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3 GRNs Underlying the Acquisition of Telencephalic, Retinal, Hypothalamic and Diencephalic Identities

3 GRNs Underlying the Acquisition of Telencephalic, Retinal, Hypothalamic and Diencephalic Identities

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F. Cavodeassi et al.

Kiecker and Lumsden 2012; Vieira et al. 2010). For instance, Fgfs promote midbrain identity in the abutting anterior neural region, thanks to the expression of

Otx2, which provides the prospective midbrain with competence to acquire its

identity (Martinez-Barbera et al. 2001). The same Fgf signals instead favour the

generation of the cerebellar region in the neural tube posterior to the MHB, because

this is primed for cerebellar identity by the expression of Gbx2 (Martinez-Barbera

et al. 2001) and Irx2 (Matsumoto et al. 2004; Rodriguez-Seguel et al. 2009).

In addition to this fundamental function, signals emanating from the MHB

contribute to forebrain patterning, in part by setting the next organizer, known as

zona limitans intrathalamica (ZLI), at the boundary between the Six3 and Irx3

expression domains, respectively repressed and activated by Wnt/ßcatenin activity

(Braun et al. 2003; Kobayashi et al. 2002; Lagutin et al. 2003). The interphase

between these two TFs is critical to establish where the ZLI forms as shifting the

Six3-Irx3 boundary changes the ZLI position accordingly (Braun et al. 2003;

Kobayashi et al. 2002). Nevertheless, recent studies have shown that other genes,

such as Otx1, Fez and Fezl, also under the influence of the MHB signals, participate

in the induction and positioning of the ZLI (Hirata et al. 2006; Jeong et al. 2007;

Scholpp et al. 2007). Once established, the ZLI becomes a source of the morphogen

Shh, which refines the pattern of the diencephalon, the most caudal of the forebrain

regions (Fig. 11.1d) (Kiecker and Lumsden 2012).

The most anterior territories of the forebrain, the eye field and the telencephalon,

are instead under the influence of the most rostral secondary organiser, known as

the anterior neural boundary (ANB), located at the anterior border of the neural

plate. Its establishment depends on global AP patterning signals, as it occurs for the

MHB. However, in this case Wnts are not sufficient, and very precise thresholds of

Bmp activity, released by the surrounding non-neural ectoderm, are important for

ANB positioning (reviewed in Cavodeassi and Houart 2012). The ANB is the

source of secreted Wnt antagonists from the Sfrp family. Repression of Wnt/

ßcatenin activity by Sfrps is required for the induction of the telencephalon and eye

field at the most anterior portion of the neural plate. Indeed, ectopic expression of

the Sfrp family member Tlc results in expansion of telencephalic fates and, conversely, its absence leads to anterior truncations (Houart et al. 2002).

Besides by antagonists-mediated repression, inhibition of Wnt/ßcatenin activity

in the rostral ANP is reinforced by transcriptional repression exerted by Six3 and

Hesx1, both specifically expressed in the telencephalon, retinal and hypothalamic

fields. Six3 directly represses ligands of the pathway, such as Wnt1 and Wnt8b,

making the anterior forebrain a “Wnt-free” region (Lagutin et al. 2003; Liu et al.

2010). Hesx1, together with TCF3, instead acts downstream in the pathway by

maintaining Wnt/ßcatenin targets in a repressed state (Andoniadou and MartinezBarbera 2013). In addition, diencephalic derived Wnt11 in zebrafish and Wnt4 in

Xenopus activate a ßcatenin-independent pathway in the eye field. This pathway, in

turn, antagonises Wnt/ßcatenin signalling, thereby refining the boundary between

the eye field and the diencephalon (Cavodeassi et al. 2005; Maurus et al. 2005).

The ANB is also a source of Fgfs, which are important only slightly later for the

maintenance and differentiation of the telencephalic fate (Rubenstein et al. 1998).


Principles of Early Vertebrate Forebrain Formation


In summary, Wnt/ßcatenin signalling has a fundamental role in the specification

of the posterior neural plate including the caudal part of the forebrain, the diencephalon, whereas its repression is critical for the combined patterning of the telencephalon, eye and, likely, hypothalamus. An outstanding question is what are the

mechanisms that promote the segregation between the telencephalon and the retinal

or the retinal and hypothalamic fields. An important advance in this respect has been

made in the zebrafish. In this species, specific levels of Bmps, expressed in the

surrounding non-neural ectoderm, are required to promote telencephalic fate at the

anterior edge of the neural plate and, at the same time, restrict the retinal fate to more

medially located portions of the ANP (Bielen and Houart 2012). Whether there are

other mechanisms contributing to this segregation and how these are coordinated

with the establishment of the diencephalon is still unclear. In a speculative view,

Sfrp1, belonging to a family of proteins that regulate Wnt, Bmp as well as Notch

signalling with different mechanisms (Bovolenta et al. 2008; Esteve et al. 2011a, b;

Kobayashi et al. 2009; Lee et al. 2006), might be an interesting candidate that could

be further investigated, as knock-down of Sfrp1 affects the eye field with a parallel

expansion of the telencephalic region (Esteve et al. 2004). The second question of

how the retinal and hypothalamic fates become separated has hardly been addressed,

as such. Nevertheless, Shh emanating from underlying axial mesoderm, the prechordal plate, is likely fundamental to establish this distinction, as in its absence,

hypothalamic induction does not occur (Blaess et al. 2014), whereas retinal field

cells, albeit abnormal, are still present (Marti and Bovolenta 2002).

Independently from the signals that contribute to segregate retinal progenitors

from the adjacent telencephalic and hypothalamic progenitors, much work has been

done to identify the transcriptional network underlying this specification (see also

Chap. 9 from Martinez-Morales). The combinatorial expression of the TFs

including Rx, Six3, Six6, Pax6 and Lhx2, is sufficient to form ectopic eye-like

structures in Xenopus, but only in the Otx2-positive neuroepithelium (Zuber et al.

2003), indicating that Otx2 confers the necessary competence for the onset of eye

development. This idea is supported by the observation that addition of Otx2 to the

above factors induces ectopic eye-like structure even in the trunk of the embryos

(Viczian et al. 2009).

Notably, this core of genes implicated in eye formation are either broadly

expressed in the forebrain, as Otx2 or Pax6, or expressed in at least two of its

regions, as in the case of Rx and Six6 in the eye and hypothalamus. We have already

discussed that the differential integration of the same gene in a specific sub-circuits

of a GRN is a key element to drive the differentiation of a group of cells towards a

fate different from that of the neighbouring cells. Increasing evidence however

indicates that the level of gene expression or dose/time of exposure to a given signal

are also important to generate different outcomes in abutting territories (Beccari

et al. 2013; Kutejova et al. 2009). In the teleost medaka, Six3.2 expression, for

example, is regulated by a network of TFs (Beccari et al. 2012, 2015), which

generates its graded distribution across the ANP. This difference is fundamental for

forebrain patterning: high levels of Six3.2 promote telencephalic development,

whereas lower levels favour retinal formation (Beccari et al. 2012). This graded


F. Cavodeassi et al.

Fig. 11.2 Schematic representation of the main GRN leading to forebrain patterning. The diagram

described in the text has been depicted using the BioTapestry software (Longabaugh et al. 2005).

This basic network patterns the vertebrate anterior neural plate (orange) into telencephalon, eye,

diencephalon and hypothalamus. The non-neural ectoderm (purple), the midbrain and the posterior

neural ectoderm (green), which cooperate in forebrain patterning, have also been included in the


expression, in turn, is maintained by a similar graded distribution of the pan-neural

determinant Sox2, so that raising or lowering the levels of either Sox2 and Six3.2

changes the proportion between telencephalic and retinal fields (Beccari et al.

2012). Similar observations apply for the expression levels of two other members of

the medaka Six family of TFs, Six3.1 and Six6, the dosage of which is critical for

setting the size balance between retinal and hypothalamic territories (Beccari et al.,


Figure 11.2 illustrates the main regulatory interactions happening during the

specification of the forebrain primordium, which we have described so far. We have

maintained this network intentionally simple, but additional detailed information has

been recently summarized in related reviews (Beccari et al. 2013; Nord et al. 2015).


From a Flat Neuroectodermal Sheet to a Complex

Three-Dimensional Structure: Morphogenetic

Transformations Leading to CNS Shaping

As mentioned in the introduction, during CNS development the acquisition of

specific cell fates is tightly linked to the generation of an accurate three-dimensional

architecture. At early developmental stages, when the neuroectoderm is still naïve,

the shape and organization of neuroepithelial cells is rather similar along the whole

extent of the neural plate. However, with progressive patterning, each CNS region


Principles of Early Vertebrate Forebrain Formation


acquires a specific shape and organization, largely under the control of the same

fundamental GRNs that implement regional fate.

At the beginning of CNS morphogenesis the flat and naïve neuroectoderm

extends along the AP axis and, at the same time, its cells compact and intercalate

medio-laterally. This process is known as “convergent extension” and culminates

with the formation of the neural plate (reviewed in Lowery and Sive 2004), which

then folds and gives rise to the neural tube. In mammals and birds, this folding

normally occurs with a specific sequence and involves the apical constriction of

neural plate cells at specific points, which favours the longitudinal bending of the

neural tube. Both convergent extension movements and neural plate folding require

the Wnt/planar cell polarity pathway (PCP), which controls similar processes in

other embryonic tissues. In the absence of Wnt/PCP function, convergent extension

is severely impaired, resulting in a wider neural plate that cannot fold (reviewed in

Sokol 2015). This is because, normally, the Wnt/PCP pathway promotes an

actomyosin-dependent contraction of neuroepithelial cells trough the upregulation

of Rho kinase at the apical adherens junctions, which ultimately favours folding and

bending of the neural plate (Nishimura et al. 2012).

In teleost fishes neural tube formation is mechanistically different, because

neural plate cells compact at the midline and form a neural rod. Neuroepithelial

cells in the rod then undergo cell division at the midline, after which the daughter

cells rearrange at both sides of the midline and orient their apical sides towards the

centre of the rod, establishing the apical (luminal) side of the forming neural tube

(Ciruna et al. 2006; Tawk et al. 2007). The molecular mechanisms involved in this

neuroepithelial condensation and lumen formation are currently unclear, but likely

involve the control of cytoskeleton dynamics and extracellular matrix remodelling

(Araya et al. 2014; Buckley and Clarke 2014; Clarke 2009).

While most of the neural plate gives rise to an almost straight tube, the anterior

part of the neural primordium undertakes a more complex reorganisation according

to the convoluted structure of the brain. The most notable initial rearrangement of

forebrain morphogenesis consists in the bulging of the eye primordia from its lateral

walls. Recent studies have exploited the advantages of teleost fish to image this

event in vivo, showing that, at least in this species, cells fated to become eye

precursors are highly cohesive and strictly segregated from those of the surrounding

domains (Cavodeassi and Houart 2012). Both phenomena, cohesion and segregation, are promoted by the combined function of Cxcr4, the Eph/Ephrin and the

Wnt/noncanonical signalling pathways (Bielen and Houart 2012; Cavodeassi et al.

2005, 2013). As they evaginate, the eye-field cells extensively rearrange, intercalate

among each other (Ivanovitch et al. 2013; Rembold et al. 2006) and, at the same

time, elongate and polarise to establish the tight neuroepithelial structure of the eye

primordia, also known as optic vesicles (Ivanovitch et al. 2013). This acquisition of

apico-basal polarity of the optic vesicle cells, marked by the onset of pard6cb

expression, an apical polarity marker, and by the localised accumulation of

Laminin1 around the eye primordium, is required for an accurate vesicle evagination as interference with polarization events disrupts proper optic vesicle formation (Ivanovitch et al. 2013).

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3 GRNs Underlying the Acquisition of Telencephalic, Retinal, Hypothalamic and Diencephalic Identities

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