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