

To clarify the relevance of Smad3 for adult neurogenesis Smad3 null mice were studied [123,124]. These mice show decreased neurogenesis in the DG and the SVZ and exhibit a thinner and more disorganized rostral migratory stream (RMS) of neuronal precursor cells (NPC). Using RMS NPCs migrate from SVZ to reach the olfactory bulb, where they differen‐ tiate into granular and periglomerular neurons. Though a decreased number of proliferating cells demonstrates the requirement of Smad3 for maintaining a proper cell division rate in

For differentiation of mesencephalic progenitors into dopaminergic (DA) neurons a concerted interaction of Smad2,3 signalling and p38 MAPK-pathways by TGFβ receptor stimulation is necessary. Accordingly, treatment of ventral mesencephalic neural progenitors with TGFβ increased the number of tyrosine-hydroxylase (TH)-positive cells [125]. Though not required for the neurogenesis, the survival of midbrain dopaminergic neurons depends on function of the homeodomain interacting protein kinase 2 (HIPK2), which interacts with R-Smads to activate TGFβ responsive genes [96]. Loss of HIPK2 increases apoptosis in DA neurons during development. Nevertheless, apoptosis of specific neural progenitors during neural differen‐

In vitro, treatment of ventral mesencephalic cells with BMP5,6 and 7 also significantly increased the number of TH-positive neurons via Smad phosphorylation and nuclear trans‐ location [128], while the neurotrophin growth/differentiation factor 5 (GDF5) induced Smad pathway promotes neuronal but not dopaminergic differentiation [129]. Remarkably, the in vitro generation of functional dopaminergic substantia nigra neurons for transplantation requires a protocol which uses the temporarily and contextually distinct roles of Smad proteins: Firstly, the dual inhibition of Smad signalling in embryonic stem cells is required to allow induction of floor plate cell state which then passes over to midbrain floor plate cell state.

tiation also involves Smad phosphorylation as shown for Smad1,5,8 [126,127].

ling network dependent on local, temporal and contextual characteristics.

SVZ, the neuronal fate is not altered by Smad3 deficiency [123].
