Simona Candiani

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.

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.

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.

Serotonin localization in Phallusia mammillata larvae and effects of 5‐HT antagonists during larval development

The neurotransmitter 5‐hydroxytryptamine (5‐HT, serotonin) plays an important role in a wide range of non‐neural processes. Using immunofluorescence with an antiserotonin antibody, 5‐HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5‐HT on development, we treated embryos with two different 5‐HT receptor subtype antagonists. Treatment at the gastrula stage with 10 μM ondansetron, an antagonist of the 5‐HT3 receptor, induced anterior truncation and a short tail. At 10 μM, ritanserin, a 5‐HT2B receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5‐HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5‐HT is suggested.

Expression of the tissue-specific transcription factor Pit-1 in the lancelet, Bbranchiostoma lanceolatum

Lancelets, known also as amphioxus, are protochordates that share common archetypal features with vertebrates. Recently, several developmental and molecular biology studies have pointed out homologies between anatomical structures of lancelets and vertebrates. We have studied the head region of the lancelet, Branchiostoma lanceolatum, by means of scanning electron microscopy, immunocytochemistry, and Western blotting techniques, to localize the pituitary‐specific transcription factor, Pit‐1. Immunoreactive Pit‐1 protein has been found in cells of two typical structures of the lancelets, the Köllikers and Hatscheks pits. Moreover, the frontal eye complex, neurons, and the rostral nerves show Pit‐1 immunoreactivity. A band of 33 kilodaltons has been resolved in lancelet extracts by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and after Western blotting, the bands have been probed by a monoclonal antibody to rat Pit‐1. Our results demonstrate that Pit‐1 is expressed in both neurones and receptosecretory epithelial cells of adult lancelets, and that the cells lining the two pits display ultrastructural and immunocytochemical features typical of chemoreceptosecretory/olfactory‐ and adenohypophyseal‐like structures. J. Comp. Neurol. 392:343–351, 1998.

A study of neural-related microRNAs in the developing amphioxus

BackgroundMicroRNAs 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.ResultsIn 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.ConclusionWe 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.

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.

Immunocytochemical localization of serotonin in embryos, larvae and adults of the lancelet, Branchiostoma floridae

Serotonin (5-hydroxytryptamine) is a biogenic amine distributed throughout the metazoans and has an old evolutionary history. It is involved as a developmental signal in the early morphogenesis of both invertebrates and vertebrates, whereas in adults it acts mainly as a neurotransmitter and gastrointestinal hormone. In vertebrates, serotonin regulates the morphogenesis of the central nervous system and the specification of serotonergic as well as dopaminergic neurons. The present study uses, as an experimental model, an invertebrate chordate, the lancelet Branchiostoma floridae, characterized by its remarkable homologies with vertebrates that allows the bauplan of the probable ancestor of vertebrates to be outlined. In particular, the involvement of serotonin as a developmental signal in embryos and larvae, as well as a neurotransmitter and gastrointestinal hormone in adult specimens of Branchiostoma floridae, gives further support to a common origin of cephalocordates and vertebrates.

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.

Extramitochondrial tricarboxylic acid cycle in retinal rod outer segments

Vertebrate retinal rod Outer Segments (OS) are the site of visual transduction, an energy demanding process for which mechanisms of ATP supply are still poorly known. Glycolysis or diffusion of either ATP or phosphocreatine from the Inner Segment (IS) does not seem to display adequate timing to supply ATP for phototransduction. We have previously reported data suggesting an aerobic metabolism in OS, which would largely account for the light-stimulated ATP need of the photoreceptor. Here, by oxymetry and biochemical analyses we show that: (i) disks isolated by Ficoll flotation consume O(2) in the presence of physiological respiring substrates either in coupled or uncoupled conditions; (ii) OS homogenates contain the whole biochemical machinery for the degradation of glucose, i.e. glycolysis and the tricarboxylic acid cycle (TCA cycle), consistently with the results of our previous proteomic study. Activities of the 8 TCA cycle enzymes in OS were comparable to those in retinal mitochondria-enriched fractions. Disk and OS preparations were subjected to TEM analysis, and while they can be considered free of inner segment contaminants, immunogold with specific antibodies demonstrate the expression therein of both the visual pigment rhodopsin and F(o)F(1)-ATP synthase. Finally, double immunofluorescence on mouse retina sections demonstrated a colocalization of some respiratory complex mitochondrial proteins with rhodopsin in rod OS. Data, suggestive of the exportability of the mitochondrial machinery for aerobic metabolism, may shed light on those retinal pathologies related to energy supply impairment in OS and to mutations in TCA enzymes.