Expression des cotransporteurs cation-chlorure KCC2 et NKCC1 au cours du développement de la moelle épinière de l’opossum Monodelphis domestica

L’inhibition est nécessaire à la génération d’outputs coordonnés entre muscles antagonistes lors de la locomotion. Une baisse de la concentration neuronale en ions chlorure au cours du développement des mammifères conduit à l’émergence de l’inhibition. Cette baisse repose sur l’équilibre entre deux cotransporteurs cation-chlorure, KCC2 et NKCC1. KCC2 expulse Cl- de la cellule alors que NKCC1 pompe Cl- dans la cellule. L’opossum Monodelphis domestica naît dans un état très immature. Le seul comportement locomoteur qu’il présente à la naissance consiste en des mouvements rythmiques et alternés des membres antérieurs pour grimper le long du ventre de la mère vers une tétine. Les membres postérieurs sont des bourgeons immobiles dont le développement est en grande partie postnatal. Pour cette raison, cette espèce constitue un modèle idéal pour l’étude du développement locomoteur. Afin d’étudier les mécanismes conduisant à l’émergence de l’inhibition durant le développement moteur, nous avons décrit l’expression développementale de KCC2 et NKCC1 chez l’opossum postnatal par immunohistochimie au niveau des renflements spinaux. Les motoneurones et afférences primaires ont été identifiés en utilisant un marquage rétrograde au TRDA. Le marquage pour KCC2 et NKCC1 est détecté dans la moelle épinière ventrale dans la matière grise et blanche présomptive dès la naissance, ce qui suggère que l’inhibition serait déjà mise en place avant la naissance, permettant subséquemment l’alternance des membres antérieurs observée chez les nouveau-nés. L’expression développementale de KCC2 et NKCC1 suit des gradients ventrodorsal et médiolatéral, tels qu’observés chez les rongeurs (rats et souris). Le patron mature d’expression de ces cotransporteurs est observé aux alentours de la 5ème semaine postnatale lorsque la locomotion de l’opossum est mature. Enfin, entre la naissance et P5, les dendrites exprimant KCC2 au niveau de la corne dorsale sont retrouvées en apposition aux afférences primaires ce qui suggère un rôle de KCC2 dans la formation des circuits sensori-moteurs.

Expression de la connexine 36 dans la moelle épinière au cours du développement postnatal de l'opossum Monodelphis domestica

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Étude de l'activité spontanée dans la moëlle épinière de l'oppossum Monodelphis domestica en développement

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Rôle du système du trijumeau dans la locomotion chez le nouveau-né d’opossum (Monodelphis domestica)

L’opossum Monodelphis domestica naît très immature et grimpe sans aide de la mère, du sinus urogénital à une mamelle où il va s’attacher pour poursuivre son développement. Des informations sensorielles sont nécessaires pour guider le nouveau-né vers la mamelle et les candidats les plus probables sont le toucher, l’équilibre et l’olfaction. Pour tester l’action des différents systèmes sur la motricité chez l’opossum nouveau-né, des régions céphaliques du trijumeau, du vestibulaire et de l’olfaction ont été stimulées électriquement sur des préparations in vitro en comparaison avec une stimulation seuil T (intensité minimale de la stimulation à la moelle épinière cervicale induisant le mouvement des membres antérieurs). Par comparaison, un mouvement similaire était induit par des stimulations à ~2T du ganglion du trijumeau, à ~20 T du complexe vestibulaire, et à ~600 T des bulbes olfactifs. L’étude de l'innervation de la peau faciale et des voies relayant les informations du trijumeau vers la moelle épinière (ME) a été approfondie en utilisant de l’immunohistochimie pour les neurofilament-200 et du traçage rétrograde avec du Texas-Red couplé à des Dextrans Aminés. De nombreuses fibres nerveuses ont été révélées dans le derme de plusieurs régions de la tête. Quelques cellules du ganglion trigéminal projettent à la ME rostrale, mais la majorité projette vers la médulla caudale où se trouvent les neurones secondaires du trijumeau ou des cellules réticulospinales. Les résultats de cette étude indiquent une influence significative des systèmes du trijumeau et du vestibulaire, mais pas de l'olfaction, sur le mouvement des membres antérieurs des opossums nouveau-nés.

Influence de la mécanoréception faciale sur les comportements moteurs chez l’opossum nouveau-né, Monodelphis domestica

L’opossum nait dans un état très immature, mais rampe avec ses membres antérieurs (MA) de l’orifice urogénital de la mère à une tétine, où il s’attache pour poursuivre son développement. Des informations sensorielles sont nécessaires pour guider le nouveau-né vers une tétine et déclencher son attachement. Des expériences précédentes ont montré que le système du trijumeau, dont dépend l’innervation somesthésique du museau, influence les mouvements précoces des MA. Le présent projet vise à déterminer si les mécanorécepteurs faciaux sont fonctionnels et exercent une influence sur les MA. On s’intéresse particulièrement aux cellules de Merkel, un mécanorécepteur épidermique innervé par des fibres à adaptation lente de type I (SA I). Ces cellules ont été localisées sur le pourtour du museau de l’opossum nouveau-né en utilisant un traceur cellulaire, l’AM1-43. Nous avons analysé les réponses musculaires des MA consécutives à l’application de forces calibrées au museau sur des préparations in vitro. Ces réponses sont bilatérales et simultanées, très variables, et leur intensité augmente avec la force de la stimulation. Lors de stimulations répétitives pendant 60 min, les réponses diminuent avec le temps. Le retrait de la peau faciale abolit presque ces réponses. De plus, l’application d’un antagoniste des récepteurs métabotropiques du glutamate, qui affecte l’activité des fibres SA I, ou d’un antagoniste des récepteurs purinergiques les diminue fortement, suggérant une participation des cellules de Merkel. Ces résultats soutiennent que le sens du toucher facial relayé par le système du trijumeau est fonctionnel chez l’opossum nouveau-né et qu’il pourrait influencer les mouvements des MA.

Co-existing squamous cell carcinoma and hemangioma on the ocular surface of a cat

A 14-year-old spayed female domestic short-haired cat was presented for evaluation of a mass in the right eye. Ophthalmic examination revealed a blind right eye and presence of two distinct masses: a pink and a red-to-brown mass, the latter occupying most of the cornea and part of the conjunctiva. Exenteration was performed under general anesthesia, and the ocular tissues were processed routinely for histopathology. Upon microscopic examination, a malignant epithelial neoplasm and a benign vascular neoplasm were present in the cornea. The conjunctiva and the third eyelid were also affected. Upon immunohistochemistry, the epithelial tumor was positive for cytokeratin and negative for vimentin and the endothelial tumor was negative for cytokeratin and positive for vimentin. A diagnosis of squamous cell carcinoma (SCC) and hemangioma was made. The SCC was affecting the cornea, bulbar conjunctiva (lateral and inferior) and the base of the third eyelid, whereas the hemangioma was affecting the cornea and medial limbus. To the authors` knowledge, this is the first report of concomitant SCC and hemangioma affecting the ocular surface in a cat.

Developmental changes of gene expression after spinal cord injury in neonatal opossums

Changes in gene expression have been measured 24 h after injury to mammalian spinal cords that can and cannot regenerate In opossums there is a critical period of development when regeneration stops being possible at 9 days postnatal cervical spinal cords regenerate, at 12 days they do not By the use of marsupial cDNA microarrays we detected 158 genes that respond differentially to injury at the two ages critical for regeneration For selected candidates additional measurements were made by real time PCR and sites of their expression were shown by immunostaining Candidate genes have been classified so as to select those that promote or prevent regeneration Up regulated by injury at 8 days and/or down regulated by injury at 13 days were genes known to promote growth, such as Mitogen activated protein kinase kinase 1 or transcripton factor TCF7L2 By contrast, at 13 days up regulation occurred of Inhibitory molecules including annexins ephrins and genes related to apoptosis and neurodegeneranve diseases Certain genes such as calmodulin 1 and NOGO changed expression similarly in animals that could and could not regenerate without any additional changes in response to injury These findings confirmed and extended changes of gene expression found in earlier screens on 9 and 12 day preparations without lesions and provide a comprehensive list of genes that serve as a basis for testing how identified molecules singly or in combination, promote and prevent central nervous system regeneration (C) 2010 Elsevier B V All rights reserved

Expression of glycoproteins in the vomeronasal organ reveals a novel spatiotemporal pattern of sensory neurone maturation

The main olfactory and the accessory olfactory systems are both anatomically and functionally distinct chemosensory systems. The primary sensory neurones of the accessory olfactory system are sequestered in the vomeronasal organ (VNO), where they express pheromone receptors, which are unrelated to the odorant receptors expressed in the principal nasal cavity. We have identified a 240 kDa glycoprotein (VNO240) that is selectively expressed by sensory neurones in the VNO but not in the main olfactory neuroepithelium of mouse. VNO240 is first expressed at embryonic day 20.5 by a small subpopulation of sensory neurones residing within the central region of the crescent-shaped VNO, Although VNO240 was detected in neuronal perikarya at this age, it was not observed in the axons in the accessory olfactory bulb until postnatal day 3.5, This delayed appearance in the accessory olfactory bulb suggests that VNO240 is involved in the functional maturation of VNO neurones rather than in axon growth and targeting to the bulb, During the first 2 postnatal weeks, the population of neurones expressing VNO240 spread peripherally, and by adulthood all primary sensory neurones in the VNO appeared to be expressing this molecule. Similar patterns of expression were also observed for NOC-1, a previously characterized glycoform of the neural cell adhesion molecule NCAM, To date, differential expression of VNO-specific molecules has only been reported along the rostrocaudal axis or at different apical-basal levels in the neuroepithelium. This is the first demonstration of a centroperipheral wave of expression of molecules in the VNO, These results indicate that mechanisms controlling the molecular differentiation of VNO neurones must involve spatial cues organised, not only about orthogonal axes, but also about a centroperipheral axis, Moreover, expression about this centroperipheral axis also involves a temporal component because the subpopulation of neurones expressing VNO240 and NOC-1 increases during postnatal maturation. (C) 2001 John Wiley & Sons, Inc.

Domestic, peridomestic and wild hosts in the transmission of Trypanosoma cruzi in the Caatinga area colonised by Triatoma brasiliensis

The role played by different mammal species in the maintenance of Trypanosoma cruzi is not constant and varies in time and place. This study aimed to characterise the importance of domestic, wild and peridomestic hosts in the transmission of T. cruzi in Tauá, state of Ceará, Caatinga area, Brazil, with an emphasis on those environments colonised by Triatoma brasiliensis. Direct parasitological examinations were performed on insects and mammals, serologic tests were performed on household and outdoor mammals and multiplex polymerase chain reaction was used on wild mammals. Cytochrome b was used as a food source for wild insects. The serum prevalence in dogs was 38% (20/53), while in pigs it was 6% (2/34). The percentages of the most abundantly infected wild animals were as follows: Thrichomys laurentius 74% (83/112) and Kerodon rupestris 10% (11/112). Of the 749 triatomines collected in the household research, 49.3% (369/749) were positive for T. brasiliensis, while 6.8% were infected with T. cruzi (25/369). In captured animals, T. brasiliensis shares a natural environment with T. laurentius, K. rupestris, Didelphis albiventris, Monodelphis domestica, Galea spixii, Wiedomys pyrrhorhinos, Conepatus semistriatus and Mus musculus. In animals identified via their food source, T. brasiliensis shares a natural environment with G. spixii, K. rupestris, Capra hircus, Gallus gallus, Tropidurus oreadicus and Tupinambis merianae. The high prevalence of T. cruzi in household and peridomiciliar animals reinforces the narrow relationship between the enzootic cycle and humans in environments with T. brasiliensis and characterises it as ubiquitous.

Evolution of mRNA expression levels of autosomal and sex-linked genes in amniotes

In addition to differences in protein-coding gene sequences, changes in expression resulting from mutations in regulatory sequences have long been hypothesized to be responsible for phenotypic differences between species. However, unlike comparison of genome sequences, few studies, generally restricted to pairwise comparisons of closely related mammalian species, have assessed between-species differences at the transcriptome level. They reported that gene expression evolves at different rates in various organs and in a pattern that is overall consistent with neutral models of evolution. In the first part of my thesis, I investigated the evolution of gene expression in therian mammals (i.e.7 placental and marsupials), based on microarray data from human, mouse and the gray short-tailed opossum (Monodelphis domestica). In addition to autosomal genes, a special focus was given to the evolution of X-linked genes. The therian X chromosome was recently shown to be younger than previously thought and to harbor a specific gene content (e.g., genes involved in brain or reproductive functions) that is thought to have been shaped by specific sex-related evolutionary forces. Sex chromosomes derive from ordinary autosomes and their differentiation led to the degeneration of the Y chromosome (in mammals) or W chromosome (in birds). Consequently, X- or Z-linked genes differ in gene dose between males and females such that the heterogametic sex has half the X/Z gene dose compared to the ancestral state. To cope with this dosage imbalance, mammals have been reported to have evolved mechanisms of dosage compensation.¦In the first project, I could first show that transcriptomes evolve at different rates in different organs. Out of the five tissues I investigated, the testis is the most rapidly evolving organ at the gene expression level while the brain has the most conserved transcriptome. Second, my analyses revealed that mammalian gene expression evolution is compatible with a neutral model, where the rates of change in gene expression levels is linked to the efficiency of purifying selection in a given lineage, which, in turn, is determined by the long-term effective population size in that lineage. Thus, the rate of DNA sequence evolution, which could be expected to determine the rate of regulatory sequence change, does not seem to be a major determinant of the rate of gene expression evolution. Thus, most gene expression changes seem to be (slightly) deleterious. Finally, X-linked genes seem to have experienced elevated rates of gene expression change during the early stage of X evolution. To further investigate the evolution of mammalian gene expression, we generated an extensive RNA-Seq gene expression dataset for nine mammalian species and a bird. The analyses of this dataset confirmed the patterns previously observed with microarrays and helped to significantly deepen our view on gene expression evolution.¦In a specific project based on these data, I sought to assess in detail patterns of evolution of dosage compensation in amniotes. My analyses revealed the absence of male to female dosage compensation in monotremes and its presence in marsupials and, in addition, confirmed patterns previously described for placental mammals and birds. I then assessed the global level of expression of X/Z chromosomes and contrasted this with its ancestral gene expression levels estimated from orthologous autosomal genes in species with non-homologous sex chromosomes. This analysis revealed a lack of up-regulation for placental mammals, the level of expression of X-linked genes being proportional to gene dose. Interestingly, the ancestral gene expression level was at least partially restored in marsupials as well as in the heterogametic sex of monotremes and birds. Finally, I investigated alternative mechanisms of dosage compensation and found that gene duplication did not seem to be a widespread mechanism to restore the ancestral gene dose. However, I could show that placental mammals have preferentially down-regulated autosomal genes interacting with X-linked genes which underwent gene expression decrease, and thus identified a novel alternative mechanism of dosage compensation.

Skull Modularity in Neotropical Marsupials and Monkeys: Size Variation and Evolutionary Constraint and Flexibility

An organism is built through a series of contingent factors, yet it is determined by historical, physical, and developmental constraints. A constraint should not be understood as an absolute obstacle to evolution, as it may also generate new possibilities for evolutionary change. Modularity is, in this context, an important way of organizing biological information and has been recognized as a central concept in evolutionary biology bridging on developmental, genetics, morphological, biochemical, and physiological studies. In this article, we explore how modularity affects the evolution of a complex system in two mammalian lineages by analyzing correlation, variance/covariance, and residual matrices (without size variation). We use the multivariate response to selection equation to simulate the behavior of Eutheria and Metharia skulls in terms of their evolutionary flexibility and constraints. We relate these results to classical approaches based on morphological integration tests based on functional/developmental hypotheses. Eutherians (Neotropical primates) showed smaller magnitudes of integration compared with Metatheria (didelphids) and also skull modules more clearly delimited. Didelphids showed higher magnitudes of integration and their modularity is strongly influenced by within-groups size variation to a degree that evolutionary responses are basically aligned with size variation. Primates still have a good portion of the total variation based on size; however, their enhanced modularization allows a broader spectrum of responses, more similar to the selection gradients applied (enhanced flexibility). Without size variation, both groups become much more similar in terms of modularity patterns and magnitudes and, consequently, in their evolutionary flexibility. J. Exp. Zool. (Mol. Dev. Evol.) 314B:663-683, 2010. (C) 2010 Wiley-Liss, Inc.

Variety matters: adaptive genetic diversity and parasite load in two mouse opossums from the Brazilian Atlantic forest

The adaptive potential of a species to a changing environment and in disease defence is primarily based on genetic variation. Immune genes, such as genes of the major histocompatibility complex (MHC), may thereby be of particular importance. In marsupials, however, there is very little knowledge about natural levels and functional importance of MHC polymorphism, despite their key role in the mammalian evolution. In a previous study, we discovered remarkable differences in the MHC class II diversity between two species of mouse opossums (Gracilinanus microtarsus, Marmosops incanus) from the Brazilian Atlantic forest, which is one of the most endangered hotspots for biodiversity conservation. Since the main forces in generating MHC diversity are assumed to be pathogens, we investigated in this study gastrointestinal parasite burden and functional associations between the individual MHC constitution and parasite load. We tested two contrasting scenarios, which might explain differences in MHC diversity between species. We predicted that a species with low MHC diversity would either be under relaxed selection pressure by low parasite diversity (`Evolutionary equilibrium` scenario), or there was a recent loss in MHC diversity leading to a lack of resistance alleles and increased parasite burden (`Unbalanced situation` scenario). In both species it became apparent that the MHC class II is functionally important in defence against gastrointestinal helminths, which was shown here for the first time in marsupials. On the population level, parasite diversity did not markedly differ between the two host species. However, we did observe considerable differences in the individual parasite load (parasite prevalence and infection intensity): while M. incanus revealed low MHC DAB diversity and high parasite load, G. microtarsus showed a tenfold higher population wide MHC DAB diversity and lower parasite burden. These results support the second scenario of an unbalanced situation.

Preferência alimentar e identificação das principais fontes de repasto sanguíneo de fêmeas Lutzomyia (Diptera: Psychodidae) em áreas endêmicas para leishmaniose visceral na grande Natal

Leishmaniasis are endemic diseases wild spread in the New and Old World, caused by the flagelated protozoan Leishmania. In the New World, the distribution of different forms of leishmaniasis is mostly in tropical regions. In the State of Rio Grande do Norte, Northeast Brazil, 85% of the captured sand flies fauna is Lutzomyia longipalpis. The distribution of the sand fly vector in the state overlaps with the disease distribution, where the presence of sand flies is associated with presence of animals shelters. The aim of this study was to analyse the blood meal preference of sand flies vector from the genus Lutzomyia spp. in laboratory conditions, to verify the vector life cicle at different temperatures sets and to identify the main blood meal source in endemic areas for visceral leishmaniasis (VL) at peri-urban regions of Natal. Sand flies samples were collected from the municipalities of São Gonçalo do Amarante and Nísia Floresta where female sand flies were grouped for the colony maintenance in the laboratory and for the analysis of the preferred source of sand fly blood meal in natural environment. The prevalence of blood meal preference and oviposition for the females sand flies was 97% for Cavia porcellus with oviposition of 19 eggs/female; 97% for Eqqus caballus with 19 eggs/female; 98% for human blood with 14 eggs/female; 71.3% for Didelphis albiventris with 8.4 eggs/female; 73% for Gallus gallus with 14 eggs/female; 86% for Canis familiaris with 10.3 eggs/female; 81.4% for Galea spixii with 26 eggs/female; 36% for Callithrix jachus with 15 eggs/female; 42.8% for Monodelphis domestica with 0% of oviposition. Female sand flies did not take a blood meal from Felis catus. Sand flies life cycle ranged from 32-40 days, with 21-50 oviposition rates approximately. This study also showed that at 32°C the life cycle had 31 days, at 28° C it had 50 days and at 22°C it increased to 79 days. Adjusting the temperature to 35°C the eggs did not hatch, thus blocking the life cycle. A total of 1540 sand flies were captured, among them, 1.310 were male and 230 were female. Whereas 86% of the sand flies captured were Lu. longipalpis as compared to 10.5% for Lu. evandroi and, 3.2% for L. lenti and 0.3% for Lu whitmani. The ratio between female and male sandfly was approximately 6 males to 1 female. In Nísia Floresta, 50.7% of the collected females took their blood meal from armadillo, 12.8% from human. Among the female sand flies captured in São Gonçalo do Amarante, 80 of them were tested for the Leishmania KDNA infectivity where 5% of them were infected with Leishmania chagasi. Female Lutzomyia spp. showed to have an opportunistic blood meal characteristic. The behavioral parameters seem to have a higher influence in the oviposition when compared to the level of total proteins detected in the host s bloodstream. A higher Lu. longipalpis life cycle viability was observed at 28°C. The increase of temperature dropped the life cycle time, which means that the life cycle is modified by temperature range, source of blood meal and humidity. Lu longipalpis was the most specie found in the inner and peridomiciliar environment. In Nísia Floresta, armadillos were the main source of blood meal for Lutzomyia spp. At São Gonçalo do Amarante, humans were the main source of blood meal due to CDC nets placed inside their houses

The discovery of Foxl2 paralogs in chondrichthyan, coelacanth and tetrapod genomes reveals an ancient duplication in vertebrates

The Foxl2 (forkhead box L2) gene is an important member of the forkhead domain family, primarily responsible for the development of ovaries during female sex differentiation. The evolutionary studies conducted previously considered the presence of paralog Foxl2 copies only in teleosts. However, to search for possible paralog copies in other groups of vertebrates and ensure that all predicted copies were homolog to the Foxl2 gene, a broad evolutionary analysis was performed, based on the forkhead domain family. A total of 2464 sequences for the forkhead domain were recovered, and subsequently, 64 representative sequences for Foxl2 were used in the evolutionary analysis of this gene. The most important contribution of this study was the discovery of a new subgroup of Foxl2 copies (ortholog to Foxl2B) present in the chondrichthyan Callorhinchus milii, in the coelacanth Latimeria chalumnae, in the avian Taeniopygia guttata and in the marsupial Monodelphis domestica. This new scenario indicates a gene duplication event in an ancestor of gnathostomes. Furthermore, based on the analysis of the syntenic regions of both Foxl2 copies, the duplication event was not exclusive to Foxl2. Moreover, the duplicated copy distribution was shown to be complex across vertebrates, especially in tetrapods, and the results strongly support a loss of this copy in eutherian species. Finally, the scenario observed in this study suggests an update for Foxl2 gene nomenclature, extending the actual suggested teleost naming of Foxl2A and Foxl2B to all vertebrate sequences and contributing to the establishment of a new evolutionary context for the Foxl2 gene. © 2013 Macmillan Publishers Limited All rights reserved.

Evolução e organização genômica do gene FOXL2 em vertebrados

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)