**2. Telencephalic neuronal cell diversity**

The neurons populating the mature telencephalon are generally classified based on their in‐ trinsic properties: neurochemical profile, morphology and electrophysiological responses. The understanding of how the telencephalic neuronal subtypes are specified encompasses not only the signaling pathways that act in spatial-temporal sequences to confer positional and molecular identity but also the location of the progenitors early in development and the migration pathways they undertake to reach their final destination in the mature brain. The main neuronal types and telencephalic domains in the adult and embryonic mouse brain are schematically presented in Figure 1.

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Schematic mouse mature (left side) and embryonic (right) brain; telencephalic subdivision: olfactory bulb (Ob), neo‐ cortex (Ncx), palleocortex (Pcx), archicortex (Acx), hippocampus (Hi), striatum (St) and globus pallidum (GP), pallium (pink) medial ganglionic eminence (MGE-violet), lateral ganglion eminence (LGE-light green), caudal ganglionic emi‐ nence, (CGE-dark green); domains and origins (as arrows) of telencephalic glutamatergic (red), GABAergic (green) and cholinergic (blue) neurons.

**Figure 1.** The domains of telencephalic neurons in adult and embryonic mouse brain

**cortex -** the most complex structure of the mammalian brain. The medial pallium devel‐ ops into the hippocampal formation (archicortex), cortical hem and the choroid field. The lateral pallium matures into the paleocortex (olfactory and some limbic areas). From the **ventral** telencephalon medial, lateral and caudal ganglionic eminences (**MGE, LGE** and **CGE**) emerge giving rise to the **basal ganglia** and parts of the **amygdala**, but also to neurons that migrate into the cortex and olfactory bulbs. The progenitor domains in the embryonic telencephalon generate specific types of neurons which finally form the com‐

Understanding the developmental ontogeny of the diverse telencephalic neuronal popula‐ tions provides an essential framework for the design of rational approaches towards pluri‐ potent stem cell differentiation for *cellular models* and *cell replacement therapies* for

In the first part, we review the stages of the mouse telencephalic development, the morpho‐ gens and the transcription factors (TF) that are intimately involved in the telencephalic pat‐

In the second part, we present recently reported protocols for differentiation of mouse and human pluripotent stem cells into telencephalic populations, following the development principles and reflecting the *in vivo* signaling pathways; we point on the relevant morpho‐ gens and TF in each stage, where the level of expression of relevant sets of TF can be consid‐

Finally, we describe our model system in which the *in vitro* differentiation of human and mouse embryonic stem (ES) cells are temporally aligned to each other and compared with mouse telencephalic neurogenesis *in vivo* [3]. Since the telencephalic development has been extensively studied in animal models, it is important to strengthen the interspecies compara‐ tive approaches in order to gain further insights into the human telencephalic development. We provide evidence for differences in the default differentiation of mouse and human plu‐ ripotent stem cells that proves the utility of the comparative system for optimizing the di‐ rected telencephalic differentiation of human pluripotent stem cells. We also exemplify how Hedgehog (Hh) signaling pathway is implied in telencephalic neuronal fate decision *in vitro*

The neurons populating the mature telencephalon are generally classified based on their in‐ trinsic properties: neurochemical profile, morphology and electrophysiological responses. The understanding of how the telencephalic neuronal subtypes are specified encompasses not only the signaling pathways that act in spatial-temporal sequences to confer positional and molecular identity but also the location of the progenitors early in development and the migration pathways they undertake to reach their final destination in the mature brain. The main neuronal types and telencephalic domains in the adult and embryonic mouse brain are

ered as a milestone between each differentiation step *in vitro* [2] (**Sections 6-8**).

plex neural networks of the mature telencephalon.

218 Trends in Cell Signaling Pathways in Neuronal Fate Decision

terning and neuronal subtype specification (**Sections 2-5**).

telencephalic related diseases.

and *in vivo* (**Section 8**).

**2. Telencephalic neuronal cell diversity**

schematically presented in Figure 1.

**Glutamatergic projection (pyramidal) neurons** comprise the majority (70-80%) of cortical neurons, they are generated in the dorsal telencephalon; have an excitatory role in the corti‐ cal and include many subtypes. Each subtype is characterized by a specific combination of laminar position, morphology, marker expression and connectivity pattern [4;5].

Cajal-Retzius neurons are a transient population expressing reelin and playing a key role in the formation of the cerebral cortex. They die during the first postnatal week [6].

**GABAergic neurons** are generated in the ventral telencephalon and also include many sub‐ types of *interneurons* and *projection neurons*.

GABAergic cortical interneurons, comprising 20-30% of the cortical neurons, have an inhibi‐ tory role in the cortical circuits; they originate in the ventral telencephalon and subsequently migrate dorsally into the developing cortex.

Other types of GABAergic neurons include the interneurons and projection neurons that populate the striatum, pallidum, olfactory bulb and other forebrain ventral regions.

Different subclasses of GABAergic interneurons arise from different progenitor domains in the subpallium: *somatostatin (Sst)* subclasses of GABAergic interneurons that ultimately re‐ side in the cortex and the basal ganglia are generated in dorsal MGE (dMGE) [7;8]. *Parvalbu‐ min (Pv)* subclasses of GABAergic interneurons, constituting the majority of the cortical interneurons, are generated in ventral MGE (vMGE) [9;10]. *Calretinin (Carl), NPY and reelin*expressing GABAergic cortical interneurons are produced primarily in CGE [11;12]. Calr ex‐ pressing GABAergic interneurons, which ultimately reside in the olfactory bulbs and amygdala, arise from the dorsal LGE (dLGE) [13].

GABAergic projection neurons, such as the medium spiny neurons (MSN) which constitute the majority of the striatal neurons, express DARPP32 and Calr and arise from the ventral LGE (vLGE) [13;14].

**Cholinergic** neurons in the telencephalon (both *interneurons* and *projection neurons*) are gen‐ erated in the MGE. First, cholinergic projection neurons are produced by vMGE, followed by the production of cholinergic interneurons from dMGE, at later time points. Cholinergic interneurons populate the striatum; cholinergic projection neurons populate the pallidum and the septum and project mainly to neocortex and hippocampus, respectively [15].

Wingless/INT proteins (WNTs), transforming growth factors (TGFs) and retinoic acid (RA). They are secreted from specific centers, named organizers, during early stages of

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Genetic evidence based on loss- and gain-of-function studies have indicated that the role of these morphogens can be rather complex. Depending on the developmental stage it ranges from establishment of general patterning characteristics to neuronal specification

A/P patterning starts to emerge in parallel with neural induction, prior to and during gas‐ trulation. At embryonic day (E) 8.5, in regions of the embryo protected from the influence of caudalizing factors, such as WNTs, BMPs, and RA, or where their antagonists are secreted, such as Dickkopf1 (DKK1), an inhibitor of the WNT signaling pathway, and Noggin, an in‐ hibitor of the BMP signaling pathway, the NE cells develop an anterior character and form the prospective forebrain (future telencephalon and diencephalon) [28;33-35]. FGFs (e.g. FGF8, FGF15, FGF3) are expressed early on at the anterior tip of the neural plate and then maintained in the anterior limit of the neural tube [32]. Although not a primary inducer of the telencephalic fate, FGF signaling influences the telencephalic gene expression [32;36].

With regard to the location and timing of telencephalic progenitor generation, different ex‐ trinsic factors are involved in their patterning and self-renewal. WNTs and BMPs pattern the telencephalic progenitors dorsally, while SHH patterns them ventrally. BMPs are ex‐ pressed dorso-medially and are required for the formation of the choroid plaque and the cortical hem [31;37;38]. WNTs are secreted from the cortical hem and promote the develop‐ ment of the hippocampus [30]. The expression of SHH is first observed at E8.5 in structures adjacent to the ventral telencephalon, and by E9.5 in the MGE and preoptic regions [29]. SHH promotes the formation of all ventral telencephalic subdivisions [29;39-41]. FGFs are involved in both ventral and dorsal patterning [27;30;32;42;43]. Activin, a TGF-related mole‐ cule, acts ventrally in the CGE patterning [44]. RA contributes to the patterning of the lateral

The balance between the signaling inputs that control NP self-renewal and differentiation is critical for the initiation of the terminal differentiation program. FGFs and SHH, in addition to their patterning activities, promote self-renewal and prevent differentiation, while RA promotes neuronal differentiation [1;47]. Notably, it has been shown that the expression of SHH is required during distinct developmental windows for the specification of neuronal identity [29]. FGF signaling may ultimately influence the generation of cell diversity within the ventral telencephalon [30;50]. WNT promotes neuronal differentiation in different late cortical progenitor cell populations [51]. BMPs inhibit neurogenesis but could participate in

telencephalon and participates in setting-up the D/V boundary [45-49].

development [28].

[5;21;23;26;29-33].

**4.2. D/V patterning**

**4.3. Neuronal specification**

**4.1. Early A/P patterning**
