Two whole genome duplications (2R) occurred early in vertebrate evolution. By using combined information from phylogenetic analyses and chromosomal location of genes, this thesis delineates the evolutionary history of two receptor-ligand systems that expanded by these large scale events. A third whole genome duplication (3R) took place in the teleost fish lineage and has also contributed to the complexity of the gene families. New members of neuropeptide Y (NPY) peptide and receptor families were generated in 2R and 3R. Evolutionary comparisons show that the ancestral teleost fish had four peptides; subsequently, differential losses of the peptide genes occurred. In zebrafish the peptides and receptors display differences in tissue distribution and have  evolved binding preferences. In the frog Silurana tropicalis three peptides and six receptors werev identified, also displaying some differences in tissue distribution and receptor-ligand preferences. The findings in these experimental animals highlight both evolutionary conservation and lineage-specific features of the NPY system. The opioid system consists of four receptors and several peptides originating from four precursors. These results show that the receptor family was formed in 2R and 3R and that 2R together with one local duplication gave rise to the peptide family. The ancestral receptor and peptide genes were located on the same chromosome, suggesting coevolution. The Hox gene clusters, important in early development, provided the first strong evidence for 2R. Several neighboring gene families were analyzed and found to have expanded in 2R and 3R. In depth analyses of insulin-like growth factor binding protein (IGFBP) and voltage-gated sodium channel (SCN) gene families illustrates the importance of local duplications in combination with whole genome duplications in the formation of gene families. These findings provide additional strong evidence for two genome duplications in early vertebrate evolution and show that these events generated many new genes that could evolve new or more specialized functions.

Recent field campaigns have measured enhanced levels of NOx (NO+NO2) and HOx precursors (i.e., H2O2, CH2O, and HONO) that can not be accounted for by gas phase chemistry alone. Snowpack emission is now considered a source of these species. Therefore, the photochemistry in the polar boundary layer is now believed to be much more complex than initially thought. Field campaigns to Summit, Greenland in the summer of 2003 and the spring of 2004 have obtained the first measurements of peroxy (HO2+RO2) and hydroxyl (OH) radicals in the Artic boundary layer. Measurements were collected with a chemical ionization mass spectrometer (CIMS). A highly constrained (ie., O3, H2O, CH4, CO, j-values, NO, H2O2,CH2O, and HONO) 0-D steady-state model was employed in order to test our current understanding of photochemistry. HO2+RO2 measurements were in excellent agreement with model predictions for both spring and summer. OH measurements were in good agreement with spring model predictions but were a factor of two greater than summer model predictions. The role of snowpack emission is also addressed in a HOx budget performed on the spring campaign. Measurements of nitric acid (HNO3) and pernitric acid (HO2NO2) were obtained with the CIMS during the Antarctic Tropospheric Chemistry Investigation (ANTCI). The linkage between HOx and NOx chemistry is examined through partitioning of reactive nitrogen between HNO3 and HO2NO2. The possible impact of reactive nitrogen partitioning on nitrate ions (NO3-) at coring sites is also investigated.

A fundamental problem in developmental and evolutionary biology is understanding the developmental genetic basis of morphological diversity. The current paradigm holds that a genetic and developmental program, or developmental genetic "toolkit", conserved across hundreds of millions of years patterns development in all metazoans. However, outside of a few well-characterized signal transduction pathways and developmental processes, overly broad strokes have been used to paint this "toolkit" metaphor as a hypothesis. Arthropoda, one of the largest groups of metazoans, represent the most morphologically diverse groups of metazoans, making them of particular interest for studies of morphological diversity and its evolution. Arthropoda is also home to one of the most well-understood model systems for developmental and genetic studies, the fruit fly Drosophila melanogaster. However, Drosophila is highly derived among arthropods with respect to the molecular genetic mechanisms that function during its development. As it is expected that all arthropods have access to the same development "toolkit", some changes are expected based on the observable differences in morphology, making arthropods extremely powerful tools for comparative genomic and molecular genetic studies. In this dissertation I characterize how modifications to the developmental "toolkit" contribute to the evolution of morphological diversity using emerging model arthropod systems. First, as part of a collaboration, I show that several genes expected to be conserved in all arthropods, belonging to the Hox family of transcription factors, have been lost from the genome of a phylogenetically basal arthropod, the two-spotted spider mite Tetranychus urticae. Second, I perform a genomic survey and find an overall reduction in the conservation of Drosophila orthologs from several major signal transduction pathways in the Tetranychus genome in comparison with findings from previous insect surveys. Third, I show that arthropod Hox genes, expected to be found in a tightly linked genomic cluster in most arthropod genomes, are not as tightly clustered as previously thought. Fourth, I show that changes in the genomic arrangement of Tetranychus Hox genes correspond with shifts in their expression and morphological change. Finally, I show the terminal Hox gene Abdominal-B is required for proper axial elongation and segment formation (both segment identity and number) during embryogenesis and metamorphosis in the red-flour beetle Tribolium castaneum. Taken together, these findings advance our knowledge of the evolution of morphological change, with a primary focus on Hox genes and their contribution to axial patterning during development.

Todos los animales, vivos y fósiles, están comprendidos dentro de 35 fila. Éstos abarcan la enorme diversidad de morfologías que se han generado durante la evolución animal, desde una medusa, hasta una mosca o nosotros mismos, el hombre. Cada una de las diferentes morfologías son en último término el resultado de un proceso de desarrollo embrionario muy finamente regulado en el que participan cientos de genes, y que forman parte de decenas de redes génicas que interactúan unas con otras. A lo largo del tiempo, estas morfologías han ido evolucionando de forma que se han ido generando nuevas especies a partir de otras preexistentes o ancestros, compartidos por las especies que derivan de él. Dado este marco, cobra fuerza una disciplina relativamente reciente, la Evo-Devo o Evolución del Desarrollo, que pretende explicar la evolución de la morfología animal buscando los cambios en los genes del desarrollo embrionario, ya que la primera es fruto del segundo. Sin embargo, la mayoría de los genes del desarrollo son los mismos en todos los animales, algo que se conoce como el toolkit genético, y esto lleva a lo que se conoce como la paradoja de la Evo-Devo: ¿cómo se explica entonces la gran diversidad morfológica, si los diferentes animales están construidos con los mismos genes? Hay que buscar por tanto las diferencias que existan en los procesos del desarrollo de los diferentes animales. Una de estas diferencias puede recaer en cómo los genes del desarrollo se regulan. En esta tesis, se han utilizado los genes Hox como modelos de evolución genómica, y se han analizado el patrón de expresión de estos genes en la especie europea de cefalocordado, Branchiostoma lanceolatum. Se ha identificado el patrón de expresión de casi todos los genes del clúster Hox de este animal, constituido por 15 genes Hox. De ellos, los genes Hox1, 2, 3, 4 y 6 ya estaban descritos en la especie americana B. floridae, aunque se ha encontrado un patrón diferente para el gen Hox6. Además, para los genes Hox6 y Hox14, se ha encontrado un patrón de regulación modular, donde parte del patrón de expresión está regulado por ácido retinóico. En otra fase de la tesis, se han identificado putativos elementos reguladores en las regiones intergénicas de estos genes mediante una estrategia de "phylogenetic footprinting". Esto ha sido realizado mediante la comparación de regiones ortólogas de los genes Hox de las dos especies de anfioxo y humando. Algunas de estas, como las que rodean el gen Hox4, están conservadas también con pez cebra, y son capaces de dirigir la expresión de un gen reporter en tejidos donde se expresan los genes endógenos. Además, siguiendo la misma estrategia, hemos identificado una región de regulación global que está presente en vertebrados, situándose su origen al menos en el ancestro de los cordados. / "Hox complex as models for genomic evolution: characterization and regulation of Hox genes expression of European Hox cluster". TEXT:Hox genes are key developmental genes involved in patterning the antero-posterior axis of most metazoans studied so far. They generally are linked in genomic cluster and expressed with spatial and temporal colinearity in amphioxus and vertebrates. The closer to the 3' extreme of the cluster the gene is, the earlier and more anteriorly it is expressed. Nonetheless, this is fully true for all genes only in vertebrate clusters, since the expression of almost all central and all posterior Hox genes of amphioxus, which represents the closest relative to the chordate ancestor, is not known, and the expression in hemichordates and tunicates is not always colinear. In this thesis, we present a complete expression profile of amphioxus Hox genes and interestingly report the breaking of both spatial and temporal colinearity of some central and posterior Hox genes. Posterior Hox genes are expressed in structures like the notochord and posterior parts of the gut. Hox14 had the most divergent expression pattern, being also present in the anterior cerebral vesicle and pharyngeal endoderm. This is the first report of Hox expression in the most anterior part of a central nervous system. We also show that Hox14 expression is partially regulated by retinoic acid (RA) (in notochord and hindgut), like it happens for more anterior Hox genes are. On the other hand, Hox14 expression in the cerebral vesicle and pharynx are not influenced by RA. The lack of constriction in the posterior part of vertebrate and cephalochordate Hox cluster may be the cause of their independent expansion and their co-option for patterning different structures, allowing the breaking of colinearity in deuterostomes.

One of the main issues in animal evolution deal with the transition from radial organisms (Cnidaria and Ctnenophora), with only one axis of symmetry, the Oral-Aboral (OA) axis, to bilateral organism (Bilateria), bearing two orthogonal body axes, the Antero-posterior (AP) and the Dorso-ventral (DV) axis.Finding the extant bilateral organism closest to the bilaterian ancestor is the first and necessary step to open new ways of analysis. Recent molecular phylogenies have convincingly shown that the acoel flatworms, traditionally classified within the turbellarian Platyhelminthes, are the sister group of the remaining Bilateria, branching out before the common ancestor of protostomes, and deuterostomes.Hox and ParaHox genes encode for transcriptional regulators involved in the control the AP body axis in all bilateral animals. Hox genes are usually organized in clusters in Bilateria, which have been originated by means of several gene tandem duplications from an original ProtoHox gene. In addition, Hox genes show a collinear correspondence between gene order within the cluster and the body levels at which these genes are expressed.On the contrary, in the phylogenetic sister group of Bilateria, the Cnidaria, Hox genes are not linked in a single cluster and do not seem to play a similar role for patterning the OA axis.Since it is still unclear when in the evolutionary history of bilaterians the Hox system first conferred positional identity along the AP-axis, the comparative study of the patterning genes Hox and ParaHox in acoel flatworms, could be crucial to understand the origin of the Hox-ParaHox axial patterning system and how the morphological transition from radial to bilateral animals took place.In this Thesis, we report on the cloning, genomic arrangement, and expression domains of Hox genes in the acoel species Symsagittifera roscoffensis. Three Hox genes were detected: one from each of the major groups of Hox genes, which are anterior, central, and posterior, named SrHox1, SrHox5 and SrHoxPost respectively. All acoel species studied to date contain the same minimal complement of three Hox genes and one Cdx ParaHox gene, suggesting that the last common bilaterian ancestor (or Urbilateria) had a simple Hox gene complement, composed of only 3 or 4 genes.In bacterial artificial chromosome cloning, sequencing, and chromosomal fluorescence in situ hybridization, Hox genes were not observed as being clustered in a unique genomic region in S. roscoffensis. Nevertheless, despite its dispersion within the genome, Hox genes are expressed in nested domains along the AP axis in the juvenile worm. The basic set of Hox genes in acoels and their coarse nested spatial deployment might be the first indicators of the role of Hox genes in the evolution of bilateral symmetry and AP positional identity from a hypothetical radial ancestor.In order to understand how the AP axis has been established over evolutionary time, the execution of functional analyses is essential. With this purpose, we have performed the knockdown of the posterior Hox, IpHoxPost, during the postembryonic development, regeneration and adulthood of the acoel species Isodiametra pulchra, using RNA interference technologies.The analysis has been done for the first time in acoels, and we demonstrate that the functions of this gene are restricted to the posterior region of the animal, within the muscular and neural tissues. We conclude, therefore, that the posterior Hox genes were used to specify and maintain defined anatomical regions within the AP axis of animals since the beginning of bilaterian evolution. / "Caracterización de los genes Hox en el acelo Symsagittifera roscoffensis"TEXTO:Los genes Hox codifican factores de transcripción que regionalizan el eje antero-posterior durante el desarrollo embrionario en todos los animales bilaterales estudiados. Los animales radiales (cnidarios y ctenóforos) poseen genes Hox, pero estos no desempeñan un rol similar al de sus homólogos en Bilateria, por lo que el sistema de regionalización Hox puede ser considerado una innovación de los Bilateria.Recientes análisis filogenéticos han demostrado que Acoelomorpha (acelos y nemertodermátidos), un grupo de gusanos clasificados tradicionalmente como platelmintos, divergieron antes del último antecesor común de protóstomos y deuteróstomos. En consecuencia, representan el grupo de organismos bilaterales idóneo para estudiar la evolución del sistema Hox entre cnidarios y bilaterales. Por este motivo, el objetivo principal de esta tesis ha sido analizar el sistema Hox en acelos.Encontramos un complemento simple de 3 genes Hox en las 2 especies de acelos estudiadas: Symsagittifera roscoffensis e Isodiametra pulchra. Estos genes no están ligados en el genoma de S. roscoffensis pero se expresan de forma colinear durante el desarrollo postembrionario, lo que representa el primer ejemplo de expresión colinear de genes Hox en Bilateria, indicando que la colinearidad estuvo presente en el ancestro de todos los Bilateria.Las funciones del Hox posterior fueron analizadas mediante RNA de interferencia en I. pulcra. El fenotipo knockdown indica que IpHoxPost está implicado en la regulación del establecimiento de las estructuras morfológicas en la parte posterior, especialmente de los músculos bucales y los situados alrededor de los aparatos copuladores; así como en el proceso de maduración de los huevos y la proliferación celular. Esto indica que el rol del Hox posterior en la regulación del desarrollo y diversificación del mesodermo postembrionario y la musculatura ha surgido tempranamente durante la evolución de los Bilateria.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22641号 / 医博第4624号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 湊谷 謙司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Für weite Teile der Weltbevölkerung stellen Übergewicht und Adipositas ein zunehmendes Gesundheitsrisiko dar. In Deutschland sind mehr als ein Viertel der Bevölkerung übergewichtig oder adipös. Gemäß der Weltgesundheitsorganisation (WHO) definieren sich Übergewicht und Adipositas über den Body-Mass-Index (BMI), der jedoch keine Aussage über den absoluten Fettgehalt oder das Fettverteilungsmuster liefert. Dass Letzteres jedoch entscheidend ist im Hinblick auf die Entwicklung Adipositas-assoziierter Komorbiditäten, wurde in den letzten Jahren immer deutlicher. Nicht nur das absolute Körpergewicht, sondern auch die individuelle Verteilung des Fettgewebes ist stark genetisch beeinflusst, wobei eine viszerale Fettakkumulation mit einem deutlich schlechteren metabolischen Risikoprofil einhergeht als eine subkutane und femoro-gluteale Fettakkumulation. Das Risiko Adipositas-assoziierte Folgeerkrankungen zu entwickeln, scheint zudem von der Funktion des Fettgewebes abzuhängen. Eine Fettgewebsdysfunktion ist dabei unter anderem durch eine Adipozytenhypertrophie, ektope Fettverteilung und die Infiltration des Fettgewebes von Entzündungszellen charakterisiert. Für die genetische Regulation der Fettgewebsverteilung kommen unter anderem Gene in Betracht, die in den einzelnen Depots (beispielsweise subkutan versus viszeral) unterschiedlich stark exprimiert werden. Hierzu zählen auch Gene, welche während der Embryonalentwicklung aktiv sind, sodass möglicherweise von einer embryonalen Determinierung der Fettverteilung ausgegangen werden kann. Ein Teil dieser Entwicklungsgene sind die sogenannten Homeobox oder kurz HOX-Gene, die eine entwicklungsbiologisch sehr alte und hoch konservierte Gruppe darstellen. Während der embryonalen Phase nehmen sie unter anderem Einfluss auf die Ausbildung der anteriorposterioren Körperachse, die Zelldifferenzierung und die Organogenese. In der vorliegenden Arbeit wurde die Expression der Gene, HOXC9 und HOXC10, in gepaarten humanen Biopsien aus abdominal subkutanem und omentalem Fettgewebe bei 636 Probanden untersucht und mit anthropometrischen und metabolischen Parametern korreliert. Dabei zeigte sich eine höhere Expression von HOXC9 und HOXC10 im subkutanen im Vergleich zum omentalen Fettgewebe. Für beide Gene zeigte sich ein statistischer Zusammenhang zu Parametern von Übergewicht/Adipositas und Körperfettgehalt, der Adipozytendysfunktion sowie des gestörten Glukosemetabolismus. Diese Daten deuten darauf hin, dass HOXC9 und HOXC10 eine zentrale Rolle in der Entwicklung von Adipositas, pathologischer Fettverteilung sowie den daraus resultierenden Adipositas-assoziierten Erkrankungen spielen.

The ParaHox cluster contains three Hox‐related homeobox genes. The evolution of this sister of the Hox‐gene clusters has been studied extensively in metazoans with a focus on its early evolution. Its fate within the vertebrate lineage, and in particular following the teleost‐specific genome duplication, however, has not received much attention. Three of the four human ParaHox loci are linked with PDGFR family tyrosine kinases. We demonstrate that these loci arose as duplications in an ancestral vertebrate and trace the subsequent history of gene losses. Surprisingly, teleost fishes have not expanded their ParaHox repertoire following the teleost‐specific genome duplication, while duplicates of the associated tyrosine kinases have survived, supporting the hypothesis of a large‐scale duplication followed by extensive gene loss.

Evolutionarily conserved non-coding genomic sequences represent a potentially rich source for the discovery of gene regulatory regions. Since these elements are subject to stabilizing selection they evolve much slower than adjacent non-functional DNA. These so-called phylogenetic footprints can be detected by comparison of the sequences surrounding orthologous genes in different species. In this paper we present a new method and an effcient software tool for the identifcation of corresponding footprints in long sequences from multiple species. This allows the evolutionary study of the origin and loss of phylogenetic footprints if suffcient number and appropriately placed species are included. We apply this method to the published sequences of HoxA clusters of shark, human, and the duplicated zebrafish and Takifugu clusters as well as the published HoxB cluster sequences. We find that there is a massive loss of sequence conservation in the intergenic region of the HoxA clusters, consistent with the finding in [Chiu et al., PNAS 99, 5492-5497 (2002)]. We further propose a simple model to estimate the loss of sequence conservation that can be attributed to gene loss and other structural reasons. We find that the loss of conservation after cluster duplication is more extensive than expected by this model. This suggests that binding site turnover and/or adaptive modification may also contribute to the loss of sequence conservation. We conclude that this method is suitable for the large scale study of the evolution of (putative) cis-regulatory elements.

The statistical analysis of phylogenetic footprints in the two known horn shark Hox clusters and the four mammalian clusters shows that the shark HoxN cluster is HoxD-like. This finding implies that the most recent common ancestor of jawed vertebrates had at least four Hox clusters, including those which are orthologous to the four mammalian Hox clusters.