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Dev Biol
2008 Dec 09;1244:32-9. doi: 10.1016/j.brainres.2008.09.067.
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Overexpression of 5-HT2B receptor results in retinal dysplasia and defective ocular morphogenesis in Xenopus embryos.
Reisoli E
,
De Lucchini S
,
Anelli T
,
Biagioni S
,
Nardi I
,
Ori M
.
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In vertebrates, eye development comprises inductive and morphogenetic events that are finely regulated by the coordinated action of many intrinsic and extrinsic factors. Recent evidence suggested that neurotransmitters could be enumerated by the extracellular signals contributing to the retinal and eye development. We showed that, among these neuromodulators, serotonin acting via the 5-HT2B receptor, is involved in the control of retinoblasts proliferation and survival in Xenopus embryogenesis. To further clarify the role of 5-HT2B receptor in ocular development, we performed a gene gain of function analysis in vitro and in vivo in Xenopus embryos. We confirmed that 5-HT2B overexpression is per se sufficient to promote cell proliferation in a neuroblastoma cell line. The in vivo experiments revealed that an over serotonin signaling, via 5-HT2B receptors, resulted in the formation of eyes with an irregular form, position and orientation. Interestingly, we showed 5-HT2B gene expression in periocular mesenchyme that represents a key signaling center required for a correct eye morphogenesis. Moreover, the 5-HT2B receptor overexpressing retina, displays a disorganization of the typical laminar structure with the presence of retinal cells scattered in ectopic positions or forming rosette like structures. On the whole our data support the idea that serotonin signalling has to be finely regulated during eye development to allow a correct retinogenesis and may participate in the correct orchestration and synergism of all the factors and events that regulate eye morphogenesis in ocular and periocular tissues.
Fig. 1.
(A) RT-PCR analysis for Xenopus (X5-HT2B) and (B) mouse (5-HT2B) serotonin 2B receptor in N18TG2 cells, and 5-HT2B-N18TG2 transfected clones. Plasmid containing the cDNA for Xenopus laevis 5-HT2B receptor, mouse brain, cerebellum and cortex were used as a control. GAPDH expression was used for normalization.
Fig. 2.
Cell number was measured with MTT photometric method in N18TG2 neuroblastoma cells, N18TG2 5-HT2B/7 and N18TG2 5-HT2B/6 transfected clones. When indicated the cultures were maintained for 4 days in the presence of the specified agonist (BW 723C86) and antagonist (Ritanserin). The observed differences are statistically significant (⁎⁎⁎P < 0.001), the values are the mean ± S.E.M. of at least 18 observations for each culture condition.
Fig. 3.
Effects of 5-HT2B receptor overexpression on Xenopus eye development. Dorsal (A) and lateral (B, C) views of injected and control side of the same stage 42 tadpole. (A) The eye in the injected side is closer to the neural tube with respect to the control and rotated dorsally (distance bars). (B) Arrow points to the optic fissure that fails to close ventrally.
Fig. 4.
(A) Coronal section of a wild-type stage 38 embryo showing X5-HT2B expression in periocular mesenchyme (arrows). (B–D’) Gene expression of cell differentiation markers and retinal delayering in X5-HT2B overexpressing eyes. In situ hybridizations were performed on cryostat cross-sections of retinas from stage 42 tadpoles. Control (left column) and injected sides (right column) are shown. Probes used for in situ hybridization are indicated. The distance bars in (B) indicate the abnormal position of the eye closer to neural tube; arrowhead in (B”) points to rosette-like structures. (C’), note the pigmented epithelium missing ventrally (arrowhead). Arrowhead in (D’) points to clusters of ectopic RGCs not arrayed in the GCL.
Fig. 5.
Apoptosis in 5-HT2B up-regulated eyes. (A–B'), TUNEL staining of retinas from a stage 42 tadpole cryo-sections overexpressing the 5-HT2B receptor. Both control side (left column) and injected side (right column) are shown. TUNEL positive nuclei are visualized by DAB staining (brown). (A), Lower magnification cross-section from the same tadpole. Note the aberrant eye phenotype. Note the pigmented epithelium missing ventrally in the injected eye. (C, C') show the Hoechst staining of the same retinas as in (B, B').
