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 etal., 1993).
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 1st
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