**5. Stage-related TF in mouse telencephalic neurogenesis**

The main TF involved in the early patterning and specification belong to **homeobox domain (HD)** and **basic helix-loop-helix (bHLH)** families, however other TF such as **zinc-finger proteins** also have essential functions.

The main HD containing TF families are: paired-box (e.g. Pax6), forkhead box (e.g. Foxg1), NK2 homeobox (e.g. Nkx2.1, Nkx6.1), orthodenthicle homeobox (e.g. Otx1/*2,*), sine-oculis homeobox (e.g. Six3), GS homeobox *(*e.g. Gsh2*)*, distal-less homeobox (e.g. Dlx1-6), LIM ho‐ meobox (e.g. Isl1, Lhx2, Lhx6, Lhx8), empty-spiracle homeobox (e.g. Emx1/2), T-box: (e.g. Tbr1/*2*). Other TF, such as Mash1, Ngn1/2 and Olig2, belong to bHLH class. Gli members are zinc-finger proteins.

#### **5.1. Early A/P patterning**

The telencephalic neuroepithelium is first characterized by the expression of **FoxG1** (also named BF1) [53], **Pax6** [54;55] and **Gli3** [56]. The anterior phenotype also expresses **Six3, Otx1** and **Otx2** [57-59].

#### **5.2. Dorsal/ventral patterning**

In the mouse **dorsal** embryonic telencephalon, Pax6, Emx1 and Emx2 are specifically ex‐ pressed in VZ and SVZ progenitor domains (Figure 2A).

Pax6 which is essential for setting up the sharp border between ventral and dorsal telencepha‐ lon is mainly expressed in the prospective neocortex, while Emx1 and Emx2 are medially ex‐ pressed in the archicortex (later hippocampus); Lhx2 is expressed in both regions [30;31;45;60;61], Tbr2 is expressed in the SVZ corresponding to basal progenitor domains [62;63].

Nkx2.1 expression is the hallmark of the MGE development. At E9.5, Nkx2.1 appears within the **ventral** telencephalic domain, defines the MGE at the molecular level and per‐ sists in this region throughout development (Figure 2A). Around E10.0 the expression of Gsh2 accompanies the emergence LGE and further to CGE, with a lower expression level in MGE (Figure 2A). Nkx6.2 is expressed along the MGE/LGE sulcus and at high levels in the dMGE [1;64].

The mutual antagonism between Pax6 and Nkx2.1 and later on between Pax6 and Gsh2 is required for the correct positioning of the D/V boundary [65-67]. FoxG1 is involved in both ventral and dorsal patterning [27;30;32;42;43;52].

The central mechanism that determines NP D/V patterning is the activity of the Gli family of transcriptional regulators–Gli1, Gli2, and Gli3. SHH promotes the formation of a ventral tel‐ encephalic subdivision by inhibiting the dorsalizing effects of Gli3 [27;35;56;68]. Gli3 is high‐ ly expressed dorsally, with lower expression in the LGE and MGE. Gli1 is expressed ventrally, at high levels in the progenitor domain of the dMGE and vLGE, whereas Gli2 is highly expressed in the progenitor domain of the dorsal telencephalon, with a lower expres‐ sion in the LGE (Figure 2B).

**Figure 2.** The domains of the main transcription factors implied in dorsal-ventral patterning in mouse embryonic telencephalon.

#### **5.3. Neuronal specification**

late neuronal specification and maturation of different subpopulations [52]. Activin is also a potent neurotrophic factor that induces differentiation of telencephalic neural precursors in‐

The main TF involved in the early patterning and specification belong to **homeobox domain (HD)** and **basic helix-loop-helix (bHLH)** families, however other TF such as **zinc-finger**

The main HD containing TF families are: paired-box (e.g. Pax6), forkhead box (e.g. Foxg1), NK2 homeobox (e.g. Nkx2.1, Nkx6.1), orthodenthicle homeobox (e.g. Otx1/*2,*), sine-oculis homeobox (e.g. Six3), GS homeobox *(*e.g. Gsh2*)*, distal-less homeobox (e.g. Dlx1-6), LIM ho‐ meobox (e.g. Isl1, Lhx2, Lhx6, Lhx8), empty-spiracle homeobox (e.g. Emx1/2), T-box: (e.g. Tbr1/*2*). Other TF, such as Mash1, Ngn1/2 and Olig2, belong to bHLH class. Gli members are

The telencephalic neuroepithelium is first characterized by the expression of **FoxG1** (also named BF1) [53], **Pax6** [54;55] and **Gli3** [56]. The anterior phenotype also expresses **Six3,**

In the mouse **dorsal** embryonic telencephalon, Pax6, Emx1 and Emx2 are specifically ex‐

Pax6 which is essential for setting up the sharp border between ventral and dorsal telencepha‐ lon is mainly expressed in the prospective neocortex, while Emx1 and Emx2 are medially ex‐ pressed in the archicortex (later hippocampus); Lhx2 is expressed in both regions [30;31;45;60;61], Tbr2 is expressed in the SVZ corresponding to basal progenitor domains

Nkx2.1 expression is the hallmark of the MGE development. At E9.5, Nkx2.1 appears within the **ventral** telencephalic domain, defines the MGE at the molecular level and per‐ sists in this region throughout development (Figure 2A). Around E10.0 the expression of Gsh2 accompanies the emergence LGE and further to CGE, with a lower expression level in MGE (Figure 2A). Nkx6.2 is expressed along the MGE/LGE sulcus and at high levels

The mutual antagonism between Pax6 and Nkx2.1 and later on between Pax6 and Gsh2 is required for the correct positioning of the D/V boundary [65-67]. FoxG1 is involved in both

to calretinin-positive cortical interneurons [44].

222 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**proteins** also have essential functions.

zinc-finger proteins.

**5.1. Early A/P patterning**

**Otx1** and **Otx2** [57-59].

[62;63].

in the dMGE [1;64].

**5.2. Dorsal/ventral patterning**

pressed in VZ and SVZ progenitor domains (Figure 2A).

ventral and dorsal patterning [27;30;32;42;43;52].

**5. Stage-related TF in mouse telencephalic neurogenesis**

The mechanisms of neuronal specification in the dorsal telencephalon have been extensively studied in the context of cerebral cortex development. The dorsal progenitors produce neu‐ rons, in a tightly controlled temporal order from E10.5 to E17.5. Pax6, Ngn1 and Ngn2 in‐ struct **glutamatergic** identity and inhibit astroglial differentiation [69-72]. The differentiation by Ngns involves the sequential activation of the expression of other TF such as NeuroD, Tbr1 and Tbr2 [69]. NeuroD has been implicated in the terminal differentiation of the hippo‐ campus [73]. The differentiation of specific populations of projection neurons is controlled by neuronal subtype-specific genes, which have only begun to be identified. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells, with differ‐ ent locations [74].

The earliest born neurons form a layered structure termed the preplate, which is later split into the superficial marginal zone and the deeply located subplate. The cortical plate, which will give rise to six-layered neocortex, begins to develop between these two layers. The later born neurons arriving at the cortical plate migrate past earlier born neurons [5;74]. During development, neurons in different layers are generated in an inside-first, outside-last order, and newly postmitotic neurons are specified to adopt the laminar positions characteristic of their birthdays [5;24;75].

Mash1 is the main neurogenic TF in the ventral telencephalon and is involved also in the neurotransmitter identity specification, being a selective instructor of **GABAergic** identity [70;76-82]. Olig1/2 can promote both neuronal and oligodendroglial fates while inhibiting astrogliogeneis [83].

of neural induction and telencephalic patterning have been reported during the follow‐

Telencephalic Neurogenesis Versus Telencephalic Differentiation of Pluripotent Stem Cells

http://dx.doi.org/10.5772/54251

225

Blocking the WNTs and BMPs pathways by applying the antagonists DKK1 and BMPR1A-FC, respectively, cause neural induction in more than 90% of mES cells and maintained around 35% of the progenitors as telencephalic, expressing FoxG1 [101]. Oth‐ er following studies has reported a massive FoxG1 expressing telencephalic progenitor generation in serum-free, RA-free conditions [96-98;100], while using a RA treatment,

SHH or a Hh agonist treatment on telencephalic progenitors suppresses the dorsal marker Pax6 and induced the ventral marker Nkx2.1. SHH application does not cause substantial

The majority of mES cell-derived progenitors exhibit by default a ventral phenotype which has been attributed to the high level of endogenous SHH signaling. Blocking the Hh signaling converts most of the ventral telencephalic precursors into dorsal progeni‐ tors, with the majority expressing typical markers of the dorsal telencephalon*:* Pax6 and

The telencephalic progenitors derived from mES cells can be directed to neurons that express the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter

The dorsal progenitors have been shown to produce mature neurons with many features of cortical pyramidal neurons in a temporal manner similar with *in vivo* corticogenesis. The first neurons generated in these cultures are reelin-positive Cajal-Retzius-like and subplatelike neurons expressing Tbr1, followed by the glutamatergic neurons generated in an insidefirst, outside-last manner. The majority of the cells generated by Gaspard *et al*. express markers of deep cortical layer V and VI neurons, like Tbr1, Otx1, Ctip2 and FoxP2 [102]. Eir‐ aku *et al.* have also generated deep layer neurons, positive for Ctip2 and Emx1 in the first 9

By transplantation of dorsally patterned progenitors into postnatal murine cerebral cortex, the production of cortical projection neurons with the correct morphology and axonal con‐ nectivity has been demonstrated [98]. The pyramidal neurons express Otx1, Emx1, and Ctip2, corresponding to deep layers neurons. They integrate and appropriately project long-

Regarding the protocols for ventral neuronal specification, it has been shown that Nkx2.1 and Gsh2 expressing progenitors give rise both to GABAergic and cholinergic neurons

ing years [96;100].

Emx1/2 [3;97;99].

GABA [3;102-106].

[3;100;101].

**6.3. Neuronal specification**

FoxG1 expression has not been detected [98].

difference in the level of Gsh2 expression [100;101].

days of neuronal differentiation of dorsalized progenitors [96].

distance axons to subcortical targets, without forming tumors [98].

**6.2. Dorsal/ventral patterning**

Dlx genes (Dlx1, 2, 5 and 6) are expressed in ventral progenitors and neurons in MGE, LGE and CGE, and are likely to play a role in neural specification [78;84;85].

Interestingly, MGE, LGE and CGE progenitor domains do not give rise to homogeneous populations of neurons, which is most likely due to a further subdivision of these domains into regions with spatially restricted expression of specific TF.

There is also a temporal control of the specification of various neuronal subtypes; as a gener‐ al pattern, the earlier- born ventral cells give rise to projection neurons while the more dor‐ sally positioned later-born cells generate interneurons [24;82;86-88].

MGE is characterized by the early production of cholinergic projection neurons from its ventral part, followed by the late production of GABAergic and cholinergic interneurons from the dorsal domains. Two TF are detected at E12.5 exclusively in the MGE: Lhx6 and Lhx8 (or Lhx7) [89], suggesting a role in the specification of MGE-derived neurons. Lhx6*-*expressing neurons have the characteristics of proto-GABAergic neurons with dual differentiation potential, while Lhx8 seems to be involved in the differentiation of specif‐ ic cholinergic neurons [15;90]. The differentiation of a common proto-GABAergic precur‐ sor into mature subtypes is regulated by the combinatorial activity of the Lhx6, Lhx8 and Isl1. Those proto-GABAergic neurons that maintain the expression of Lhx6 differen‐ tiate into mature GABAergic striatal interneurons. By contrast, induction of Isl1 and the combined activity of Lhx7/8 and Isl1 results in down-regulation of Lhx6 and commit‐ ment along the cholinergic interneuron sublineage [90-92]. Thus, it appears that a LIM HD transcriptional code determines cell-fate specification and neurotransmitter identity in neuronal subpopulations of the ventral telencephalon.

Ventral LGE generate GABAergic projection neurons that also express Isl1 during early specification, followed by the expression of other striatal-specific TF such as FoxP1, FoxP2, and Ctip2 [93-95]. Later in development, dLGE generates interneurons that migrate to the olfactory bulbs [13;95].
