Diana Oliveri

Expression of the amphioxus Pit-1 gene (AmphiPOU1F1/Pit-1) exclusively in the developing preoral organ, a putative homolog of the vertebrate adenohypophysis

For the Florida amphioxus (Branchiostoma floridae), the full-length sequence and developmental expression of AmphiPOU1F1/Pit-1 are described. This gene, which is present in a single copy in the genome, is homologous to Pit-1 genes of vertebrates that play key roles in the development of the adenohypophysis. During amphioxus development, AmphiPOU1F1/Pit-1 transcripts are limited to Hatscheks left diverticulum and the larval tissue developing from it--namely the concave portion of the preoral organ. No other expression domains for this gene were detected during embryonic and larval development. From data currently available for hemichordates, amphioxus and ascidians, the best supported homologs for the vertebrate adenohypophysis are the preoral ciliary organ of hemichordates, preoral organ/Hatscheks pit of amphioxus and the neural gland/duct complex of ascidians. Better insights into pituitary evolution will require additional information: for invertebrate deuterostomes, more of the key pituitary genes in hemichordates and tunicates need to be studied; for the more basal groups vertebrates, it will be important to determine whether the source of the adenohypophysis is endodermal or ectodermal and to demonstrate what, if any, contribution mesodermal head coeloms might make to the developing pituitary.

Ci-POU-IV expression identifies PNS neurons in embryos and larvae of the ascidian Ciona intestinalis

Several lines of evidence suggest that members of the POU domain gene family may regulate invertebrate and vertebrate neurogenesis. In particular, POU IV genes appear to be neural genes involved in differentiation of sensory neurons, as demonstrated in mollusc, Drosophila, Caenorhabditis elegans and vertebrates. In the present work, we describe the developmental expression of a homologue of POU IV genes, Ci-POU-IV, in the ascidian Ciona intestinalis. Ci-POU-IV is expressed in the precursor cells of the neural system during development and in the neural system of the larva. In particular, transcripts are prevalent in the peripheral nervous system (PNS), with expression in the central nervous system (CNS) restricted to the posterior sensory vesicle. Therefore, the evolution of a complex sensory system seems to be under the control of a common genetic mechanism.

Developmental expression of glutamic acid decarboxylase and of γ‐aminobutyric acid type B receptors in the ascidian Ciona intestinalis

We describe Ciona intestinalis γ‐aminobutyric acid (GABA)‐ergic neurons during development, studying the expression pattern of Ci‐GAD (glutamic acid decarboxylase: GABA synthesizing enzyme) by in situ hybridization. Moreover, we cloned two GABAB receptor subunits (Ci‐GABABRs), and a phylogenetic analysis (neighbor‐joining method) suggested that they clustered with their vertebrate counterparts. We compared Ci‐GAD and Ci‐GABABRs expression patterns in C. intestinalis embryos and larvae. At the tailbud stage, Ci‐GAD expression was widely detected in central and peripheral nervous system (CNS/PNS) precursors, whereas Ci‐GABABRs expression was evident at the level of the precursors of the visceral ganglion. GABA was localized by immunohistochemistry at the same developmental stage. In the larva, Ci‐GAD transcripts and GABA immunofluorescence were also detected throughout the CNS and in some neurons of the PNS, whereas transcripts of both GABAB receptor subunits were found mainly in the CNS. The expression pattern of Ci‐GABABRs appeared restricted to Ci‐GAD‐positive territories in the sensory vesicle, whereas, in the visceral ganglion, Ci‐GABABRs transcripts were found in ventral motoneurons that did not express Ci‐GAD. Insofar as GABAergic neurons are widely distributed also in the CNS and PNS of vertebrates and other invertebrate chordates, it seems likely that GABA signaling was extensively present in the protochordate nervous system. Results from this work show that GABA is the most widespread inhibitory neurotransmitter in C. intestinalis nervous system and that it can signal through GABAB receptors both pre‐ and postsynaptically to modulate different sensory inputs and subsequent swimming activity. J. Comp. Neurol. 506:489–505, 2008.

Expression of AmphiPOU-IV in the developing neural tube and epidermal sensory neural precursors in amphioxus supports a conserved role of class IV POU genes in the sensory cells development

POU genes play a prominent role in the nervous system differentiation of several organism models, and in particular, they are involved in the differentiation of sensory neurons in numerous invertebrate and vertebrate species. In the present report, cloning and expression profile of a class IV POU gene in amphioxus was assessed for understanding its role in the sensory systems development. A single class IV gene, AmphiPOU-IV was isolated from the amphioxus Branchiostoma floridae. From a phylogenetic point of view, AmphiPOU-IV appears to be strictly related to the vertebrate one, sharing a high homology ratio especially with all vertebrate POU-IV proteins Brn-3a, Brn-3b, and Brn-3c. AmphiPOU-IV was found in the most anterior neural plate and in scattered ectodermic cells on the flanks of neurula, such ectodermic cells resemble the characteristic morphology and position of AmphiCoe and AmphiTrk developing sensory cells. Later on, the expression was confined in some motoneurons at level of the PMC and in some segmental arranged motoneurons in the hindbrain. Such expression is also maintained in larvae, and a new site of AmphiPOU-IV expression was also found in rostrum and mouth edge epidermal sensory cells of the larva. In conclusion, our data suggest an evolutionary conserved role of POU-IV transcription factors in the specification and differentiation of the sensory system in both vertebrates and invertebrates and underline the importance of amphioxus as linking step between them.

AmphiD1/β, a dopamine D1/β-adrenergic receptor from the amphioxus Branchiostoma floridae: evolutionary aspects of the catecholaminergic system during development

Catecholamine receptors mediate wide-ranging functions in vertebrates and invertebrates but are largely unknown in invertebrate chordates such as amphioxus. Catecholaminergic cells have been described in amphioxus adults, but few data are known about the transmembrane signal transduction pathways and the expression pattern of related receptors during development. In Branchiostoma floridae, we cloned a full-length cDNA (AmphiD1/β) that corresponds to the dopamine D1/β receptor previously cloned from a related species of amphioxus, Branchiostoma lanceolatum, but no expression studies have been performed for such receptor in amphioxus. In B. floridae, AmphiD1/β encodes a polypeptide with typical G-protein-coupled receptor features, characterized by highest sequence similarity with D1 dopamine and β-adrenergic receptors. The expression of AmphiD1/β mRNA in different regions of the cerebral vesicle corresponds to that of D1-like receptors in vertebrate homologous structures. Furthermore, in situ experiments show that during development, the expression in the nervous system is restricted to cells located anteriorly. A further expression was found in larvae at the level of the endostyle, but it has no counterpart in the predominant expression domains of vertebrate dopamine and/or adrenergic receptor genes. At the same time, we compared the dopaminergic system, consisting of AmphiTH-expressing cells, with the AmphiD1/β expression. In conclusion, the identification of the AmphiD1/β receptor provides further basis for understanding the evolutionary history of the dopaminergic system at the transition from invertebrates and vertebrates.

Cloning and developmental expression of amphioxus Dachschund

The nuclear factor dachshund (dac) is a key regulator of eye and leg development in Drosophila. We have cloned a Dachshund homologue from an invertebrate chordate amphioxus (Branchiostoma floridae). Sequence comparison reveals a high degree of similarity of amphioxus Dachshund (AmphiDach) to the known vertebrate and Drosophila dachshund genes. AmphiDach is first expressed in the prospective paraxial mesoderm at the gastrula stage. At the early neurula stage, expression is detected in developing somites and anterior endoderm, but in late neurula transcripts are present exclusively in the most posterior region of the cerebral vesicle and the anterior pharynx endoderm. Then, in larva, AmphiDach is localized in photoreceptive neurons of the frontal eye, infundibular organ, and endostyle as well as in Hesse organs and in nerve cells scattered along the nerve cord. Comparison of Dach expression in amphioxus and vertebrates suggests that such patterns are relatively similar (because they are expressed in somites, photoreceptor cells and CNS), even if expression of AmphiDach in the endostyle has no counterparts in its vertebrate homolog, the thyroid.

Cloning and developmental expression of AmphiBrn1/2/4, a POU III gene in amphioxus

The large family encoding POU transcription factors has been described in several species. In particular, class III POU genes regulate critical steps of vertebrate and invertebrate neurogenesis. A novel amphioxus class III POU gene, AmphiBrn1/2/4, has been isolated and its spatio-temporal expression has been reported. AmphiBrn1/2/4 is first expressed in the dorsal epiblast, then throughout the neural plate except for a gap at level of the anterior region of the cerebral vesicle. Transcripts are also detected in the primordium of gill slits, pharynx and left Hatscheks diverticulum.

The cholinergic gene locus in amphioxus: Molecular characterization and developmental expression patterns

The cholinergic gene locus (CGL), consisting of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT) gene, encodes two specific cholinergic neuronal markers used extensively to study cholinergic transmission. In the present work, we isolated the amphioxus homologs of VAChT and ChAT and examined their expression during development. Analysis of the 5′ untranslated region of VAChT and ChAT suggests that the splicing of the VAChT/ChAT mRNA has been evolutionarily conserved in amphioxus and mammals. By double whole‐mount in situ hybridization, we demonstrate that VAChT and ChAT are coexpressed in the same cells. They are first expressed in four pairs of differentiating cells in the neural plate. Their later expression is primarily in the anterior nerve cord in several types of motoneurons, some of the interneurons and in the receptor cells of the larval ocellus. Developmental Dynamics 237:1399‐1411, 2008.

Developmental expression of tryptophan hydroxylase gene in Ciona intestinalis

To describe the serotonergic system in a tunicate larva, we cloned a gene encoding for tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, in the ascidian Ciona intestinalis and studied its expression pattern during development. Ci-TPH expression was found from tailbud stage in the precursor cells of the visceral ganglion and in the tail. In the larva, TPH-expressing neurons formed two clusters in the anterior central nervous system at the level of the visceral ganglion. Moreover, we found Ci-TPH expression at the level of the muscle cells of the tail and suggested that this localisation might be at the level of neuro-muscolar junctions. Moreover, we discussed the involvement of serotonin in the control of larval locomotory activity.

Nitric oxide-evoked cGMP production in Purkinje cells in rat cerebellum : An immunocytochemical and pharmacological study

The cerebellar cells that account for glutamate-dependent cyclic GMP (cGMP) production, involving activation of the ionotropic glutamate receptors/nitric oxide synthase/soluble guanylyl cyclase pathway, are not fully established. In the present paper we have searched for the localisation of the cGMP response to the nitric oxide (NO) donor S-nitroso-penicillamine (SNAP 1muM), expected to generate local NO concentrations in the low nanomolar physiological range and evoking a cGMP response dependent on glutamate release and on the consequent activation of ionotropic glutamate NMDA/non-NMDA receptors, in cerebellar slices from adult rat. We have found that low concentration of exogenous NO evoked cGMP accumulation in Purkinje cells in an ionotropic glutamate receptor-dependent and tetrodotoxin-sensitive manner. Such immunocytochemical localisation appears consistent with functional evidence for physiologically relevant glutamate-dependent cGMP production in Purkinje cells in rat cerebellar cortex.