Michela Ori

Distribution and cellular localization of the serotonin type 2C receptor messenger RNA in human brain

The regional and cellular distribution of serotonin type 2C receptor messenger RNA was investigated in autopsy samples of human brain by in situ hybridization histochemistry. The main sites of serotonin receptor type 2C messenger RNA expression were the choroid plexus, cerebral cortex, hippocampus, amygdala, some components of the basal ganglia, the substantia nigra, the substantia innominata and the ventromedial hypothalamus, suggesting that this receptor might be involved in the regulation of different brain functions. Interestingly, in all regions examined, the serotonin type 2C receptor messenger RNA was always restricted to subpopulations of cells, suggesting a specific role, perhaps determined by regionality. A comparison of the in situ hybridization results with those previously obtained by means of radioligand binding experiments suggested that in most of the areas analysed the serotonin type 2C receptors were located at axon terminals.

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.

Distribution of the 5-HT5A serotonin receptor mRNA in the human brain

The 5-HT5A receptor is a member of a new subfamily of serotonin [5-hydroxytryptamine (5-HT)] receptors recently cloned from the human and rodent brain. The role of this receptor in normal brain functions as well as its possible involvement in pathological states is still to be determined. We therefore studied the regional distribution and cellular localization of 5-HT5A receptor mRNA in human brain sections from autopsy samples by in situ hybridization histochemistry, in order to obtain anatomical information which might be useful in formulating hypotheses on possible functions subserved by this receptor in the central nervous system (CNS). Our results showed that the main sites of 5-HT5A mRNA expression were the cerebral cortex, hippocampus and cerebellum. In the neocortical regions, the 5-HT5A receptor mRNA was mainly distributed in the layers II-III and V-VI. In the hippocampus, the dentate gyrus and the pyramidal cell layer of the CA1 and CA3 fields expressed 5-HT5A mRNA at high levels. The broad distribution in the neocortex and hippocampus supports the view that the 5-HT5A receptor in these areas might be implicated in high cortical and limbic functions. The 5-HT5A mRNA was widely distributed in the cerebellum where it was highly expressed in the Purkinje cells, in the dentate nucleus and, at a lower level, in the granule cells. Since the cerebellum receives diffuse serotonergic afferents, this finding suggests that the 5-HT5A receptor may have an important role in mediating the effects of 5-HT on cerebellar functions.

Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation.

In vertebrate embryos, retinoic acid (RA) synthesized in the mesoderm by Raldh2 emanates to the hindbrain neuroepithelium, where it induces anteroposterior (AP)-restricted Hox expression patterns and rhombomere segmentation. However, how appropriate spatiotemporal RA activity is generated in the hindbrain is poorly understood. By analyzing Pbx1/Pbx2 and Hoxa1/Pbx1 null mice, we found that Raldh2 is itself under the transcriptional control of these factors and that the resulting RA-deficient phenotypes can be partially rescued by exogenous RA. Hoxa1-Pbx1/2-Meis2 directly binds a specific regulatory element that is required to maintain normal Raldh2 expression levels in vivo. Mesoderm-specific Xhoxa1 and Xpbx1b knockdowns in Xenopus embryos also result in Xraldh2 downregulation and hindbrain defects similar to mouse mutants, demonstrating conservation of this Hox-Pbx-dependent regulatory pathway. These findings reveal a feed-forward mechanism linking Hox-Pbx-dependent RA synthesis during early axial patterning with the establishment of spatially restricted Hox-Pbx activity in the developing hindbrain.

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. Developmental Dynamics 234:858–867, 2005.

RNA-seq of the aging brain in the short-lived fish N. furzeri – conserved pathways and novel genes associated with neurogenesis

The brains of teleost fish show extensive adult neurogenesis and neuronal regeneration. The patterns of gene regulation during fish brain aging are unknown. The short‐lived teleost fish Nothobranchius furzeri shows markers of brain aging including reduced learning performances, gliosis, and reduced adult neurogenesis. We used RNA‐seq to quantify genome‐wide transcript regulation and sampled five different time points to characterize whole‐genome transcript regulation during brain aging of N. furzeri. Comparison with human datasets revealed conserved up‐regulation of ribosome, lysosome, and complement activation and conserved down‐regulation of synapse, mitochondrion, proteasome, and spliceosome. Down‐regulated genes differ in their temporal profiles: neurogenesis and extracellular matrix genes showed rapid decay, synaptic and axonal genes a progressive decay. A substantial proportion of differentially expressed genes (~40%) showed inversion of their temporal profiles in the last time point: spliceosome and proteasome showed initial down‐regulation and stress‐response genes initial up‐regulation. Extensive regulation was detected for chromatin remodelers of the DNMT and CBX families as well as members of the polycomb complex and was mirrored by an up‐regulation of the H3K27me3 epigenetic mark. Network analysis showed extensive coregulation of cell cycle/DNA synthesis genes with the uncharacterized zinc‐finger protein ZNF367 as central hub. In situ hybridization showed that ZNF367 is expressed in neuronal stem cell niches of both embryonic zebrafish and adult N. furzeri. Other genes down‐regulated with age, not previously associated with adult neurogenesis and with similar patterns of expression are AGR2, DNMT3A, KRCP, MEX3A, SCML4, and CBX1. CBX7, on the other hand, was up‐regulated with age.

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.

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 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.

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.