Hervé Tostivint

Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis

To define the role that repeat expansions of a GGGGCC hexanucleotide sequence of the C9orf72 gene play in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A genetic model for ALS was developed to determine whether loss of function of the zebrafish orthologue of C9orf72 (zC9orf72) leads to abnormalities in neuronal development.

Cloning, sequence analysis and tissue distribution of the mouse and rat urotensin II precursors.

Urotensin II (UII) is a cyclic neuropeptide initially isolated from the caudal neurosecretory system of teleost fish. The recent cloning of the UII precursor in frog and human has demonstrated that the peptide is not restricted to the fish urophysis but that it is also expressed in the central nervous system of tetrapods. Here, we describe the characterization of the cDNAs encoding prepro‐UII in mouse and rat. A comparison of the primary structures of mouse and rat UII with those of other vertebrate UII reveals that the sequence of the cyclic region of the molecule (CFWKYC) has been fully conserved. In contrast, the N‐terminal flanking domain of prepro‐UII has markedly diverged with only 48% sequence identity between the mouse or rat and the human precursors. In situ hybridization histochemistry showed that the prepro‐UII gene is predominantly expressed in motoneurons of the brainstem and spinal cord, suggesting that UII may play a role in the control of neuromuscular functions.

Somatostatin- and urotensin II-related peptides: molecular diversity and evolutionary perspectives

Recent advances in the fields of molecular cloning and peptide purification necessitate a reappraisal of our views concerning the evolution of the genes encoding somatostatin-related peptides. The currently widely held view that the genomes of tetrapods contain only the preprosomatostatin-I (PSS-I) gene, encoding somatostatin-14, with a second preprosomatostatin gene being expressed only in teleost fish is no longer tenable. Identification of genes encoding both somatostatin-14 and the somatostatin-related peptide, cortistatin in mammals, identification of the PSS-I and PSS-II preprosomatostatin genes in amphibia, and the isolation of gene products from at least two non-allelic preprosomatostatin genes in lampreys suggests the alternative hypothesis that duplication of the PSS-I gene occurred early in evolution, predating or concomitant with the appearance of the chordates. We speculate that at least two somatostatin genes are expressed in all classes of vertebrates but these genes have evolved at very different rates. It is probable that the preprosomatostatin-II (PSS-II) gene, encoding [Tyr7, Gly10] somatostatin-14 or a related peptide, arose from a second independent duplication of the PSS-I gene in the ancestor of present-day teleost fish at a time after the divergence of the teleost stock from the line of evolution leading to tetrapods. The recent isolation of urotensin II, a peptide which contains a region of structural similarity but is not evolutionarily related to somatostatin-14, from the central nervous systems of lampreys, elasmobranchs and amphibia necessitates that we modify the accepted view that urotensin II is exclusively a product of the caudal neurosecretory system of teleost fish.

Urotensin II, from fish to human

The cyclic peptide urotensin II (UII) was originally isolated from the urophysis of teleost fish on the basis of its ability to contract intestinal smooth muscle. The UII peptide has subsequently been isolated from frog brain and, later on, the pre‐proUII cDNA has been characterized in mammals, including humans. A UII paralog called urotensin II‐related peptide (URP) has been identified in the rat brain. The UII and URP genes originate from the same ancestral gene as the somatostatin and cortistatin genes. In the central nervous system (CNS) of tetrapods, UII is expressed primarily in motoneurons of the brainstem and spinal cord. The biological actions of UII and URP are mediated through a G protein–coupled receptor, termed UT, that exhibits high sequence similarity with the somatostatin receptors. The UT gene is widely expressed in the CNS and in peripheral organs. Consistent with the broad distribution of UT, UII and URP exert a large array of behavioral effects and regulate endocrine, cardiovascular, renal, and immune functions.

Localization of the urotensin II receptor in the rat central nervous system.

The vasoactive peptide urotensin II (UII) is primarily expressed in motoneurons of the brainstem and spinal cord. Intracerebroventricular injection of UII provokes various behavioral, cardiovascular, motor, and endocrine responses in the rat, but the distribution of the UII receptor in the central nervous system (CNS) has not yet been determined. In the present study, we have investigated the localization of UII receptor (GPR14) mRNA and UII binding sites in the rat CNS. RT‐PCR analysis revealed that the highest density of GPR14 mRNA occurred in the pontine nuclei. In situ hybridization histochemistry showed that the GPR14 gene is widely expressed in the brain and spinal cord. In particular, a strong hybridization signal was observed in the olfactory system, hippocampus, olfactory and medial amygdala, hypothalamus, epithalamus, several tegmental nuclei, locus coeruleus, pontine nuclei, motor nuclei, nucleus of the solitary tract, dorsal motor nucleus of the vagus, inferior olive, cerebellum, and spinal cord. Autoradiographic labeling of brain slices with radioiodinated UII showed the presence of UII‐binding sites in the lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, anteroventral thalamus, anterior pretectal nucleus, pedunculopontine tegmental nucleus, pontine nuclei, geniculate nuclei, parabigeminal nucleus, dorsal endopiriform nucleus, and cerebellar cortex. Intense expression of the GPR14 gene in some hypothalamic nuclei (supraoptic, paraventricular, ventromedian, and arcuate nuclei), in limbic structures (amygdala and hippocampus), in medullary nuclei (solitary tract, dorsal motor nucleus of the vagus), and in motor control regions (cerebral and cerebellar cortex, substantia nigra, pontine nuclei) provides the anatomical substrate for the central effects of UII on behavioral, cardiovascular, neuroendocrine, and motor functions. The occurrence of GPR14 mRNA in cranial and spinal motoneurons is consistent with the reported autocrine/paracrine action of UII on motoneurons. J. Comp. Neurol. 495:21–36, 2006.

Evolution of the gonadotropin-releasing hormone (GnRH) gene family in relation to vertebrate tetraploidizations

The neuropeptide gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Vertebrates possess multiple GnRH isoforms that are classified into three main groups, namely GnRH1, GnRH2 and GnRH3. In the present study, we show that the chromosomal organization of the three GnRH loci is very well conserved among gnathostome species. We analyzed genes belonging to several other multigenic families that are present in the vicinity of GnRH genes. Five of them were seen to occur in four chromosomal regions that clearly form a paralogon. Moreover, we show that the homologous regions in the amphioxus genome are present on a single locus. Taken together, these observations indicate that GnRH1, GnRH2 and GnRH3 genes represent three paralogous genes that resulted from the two rounds of tetraploidization that took place early in vertebrate evolution. They confirm that the GnRH3 gene which is currently known only in teleost has most likely been lost in the tetrapod lineage. Finally, they suggest the existence of a fourth GnRH gene, named GnRH4. Whether the GnRH4 gene still exists in extant vertebrates is currently unknown. A search for this putative gene would be particularly useful in basal groups such as agnathans and cartilaginous fish.

Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates

Over 90 years ago, Kolmer and Agduhr identified spinal cerebrospinal fluid-contacting neurons (CSF-cNs) based on their morphology and location within the spinal cord. In more than 200 vertebrate species, they observed ciliated neurons around the central canal that extended a brush of microvilli into the cerebrospinal fluid (CSF). Although their morphology is suggestive of a primitive sensory cell, their function within the vertebrate spinal cord remains unknown. The identification of specific molecular markers for these neurons in vertebrates would benefit the investigation of their physiological roles. PKD2L1, a transient receptor potential channel that could play a role as a sensory receptor, has been found in cells contacting the central canal in mouse. In this study, we demonstrate that PKD2L1 is a specific marker for CSF-cNs in the spinal cord of mouse (Mus musculus), macaque (Macaca fascicularis) and zebrafish (Danio rerio). In these species, the somata of spinal PKD2L1+ CSF-cNs were located below or within the ependymal layer and extended an apical bulbous extension into the central canal. We found GABAergic PKD2L1-expressing CSF-cNs in all three species. We took advantage of the zebrafish embryo for its transparency and rapid development to identify the progenitor domains from which pkd2l1+ CSF-cNs originate. pkd2l1+ CSF-cNs were all GABAergic and organized in two rows—one ventral and one dorsal to the central canal. Their location and marker expression is consistent with previously described Kolmer–Agduhr cells. Accordingly, pkd2l1+ CSF-cNs were derived from the progenitor domains p3 and pMN defined by the expression of nkx2.2a and olig2 transcription factors, respectively. Altogether our results suggest that a system of CSF-cNs expressing the PKD2L1 channel is conserved in the spinal cord across bony vertebrate species.

Comparative Evolutionary Histories of Kisspeptins and Kisspeptin Receptors in Vertebrates Reveal Both Parallel and Divergent Features

During the past decade, the kisspeptin system has been identified in various vertebrates, leading to the discovery of multiple genes encoding both peptides (Kiss) and receptors (Kissr). The investigation of recently published genomes from species of phylogenetic interest, such as a chondrichthyan, the elephant shark, an early sarcopterygian, the coelacanth, a non-teleost actinopterygian, the spotted gar, and an early teleost, the European eel, allowed us to get new insights into the molecular diversity and evolution of both Kiss and Kissr families. We identified four Kissr in the spotted gar and coelacanth genomes, providing the first evidence of four Kissr genes in vertebrates. We also found three Kiss in the coelacanth and elephant shark genomes revealing two new species, in addition to Xenopus, presenting three Kiss genes. Considering the increasing diversity of kisspeptin system, phylogenetic, and synteny analyses enabled us to clarify both Kiss and Kissr classifications. We also could trace back the evolution of both gene families from the early steps of vertebrate history. Four Kissr and four Kiss paralogs may have arisen via the two whole genome duplication rounds (1R and 2R) in early vertebrates. This would have been followed by multiple independent Kiss and Kissr gene losses in the sarcopterygian and actinopterygian lineages. In particular, no impact of the teleost-specific 3R could be recorded on the numbers of teleost Kissr or Kiss paralogs. The origin of their diversity via 1R and 2R, as well as the subsequent occurrence of multiple gene losses, represent common features of the evolutionary histories of Kiss and Kissr families in vertebrates. In contrast, comparisons also revealed un-matching numbers of Kiss and Kissr genes in some species, as well as a large variability of Kiss/Kissr couples according to species. These discrepancies support independent features of the Kiss and Kissr evolutionary histories across vertebrate radiation.

Multiple kisspeptin receptors in early osteichthyans provide new insights into the evolution of this receptor family.

Deorphanization of GPR54 receptor a decade ago led to the characterization of the kisspeptin receptor (Kissr) in mammals and the discovery of its major role in the brain control of reproduction. While a single gene encodes for Kissr in eutherian mammals including human, other vertebrates present a variable number of Kissr genes, from none in birds, one or two in teleosts, to three in an amphibian, xenopus. In order to get more insight into the evolution of Kissr gene family, we investigated the presence of Kissr in osteichthyans of key-phylogenetical positions: the coelacanth, a representative of early sarcopterygians, the spotted gar, a non-teleost actinopterygian, and the European eel, a member of an early group of teleosts (elopomorphs). We report the occurrence of three Kissr for the first time in a teleost, the eel. As measured by quantitative RT-PCR, the three eel Kissr were differentially expressed in the brain-pituitary-gonadal axis, and differentially regulated in experimentally matured eels, as compared to prepubertal controls. Subfunctionalisation, as shown by these differences in tissue distribution and regulation, may have represented significant evolutionary constraints for the conservation of multiple Kissr paralogs in this species. Furthermore, we identified four Kissr in both coelacanth and spotted gar genomes, providing the first evidence for the presence of four Kissr in vertebrates. Phylogenetic and syntenic analyses supported the existence of four Kissr paralogs in osteichthyans and allowed to propose a clarified nomenclature of Kissr (Kissr-1 to -4) based on these paralogs. Syntenic analysis suggested that the four Kissr paralogs arose through the two rounds of whole genome duplication (1R and 2R) in early vertebrates, followed by multiple gene loss events in the actinopterygian and sarcopterygian lineages. Due to gene loss there was no impact of the teleost-specific whole genome duplication (3R) on the number of Kissr paralogs in current teleosts.

Polygenic expression of somatostatin in the sturgeon Acipenser transmontanus: molecular cloning and distribution of the mRNAs encoding two somatostatin precursors.

The sequence of somatostatin‐14 (SS1) has been strongly preserved throughout the evolution of vertebrates from agnathans to mammals. In Acipenseridae (sturgeons), two isoforms of somatostatin have been characterized to date: somatostatin‐14 has been identified from the gastrointestinal tract of the pallid sturgeon Scaphirhynchus albus and [Pro2]somatostatin‐14 has been identified from the pituitary of the Russian sturgeon Acipenser gueldenstaedti. In the present study, we report the cloning of two distinct somatostatin cDNAs from the brain of the sturgeon Acipenser transmontanus. One of the cDNAs encodes a 116‐amino acid protein (PSS1) that contains the SS1 sequence at its C‐terminal extremity and, thus, is clearly orthologous to other vertebrate PSS1. The other cDNA encodes a 111‐amino acid protein that contains the somatostatin variant [Pro2]somatostatin‐14 at its C‐terminal extremity. This second precursor exhibits more than 67% identity with the recently characterized lungfish PSS2 and goldfish PSS2. Reverse transcriptase‐polymerase chain reaction analysis revealed that PSS1 is expressed in the central nervous system, the pancreas and the gut, whereas PSS2 is found in the central nervous system but not in the digestive system. In situ hybridization histochemistry showed that the PSS1 and PSS2 genes are differently expressed in numerous regions of the sturgeon brain. Interestingly, PSS1 and PSS2 mRNAs are present in the hypothalamus suggesting that, in sturgeon, both SS1 and SS2 may play hypophysiotropic functions. The PSS2 mRNA but not the PSS1 mRNA was found in the intermediate lobe of the pituitary. The present data demonstrate that two somatostatin genes are expressed in the sturgeon brain: one precursor generates somatostatin‐14 and the other one gives rise to a [Pro2]somatostatin‐14 variant, which is orthologous to goldfish, lungfish, and frog SS2. J. Comp. Neurol. 443:332–345, 2002.