Search

Serotonin (5-HT) is an ancient molecule that appeared very early during evolution, and it is present in different phyla. The 5-HT signaling system includes several G-coupled receptors and it is widely conserved in vertebrates. 5-HT is implicated in an astonishing number of biological processes and it has a key role as a morphogen in several complex networks during development before it can act as a neurotransmitter. Recent advances on how serotonin signaling can influence early development and its role in vertebrate morphogenesis come from mice and Xenopus. The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates (agnathans) represents a major event in the evolution of vertebrates. The acquisition of a jaw is assumed to have occurred after the split between gnathostomes and jawless vertebrates. A crucial question concerns what changes were introduced in the developmental patterning programme to obtain a jaw joint that is one of the most innovative inventions in the history of vertebrates. Molecular and developmental studies performed in Xenopus revealed for the first time that serotonin, through the 5-HT2B receptor signaling, is both sufficient and necessary to modulate the shape and functionality of the jaw, including the jaw joint. Accordingly, serotonin can be added to the complex interactive network of extrinsic factors that regulates mandibular arch development, thus contributing to one of the major vertebrate successes in evolution.

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.

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.

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.

In addition to tyrosine sites, FAK (focal adhesion kinase) is phosphorylated on multiple serine residues. In the present study, the regulation of two of these sites, Ser-722 (S1) and Ser-911 (S4), was investigated. Phosphorylation of S1 (but not S4) decreased in resuspended cells, and recovered during spreading on fibronectin, indicating adhesion-dependent regulation. GSK3 (glycogen synthase kinase 3) inhibitors decreased S1 phosphorylation, and siRNA (short interfering RNA) silencing indicated further the involvement of GSK3beta. Furthermore, GSK3beta was found to become activated during cell spreading on fibronectin, and to physically associate with FAK. S1 phosphorylation was observed to decrease in wounded cell monolayers, while GSK3beta underwent inactivation and later was observed to increase to the original level within 24 h. Direct phosphorylation of S1, requiring pre-phosphorylation of Ser-726 in the +4 position, was demonstrated using purified GSK3 and a synthetic peptide containing FAK residues 714-730. An inhibitory role for S1 phosphorylation in FAK signalling was indicated by findings that both alanine substitution for S1 and dephosphorylation of S1 by PP1 (serine/threonine protein phosphatase type-1) resulted in an increase in FAK kinase activity; likewise, this role was also shown by cell treatment with the GSK3 inhibitor LiCl. The inhibitory role was confirmed by the finding that cells expressing FAK with alanine substitution for S1 displayed improved cell spreading and faster migration in wound-healing and trans-well assays. Finally, the finding that S1 phosphorylation increased in cells treated with the PP1 inhibitor okadaic acid indicated targeting of this site by PP1. These results indicate an additional mechanism for regulation of FAK activity during cell spreading and migration, involving Ser-722 phosphorylation modulated through the competing actions of GSK3beta and PP1.

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.

Protein phosphatase 1delta (PP1delta) localizes to focal adhesions and associates with the focal adhesion kinase (FAK). In the present work we used deletion mutants of PP1delta and FAK to detect their reciprocally interacting domains. Dissection of PP1delta indicated 194-260 as the shortest FAK-interacting domain among those tested. Domain 194-260 encompasses several sites involved in catalysis, indirectly confirming that FAK is a PP1 substrate. Mutation of one of these sites, R220 (R220S or R220Q), did not abolish but on the contrary increased the ability of 194-260 to pull-down FAK. Such property might be exploited to detect new potential PP1 substrates. Among the FAK deletion mutants, only the C-terminal domain (684-1053, also known as FRNK) pulled-down a significant amount of PP1. The PP1 eluted from a GST-FRNK affinity column displayed Mr of 35,000 when analyzed by gel-filtration on FPLC Superose 12, indicating the presence of an isolated PP1 catalytic subunit.

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.

We cloned the 5-HT2B serotonin receptor from the puffer fish Tetraodon fluviatilis. Two cDNAs differing in length because of the use of alternative polyadenylation sites were isolated. We partly characterized the genomic organization of the 5-HT2B gene and we found two introns conserved in position between the puffer fish and mammals. In addition, four splice variants which would generate truncated forms of the receptor were detected.

Ribosomal DNA (rDNA) variation in the species Triturus vulgaris meridionalis (Amphibia, Urodela) is remarkable because of unusually high intraspecific variability in the number and distribution of ribosomal loci in the karyotype; in addition, portions of the intergenic spacer (IGS) are clustered at chromosomal loci where they are not associated with ribosomal 18S and 28S RNA genes. These clusters are referred to as extraribosomal, and they appear to consist mostly of repetitive BamHI elements. In this paper, we report the complete nucleotide sequence of an IGS of T. v. meridionalis; this structural analysis is aimed to get insight into the molecular mechanism(s) of spreading of the ribosomal cistrons as well as its possible functional significance. We found that the IGS of T. vulgaris has a modular structure: modular repetitive elements contain sequences possibly related to the regulation of transcription of the ribosomal units. In particular, both ribosomal and extraribosomal IGS elements contain presumptive enhancers. Interestingly, the enhancer-containing region is mostly conserved between ribosomal and extraribosomal elements, while mutations accumulate in a region characterized by repetitions of a simple sequence motif, that we consider as a possible recombination hotspot. Our data suggest that extraribosomal elements most probably originated from ribosomal enhancer-containing elements able to move independently from the ribosomal unit at novel chromosomal positions, perhaps with the aid of the simple repetitive motif. We argue that a similar mechanism may lead to the spreading of complete repetition units as well, giving rise to multiple, and variable, ribosomal sites. We propose that hypervariability in the number and distribution of the rDNA loci, as seen in T. vulgaris, is a further mechanism to ensure redundancy, which seems to be an intrinsic property of rDNA biology, the occurrence of IGS elements independently clustered at separate chromosomal loci being a by-product of this mechanism.

In this paper we address the question of how genes can control development by using Xenopus as a model system, since it combines the classical advantages of amphibian embryology with advanced molecular techniques. Several developmental regulator genes have been shown to encode for transcription factors which trigger the activation of downstream genes, thus resulting in a cascade of regulatory events. In the first two examples, we deal with regulatory events that underlie early body patterning in vertebrates, and with the role of homeobox transcription factors in deciphering positional information along the body axis. In the third example, we address the question of the role of post-transcriptional regulation in development by studying the possible regulatory role of a cytoplasmic zinc finger protein, presumably acting through RNA-protein interactions. The general idea is that understanding how genes can control development will hopefully lead to understanding the construction of a shape, and eventually of an organism.

Xfin is a Xenopus zinc finger protein which is expressed in the cytoplasm of the oocyte and throughout embryogenesis, as well as in the cytoplasm of some specific and highly differentiated cell types (De Lucchini et al., Mech. Dev. 36, 31-40, 1991). In this paper we present a characterization of some structural features of the protein and of its nucleic acid binding properties. We found that Xfin is a phosphoprotein, is present in the soluble fraction of the cytoplasm, and is actively phosphorylated in cytosolic extracts. Several putative phosphorylation sites are present in the cDNA-derived protein sequence, mostly located at specific positions within the Zn-fingers. In an in vitro assay a fusion protein containing part of the finger region of Xfin exhibits specific binding to a poly (G) RNA homopolymer, while it does not bind DNA. The RNA binding activity of the protein is significantly enhanced by phosphorylation. A putative Xfin homolog, which appears to be evolutionarily conserved with regard to size, cytoplasmic expression and antigenic specificity, is present in representatives of five Vertebrate classes. Taken together, these results may suggest that, by virtue of its RNA binding activity modulated through phosphorylation, Xfin could serve some evolutionarily conserved function in post-transcriptional regulation processes.

Xfin is a member of a Zn-finger multigene family that shares homology with the Drosophila segmentation gene, Krüppel. This paper reports on our identification and cellular localization, through the utilization of specific antibodies, of the expression product of Xfin in the Xenopus laevis retina, and its pattern of expression during the retinal developmental stages. By immunostaining sections of the retina, we show that the major staining is localized in the cytoplasm of the cones. The protein appears at an early differentiation stage of the cones, when they can not be univocally identified by morphological criteria, and is maintained up to the adult retina. The same antigenicity pattern is detectable in the retina of the anuran genus Bufo. The immunostaining data are confirmed by Western blot analysis on Xenopus eye protein extracts. Because of its cytoplasmic localization, and because of the ability of Zn-finger proteins to bind nucleic acids, we think that Xfin may be involved in the terminal differentiation of the cones through RNA-protein interactions.

Xfin is a member of a large family of Krüppel-type transcripts stored in the Xenopus egg, whose function is unknown. By using polyclonal antibodies raised against fusion proteins containing different portions of Xfin, we have identified the Xfin gene product and established its pattern of expression in some adult tissues and during oogenesis and embryogenesis. The corresponding mRNA localization has been studied by in situ hybridization on ovary and testis sections. The Xfin product is found in the cytoplasm, both during oogenesis and adulthood; in adult tissues, it is differentially expressed in a cell-type specific fashion. The expression of the protein in specialized cell types and its cytoplasmic localization may favour the hypothesis that it could be involved in cell differentiation events through protein-RNA interactions.

The mitotic chromosomes of six specimens from Triturus vulgaris meridionalis have been examined by both in situ hybridization with 3H 18S + 28S rRNA and AS-SAT staining method. The results of these two sets of experiments can be summarized as follows: 1) in each specimen the NORs and the additional ribosomal sites, which react positively to in situ hybridization with 3H 18S + 28S rRNA, are also stained by silver; 2) other chromosomal regions, which do not hybridize in situ with 3H 18S + 28S rRNA, are on the other hand stained by the AS-SAT method. These latter AG-positive sites show a species-specific pattern of chromosomal distribution.

The ribosomal RNA genes ofTriturus vulgaris meridionalis are clustered at variable and often multiple chromosomal loci. The rDNA repeats exhibit only a limited and discrete length heterogeneity which is accounted for by the non-transcribed spacer (NTS). Interestingly, sequences homologous to the NTS are clustered outside the ribosomal loci. Clones containing such 'non ribosomal sequences' have been isolated from a genomic library ofT. v. meridionalis and analyzed by restriction mapping. These sequences appear to consist mostly of repetitive Bam HI fragments ranging from 500 bp to 1000 bp. The Bam HI fragments are internally repetitious and highly homologous to each other.

The ribosomal RNA genes of Triturus vulgaris meridionalis (Amphibia, Urodela) show the peculiar feature of being clustered not only at the nucleolar organizer, present in the species at a definite chromosome location, but also at "additional ribosomal sites" which are highly variable in number and chromosomal distribution among individuals. The additional ribosomal sites are most often found at specific chromosome regions, such as telomeres, C-bands and centromeres, in virtually all the chromosomes. With increasing numbers of additional clusters, the genomic dosages of ribosomal RNA genes are found to increase over a tenfold range, though not linearly. At a molecular level, the ribosomal DNA repeats differ in size because of discrete variations in the length of the non-transcribed spacers. However, the resulting length heterogeneity of the gene family is rather limited within a single genome as well as within the species. Many of the ribosomal loci appear to be internally homogeneous with respect to the repeat length. Moreover, separate clusters from distant genomic regions can share the same size class of ribosomal repeats even in the same specimen. The nucleolar organizer is mostly endowed with "shorter" ribosomal repeating units, ranging in size from 13.7 X 10(3) to 15.2 X 10(3) base-pairs. The additional ribosomal sites are characterized by the occurrence of "longer" repeats, ranging in size from 16.2 X 10(3) to 19.7 X 10(3) base-pairs. The "shorter" class of ribosomal repeats is always detected in the amplified ribosomal DNA, suggesting that the nucleolar organizer locus is involved in the amplification process in most oocytes. "Longer" ribosomal repeats are also detectable in the amplified ribosomal DNA of a few females.

We show that, in Triturus vulgaris meridionalis, sequences homologous to the rDNA "non-transcribed" spacer (NTS) are clustered at chromosomal loci where they are not associated with 18 S or 28 S rDNA genes: these sequences are referred to as the extra-ribosomal spacer sequences. Genomic clones containing such extra-ribosomal spacer sequences have been isolated. As shown by restriction mapping, these clones appear to consist mostly of repetitive BamHI fragments that are, in turn, internally repetitious and highly homologous to each other. The structure of the clones was confirmed by nucleotide sequence analysis, which also demonstrates the high degree of conservation between the BamHI elements and the homologous NTS sequences. An intriguing 12 base-pair homology between the extra-ribosomal spacer sequences and a Xenopus NTS enhancer sequence is reported. The possibility that a repetitive octanucleotide motif found within the BamHI elements could act as a recombination hotspot by virtue of its similarity with the Escherichia coli chi sequence is discussed.