42
RESULTS
1.5-HT2B loss of function during Xenopus laevis ocular morphogenesis
To investigate the effects of a spatial abrogation of the serotonin receptor 2B, I injected only one side of two-cell stage embryos with X5-HT2B oligonucleotide antisense morpholino (5-HT2BMO). The injected side of each embryo was revealed thanks to the co-injection of mRNAs encoding the vital lineage tracers nuclear-β-galactosidase (β-gal) and/or green or red fluorescent protein (GFP or RPF) to assess the correlation between spatial restriction of injected X5-HT2B mRNA and the observed phenotype. Control embryos, injected only with β-gal or GFP mRNAs, developed normally, demonstrating the absence of toxic effects (data not shown). All embryos were allowed to develop till stage 37, when the eyes are correctly formed; they were selected for β-gal or fluorescent protein expression and subsequently analyzed at morphological and molecular level.
1.1 Morphological analysis
As evaluated by in vivo external morphological analysis, loss of function of 5-HT2B receptor resulted in a ventral failure of the optic fissure closure noticeable from stage
43 37 and maintained in the late stages. Moreover the neural epithelium was not uniformly distributed around the neural retina appearing missing ventrally (Fig.3.1). In all the analyzed phenotypes the retina appeared correctly specified. In each analysis, the un-injected sides of the embryos did not present any morphological alterations and were used as negative controls.
Fig.3.1 Effects of 5-HT2B receptor knockdown on Xenopus eye development
Lateral view of injected (A,A’,B,B’) and control (A’’,B’’) side of the same tadpole at stages 37 and 45. The injected side recognized by RFP (A,B) presents the optic fissure that fails to close ventrally (black arrow)
1.2 Abrogation of 5-HT2B causes defects in the optic nerve
In order to verify whether the 5-HT2BMO injected embryos present other phenotypes correlated to the failure of optic fissure closure, I performed a molecular analysis by monitoring the effects of 5-HT2B loss of
44 function on the optic nerve. So I analyzed the optic nerve formation at different stages of development. In situ hybridizations at stage 37 have been performed by using Vax2 and Pax2 molecular markers and they revealed that the optic nerve is correctly specified in the injected side compared to the wild type side(Fig.3.2). At stage 45 I analyzed the optic nerve arrangement by immuno-histochemistry experiments with the monoclonal neurofilament antibody 3A10. Embryos injected with 5-HT2BMO revealed that the optic nerve is shorter compared to the wild type sides of the embryos (66% n=54) (Fig.3.2).
45 Fig.3. Effects of 2 5-HT2B depletion on optic nerve development Lateral view
(A-B’) and frontal(A’’) view of embryos at stage 37 with Pax2 and Vax2 molecular markers. The embryos present sides the optic nerve correctly specified in wild type sides and in injected sides, where the failure of optic fissure closure is present (black and red arrows). (D) Frontal view of embryo at stage 45, immunohistochemistry experiments show a shorter optic nerve in the injected side compare t wild type side. (A’’) Frontal view of embryo hybridizated using Pax2 marker show a shorter optic nerve in the injected side compare wild type side (red harrows)
46 2. 5-HT2B signaling pathway interacts with Retinoic Acid pathway during ocular morphogenesis
2.1 5-HT2B abrogation results in a disregulation of gene expression involved in Retinoic Acid synthesis The morphological analysis of 5-HT2B loss of function embryos revealed a specific defect in the ocular morphogenesis: the failure of the optic fissure closure. In humans this is a common developmental ocular defect that is named coloboma. Therefore I focused my attention on a possible interaction between 5-HT2B and retinoic acid (RA) signaling pathways. RA signaling has in fact been shown to be involved in the optic fissure closure (Matt et al. 2005).
In order to analyze the RA expression domain in the ventral part of the retina in the 5-HT2B morphants, I performed in situ hybridization by using Raldh3 (the enzyme that produces RA in the ventral retina) as a marker. These experiments revealed that in the injected side the Raldh3 expression domain was enlarged with respect to the wild type side (53% n=115) (Fig. 3.3). To quantify the increase of Raldh3 mRNA expression in the injected embryos, I carried out REALTIME qPCR experiments in dissected heads of embryos injected in both blastomeres of two-cell stage. The qPCRs showed a 6-time increase of the Raldh3 expression in the morphants compared with the wild type embryos (Fig. 3.4A).
47 To gain further support to this result I analyzed others key genes involved in RA metabolism by qPCR: Rdh10 and Dhrs3a. In fact, RA levels are strictly regulated by a self-performing RA feedback. Rdh10 encodes a retinol oxidase that metabolizes vitamin A into retinaldehyde which is the immediate precursor of RA (Wu et al., 2002). When RA is present at high levels, Rdh10 expression is lowered by RA itself (Strate et al., 2009). Dhrs3a instead, encodes an enzyme with retinaldehyde reductase activity on retinoids. In particular it reduces retinal to vitamin A and it is a RA-inducible gene which is expressed in a RA-dependent manner functioning as a feedback inhibitor of RA biosynthesis. As expected, embryos, in which 5-HT2B function has been abrogated, showed a significant decrease of Rdh10 mRNA (Fig. 3.4B) and an increase of Dhrs3a mRNA (Fig. 3.4C). On the whole these results point to an increase of RA production in the ventral retina.
48 Fig.3.3 Raldh3 expression in 5-HT2B morphants Lateral view of
embryo at stage 37. Raldh3 molecular marker shows an enlargment of its espression domain in the ventral retina in the injected side (A’) of the embryo where is present a failure of the optic fissure closure compare to the wild type side (A) with the optic fissure correctly closed
Fig.3.4 qPCR analysis of genes involved in Retinoic Acid metabolism Graphics
show as 5-HT2B depletion influences gene expression levels of enzymes involved in RA metabolism. In the morphants (A) Raldh3 and (C) Dhrs3a present a 6-time increase, while (B) Rdh10 decreases compared to wild type embryos which expression
49
has been shown in A,B,C graphs with the blue histogram. *= p>0,05; **= p>0,01
2.2 5-HT2B loss of function interferes with the expression of Pitx2 and FoxC1, key genes involved in ocular morphogenesis
RA produced in the ventral retina acts in a paracrine manner and it is able to direct POM development by regulating two key genes: Pitx2 and FoxC1. These genes are expressed both in NCC and mesoderm derived cells of the POM. By in situ hybridization experiments I analyzed the expression patterns of these genes in the 5-HT2BMO injected embryos. In the control eyes positive stained cells were localized both around the eye and on its surface. In the injected sides stained cells were visible mainly around the eye (Pitx2 34% n=227; FoxC1 32% n=184) (Fig.3.5). To further visualize the pitx2 expression pattern I performed vibratome (Fig.3.6A) and microtome (Fig.3.6B) histological sections on whole mount hybridized embryos.
50 Fig.3.5 Pitx2 and Foxc1 expression patterns in the 5-HT2B morphants Lateral
view of embryo at stage 37 show an altered gene expression pattern of key genes involved in POM development such as Pitx2 (A-A’) and FoxC1 (B-B’). Hybridized cells are mainly localized around the eye in the injected side (A’-B’) while they appear also over the eye surface in the wild side of the embryo (A-B).
Fig.3.6 Pitx2 expression pattern visualized on histological sections
Vibratome (A,A’) and microtome (B,B’) sectioned embryos at stage 37 after Pitx2 in situ hybridization. Cells expressing pitx2 are located in the inner layer the cornea in the wild type side, while they accumulate in the ventral region of the eye in the injected side.
51 To verify whether the number of Pitx2 and FoxC1 expressed cells decreases in the injected side due to an altered apoptosis rate, I performed TUNEL experiments. The results showed a same rate of cell death in both sides of the embryos (Fig.3.7).
Fig.3.7 Apoptosis in 5-HT2B knockdown eyes Lateral view of embryo at stage 37
processed by TUNEL staining. The results don’t present a significant rate of cell death increase in the injected side compare to wild type side. In the injected side the apoptosis rate appear slightly increased as a normal effect of morpholino injection.
Since FoxC1 and Pitx2 expression are regulated by RA I carried out qPCR experiments in dissected heads of embryos injected in both blastomeres of two-cell stage embryos. The results showed a 3,8-time increase of Pitx2 expression levels (Fig.3.8A) and approximately a 2-time increase of FoxC1 expression levels (Fig. 3.8B) in the morphants with respect to the wildtype embryos.
52
Fig.3.8 qPCR analysis of Pitx2 and Foxc1 genes Graphics showing that Pitx2 gene
expression presents a 3,8-time increase in the morphants (A), while FoxC1 gene expression in 5-HT2B depleted embryos increases about 2-time (B) compared to wild type embryos which expression has been shown in A,B,C graphs with the blue histogram. *= p>0,05
3. 5-HT2B loss of function influences NCCs migration during ocular morphogenesis
To better visualize the POM NCCs behavior I performed in situ hybridization using Twist as specific marker of cranial NCCs. These experiments revealed that while in the control side the NCCs migrate neatly inside the optic fissure, in the injected side they accumulate in the ventral part of the eye (45% n=102) (Fig.3.9B).
Dct was used as a marker to visualize the retinal pigmented epithelium (RPE). This marker showed a RPE gap in the ventral part of the retina in the 5-HT2B injected embryo (33% n=150) (Fig.3.9A).
53 To assess whether the widespread loss of function of 5-HT2B receptor is involved in the correct NCC migration I performed a homochronic and homotopic cranial NCCs transplantation assay (Borchers et al.,2000). I co-injected 5-HT2BMO and GFP mRNAs in one blastomere of two-cell stage Xenopus embryos and once they reached neurula stage -when NCCs are specificied but still attached to the neural tube- I transplanted the injected cranial NCCs from a donor neurula to a host wild type embryo at the same stage. I then performed time lapse analysis to visualize NCCs behavior in later developmental stages. This kind of assay allows transplanted NCCs, whose 5-HT2B function was abrogated, to develop in a wild type context. As control Fig.3.9 Molecular
characterization of the NCCs behaviour Lateral
view of embryo at stage 37. (A,A’). A specific marker of NCCs such as Twist reveals that NCCs remain gathered in the ventral part of the eye failing to conclude their migration within the optic fissure in 5-HT2B morphants, while in the wild type Twist marked cells enter the optic fissure(black arrow). (B,B’) Retinal pigmented epithelium marked by Dct hybridization shows a gap in the ventral optic cup in injected embryos
54 experiment, in order to visualize normal NCCs behavior, I transplanted GFP-NCCs from a donor embryo to a host wild type.
The time lapse analysis demonstrates that in the wild type context GFP-NCCs migrate correctly along their routes, and particularly, the NCCs that migrate in the first branchial arch migrate around the eye and enter the optic fissure. At the end of their migration the eye turns out to be covered by NCCs. In the morphants the NCCs transplanted in a wild type context show a correct migration around the eye but a failure to cover the eye and to enter the optic fissure in the further migration. These demonstrate that 5-HT2B receptor is involved in the NCCs correct migration.
3.1 5-HT2B abrogation does not influence structures derived by the mesodermal cells component of the POM The POM is composed by NCCs migrating in the first pharyngeal arch and by mesodermal cells. I analyzed blood vessels and extra-ocular muscles derived by mesodermal cells to verify whether 5-HT2B depletion influenced both components of the POM.
By in situ hybridization using Fli as endothelial cells marker I was able to visualize blood vessels that enter the optic fissure in hystological sections of stage 37 embryos. No appreciable differences between injected and wild type side were revealed (Fig.3.10).
55 The extra-ocular muscles visualized by immunohistochemistry experiments are present and correctly specified in both sides of the embryo, but in the 5-HT2BMO injected side they showed a disorganized attachment to the orbital cartilages (34% n=53) (Fig.3.11). Since NCCs give rise to the connective tissue surrounding the muscles (fascia cells), these results suggest that 5-HT2B depletion influences only the NCCs component of the POM.
Fig. 3.10 5-HT2B loss of function doesn’t affect blood vessels development In situ hybridization
was performed on cryostat sections of embryo at stage 37 using Fli, as endothelial cells marker. Control and injected sides are shown. Arrowheads indicate the blood vessel entering the optic fissure. The level of Fli expression is similar in both sides.
56 4.Validation of Morpholino results by rescue experiments and by a pharmacological approach with a 5-HT2B high selective antagonist
The main difficulty in interpreting the morpholino experiments is the possibility of ‘off-target’ effects; that is the possibility that the MO inhibits the function of an irrelevant gene instead of, or in addition to, the gene of interest.
Fig. 3.11 5-HT2B depletion causes alteration of the skeletomuscular connectivity in extra-ocular muscles Immunohistochemistry experiments were performed using 12/101 antibody. Dorsal views of tadpoles at stage 45 are shown. In 5-HT2B morphants the extraocular muscles, derived by mesoderm cells, are present but they have lost their sterotipated attachment points on periocular cartilages.
57 In order to confirm the specificity of 5-HT2BMO effects, I co-injected the 5-HT2BMO and a 5-HT2B mRNA that is not recognized by the morpholino. In this experiment, named RNA rescue, the co-injected embryos rescued the normal activity of 5-HT2B. Then I performed in situ hybridization experiments using Pitx2 and Raldh3 markers to verify the rescue of the wild type phenotype. In the embryos injected with only the 5-HT2BMO I observed the phenotype (already described) in the 57% (n=54) using Pitx2 as marker, and 41% (n=56) using Raldh3. In the co-injected embryos the percentage of the observed phenotype decreases: 17% (n=58) using Pitx2 as marker and 17% (n=47) using Raldh3 (Fig.3.12). Furthermore, the same phenotype observed using the Morpholino approach was obtained using a pharmacological approach. I used a new selective high affinity HT2B antagonist, RS-127445, to block the 5-HT2B signaling pathway from stage 20 to stage 37. The treated embryos were analyzed by in situ hybridization using Dct and Pitx2. The embryos showed the same altered expression pattern present in the 5-HT2B morphants (Fig.).
58 Fig.3.12 The coinjection of the X5-HT2B mRNA together with the 5-HT2BMO
causes a rescue of 5-HT2B function. The injected sides of embryos at stage 37 processed by in situ hybridization using Pitx2 and Raldh3 confirm the normal phenotype in the eye of injected side (B’ sides) compared to the control side (B sides). In embryos injected with 5-HT2B morpholino the injected side present phenotypes (A’ sides) previously described compare to wild type side (A sides). Lateral view of embryos at stage 37
59 Fig. 3.13 Pharmacological approach to block the 5-HT2B signaling Embryos treated with high selective 5-HT2B antagonist from stage 20 to 37 were analyzed by
in situ hybridization with Raldh3 and Dct. The treated embryos show the failure of
optic fissure closure and the same phenotypes present in the 5-HT2B depleted embryos.
