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4 From a Flat Neuroectodermal Sheet to a Complex Three-Dimensional Structure: Morphogenetic Transformations Leading to CNS Shaping

4 From a Flat Neuroectodermal Sheet to a Complex Three-Dimensional Structure: Morphogenetic Transformations Leading to CNS Shaping

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11



Principles of Early Vertebrate Forebrain Formation



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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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As optic vesicles form, telencephalic cells also undergo morphogenetic changes

with at least two differences from eye precursors: they converge fast towards the

midline and polarize later (Rembold et al. 2006). This differential cell migration is

effected, at least in part, by the cadherin-like molecule Nlcam, which is respectively

expressed at high and low levels in the telencephalic and eye precursors (Brown

et al. 2010). Increasing Nlcam expression in the eye field makes its cells converging

towards the midline instead of evaginating, thus behaving like telencephalic progenitors. Polarisation and migration differences between eye and telencephalic cells

are ultimately controlled by the GRN that directs their respective fate. For example,

Rx3, one of the “kernels” for eye specification and morphogenesis, regulates the

expression of pard6 and nlcam (Brown et al. 2010; Ivanovitch et al. 2013), as well

as that of cxcr4a, ephA4/B4 and the Wnt/noncanonical receptor fzd5, therefore

forming a GRN necessary to control the segregation of the eye field from the

surrounding neural plate territories (Fig. 11.3) (Bielen and Houart 2012;

Cavodeassi et al. 2013 and our unpublished results; see also Chap. 9).

Forebrain morphogenesis in organisms others than teleost fishes has not been

analysed in detail mainly due to imaging limitations. It is thus unclear whether

similar mechanisms operate across species. As neural tube formation in birds and

mammals involves neural plate folding instead of rod cavitation, it is possible that

cell specification/polarization/movement occur with a different sequence and certainly at a different pace. Nevertheless, mouse embryonic stem cells have the

spontaneous ability to generate optic cups, when cultured in the presence of the

appropriate factors and in a three-dimensional matrix rich in Laminin1 (Eiraku et al.

2011). This process involves the polarisation of the actin cytoskeleton in the

evaginating cells (Eiraku et al. 2011), suggesting some mechanistic conservation

across species. Our increasing ability to reproduce organ morphogenesis in vitro

constitutes an excellent opportunity to fully understand conserved and divergent

aspects of vertebrate forebrain formation (reviewed in Sasai et al. 2012).



Fig. 11.3 A basic and simplified GRN involved in eye field morphogenesis. The network has

been depicted with BioTapestry software (Longabaugh et al. 2005) using information from

zebrafish. Rx3 is represented as a “kernel” gene that coordinates eye morphogenesis and its

segregation from the telencephalic and diencephalic territories



11



Principles of Early Vertebrate Forebrain Formation



11.5



309



Post-transcriptional Control: The Role of miRNAs



The networks described so far are linked to the transcriptional control of gene

expression. However, there is mounting evidence that post-transcriptional mechanisms are additional essential pieces of the regulatory landscape in both embryonic

development and tissue homeostasis. The mechanism that is currently receiving

most attention implicates the activity of microRNAs, a family of non-coding RNAs.

miRNAs are single-stranded RNAs of around *22 nucleotides, the generation of

which involves the transcription of their respective genes into a primary transcript

that is processed in the nucleus into an approximate 70 nucleotides long

pre-miRNA (Lee et al. 2002). This pre-miRNA is further sequentially processed

into a mature double-stranded RNA by two endoribonucleases, known as Drosha

and Dicer (Hutvagner et al. 2001; Lee et al. 2003). One of the strands of the mature

miRNA is then incorporated into a silencing ribonucleo-protein complex called

RISC (RNA-induced silencing complex) that binds to the complementary seed

sequences present in the 3′ UTR of the multiple target mRNAs (Hammond et al.

2000; Schwarz et al. 2003). Once bound to the target mRNAs, the catalytic activity

of the RISC, known as Argonaute, destabilises the mRNAs or inhibits their

translation (Makeyev et al. 2007; Valencia-Sanchez et al. 2006), thereby controlling

the amount of the corresponding protein finally available in a tissue.

miRNAs show very dynamic expression patterns from early embryogenesis to

adult tissues in most organs and species so far analysed (Kapsimali et al. 2007).

Their function is essential for early development as demonstrated by the evolutionary conserved existence of both maternal and zygotic Dicer transcripts, in the

absence of which the formation of mature miRNAs is abolished. Zygotic dicer null

mutant mice show an embryonic lethal phenotype at gastrulation stages (Bernstein

et al. 2003), whereas in absence of maternal Dicer mouse zygotes do not complete

the first cell division (Tang et al. 2007). In zebrafish instead loss of function of both

maternal and zygotic Dicer leads to abnormal gastrulation and severe alterations in

morphogenesis (Giraldez et al. 2005), whereas zygotic dicer mutants develop

normally until late larva stages, suggesting that the requirement of miRNAs

function during gastrulation is not conserved in teleosts (Wienholds et al. 2003).

About 70 % of all known miRNAs are expressed in the CNS (Diaz et al. 2014),

although most of them seem to play roles at late stages of CNS differentiation

(Kawase-Koga et al. 2009), when the fine regulation of mRNA products is perhaps

most necessary to generate neuronal diversity. A few studies have nevertheless

identified miRNAs involved in early stages of CNS specification, patterning and

morphogenesis. miR-96, miR-290–295 and miR-200 promote ectoderm versus

neuroectoderm fate specification by limiting the amount of the neural TF Pax6 (Du

et al. 2013; Kaspi et al. 2013), which, in turn, activates miR-135b. The latter then

down-regulates TGF-b/BMP signalling and therefore locks neuroectodermal fate

(Bhinge et al. 2014). Components of the Wnt/b-catenin pathway that, as we have

already mentioned, controls all these events, are also conserved miRNAs targets.

For instance, miR-34 targets b-catenin in Xenopus, thereby regulating the



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expression of downstream genes and the correct establishment of axis polarity (Kim

et al. 2011). An additional example of how miRNAs impact on AP patterning is

provided by their role in refining and maintaining the function of secondary

organisers, such as in the case of the MHB. miR-9 is expressed around this organizing boundary, where it targets several transducers of the FGF signalling pathway

(Leucht et al. 2008), limiting its signalling effects. As miRNAs usually bind several

distinct mRNAs, miR-9 seems also to reduce the activity of neurogenic genes at the

MHB, maintaining this territory in an undifferentiated state essential for its function

as an organiser (Leucht et al. 2008).

Quite likely miRNAs participate in the regionalization of the entire forebrain

primordium, but at the moment, most studies have focused on the eye primordium,

which expresses several miRNAs and is severely affected by Dicer inactivation.

Dicer deletion causes microphthalmia (reduction of the eye size) affecting the lens

placode, the neural retina, the pathfinding of the retinal ganglion cell axons as well

as the pigmentation and adhesion of the Retinal Pigment Epithelium (RPE), which,

in turn, affect photoreceptors’ maturation (Conte et al. 2013; Ohana et al. 2015).

Besides the general demonstration that mRNA silencing is relevant for eye specification, knock-down/out studies are beginning to delineate the specific function of

each miRNA in eye formation. Among them, miR-124 and miR-204 are particularly important. miR-124 maintains optic vesicle cell proliferation at early stages of

development by turning off the proneural gene neuroD1. This early function prevents the onset of neurogenesis (Liu et al. 2011). Later on miR-124 promotes

differentiated cone photoreceptor survival by targeting the TF Lhx2 (Sanuki et al.

2011). miR-204 instead modulates the levels of the TF Meis2, which is upstream of

Pax6 in the GRNs controlling morphogenesis and specification of both the lens and

the retina (Conte et al. 2010). Consistent with the general observation that miRNAs

have rather heterogeneous targets, slightly later, miR-204 targets EphB2 and EfnB3

(Conte et al. 2014), a signalling system implicated in retinal ganglion cell axon

pathfinding, as well as effector genes of RPE differentiation (Adijanto et al. 2012).

Many more studies are needed to fully understand how miRNAs contribute to

forebrain development. Nevertheless, it is becoming apparent that many miRNAs

can contribute to the regulation of the same process and also that each miRNA, is

recurrently used during development for different purposes, further contributing to

diversify the GRNs that lead to a mature forebrain.



11.6



Conclusion/Perspectives



In conclusion, in this chapter we have provided a general and simplified view of the

principles that govern early forebrain development. This information derives from a

huge number of studies based on experimental manipulations of gene activity in

different vertebrate species, of which unfortunately we could not give a full account

here. These studies have been facilitated by the sequencing of several genomes,

which also led to the identification of a large number of non-coding RNAs, as well



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