**6. Concluding remarks**

Overexpression of Igf-1 increases β-catenin-levels and stimulates progenitor proliferation [119]. These experiments clearly establish an important cross-talk to regulate progenitor

IIS has also been described to cross-talk with the Tgfβ-pathway in numerous systems [120]. However, a comprehensive analysis of the multiple points of intersections was not published yet for the nervous system. In a general view, Tgfβ-ligands 1, 2, and 3 exert opposing effects compared to IIS. This has been shown for the potential of NPC proliferation and differentiation [121] upon Tgfβ-treatment, since these cells stop proliferating and initiate neuronal differen‐ tiation in mouse E16.5 hippocampus and cerebral cortex, *in vitro* and *in vivo*. Proliferation and differentiation of ESCs are also influenced in opposite ways through IIS or Tgfβ-signalling. Smad4-deficient ESC show increased neuronal differentiation, and hereby preferentially in neuronal lineages of the mid- and anterior hindbrain [122]. Further, more serotonergic neurons of the dorsal midbrain are observed in *in vitro* differentiated Smad4-deficient cultures. However, Smad4 is a transcriptional mediator of several members of the Tgfβ-superfamiliy, including Activin-, Gdf-, Nodal-, Tgfβ- as well as Bmp-signalling. Thus, direct correlation to one specific signalling pathway is not achieved in this study. Neural induction of human ESCs is also increased in the presence of the Alk4,5,7 inhibitor SB431542 that interferes with Activin-, Gdf- and Nodal-signalling apart from Tgfβ. Increased differentiation into the neural lineage is assessed by Pax-6 expression which is accompanied by decreased Bmp-4 and increased Noggin expression. These dynamics point to a contribution of several members of the Tgfβsuperfamily of which the Bmp branch has a major contribution [123], but certainly does not act alone. Of further note, neuronal differentiation of ESC cells is not solely dependent on signalling of the above mentioned members of the Tgfβ-superfamily, since deletion of the common effector Smad4 in ESC does not completely abolish the potential for neuronal differentiation [122]. In contrast, IIS keeps ESCs in a proliferative state and is necessary for selfrenewal. Whether both pathways cross-talk to suppress the respective other one is not investigated so far, but might be a likely scenario, given the connections described in other

FoxO and Smad proteins interact and the FoxO/Smad complex is implicated in transcriptional control in early mouse cortical NPCs, thereby defining one point of intersection of IIS and Tgfβ. Smad2 and 3 transcriptions factors are part of the canonical Tgfβ-signalling pathway and are phosphorylated upon binding of Tgfβ-ligands to the Tgfβ-receptor 1 and 2. The phos‐ phorylated proteins bind to Smad4 and together translocate to the nucleus. This Smad complex interacts with FoxO1, 3a, and 4, three TF of the forkhead family that are under influence of IIS. Upon activation of the Igf-1r, FoxO TF are phosphorylated in a PI3K-Akt-dependent manner. This phosphorylation leads to retention of FoxO proteins in the cytoplasm. Thus, IIS opposes transcriptional control through the Tgfβ-pathway by interfering with translocation of cofactors

While survival of cerebellar granule precursors depends on IIS, control of CGP proliferation indicates cross-talk of IIS and Shh-signalling [34,55,125]. Igf/Shh interaction is reported to occur on various levels. The study of Fernandez et al. (2010) suggests that IIS via Igf-1r increases Shhmediated proliferation, but IIS-mediated proliferation is independent on Shh-signalling. In

proliferation in the developing brain.

60 Trends in Cell Signaling Pathways in Neuronal Fate Decision

cellular lineages.

into the nucleus [124].

As pointed out through the many studies of IIS in the CNS, this signalling pathway has an essential role for development and function of this organ. Albeit studied in many laboratories, our understanding of this central signalling pathway is far from being complete. Still we do not understand how the same biological function is achieved by activating different down‐ stream cascades and why it is necessary to have these multiple pathways that respond to one stimulus. We also do not know why different biological functions are the consequence of IIS at different developmental time points in cells from the same part of the brain. In this light it is of major interest that studies are emerging that focus on cross-talks of various stimuli. Exposure to multiple stimuli at a time probably reflects more the natural environment of a developing and mature neural cell than exposure to one single growth factor. It is also of major interest to dissect different functions during development and to define the molecular players involved into functional switches. In this regard it will be important to dissect different subcomplexes of PI3Ks or Akts that might be implicated in IIS. It will be also needed to carefully analyse the balances between the different branches of the signalling cascades and to identify and close further gaps that might be still in the pathways as they are commonly described today. Given the central role of IIS in body development, growth and maintenance, this knowledge will be of utmost interest and will also be exploited for therapeutic medicine in various contexts.

### **Nomenclature and abbreviations**

CA - Cornu ammonis, CGN - Cerebellar granule neurons, CGP - Cerebellar granule precursor, CNS - Central nervous system, CSF - Cerebrospinal fluid, DG - dentate gyrus, ESC - Embryonic Stem Cell, GH - Growth hormone, IIS - Insulin/Igf-signalling, Igf - Insulin like growth factor 1, Igfbp - Insulin like growth factor binding protein, Ir - Insulin receptor, mTOR - mammalian target of rapamycin, NSC - Neural Stem Cell, NPC - Neural Progenitor cell, OB - Olfactory Bulb, OBSC - Olfactory Bulb stem cell, p - phospho-, SGZ - Subgranular zone, SVZ - Subven‐ tricular zone, TF - Transcription factor.

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