Unraveling new roles for serotonin receptor 2B in development: key findings from Xenopus.

The serotonin receptor 5-HT2B has been shown to be critically important during embryogenesis as the knockout of this gene in mice causes heart defects and embryonic lethality that impairs further analyses on other embryonic cell and tissue types. In the present review, we highlight how the use of Xenopus laevis, an alternative vertebrate model suitable for gene loss and gain of function analyses, has contributed to our understanding of the role of 5-HT2B signaling during development. In vivo studies showed that 5-HT2B signaling is not only required for heart development, but that it also has a crucial role in ocular and craniofacial morphogenesis, being involved in shaping the first branchial arch and the jaw joint, in retinogenesis and possibly in periocular mesenchyme development. These findings may be relevant for our understanding of congenital defects including human birth malformations. In addition, 5-HT2B appears to be required for the therapeutic actions of selective serotonin reuptake inhibitors commonly prescribed as antidepressant drugs to pregnant and lactating women. We discuss how the understanding of the molecular basis of serotonin signaling in a suitable animal embryogenesis model may open new lines of investigations and therapies in humans.

5-Hydroxytryptamine 1A and 2B serotonin receptors in neurite outgrowth: involvement of early growth response protein 1.

Neurotransmitters play important roles in neurogenesis; in particular, acetylcholine and serotonin may regulate neurite elongation. Acetylcholine may also activate transcription factors such as early growth response protein 1 (EGR-1), which plays a role in neurite extension. N18TG2 neuroblastoma cells (which do not produce neurotransmitters and constitutively express muscarinic acetylcholine receptors) were transfected with constructs containing the cDNA for choline acetyltransferase, 5-hydroxytryptamine 1A (5-HT1A) and 5-HT2B serotonin receptors to study acetylcholine and serotonin interplay in neurite outgrowth. 5-HT1A receptor stimulation causes a decrease in EGR-1 levels and inhibition of neurite outgrowth; 5-HT2B stimulation, however, has no effect. Muscarinic cholinergic stimulation, on the other end, increases EGR-1 levels and fiber outgrowth. Inhibition of EGR-1 binding reduces fiber outgrowth activity. When both cholinergic and 5-HT1A receptors are stimulated, fiber outgrowth is restored; therefore, acetylcholine counterbalances the inhibitory effect of serotonin on neurite outgrowth. These results suggest that EGR-1 plays a role in the interplay of acetylcholine and serotonin in the regulation of neurite extension during development.

Hyaluronan is required for cranial neural crest cells migration and craniofacial development.

BACKGROUND: Hyaluronan is a crucial glycosaminoglycan of the vertebrate embryonic extracellular matrix able to influence cell behaviour, both by assembling the pericellular matrices and by activating signal transducing receptors such as CD44. RESULTS: We showed that the hyaluronan synthases, Has1 and Has2, and CD44 display a dynamic expression pattern during cranial neural crest cells (NCC) development. By knocking down Has1 and Has2 gene functions, we revealed that hyaluronan synthesized by Has1 and Has2 is necessary for the proper development of the visceral skeleton. CONCLUSIONS: The data suggest that hyaluronan helps to maintain the active migratory behaviour of cranial NCC, and that its presence around pre-chondrogenic NCC is crucial for their survival. CD44 knock down also suggests that the role of hyaluronan in cranial NCC migration could be mediated, at least in part, by the activation of CD44. These findings contribute to the unveiling of the functional relation between NCC and their extracellular environment during craniofacial development.

Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus.

Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. We used Xenopus laevis as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, we demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, we analyzed the function of phospholipase C beta 3 (PLC) in Xenopus development and showed that PLC is the effector of 5-HT2B during craniofacial development. Our results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to our understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch.

Identification and gene expression of versican during early development of Xenopus.

The chondroitin sulfate proteoglycan (PG) PG-M/versican is known to be a primary component of the vertebrate embryonic extracellular matrix and, in the mouse, functional abrogation of the versican gene leads to severe cardiovascular malformations and embryonic lethality. In order to provide a means for approaching the study of the role of versican during embryogenesis, we have cloned the Xenopus versican cDNA and we have performed in situ hybridization on embryos at different stages of development. We showed maternal Xversican transcription, as well as a previously undocumented early expression of the PG during gastrulation and neurulation. At later stages of development, spatial transcription of Xversican correlates with the patterns of migrating neural crest cells (NCC) and it is expressed in embryonic regions representing the final sites of arrest of NCC. Xversican mRNA was also detected in a subpopulation of trunk NCC migrating into the fin, in tissues flanking the trunk NCC ventral migratory pathway and in post-migratory cranial skeletogenic NCC. Further embryonic sites expressing Xversican were the pronephros, pronephric ducts, heart anlage and branchial pouches. These findings constitute the basis for future studies aimed at clarifying unresolved aspects of versican function during embryogenesis.

Overexpression of 5-HT2B receptor results in retinal dysplasia and defective ocular morphogenesis in Xenopus embryos.

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.

Molecular cloning and characterization of UDP-glucose dehydrogenase from the amphibian Xenopus laevis and its involvement in hyaluronan synthesis.

UDP-glucose dehydrogenase (UGDH) supplies the cell with UDP-glucuronic acid (UDP-GlcUA), a precursor of glycosaminoglycan and proteoglycan synthesis. Here we reported the cloning and the characterization of the UGDH from the amphibian Xenopus laevis that is one of the model organisms for developmental biology. We found that X. laevis UGDH (xUGDH) maintained a very high degree of similarity with other known UGDH sequences both at the genomic and the protein levels. Also its kinetic parameters are similar to those of UGDH from other species. During X. laevis development, UDGH is always expressed but clearly increases its mRNA levels at the tail bud stage (i.e. 30 h post-fertilization). This result fits well with our previous observation that hyaluronan, a glycosaminoglycan that is synthesized using UDP-GlcUA and UDP-N-acetylglucosamine, is abundantly detected at this developmental stage. The expression of UGDH was found to be related to hyaluronan synthesis. In human smooth muscle cells the overexpression of xUGDH or endogenous abrogation of UGDH modulated hyaluronan synthesis specifically. Our findings were confirmed by in vivo experiments where the silencing of xUGDH in X. laevis embryos decreased glycosaminoglycan synthesis causing severe embryonic malformations because of a defective gastrulation process.

XHas2 activity is required during somitogenesis and precursor cell migration in Xenopus development.

In vertebrates, hyaluronan biosynthesis is regulated by three transmembrane catalytic enzymes denoted Has1, Has2 and Has3. We have previously cloned the Xenopus orthologues of the corresponding genes and defined their spatiotemporal distribution during development. During mammalian embryogenesis, Has2 activity is known to be crucial, as its abrogation in mice leads to early embryonic lethality. Here, we show that, in Xenopus, morpholino-mediated loss-of-function of XHas2 alters somitogenesis by causing a disruption of the metameric somitic pattern and leads to a defective myogenesis. In the absence of XHas2, early myoblasts underwent apoptosis, failing to complete their muscle differentiation programme. XHas2 activity is also required for migration of hypaxial muscle cells and trunk neural crest cells (NCC). To approach the mechanism whereby loss of HA, following XHas2 knockdown, could influence somitogenesis and precursor cell migration, we cloned the orthologue of the primary HA signalling receptor CD44 and addressed its function through an analogous knockdown approach. Loss of XCD44 did not disturb somitogenesis, but strongly impaired hypaxial muscle precursor cell migration and the subsequent formation of the ventral body wall musculature. In contrast to XHas2, loss of function of XCD44 did not seem to be essential for trunk NCC migration, suggesting that the HA dependence of NCC movement was rather associated with an altered macromolecular composition of the ECM structuring the cells' migratory pathways. The presented results, extend our knowledge on Has2 function and, for the first time, demonstrate a developmental role for CD44 in vertebrates. On the whole, these data underlie and confirm the emerging importance of cell-ECM interactions and modulation during embryonic development.

Hoxa2 knockdown in Xenopus results in hyoid to mandibular homeosis.

The skeletal structures of the face and throat are derived from cranial neural crest cells (NCCs) that migrate from the embryonic neural tube into a series of branchial arches (BAs). The first arch (BA1) gives rise to the upper and lower jaw cartilages, whereas hyoid structures are generated from the second arch (BA2). The Hox paralogue group 2 (PG2) genes, Hoxa2 and Hoxb2, show distinct roles for hyoid patterning in tetrapods and fishes. In the mouse, Hoxa2 acts as a selector of hyoid identity, while its paralogue Hoxb2 is not required. On the contrary, in zebrafish Hoxa2 and Hoxb2 are functionally redundant for hyoid arch patterning. Here, we show that in Xenopus embryos morpholino-induced functional knockdown of Hoxa2 is sufficient to induce homeotic changes of the second arch cartilage. Moreover, Hoxb2 is downregulated in the BA2 of Xenopus embryos, even though initially expressed in second arch NCCs, similar to mouse and unlike in zebrafish. Finally, Xbap, a gene involved in jaw joint formation, is selectively upregulated in the BA2 of Hoxa2 knocked-down frog embryos, supporting a hyoid to mandibular change of NCC identity. Thus, in Xenopus Hoxa2 does not act redundantly with Hoxb2 for BA2 patterning, similar to mouse and unlike in fish. These data bring novel insights into the regulation of Hox PG2 genes and hyoid patterning in vertebrate evolution and suggest that Hoxa2 function is required at late stages of BA2 development.

5-HT2B-mediated serotonin signaling is required for eye morphogenesis in Xenopus.

In this paper, we show that serotonin, via 5-HT2B receptor, is involved in Xenopus retinal histogenesis and eye morphogenesis by supporting cell proliferation and survival. To analyze the 5-HT2B function in retinal development, we performed a loss-of-function study using both a pharmacological and a morpholino antisense oligonucleotide approach. Gain-of-function experiments were made by microinjecting 5-HT2B mRNA. Misregulation of the 5-HT2B receptor activity causes alterations in the proliferation rate and survival of retinal precursors, resulting in abnormal retinal morphology, where lamination is severely compromised. Clones derived from lipofected retinoblasts that overexpress 5-HT2B show an increase in the relative percentage of ganglion cells, possibly due to protection from apoptosis. This effect is reversed in clones lipofected with a 5-HT2B-specific morpholino. We hypothesize that the survival of the correct number of ganglion cells is controlled by 5-HT/5-HT2B signaling. Serotonin, acting as a neurotrophic factor, may contribute by refining retinal connectivity and cytoarchitecture.

Association of multiple DRD2 polymorphisms with anorexia nervosa.

To investigate whether the dopaminergic system plays a role in the etiology of anorexia nervosa (AN) via the dopamine D2 receptor, we investigated association and transmission disequilibrium at seven single-nucleotide polymorphisms (SNPs) spanning about 75 kbp of the gene DRD2. We studied 191 probands with a DSM-IV diagnosis of AN, 457 parents and affected relatives with a DSM-IV eating disorder diagnosis, and 98 unrelated, female, normal weight controls. The -141 C/- insertion/deletion (-141 Indel), previously shown to affect DRD2 transcription efficiency, and multiple exon seven polymorphisms, one of which has previously been shown to affect DRD2 transcript stability, exhibited statistically significant association with diagnosis in haplotype transmission disequilibrium and in haplotype case : control analyses. Significant linkage disequilibrium between the -141 Indel and two exon seven SNPs (939Y and 957Y) was observed over a distance of >50 kbp in the AN probands but not in the controls. Genetically transmitted variation in D2 dopamine receptor expression mediated by functional polymorphisms affecting transcription and translation efficiency may play a role in vulnerability to AN.

Regulated gene expression of hyaluronan synthases during Xenopus laevis development.

Here reported is the developmental gene expression pattern of the three known vertebrate hyaluronan synthases (XHas1, XHas2 and XHas3) and a comparative analysis of their mRNAs spatio-temporal distribution during Xenopus laevis development. We found that while XHas2 shows a steady-state expression from gastrula to late tailbud stage, XHas1 is mainly present in the early phases of development while XHas3 is predominantly transcribed in tailbud embryos. XHas1, XHas2 and XHas3 show distinct tissue expression patterns. In particular, XHas1 is localized in ectodermal derivatives and in cranial neural crest cells, whereas XHas2 is mainly found in mesoderm-derived structures and in trunk neural crest cells. Moreover, the expression pattern of XHas2 overlaps that of MyoD in cells committed to a muscle fate. Unlike the other hyaluronan synthases, XHas3 mRNA distribution is very restricted. In particular, XHas3 is expressed in the otic vesicles and closely follows the inner ear development. In conclusion, XHas1, XHas2 and XHas3 mRNAs have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis.

Molecular cloning, genomic organization and developmental expression of the Xenopus laevis hyaluronan synthase 3.

The content of hyaluronan (HA), a polymer of the extracellular matrix involved in a variety of physiological and pathological processes, depends on the activity of synthetic (HAS) and degrading enzymes. Since HA is also involved in embryogenesis, we have used Xenopus as a model organism because information is available for HAS1 and HAS2, but not for HAS3. We report the sequence of xlHAS3 mRNA, its genomic organization and its expression in adult tissues as well as during embryonic development. Interestingly, evidence from in situ hybridization indicates that xlHAS3 expression is restricted to the developing inner ear and cement gland. In addition, we have correlated the expression pattern of the enzymes involved in HA metabolism with the HA content during development.

Identification of circadian brain photoreceptors mediating photic entrainment of behavioural rhythms in lizards.

We have shown previously that in ruin lizards (Podarcis sicula) the ablation of all known photoreceptive structures (lateral eyes, pineal and parietal eye) in the same individual animal does not prevent entrainment of their circadian locomotor rhythms to light. The present study was aimed at identifying the circadian brain photoreceptors mediating entrainment. For this purpose, we looked for opsin expression in the brain by means of immunocytochemistry. Using anti-cone-opsin antiserum CERN 874 we have localized photoreceptors in the periventricular area of hypothalamus, near the third cerebral ventricle. We also cloned a brain opsin cDNA that, on the basis of the deduced amino acid sequence, appears to belong to the RH2 class of cone-opsins. We named the cloned cone-opsin Ps-RH2. To examine whether brain cone-opsins mediate photic entrainment of circadian locomotor rhythms, we performed post-transcriptional inactivation experiments by injecting an expression eukaryotic vector transcribing the antisense cone-opsin Ps-RH2 mRNA in the third cerebral ventricle of pinealectomized-retinectomized lizards previously entrained to a light-dark (LD) cycle. Injections of the antisense construct abolished photic entrainment of circadian locomotor rhythms of pinealectomized-retinectomized lizards to the LD cycle for 6-9 days. CERN 874 completely failed to label cells within the periventricular area of hypothalamus of brains injected with antisense construct. Thus, abolishment of photic entrainment is due to inactivation of endogenous brain cone-opsins mRNA. The present results demonstrate for the first time in a vertebrate that brain cone-opsins are part of a true circadian brain photoreceptor participating in photic entrainment of behavioural rhythms.

Expression of 5-HT2B and 5-HT2C receptor genes is associated with proliferative regions of Xenopus developing brain and eye.

Here we clone the Xenopus 5-HT2B receptor cDNA and describe its spatio-temporal mRNA expression within the developing larval brain and visual system. Expression of the 5-HT2B transcripts is compared to that of 5-HT2C as well as proliferation and neurogenic markers. In developing brain and retina, 5-HT2B and 2C mRNAs are mainly expressed in proliferative regions. We suggest that these receptors may play a role in the larval secondary neurogenesis by mediating mitogenic effects of serotonin.

[3H]-ketanserin binding sites in different psychiatric disorders.

The results of the present study showed the presence of a high-affinity and saturable binding of [3H]-ketanserin to frontal and parietal brain membranes obtained postmortem from bipolar, depressed, schizophrenic patients and normal controls. The human brain samples (60 frontal cortex and 51 parietal cortex), were donated by the Stanley Foundation Brain Collection. The overall data showed that normal controls, depressed and schizophrenic patients had a higher density in the frontal than in the parietal cortex, while bipolar patients did not show any difference. When the data were analysed according to the two hemispheres, some additional, intriguing observations were made: it emerged that [3H]-ketanserin binding sites did not show any difference in the two frontal cortices, while they were less represented in the right parietal cortex of normal and bipolar patients and more dense in schizophrenic patients. In conclusion, our study has demonstrated the presence of heterogenous alterations of [3H]-ketanserin binding sites in healthy controls and different psychiatric disorders that may be of help in a further elucidation of the specific role that 5-HT(2A) receptors may play in these disorders.

Xdtx1, a Xenopus Deltex homologue expressed in differentiating neurons and in photoreceptive organs.

We report the isolation of Xdtx1, a Xenopus homologue of the Drosophila Deltex gene. Starting from tailbud stage, Xdtx1 transcripts are detected in the olfactory bulbs, pineal complex and along the neural tube according to an antero-posterior gradient showing a gap at the midbrain-hindbrain boundary. At tadpole stage, Xdtx1 expression is activated in the differentiating retina, where it is also found in the neuronal fibres of the outer and inner plexiform layers, while its expression in the pineal complex becomes restricted to the photosensitive frontal organ. Differently from other vertebrate Deltex homologues, Xdtx1 is exclusively expressed in regions undergoing neuronal differentiation as shown by complementarity with X-Notch-1 expression.

Xdtx1, a Xenopus Deltex homologue expressed in differentiating neurons and in photoreceptive organs.

We report the isolation of Xdtx1, a Xenopus homologue of the Drosophila Deltex gene. Starting from tailbud stage, Xdtx1 transcripts are detected in the olfactory bulbs, pineal complex and along the neural tube according to an antero-posterior gradient showing a gap at the midbrain-hindbrain boundary. At tadpole stage, Xdtx1 expression is activated in the differentiating retina, where it is also found in the neuronal fibres of the outer and inner plexiform layers, while its expression in the pineal complex becomes restricted to the photosensitive frontal organ. Differently from other vertebrate Deltex homologues, Xdtx1 is exclusively expressed in regions undergoing neuronal differentiation as shown by complementarity with X-Notch-1 expression.