Most of these defects have been supposed to be

60

DISCUSSION

Eye morphogenetics events are highly dependent upon precisely coordinated interactions between different tissues, such as neuroectoderm, neural crest-derived- POM, mesoderm and surface ectoderm (Adler and Canto-Soler 2007) (Gage and Zacharias 2009).

A role of 5-HT during morphogenesis has long been suggested on the basis of malformations caused by exposure of cultured mouse embryos to selective 5-HT reuptake inhibitors like fluoxetine (Prozac) and 5-HT receptors antagonists (Shuey et al., 1992; Shuey et al., 1993; Moiseiwitsch, 2000). In particular, mouse embryos treated with ritanserin, a 5-HT2R class specific antagonist, presented strong growth retardation, underdeveloped forebrain and hindbrain, hypoplastic pharyngeal arches and cardiac defects (Choi et al., 1997).

Most of these defects have been supposed to be

dependent on 5-HT2B receptor inactivation because 5-

HT2B mRNA is expressed in the affected tissues and

only other antagonists of this class with high affinity for

5-HT2B receptor gave similar phenotypes. 5-HT2B KO

mice die early during development because of heart

abnormalities, demonstrating the importance of this

receptor during cardiac morphogenesis but its role during

development is wide and still open.

61 During my thesis work I investigated the role of 5-HT2B in ocular morphogenesis through a loss of function approach in Xenopus laevis.

1.1 5-HT2B role during ocular morphogenesis

In all vertebrates the neural crest is a pluripotent, mesenchymal population which emerges from the dorsal neural tube and migrates in a conserved and characteristic pattern of three distinct streams termed mandibular, hyoid and branchial, into the 1st, 2nd, 3rd and 4th pharyngeal arches respectively where it gives rise to different derivatives including nerves, ganglia, melanocytes, as well as craniofacial skeleton (Couly et

In particular a NCCs subpopulation that migrates in the first pharyngeal arch contributes to the formation of the POM that influences the correct eye morphogenesis.

To contribute to ocular morphogenesis the cranial NCCs component of the POM respond to patterning cues provided by extrinsic factors and by their environment.

In such a complex scenario, my study provides novel

insights into the molecular mechanisms underlying

ocular morphogenesis. By using a loss of function

approach in Xenopus embryos I demonstrated in vivo, for

the first time, the capacity of serotonin, via 5-HT2B

receptor, to act as a morphogen during ocular

development by influencing post-migrated 1

pharingeal

62 arch NCCs behavior. We found that 5-HT2B abrogation causes ocular defects such as the failure of the optic fissure closure.

These results have developmental implications for understanding the control of vertebrate eye morphogenesis.

1.2 5-HT2B loss of function results in ocular defects due to the NNC component of the POM

The 5-HT2B depletion resulted in a morphological defect during ocular morphogenesis characterized by the failure of the optic fissure closure. This phenotype named coloboma is specific and reproducible.

The 5-HT2B loss of function also resulted in a change of

extra-ocular muscles connectivity but not in their

specification. Since it has been demonstrated that the

cranial neural crest controls the patterns of muscle

arrangement by forming the connective tissue of

individual muscles (Noden, 1986), these data suggest that

5-HT2B abrogation may affect only the NCCs

component of the POM. That the POM mesoderm

component is not affected in the 5-HT2B morphants is

supported by in situ hybridization experiments using Fli

as a marker. In fact, the fli gene is expressed in

mesodermal derived POM cells which migrate inside the

optic fissure to form retinal blood vessels (Etchevers et

al. 2001) and we did not observe any defect in the

63 migration pattern of these cells in the morphants. Since the coloboma phenotype can arise as a consequence of impaired dorso-ventral patterning of the eye (Chang et al.

2006) and mutations in genes such as Pax2, a marker of the optic stalk, and Vax2, a marker of the ventral retina (Take-uchi et al. 2003) cause coloboma in humans (Evans et al. 2004), I

examinened a possible role of 5-HT2B abrogation on the expression of these genes. No changes in their expression domains were detected in the injected embryos, suggesting that these regions are correctly specified.

We also analyzed the optic nerve to see whether 5-HT2B receptor abrogation could influence its arrangement. The optic nerve originates from the brain such as other cranial nerves but POM influences its correct elongation. In the morphants the optic nerve is correctly specified but it appears shorter compare to the wild type.

1.3 5-HT2B signaling interferes with the retinoic acid metabolism in the developing eye

The NCCs component of the POM is a retinoic acid

(RA) target in the developing eye (Matt et al. 2005). RA

is a metabolite of Vitamin A and its metabolism has a

fundamental role in visual function. It also plays crucial

roles in ocular morphogenesis during embryonic

development. Previous works suggested a possible

64 functional relationship between 5-HT2B receptor and RA signaling during embryogenesis(Choi et al., 1997; Lauder et al., 2000). Bhasin and colleagues (2004) established that even if 5-HT2B promoter contains several potential retinoids response elements, RA does not regulate 5- HT2B transcription but they showed that 5-HT2B receptor and RA promotes and inhibits proliferation in mouse frontonasal mass respectively, further supporting their hypothesis that RA and 5-HT2B signals may act as opposing signals for common cellular mechanisms.

Therefore, I focused my attention on a possible interaction between 5-HT2B receptor and retinoic acid signaling during eye morphogenesis. Raldh3 is the enzyme involved in the synthesis of RA in the ventral retina and it acts on the POM in a paracrine manner (Gage and Zacharias, 2009; Lupo et al., 2011). In situ hybridization experiments showed an enlargement of Raldh3 expression domain in the morphants. Realtime qPCR experiments confirmed a 6-time increase of Raldh3 expression level in the injected embryos. In the morphants other key genes involved in RA metabolism such as Rdh10 and Dhrs3a were regulated in a manner compatible with an increase of RA. On the whole these results point to an increase of RA production in the ventral retina as a consequence of abrogation of 5-HT2B signaling.

Also key POM genes, known to be regulated in a positive

manner by Retinoic Acid such as Pitx2 and FoxC1,

present a higher expression level in the morphants. On

65 the basis of these results, I can suggest a cross-talk between 5-HT2B and RA signaling during eye morphogenesis.

1.4 5-HT2B abrogation alters the migration of the NC

POM cells in the eye

A first demonstration of the role of serotonergic signaling in cranial NCCs early development has been provided by Moiseiwitsch and Lauder in 1995. By means of in vitro assays these authors demonstrated that 5-HT produces a dose-dependent inhibition of migration. In particular they pointed out that these effects are mediated by 5-HT1A receptor (Moiseiwitsch and Lauder, 1995).

Therefore I investigated whether the 5- HT2B receptor loss of function could act on the migration of the POM NCCs in the ocular districts.

By in situ hybridization experiment with a specific NCCs marker, Twist, we have observed a failure of these cells to migrate into the optic fissure: these cells remain in fact gathered at the borders of the fissure.

Defects of NCCs migration ocular districts have also

been observed in cell trasplant assays. I showed that

when pre-migrating NCCs in which 5-HT2B has been

abrogated were transplanted in wild type embryos, they

migrate correctly in the pharyngeal arches and around the

eye, but failed to complete their migration into the

anterior eye segment and within the choroid fissure.

66

In this PhD thesis I describe the effects of 5-HT2B knockdown in Xenopus ocular morphogenesis.

On the basis of the obtained results I can hypothesize a double role of the 5-HT2B signaling in eye morphogenesis. First, in line with its expression in the retina (De Lucchini et al., 2003), the 5-HT2B receptor signaling appears to be involved in the RA metabolism.

In particular its abrogation causes an enlargement of the Raldh3 gene expression domain and consequently an increase in RA level. Since dis-regulation of the RA signaling in late phases of eye development has been implicated in ocular defects such as coloboma (Lupo et al., 2011) I suggest that the failure of optic fissure closure in 5-HT2B morphants may be due to an indirect action of 5-HT2B signaling on POM by altering RA levels. Although a possible cross talk between these two signaling pathways have been previously suggested during development (Bashin et al., 2004a, Bashin et al., 2004b), these results show for the first time a direct action of a 5-HT signaling on RA synthesis in ocular morphogenesis.

On the other hand the NCCs transplant experiments

suggest an autonomous effect of 5-HT2B receptor on

these cells behavior. As previously shown, the 5-HT2B

receptor mRNA is expressed in cranial NCCs and

especially in the NCCs that migrate in the first

pharyngeal arch and that contribute to the POM (De

67 Lucchini et al., 2005). The 5-HT2B receptor is not involved in the early migration of NCCs from the neural tube (Reisoli et al., 2010), but once they have migrated into the POM surrounding the eye, the autonomous action of the 5-HT2B receptor would direct the final migration of NCCs into the anterior eye segment and within the choroid fissure.

The demonstration that a new signaling pathway such as

the one activated by 5-HT2B may be involved in the

development of the POM that contributes to ocular

structures development could open new lines of

investigations also aimed at understanding the molecular

basis of some ocular diseases.