Functional Conservation and Genetic Divergence of Chordate Glycinergic Neurotransmission: Insights from Amphioxus Glycine Transporters.

Glycine is an important neurotransmitter in vertebrates, performing both excitatory and inhibitory actions. Synaptic levels of glycine are tightly controlled by the action of two glycine transporters, GlyT1 and GlyT2, located on the surface of glial cells and neurons, respectively. Only limited information is available on glycinergic neurotransmission in invertebrates, and the evolution of glycinergic neurotransmission is poorly understood. Here, by combining phylogenetic and gene expression analyses, we characterized the glycine transporter complement of amphioxus, an important invertebrate model for studying the evolution of chordates. We show that amphioxus possess three glycine transporter genes. Two of these (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, whereas the third (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. GlyT2.2 expression is predominantly non-neural, whereas GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are differentially expressed, respectively, in neurons and glia. Vertebrate glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by a paralog-specific inversion of gene expression. Despite this genetic divergence between amphioxus and vertebrates, we found strong evidence for conservation in the role glycinergic neurotransmission plays during larval swimming, the implication being that the neural networks controlling the rhythmic movement of chordate bodies may be homologous.

Amphioxus neuroglia: Molecular characterization and evidence for early compartmentalization of the developing nerve cord.

Glial cells play important roles in the development and homeostasis of metazoan nervous systems. However, while their involvement in the development and function in the central nervous system (CNS) of vertebrates is increasingly well understood, much less is known about invertebrate glia and the evolutionary history of glial cells more generally. An investigation into amphioxus glia is therefore timely, as this organism is the best living proxy for the last common ancestor of all chordates, and hence provides a window into the role of glial cell development and function at the transition of invertebrates and vertebrates. We report here our findings on amphioxus glia as characterized by molecular probes correlated with anatomical data at the transmission electron microscopy (TEM) level. The results show that amphioxus glial lineages express genes typical of vertebrate astroglia and radial glia, and that they segregate early in development, forming what appears to be a spatially separate cell proliferation zone positioned laterally, between the dorsal and ventral zones of neural cell proliferation. Our study provides strong evidence for the presence of vertebrate-type glial cells in amphioxus, while highlighting the role played by segregated progenitor cell pools in CNS development. There are implications also for our understanding of glial cells in a broader evolutionary context, and insights into patterns of precursor cell deployment in the chordate nerve cord.

Alexander Disease Modeling in Zebrafish: An In Vivo System Suitable to Perform Drug Screening.

Alexander disease (AxD) is a rare astrogliopathy caused by heterozygous mutations, either inherited or arising de novo, on the glial fibrillary acid protein (GFAP) gene (17q21). Mutations in the GFAP gene make the protein prone to forming aggregates which, together with heat-shock protein 27 (HSP27), αB-crystallin, ubiquitin, and proteasome, contribute to form Rosenthal fibers causing a toxic effect on the cell. Unfortunately, no pharmacological treatment is available yet, except for symptom reduction therapies, and patients undergo a progressive worsening of the disease. The aim of this study was the production of a zebrafish model for AxD, to have a system suitable for drug screening more complex than cell cultures. To this aim, embryos expressing the human GFAP gene carrying the most severe p.R239C under the control of the zebrafish gfap gene promoter underwent functional validation to assess several features already observed in in vitro and other in vivo models of AxD, such as the localization of mutant GFAP inclusions, the ultrastructural analysis of cells expressing mutant GFAP, the effects of treatments with ceftriaxone, and the heat shock response. Our results confirm that zebrafish is a suitable model both to study the molecular pathogenesis of GFAP mutations and to perform pharmacological screenings, likely useful for the search of therapies for AxD.

Evidence of Oxidative Phosphorylation in Zebrafish Photoreceptor Outer Segments at Different Larval Stages.

Previous studies on purified bovine rod outer segments (OS) disks pointed to Oxidative Phosphorylation (OXPHOS) as being the most likely mechanism involved in ATP production, as yet not fully understood, to support the first phototransduction steps. Bovine and murine rod OS disks, devoid of mitochondria, would house respiratory chain complexes I to IV and ATP synthase, similar to mitochondria. Zebrafish ( Danio rerio) is a well-suited animal model to study vertebrate embryogenesis as well as the retina, morphologically and functionally similar to its human counterpart. The present article reports fluorescence and Transmission Electron Microscopy colocalization analyses of respiratory complexes I and IV and ATP synthase with zpr3, the rod OS marker, in adult and larval zebrafish retinas. MitoTracker Deep Red 633 staining and assays of complexes I and III-IV activity suggest that those proteins are active in OS. Results show that an extramitochondrial aerobic metabolism is active in the zebrafish OS at 4 and 10 days of larval development, as well as in adults, suggesting that it is probably maintained during embryogenesis. Data support the hypothesis of an extramitochondrial aerobic metabolism in the OS of zebrafish.

The HMGA gene family in chordates: evolutionary perspectives from amphioxus.

High mobility group A proteins of vertebrates, HMGA1 and 2, are chromatin architectural factors involved in development, cell differentiation, and neoplastic transformation. Here, we characterize an amphioxus HMGA gene ortholog and analyze its expression. As a basal chordate, amphioxus is well placed to provide insights into the evolution of the HMGA gene family, particularly in the transition from invertebrates to vertebrates. Our phylogenetic analysis supports the basal position of amphioxus, echinoderm, and hemichordate HMGA sequences to those of vertebrate HMGA1 and HMGA2. Consistent with this, the genomic landscape around amphioxus HMGA shares features with both. Whole mount in situ hybridization shows that amphioxus HMGA mRNA is detectable from neurula stage onwards in both nervous and non-nervous tissues. This correlates with protein expression monitored immunocytochemically using antibodies against human HMGA2 protein, revealing especially high levels of expression in cells of the lamellar body, the amphioxus homolog of the pineal, suggesting that the gene may have, among its many functions, an evolutionarily conserved role in photoreceptor differentiation.

The origin of dopaminergic systems in chordate brains: insights from amphioxus.

The basic anatomy of the central nervous system (CNS) is well conserved within the vertebrates and differs in significant ways from that of non-vertebrate chordates. Of the latter, amphioxus is of special interest, being the best available stand-in for the basal chordate condition. Immunohistochemical and gene expression studies on the developing CNS of amphioxus embryos and larvae are now sufficiently advanced that we can begin to assign specific neurotransmitter phenotypes to neurons identified by transmission electron microscopy (TEM), and then compare the distribution of cell types to that in vertebrate brains. Here, by monitoring tyrosine hydroxylase (TH) transcripts and protein, along with serial TEM, we identify a population of catecholamine-containing neurons in the anterior nerve cord of amphioxus larvae and describe their pattern of synaptic inputs and outputs. Inputs parallel those to the large paired neurons that control the larval escape response, suggesting that the TH+ system functions as an accessory excitatory and perhaps modulatory pathway in larval locomotion, with the added feature of recruiting an assortment of additional interneurons to the circuitry. The TH+ cells probably contain either L-DOPA or dopamine, and correspond closely with a cell population known from previous work on adult amphioxus to be dopaminergic. This population lies in a CNS domain now thought to comprise a combined vertebrate diencephalon plus mesencephalon, the implication being that dopaminergic nuclei in both of these brain regions could derive from a single dien-mesencephalic population in the last common ancestor of amphioxus and vertebrates.

Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?

Acetylcholinesterase (ACHE) is a glycoprotein with a key role in terminating synaptic transmission in cholinergic neurons of both vertebrates and invertebrates. ACHE is also involved in the regulation of cell growth and morphogenesis during embryogenesis and regeneration acting through its non-cholinergic sites. The mollusk Octopus vulgaris provides a powerful model for investigating the mechanisms underlying tissue morphogenesis due to its high regenerative power. Here, we performed a comparative investigation of arm morphogenesis during adult arm regeneration and embryonic arm development which may provide insights on the conserved ACHE pathways. In this study, we cloned and characterized O. vulgaris ACHE, finding a single highly conserved ACHE hydrophobic variant, characterized by prototypical catalytic sites and a putative consensus region for a glycosylphosphatidylinositol (GPI)-anchor attachment at the COOH-terminus. We then show that its expression level is correlated to the stage of morphogenesis in both adult and embryonic arm. In particular, ACHE is localized in typical neuronal sites when adult-like arm morphology is established and in differentiating cell locations during the early stages of arm morphogenesis. This possibility is also supported by the presence in the ACHE sequence and model structure of both cholinergic and non-cholinergic sites. This study provides insights into ACHE conserved roles during processes of arm morphogenesis. In addition, our modeling study offers a solid basis for predicting the interaction of the ACHE domains with pharmacological blockers for in vivo investigations. We therefore suggest ACHE as a target for the regulation of tissue morphogenesis.

A neurochemical map of the developing amphioxus nervous system.

BACKGROUND: Amphioxus, representing the most basal group of living chordates, is the best available proxy for the last invertebrate ancestor of the chordates. Although the central nervous system (CNS) of amphioxus comprises only about 20,000 neurons (as compared to billions in vertebrates), the developmental genetics and neuroanatomy of amphioxus are strikingly vertebrate-like. In the present study, we mapped the distribution of amphioxus CNS cells producing distinctive neurochemicals. To this end, we cloned genes encoding biosynthetic enzymes and/or transporters of the most common neurotransmitters and assayed their developmental expression in the embryo and early larva. RESULTS: By single and double in situ hybridization experiments, we identified glutamatergic, GABAergic/glycinergic, serotonergic and cholinergic neurons in developing amphioxus. In addition to characterizing the distribution of excitatory and inhibitory neurons in the developing amphioxus CNS, we observed that cholinergic and GABAergic/glycinergic neurons are segmentally arranged in the hindbrain, whereas serotonergic, glutamatergic and dopaminergic neurons are restricted to specific regions of the cerebral vesicle and the hindbrain. We were further able to identify discrete groups of GABAergic and glutamatergic interneurons and cholinergic motoneurons at the level of the primary motor center (PMC), the major integrative center of sensory and motor stimuli of the amphioxus nerve cord. CONCLUSIONS: In this study, we assessed neuronal differentiation in the developing amphioxus nervous system and compiled the first neurochemical map of the amphioxus CNS. This map is a first step towards a full characterization of the neurotransmitter signature of previously described nerve cell types in the amphioxus CNS, such as motoneurons and interneurons.

A study of neural-related microRNAs in the developing amphioxus.

BACKGROUND: MicroRNAs are small noncoding RNAs regulating expression of protein coding genes at post-transcriptional level and controlling several biological processes. At present microRNAs have been identified in various metazoans and seem also to be involved in brain development, neuronal differentiation and subtypes specification. An approach to better understand the role of microRNAs in animal gene expression is to determine temporal and tissue-specific expression patterns of microRNAs in different model organisms. Therefore, we have investigated the expression of six neural related microRNAs in amphioxus, an organism having an important phylogenetic position in terms of understanding the origin and evolution of chordates. RESULTS: In amphioxus, all the microRNAs we examined are expressed in specific regions of the CNS, and some of them are correlated with specific cell types. In addition, miR-7, miR-137 and miR-184 are also expressed in endodermal and mesodermal tissues. Several potential targets expressed in the nervous system of amphioxus have been identified by computational prediction and some of them are coexpressed with one or more miRNAs. CONCLUSION: We identified six miRNAs that are expressed in the nervous system of amphioxus in a variety of patterns. miR-124 is found in both differentiating and mature neurons, miR-9 in differentiated neurons, miR-7, miR-137 and miR-184 in restricted CNS regions, and miR-183 in cells of sensory organs. Therefore, such amphioxus miRNAs may play important roles in regional patterning and/or specification of neuronal cell types.

The synapsin gene family in basal chordates: evolutionary perspectives in metazoans.

BACKGROUND: Synapsins are neuronal phosphoproteins involved in several functions correlated with both neurotransmitter release and synaptogenesis. The comprehension of the basal role of the synapsin family is hampered in vertebrates by the existence of multiple synapsin genes. Therefore, studying homologous genes in basal chordates, devoid of genome duplication, could help to achieve a better understanding of the complex functions of these proteins. RESULTS: In this study we report the cloning and characterization of the Ciona intestinalis and amphioxus Branchiostoma floridae synapsin transcripts and the definition of their gene structure using available C. intestinalis and B. floridae genomic sequences. We demonstrate the occurrence, in both model organisms, of a single member of the synapsin gene family. Full-length synapsin genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans. Comparative genome analysis reveals extensive conservation of the SYN locus in several metazoans. Moreover, developmental expression studies underline that synapsin is a neuronal-specific marker in basal chordates and is expressed in several cell types of PNS and in many, if not all, CNS neurons. CONCLUSION: Our study demonstrates that synapsin genes are metazoan genes present in a single copy per genome, except for vertebrates. Moreover, we hypothesize that, during the evolution of synapsin proteins, new domains are added at different stages probably to cope up with the increased complexity in the nervous system organization. Finally, we demonstrate that protochordate synapsin is restricted to the post-mitotic phase of CNS development and thereby is a good marker of postmitotic neurons.

Expression of the orphan nuclear receptor NR4A in a putative adenohypophyseal homologue of amphioxus.

Nur77, Nurr1, and NOR-1 form a subfamily (the NR4A/Nur subfamily) of transcription factors belonging to the family of nuclear receptors. In this report, we characterized the single member of such a subfamily in the amphioxus Branchiostoma floridae. Developmental expression analysis of AmphiNR4A showed transcripts limited to the putative adenohypophyseal homologue of amphioxus.

Comprehensive survey and classification of homeobox genes in the genome of amphioxus, Branchiostoma floridae.

The homeobox genes comprise a large and diverse gene superfamily, many of which encode transcription factors with pivotal roles in the embryonic development of animals. We searched the assembled draft genome sequence of an amphioxus, Branchiostoma floridae, for genes possessing homeobox sequences. Phylogenetic analysis was used to divide these into gene families and classes. The 133 amphioxus homeobox genes comprise 60 ANTP class genes, 29 PRD genes (excluding Pon and Pax1/9), nine TALE genes, seven POU genes, seven LIM genes, five ZF genes, four CUT genes, four HNF genes, three SINE genes, one CERS gene, one PROS gene, and three unclassified genes. Ten of the 11 homeobox gene classes are less diverse in amphioxus than humans, as a result of gene duplication on the vertebrate lineage. Amphioxus possesses at least one member for all of the 96 homeobox gene families inferred to be present in the common ancestor of chordates, including representatives of the Msxlx, Bari, Abox, Nk7, Ro, and Repo gene families that have been lost from tunicates and vertebrates. We find duplication of several homeobox genes in the cephalochordate lineage (Mnx, Evx, Emx, Vent, Nk1, Nedx, Uncx, Lhx2/9, Hmbox, Pou3, and Irx) and several divergent genes that probably originated by extensive sequence divergence (Hx, Ankx, Lcx, Acut, Atale, Azfh, Ahbx, Muxa, Muxb, Aprd1-6, and Ahnf). The analysis reveals not only the repertoire of amphioxus homeobox genes but also gives insight into the evolution of chordate homeobox genes.

The amphioxus genome illuminates vertebrate origins and cephalochordate biology.

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

In vitro modulation of gamma amino butyric acid (GABA) receptor expression by bone marrow stromal cells.

Bone marrow stromal cells (BMSC) have the capability to undergo a change of morphology, reminiscent of neuronal cells, after exposure to an inductive medium. These induced BMSC-derived neuron-like (BDNL) cells express several neuronal markers, including Microtubule-Associated Protein Tau, Neurofilament M, and Nestin as revealed by immunocytochemistry analysis. To assess whether the induction process has possible functional relevance, we have focused our attention on the expression of neurotransmitter receptors. In particular, we show that the expression of GABA(A) subunits alpha1, beta2/3, epsilon and GABA(B1) mRNAs is greatly enhanced in BMSC by the induction treatment. Similar results were obtained from rat skin fibroblasts subjected to the same induction protocol, with the exception for the GABA(B2) transcript that was expressed only by BMSC and BDNL. The presence of both GABA(B1) and GABA(B2) subunits in BDNL cells suggests that functional GABA(B) receptors might be assembled: we indeed found that a functional GABA(B) receptor, negatively linked to cyclic AMP production, is expressed in BDNL. Therefore, we suggest that BMSC can be converted into cells equipped with appropriate receptors coupled to transduction mechanisms, potentially responding to a specific neurotransmitter.

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.

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 Hatschek's 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/Hatschek's 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.

Developmental expression of glutamic acid decarboxylase and of gamma-aminobutyric acid type B receptors in the ascidian Ciona intestinalis.

We describe Ciona intestinalis gamma-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 GABA(B) receptor subunits (Ci-GABA(B)Rs), and a phylogenetic analysis (neighbor-joining method) suggested that they clustered with their vertebrate counterparts. We compared Ci-GAD and Ci-GABA(B)Rs 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-GABA(B)Rs 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 GABA(B) receptor subunits were found mainly in the CNS. The expression pattern of Ci-GABA(B)Rs appeared restricted to Ci-GAD-positive territories in the sensory vesicle, whereas, in the visceral ganglion, Ci-GABA(B)Rs 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 GABA(B) receptors both pre- and postsynaptically to modulate different sensory inputs and subsequent swimming activity.

Characterization, developmental expression and evolutionary features of the huntingtin gene in the amphioxus Branchiostoma floridae.

BACKGROUND: Huntington's disease is an inherited neurodegenerative disorder that is caused by the expansion of an N-terminal polyQ stretch in the huntingtin protein. In order to investigate the hypothesis that huntingtin was already involved in development of the nervous system in the last common ancestor of chordates, we isolated and characterised the huntingtin homologue from the amphioxus Branchiostoma floridae. In the present paper the amphioxus general term must be referred to Branchiostoma floridae. RESULTS: In this report, we show that the exon-intron organization of the amphioxus huntingtin gene is highly conserved with that of other vertebrates species. The AmphiHtt protein has two glutamine residues in the position of the typical vertebrate polyQ tract. Sequence conservation is greater along the entire length of the protein than in a previously identified Ciona huntingtin. The first three N-terminal HEAT repeats are highly conserved in vertebrates and amphioxus, although exon rearrangement has occurred in this region. AmphiHtt expression is detectable by in situ hybridization starting from the early neurula stage, where it is found in cells of the neural plate. At later stages, it is retained in the neural compartment but also it appears in limited and well-defined groups of non-neural cells. At subsequent larval stages, AmphiHtt expression is detected in the neural tube, with the strongest signal being present in the most anterior part. CONCLUSION: The cloning of amphioxus huntingtin allows to infer that the polyQ in huntingtin was already present 540 million years ago and provides a further element for the study of huntingtin function and its evolution along the deuterostome branch.

Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum).

The cephalochordate amphioxus (Branchiostoma sp.) is an important animal model for studying the evolution of chordate developmental mechanisms. Obtaining amphioxus embryos is a key step for these studies. It has been shown that an increase of 3-4 degrees C in water temperature triggers spawning of the European amphioxus (Branchiostoma lanceolatum) in captivity, however, very little is known about the natural spawning behavior of this species in the field. In this work, we have followed the spawning behavior of the European amphioxus during two spawning seasons (2004 and 2005), both in the field and in captivity. We show that animals in the field spawn approximately from mid-May through early July, but depending on the year, they show different patterns of spawning. Thus, even if temperature has a critical role in the induction of the spawning in captivity, it is not the major factor in the field. Moreover, we report some improvements on the methodology for inducing spawning in captivity (e.g. in maintenance, light cycle control and induction of spawning in a laboratory without running sea water system). These studies have important implications for amphioxus animal husbandry and for improving laboratory techniques to develop amphioxus as an experimental animal model.