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Cilia, either motile or non-motile (a.k.a primary or sensory), are complex evolutionarily conserved eukaryotic structures composed of hundreds of proteins required for their assembly, structure and function that are collectively known as the ciliome. Ciliome gene mutations underlie a group of pleiotropic genetic diseases known as ciliopathies. Proper cilium function requires the tight coregulation of ciliome gene transcription, which is only fragmentarily understood. RFX transcription factors (TF) have an evolutionarily conserved role in the direct activation of ciliome genes both in motile and non-motile cilia cell-types. In vertebrates, FoxJ1 and FoxN4 Forkhead (FKH) TFs work with RFX in the direct activation of ciliome genes, exclusively in motile cilia cell-types. No additional TFs have been described to act together with RFX in primary cilia cell-types in any organism. Here we describe FKH-8, a FKH TF, as a direct regulator of the sensory ciliome genes in Caenorhabditis elegans. FKH-8 is expressed in all ciliated neurons in C. elegans, binds the regulatory regions of ciliome genes, regulates ciliome gene expression, cilium morphology and a wide range of behaviors mediated by sensory ciliated neurons. FKH-8 and DAF-19 (C. elegans RFX) physically interact and synergistically regulate ciliome gene expression. C. elegans FKH-8 function can be replaced by mouse FOXJ1 and FOXN4 but not by other members of other mouse FKH subfamilies. In conclusion, RFX and FKH TF families act jointly as direct regulators of ciliome genes also in sensory ciliated cell types suggesting that this regulatory logic could be an ancient trait predating functional cilia sub-specialization.

Neuronal programming by forced expression of transcription factors (TFs) holds promise for clinical applications of regenerative medicine. However, the mechanisms by which TFs coordinate their activities on the genome and control distinct neuronal fates remain obscure. Using direct neuronal programming of embryonic stem cells, we dissected the contribution of a series of TFs to specific neuronal regulatory programs. We deconstructed the Ascl1-Lmx1b-Foxa2-Pet1 TF combination that has been shown to generate serotonergic neurons and found that stepwise addition of TFs to Ascl1 canalizes the neuronal fate into a diffuse monoaminergic fate. The addition of pioneer factor Foxa2 represses Phox2b to induce serotonergic fate, similar to in vivo regulatory networks. Foxa2 and Pet1 appear to act synergistically to upregulate serotonergic fate. Foxa2 and Pet1 co-bind to a small fraction of genomic regions but mostly bind to different regulatory sites. In contrast to the combinatorial binding activities of other programming TFs, Pet1 does not strictly follow the Foxa2 pioneer. These findings highlight the challenges in formulating generalizable rules for describing the behavior of TF combinations that program distinct neuronal subtypes.

[EN] Cilia are complex evolutionary conserved eukaryotic structures that, projecting from cell surfaces, perform a variety of biological roles. Cilia are traditionally classified into motile or sensory and hundreds of proteins take part in their composition. This set of genes coding for ciliary components is known as the ciliome. Mutations in the ciliome underlie an ever-growing group of highly pleiotropic multisystemic diseases globally termed as ciliopathies. These diseases are characterized, among other symptoms, by mental retardation, sensory defects and/or metabolic disorders. Despite an estimated 1 in 1,000 people affected by these diseases, the molecular bases of the ciliopathies are still poorly understood. Proper cilium assembly and functionality requires the tightly co-regulated expression of ciliary components; however, little is known about the regulatory logic controlling ciliome transcription. Most ciliome genes are shared between motile and sensory cilia. RFX transcription factors (TFs) have an evolutionarily conserved role in the transcriptional regulation of both motile and sensory ciliome. In vertebrates, transcription of motile ciliome is also directly regulated by FoxJ1, a Forkhead (FKH) TF. However, to date, TFs working together with RFX in the transcription of the sensory ciliome are unknown in any organism. In this work, we have identified FKH-8, a FKH TF, as a terminal selector of the sensory ciliome in C. elegans. fkh-8 is consistently expressed within the sixty ciliated sensory neurons of C. elegans, it binds the regulatory regions of the sensory ciliome genes, it is also required for correct ciliome gene expression and acts synergistically with the known master regulator of the ciliogenesis DAF-19/RFX. Accordingly, fkh-8 mutants display a wide range of behavioural defects in a plethora of sensory mediated paradigms, including olfaction, gustation, and mechano-sensation. Thus, we have identified, for the first time, a TF that acts together, [ES] Los cilios son estructuras eucariotas complejas conservadas evolutivamente que, proyectando desde la superficie de las células, desempeñan un gran número de funciones biológicas. Los cilios se clasifican tradicionalmente en móviles o sensoriales y en su composición intervienen cientos de proteínas. Este conjunto de genes que codifican para los componentes ciliares se conoce como cilioma. Las mutaciones en el cilioma subyacen a un grupo cada vez mayor de enfermedades multisistémicas altamente pleiotrópicas denominadas globalmente como ciliopatías. Estas enfermedades se caracterizan, entre otros síntomas, por retraso mental, defectos sensoriales y/o trastornos metabólicos. A pesar de que se estima que 1 de cada 1.000 personas está afectada por estas enfermedades, las bases moleculares de las ciliopatías son todavía poco conocidas. El adecuado ensamblaje y funcionalidad del cilio requieren de la expresión estrechamente coordinada de los componentes del cilio; sin embargo, se sabe poco sobre la lógica reguladora que controla la transcripción del cilioma. La mayoría de los genes del cilioma son compartidos tanto por cilios móviles como sensoriales. Los factores de transcripción (FTs) de la familia RFX tienen un papel evolutivamente conservado en la regulación transcripcional del cilioma tanto móvil como sensorial. En los vertebrados, la transcripción del cilioma móvil también está regulada directamente por FoxJ1, un FT de la familia forkhead (FKH). Sin embargo, hasta la fecha, se desconocen los FTs que actúan junto a RFX en la transcripción del cilioma sensorial en cualquier organismo. En este trabajo, hemos identificado a FKH-8, un FT de la familia FKH, como selector terminal del cilioma sensorial de C. elegans. fkh-8 se expresa de forma consistente en las sesenta neuronas sensoriales ciliadas de C. elegans, se une a las regiones reguladoras de los genes del cilioma sensorial, también es necesario para la correcta expresión de los genes del cilioma y actúa de forma

To systematically investigate the complexity of neuron specification regulatory networks, we performed an RNA interference (RNAi) screen against all 875 transcription factors (TFs) encoded in Caenorhabditis elegans genome and searched for defects in nine different neuron types of the monoaminergic (MA) superclass and two cholinergic motoneurons. We identified 91 TF candidates to be required for correct generation of these neuron types, of which 28 were confirmed by mutant analysis. We found that correct reporter expression in each individual neuron type requires at least nine different TFs. Individual neuron types do not usually share TFs involved in their specification but share a common pattern of TFs belonging to the five most common TF families: homeodomain (HD), basic helix loop helix (bHLH), zinc finger (ZF), basic leucine zipper domain (bZIP), and nuclear hormone receptors (NHR). HD TF members are overrepresented, supporting a key role for this family in the establishment of neuronal identities. These five TF families are also prevalent when considering mutant alleles with previously reported neuronal phenotypes in C. elegans, Drosophila, and mouse. In addition, we studied terminal differentiation complexity focusing on the dopaminergic terminal regulatory program. We found two HD TFs (UNC-62 and VAB-3) that work together with known dopaminergic terminal selectors (AST-1, CEH-43, CEH-20). Combined TF binding sites for these five TFs constitute a cis-regulatory signature enriched in the regulatory regions of dopaminergic effector genes. Our results provide new insights on neuron-type regulatory programs in C. elegans that could help better understand neuron specification and evolution of neuron types.

Neuronal diversity is an intrinsic feature of the nervous system. Transcription factors (TFs) are key regulators in the establishment of different neuronal identities; how are the actions of different TFs coordinated to orchestrate this diversity? Are there common features shared among the different neuron types of an organism or even among different animal groups? In this review, we provide a brief overview on common traits emerging on the transcriptional regulation of neuron type diversification with a special focus on the comparison between mouse and Caenorhabditis elegans model systems. In the first part, we describe general concepts on neuronal identity and transcriptional regulation of gene expression. In the second part of the review, TFs are classified in different categories according to their key roles at specific steps along the protracted process of neuronal specification and differentiation. The same TF categories can be identified both in mammals and nematodes. Importantly, TFs are very pleiotropic: Depending on the neuron type or the time in development, the same TF can fulfil functions belonging to different categories. Finally, we describe the key role of transcriptional repression at all steps controlling neuronal diversity and propose that acquisition of neuronal identities could be considered a metastable process.

The serotonergic system of mammals innervates virtually all the central nervous system and regulates a broad spectrum of behavioral and physiological functions. In mammals, serotonergic neurons located in the rostral raphe nuclei encompass diverse sub-systems characterized by specific circuitry and functional features. Substantial evidence suggest that functional diversity of serotonergic circuits has a molecular and connectivity basis. However, the landscape of intrinsic developmental mechanisms guiding the formation of serotonergic sub-systems is unclear. Here, we employed developmental disruption of gene expression specific to serotonergic subsets to probe the contribution of the tyrosine kinase receptor ErbB4 to serotonergic circuit formation and function. Through an in vivo loss-of-function approach, we found that ErbB4 expression occurring in a subset of serotonergic neurons, is necessary for axonal arborization of defined long-range projections to the forebrain but is dispensable for the innervation of other targets of the serotonergic system. We also found that Erbb4-deletion does not change the global excitability or the number of neurons with serotonin content in the dorsal raphe nuclei. In addition, ErbB4-deficiency in serotonergic neurons leads to specific behavioral deficits in memory processing that involve aversive or social components. Altogether, our work unveils a developmental mechanism intrinsically acting through ErbB4 in subsets of serotonergic neurons to orchestrate a precise long-range circuit and ultimately involved in the formation of emotional and social memories.

During development, signal-regulated transcription factors (TFs) act as basal repressors and upon signalling through morphogens or cell-to-cell signalling shift to activators, mediating precise and transient responses. Conversely, at the final steps of neuron specification, terminal selector TFs directly initiate and maintain neuron-type specific gene expression through enduring functions as activators. C. elegans contains 3 types of serotonin synthesising neurons that share the expression of the serotonin biosynthesis pathway genes but not of other effector genes. Here, we find an unconventional role for LAG-1, the signal-regulated TF mediator of the Notch pathway, as terminal selector for the ADF serotonergic chemosensory neuron, but not for other serotonergic neuron types. Regulatory regions of ADF effector genes contain functional LAG-1 binding sites that mediate activation but not basal repression. lag-1 mutants show broad defects in ADF effector genes activation, and LAG-1 is required to maintain ADF cell fate and functions throughout life. Unexpectedly, contrary to reported basal repression state for LAG-1 prior to Notch receptor activation, gene expression activation in the ADF neuron by LAG-1 does not require Notch signalling, demonstrating a default activator state for LAG-1 independent of Notch. We hypothesise that the enduring activity of terminal selectors on target genes required uncoupling LAG-1 activating role from receiving the transient Notch signalling.

Neuronal specification is a protracted process that begins with the commitment of progenitor cells and culminates with the generation of mature neurons. Many transcription factors are continuously expressed during this process but it is presently unclear how these factors modify their targets as cells transition through different stages of specification. In olfactory bulb adult neurogenesis, the transcription factor PBX1 controls neurogenesis in progenitor cells and the survival of migrating neuroblasts. Here, we show that, at later differentiation stages, PBX1 also acts as a terminal selector for the dopaminergic neuron fate. PBX1 is also required for the morphological maturation of dopaminergic neurons and to repress alternative interneuron fates, findings that expand the known repertoire of terminal-selector actions. Finally, we reveal that the temporal diversification of PBX1 functions in neuronal specification is achieved, at least in part, through the dynamic regulation of alternative splicing. In Caenorhabditis elegans, PBX/CEH-20 also acts as a dopaminergic neuron terminal selector, which suggests an ancient role for PBX factors in the regulation of terminal differentiation of dopaminergic neurons.

Introduction The complexity of animal morphology, from body plans to the smallest appendage, and the functions that different organs and cell types are able to do, all arise from a set of tightly coordinated processes that together constitute development. Understanding these processes, i.e., understanding how a fertilized egg develops into a multicellular animal, has been acknowledged as one of the greatest challenges in biology. The aim of this thesis is to gain knowledge on the mechanisms controlling the final step of development, terminal differentiation and how they change through evolution. During differentiation, cells gradually restrict their potential to become different cell types. Usually, cells up to the step of gastrulation are regarded as pluripotent, which means that they have the potential to become any cell type in each of the three germ layers. Afterwards, cells traverse different stages of decreasing multipotency. The term ‘stem cell’ refers to undifferentiated cells that are capable of self-renewal. Tissue-specific stem cells are usually multipotent, such as haematopoietic stem cells, or unipotent, such as myosatellite cells of muscle. Tissue-specific stem cells give rise to committed progenitors, generally regarded as uni- or oligopotent, which are capable of a finite number of divisions and give rise to immature unipotent cells, which eventually maturate and become terminally differentiated cells of a given type. Each terminally differentiated cell type is defined by a particular combination of differentially expressed effector genes, which are responsible for its unique properties and behaviours.[...]

Developmental programs that generate the astonishing neuronal diversity of the nervous system are not completely understood and thus present a major challenge for clinical applications of guided cell differentiation strategies. Using direct neuronal programming of embryonic stem cells, we found that two main vertebrate proneural factors, Ascl1 and neurogenin 2 (Neurog2), induce different neuronal fates by binding to largely different sets of genomic sites. Their divergent binding patterns are not determined by the previous chromatin state, but are distinguished by enrichment of specific E-box sequences that reflect the binding preferences of the DNA-binding domains. The divergent Ascl1 and Neurog2 binding patterns result in distinct chromatin accessibility and enhancer activity profiles that differentially shape the binding of downstream transcription factors during neuronal differentiation. This study provides a mechanistic understanding of how transcription factors constrain terminal cell fates, and it delineates the importance of choosing the right proneural factor in neuronal reprogramming strategies.

[EN] Neuronal specification is a protracted process that begins with progenitor commitment and culminates with the generation of mature and functional neurons. Transcription factors (TF) are the main orchestrators in the acquisition of neuron type-specific transcriptomes which confer neuron-type specific functionalities. Terminal selectors are TF that directly activate the expression of the genes that allow for neuron-type-specific functions such as ion channels, neurotransmitter receptors, biosynthesis enzymes (called effector genes). Terminal selector expression is necessary throughout the life of the neuron not only for the establishment, but also for the maintenance of the neuronal gene expression profile. In this work, we studied dopaminergic (DA) neuron terminal differentiation. DA neurons synthetize and use dopamine as neurotransmitter. In mammals, these cells have a crucial role in the central nervous system regulating several cerebral circuits, thereby alteration of this homeostasis result in psychiatric disorders such as schizophrenia or depression. DA neurons are present in all animal groups, and their effector genes are also phylogenetically conserved. Studies in the nematode C. elegans have shown that least three terminal selectors are needed for DA terminal differentiation; CEH-20 (PBX Homeodomain family), AST-1 (ETS transcription factor family) and CEH-43 (Distalless homeodomain family). In mammals, DA neurons are located in the midbrain, diencephalon and the telencephalic structure known as the olfactory bulb (OB). OB dopaminergic neurons are the most ancestral in evolution. Importantly, these neurons have the particularity of being, generated throughout the life of the animals by adult neurogenesis. Previous work described the expression and function of AST-1 and CEH-43 orthologs (ER81 and DLX2 respectively) in the differentiation of OB DA neurons. Here we aimed to test if mammalian orthologs for CEH-20, the third terminal select for nematode DA neu, [ES] La especificación neuronal es un proceso que se inicia con la determinación de los progenitores hacia su destino neuronal y que culmina con la generación de las neuronas maduras y funcionales. Los factores de transcripción (FT) tienen un papel crucial en el proceso de activación del transcriptoma específico de cada tipo neuronal que es el encargado de conferir sus funcionalidades concretas. Los selectores terminales son FT que regulan de manera directa y coordinada la expresión de los genes necesarios para la adquisición de las características fenotípicas de cada tipo de neurona madura (genes efectores). Estos FT mantienen su expresión a lo largo de la vida de la neurona siendo necesarios, no solo para el establecimiento, sino también para el mantenimiento del perfil de expresión génica de la neurona. En este trabajo nos hemos centrado en el estudio del programa de diferenciación terminal de las neuronas dopaminérgicas (DA). Las neuronas DA sintetizan y usan el neurotransmisor dopamina. En mamíferos, las neuronas dopaminérgicas tienen gran importancia en el sistema nervioso central ya que regulan muchos de los circuitos cerebrales, siendo la alteración de la homeóstasis de la DA una de las causas relacionadas con trastornos psiquiátricos como la esquizofrenia o la depresión, por lo que estudiar cómo se adquieren las características de estas neuronas es de vital importancia. Las neuronas DA están presenten en todos lo grupos animales. Además, los genes efectores encargados de la biosíntesis y captación de DA están también filogenéticamente muy conservados. Estudios en el nemátodo C. elegans han revelado que la diferenciación terminal de las neuronas dopaminérgicas (DA) necesita al menos de tres FT que actúan como selectores terminales; CEH-20 (Familia Homeodominio PBX), AST-1 (Familia ETS) y CEH-43 (Familia homeodominio distalless). En mamíferos, diferentes núcleos del mesencéfalo, diencéfalo y el bulbo olfatorio (BO) contienen neuronas DA. Sin embargo, las que

En el marco de la celebración del Día Internacional de la Mujer y la Niña en la Ciencia, la Vicepresidencia de Relaciones Internacionales del CSIC ha organizado para el viernes 12 de febrero, a las 13.00h la mesa redonda online: Charla con investigadoras sobre la perspectiva de género en la I+I internacional, en la que se tratarán temas como los efectos de las políticas de igualdad en el ámbito científico, o las dificultades que encuentran las mujeres investigadoras, especialmente a nivel europeo. La mesa estará presentada por Elena Domínguez, Vicepresidenta de Relaciones Internacionales del CSIC, y moderada por Lina Gálvez, europarlamentaria. Contará con la participación de Mercedes García-Arenal (Centro de Ciencias Humanas y Sociales - CCHS), Nuria Flames (Instituto de Biomedicina de Valencia - IBV), Pilar Tiemblo (Instituto de Ciencia y Tecnología de Polímeros - ICTP) y Lourdes Verdes-Montenegro (Instituto de Astrofísica de Andalucía - IAA).

El Consejo Europeo de Investigación (ERC, por sus siglas en inglés) cumplió 10 años el 24 de marzo de 2017. A lo largo de esta década de vida, se ha financiado a unos 7.000 investigadores, permitiendo la publicación de cerca de 100.000 artículos en revistas científicas internacionales. En España, cerca de 400 investigadores han recibido un total de 650 millones de euros y el Consejo Superior de Investigaciones Científicas (CSIC) destaca por ser la institución española con mayor número de ayudas, por delante de la Universidad Pompeu Fabra, el Instituto de Ciencias Fotónicas, la Universidad de Barcelona y el Centro de Regulación Genómica. Entre los más de 400 proyectos que han obtenido una ayuda del Consejo Europeo de Investigación en España, más de un centenar han recaído en el CSIC.

[EN] Specific cell identities are defined by the expression of selected transcriptomes that allow the cell to fulfill its distinctive functions. It is broadly stablished that transcriptomes are selected by specific combinations of transcription factors (TF) that bind selectively to the regulatory regions of the DNA, promoters and enhancers. In fact, it is the combinatorial and cooperative binding of transcription factors what promotes the expression of specific cell features. However, the mechanisms that allow TFs to identify regulatory regions remain poorly understood. One of the niches that gathers more cell diversity is the nervous system, thus, this tissue becomes one of the best scenarios to study how distinct cell fates are acquired. In this Thesis we focus on monoaminergic system (MA), composed of distinct subpopulations of neurons that use biogenic amines as neurotransmitters, such as dopamine (DA), serotonin (5-HT) and adrenaline (A) and noradrenaline (NA), in vertebrates, or octopamine (OA) and tyramine (TA) in invertebrates. Monoaminergic system is conserved in all eumetazoans and regulates a plethora of complex behaviors, from locomotion to mood or appetite. An unbalance in the production or release of these neurotransmitters leads to diseases such as Parkinson or mental disorders such as bipolar disorder, depression or anxiety. In this Thesis we took advantage of the phylogenetic conservation of monoaminergic neurons to use the nematode Caenorhabidtis elegans as a model system. C. elegans monoaminergic system is composed of four pairs of DA neurons (CEPV/D, ADE and PDE), four pairs (NSM, ADF, HSN and AIM) and a unilateral (RIH) serotonergic neurons; a pair of TA neurons (RIM) and another OA pair (RIC)., All these neurons express a battery of genes that encode for enzymes and transporters that produce, carry and release the neurotransmitter which are also phylogenetically conserved. Using this nematode, our goal in this Thesis was to unravel which, among the 876 transcription factors present in C. elegans genome, play a key role in specification of monoaminergic neurons. To accomplish this aim, we optimized the RNAi strategy to perform an RNAi screen to knockdown the expression of all C. elegans TFs and look for defects in specific features of MA neurons, such us in the battery of genes required to synthesized the neurotransmitter. This strategy provided 91 TFs candidates involved in MA specification, we selected the ones that retrieved the most penetrant phenotypes and validate the results according to the MA subpopulation affected. Our results showed that, for DA population, unc-62, vab-3 and unc-55 are essential in the regulatory complex that drives the specification of these neurons. Regarding 5-HT neurons, the combined action of lag-1 and hlh-14 promote the expression of ADF mature features and are determining in the behaviors exhibited by this neuron. Finally, the bZIP factor, zip-5, has been identified as the first TF known to be involved in the specification of RIC and RIM neurons. To conclude, our work shed light into the transcriptional regulation of monoaminergic neurons, identifying key TFs that control the expression of identity genes of this population and that can be used to find functional orthologs in more evolved organisms that control monoaminergic specification., [ES] La identidad celular se define mediante la expresión de transcriptomas seleccionados que permiten a la célula cumplir sus funciones características. Los estudios en este campo señalan que un transcriptoma se selecciona mediante combinaciones específicas de factores de transcripción (FT) que se unen selectivamente a las regiones reguladoras del ADN, promotores y enhancers. De hecho, es la unión cooperative y en combinación de los factores de transcripción lo que promueve la expresión de características celulares específicas. Sin embargo, los mecanismos que permiten a los FT identificar regiones reguladoras siguen siendo poco conocidos. Uno de los nichos que reúne más diversidad celular es el sistema nervioso, por lo tanto, este tejido se convierte en uno de los mejores escenarios para estudiar cómo se especifican los distintos destinos celulares. En esta Tesis nos centramos en el estudio del sistema monoaminérgico (MA), compuesto por distintas subpoblaciones de neuronas que utilizan aminas biógenas como neurotransmisores, entre los que se incluyen la dopamina (DA), la serotonina (5-HT) y la adrenalina (A) y noradrenalina (NA), en vertebrados, u octopamina (OA) y tiramina (TA), en invertebrados. El sistema monoaminérgico está conservado en todos los eumetazoos y regula una gran variedad de comportamientos complejos, desde la locomoción hasta el estado de ánimo o el apetito. Un desequilibrio en la producción o liberación de estos neurotransmisores conduce a enfermedades como el Parkinson o trastornos mentales como el trastorno bipolar, la depresión o la ansiedad. En esta Tesis, aprovechamos la conservación filogenética de las neuronas monoaminérgicas para utilizar el nematodo Caenorhabidtis elegans como sistema modelo. El sistema monoaminérgico de C. elegans se compone de cuatro pares de neuronas DA (CEPV / D, ADE y PDE), cuatro pares (NSM, ADF, HSN y AIM) y una neurona serotoninérgica unilateral (RIH); un par de neuronas TA (RIM) y otro par de OA (RIC)., Todas estas neuronas expresan una batería de genes que codifican enzimas y transportadores que producen, transportan y liberan el neurotransmisor y que también está filogenéticamente conservada. Usando este nematodo, nuestro objetivo en esta Tesis fue desentrañar cuáles, entre los 876 factores de transcripción presentes en el genoma de C. elegans, juegan un papel clave en la especificación de las neuronas monoaminérgicas. Para lograr este objetivo, optimizamos la estrategia de ARN de interferencia (ARNi) para realizar un cribado de ARNi con el fin de disminuir la expresión de todos los FT de C. elegans y buscar defectos en las características específicas de las neuronas MA, como la batería de genes necesarios para sintetizar el neurotransmisor. Esta estrategia proporcionó 91 FT candidatos implicados en la especificación MA. Posteriormente seleccionamos aquellos que proporcionaron los fenotipos más penetrantes y validamos los resultados de acuerdo con la subpoblación MA afectada. Nuestros resultados mostraron que, para la población DA, unc-62, vab-3 y unc-55 son esenciales en el complejo regulador que impulsa la especificación de estas neuronas. En cuanto a las neuronas 5-HT, la acción combinada de lag-1 y hlh-14 promueve la expresión de las características de madurez de la neurona ADF y son determinantes en los comportamientos exhibidos por la misma. Finalmente, el factor bZIP, zip-5, ha sido identificado como el primer FT relacionado con la especificación de las neuronas RIC y RIM. Para concluir, nuestro trabajo arrojó luz sobre la regulación transcripcional de las neuronas monoaminérgicas, identificando FT clave que controlan la expresión de genes de identidad de esta población y que pueden usarse para encontrar ortólogos funcionales en organismos más evolucionados que también controlen la especificación monoaminérgica.

Cell differentiation is controlled by individual transcription factors (TFs) that together activate a selection of enhancers in specific cell types. How these combinations of TFs identify and activate their target sequences remains poorly understood. Here, we identify the cis-regulatory transcriptional code that controls the differentiation of serotonergic HSN neurons in Caenorhabditis elegans. Activation of the HSN transcriptome is directly orchestrated by a collective of six TFs. Binding site clusters for this TF collective form a regulatory signature that is sufficient for de novo identification of HSN neuron functional enhancers. Among C. elegans neurons, the HSN transcriptome most closely resembles that of mouse serotonergic neurons. Mouse orthologs of the HSN TF collective also regulate serotonergic differentiation and can functionally substitute for their worm counterparts which suggests deep homology. Our results identify rules governing the regulatory landscape of a critically important neuronal type in two species separated by over 700 million years.

[EN] Dopamine signalling regulates a variety of complex behaviours and defects in dopaminergic neuron function or survival result in severe human pathologies such as Parkinson´s disease. All dopaminergic neurons are characterized by the expression of a battery of genes that code for a set of proteins required for the synthesis, release and re-uptake of dopamine and all together are called the DA pathway genes. Therefore, understanding how this set of genes are transcriptionally activated in a coordinated manner is the first step to discover the clues of dopaminergic terminal differentiation. The dopamine pathway genes are highly conserved throughout evolution. Studies in the nematode C.elegans have shown that three transcription factors, CEH-20 (PBX transcription factor family), AST-1 (ETS transcription factor family) and CEH-43 (Homeodomain transcription factor family) are necessary for the terminal differentiation of DA neurons. These factors act in a coordinated manner to bind the regulatory regions and activate the expression not only of the DA pathway but of most of the mature dopaminergic neuron transcriptome. The dopaminergic system in mammals is distributed in nuclei of the mesencephalon, diencephalon and olfactory bulb. However, in this project we have focused on the olfactory bulb dopaminergic neurons since it is the most ancestral population and also because it requires AST-1 and CEH -43 orthologs (ER81 and DLX2 respectively) for their correct differentiation. Our main interest in this PhD project has therefore been to study whether homologues of CEH-20, the third known factor in the worm, directly participates in the dopaminergic specification of the bulb in higher organisms such as the mouse. In mammals, there are four members in the PBX family: PBX1, PBX2, PBX3 and PBX4. The study of its expression in the bulb, by double immunostaining against tyrosine hydroxylase (DA marker) and PBX factors, determined that both PBX1 (isoform 1a but not 1b) and PBX2 a, Studies of cell lineage tracing in which the neurons of the dopaminergic lineage are permanently marked with green fluorescent protein demonstrate that, in Pbx1 mutants, the neurons are present but they are unable to differentiate into DA neurons. On the other hand, it was also observed that a small percentage of the DA lineage neurons in mutants for Pbx1 ectopically express Calretinin and / or Calbindin suggesting a change in their cell fate from DA to other lineages. In addition, experiments indicated that PBX1 regulates the expression of other transcription factors required for dopaminergic differentiation such as MEIS2 and COUP-TF1. Moreover, in vivo electroporation experiments in neonatal mice confirm that the phenotype observed in the olfactory bulb is not an indirect effect derived from possible defects in proliferation of progenitors but is due to terminal differentiation defects and that it can be rescued specifically by the Pbx1a isoform but not by Pbx1b. Furthermore, overexpression experiments show that Pbx1a and to a lesser extent Pbx1b increase the dopaminergic differentiation in electroporated cells. Finally, we have detected that PBX1 directly binds to the cis regulatory sequences of the Th gene supporting a direct effect of this factor on its expression. In this way we propose PBX1a as functional homolog of CEH-20 thus reinforcing the hypothesis of a possible evolutionary conservation in the differentiation to dopaminergic neuron between nematodes and mammals., [ES]Las neuronas dopaminérgicas (DA) regulan una amplia variedad de comportamientos; defectos en la función o supervivencia de estas neuronas dan lugar a patologías tan importantes como el Parkinson. Todas las neuronas dopaminérgicas se caracterizan por la expresión de una batería de genes que codifican para un grupo de proteínas implicadas en la síntesis, transporte y recaptación de la dopamina y que en su conjunto denominamos genes de la vía de la dopamina. Por tanto, conocer los factores de transcripción que activan de forma directa la transcripción de estos genes es el primer paso para descubrir las claves de la diferenciación terminal de las neuronas dopaminérgicas. Los genes de la vía de la dopamina están altamente conservados a lo largo de la evolución. Estudios en el nematodo C.elegans han demostrado que tres factores de transcripción, CEH-20 (factor de la familia PBX), AST-1( factor de la familia ETS) y CEH-43 (factor de la familia Homeodominio) son necesarios para la diferenciación terminal de las neuronas DA. Estos factores actúan de forma coordinada uniéndose a las regiones reguladoras y activando la expresión, no sólo de los genes de la vía de la DA, sino de la mayor parte del transcriptoma de la neurona dopaminérgica madura. El sistema dopaminérgico en mamíferos se distribuye en núcleos del mesencéfalo, diencéfalo y bulbo olfatorio. Sin embargo, en este proyecto nos hemos centrado en el estudio de las neuronas dopaminérgicas del bulbo olfatorio ya que se trata de la población más ancestral en evolución y además porque las neuronas DA del bulbo requieren para su correcta diferenciación los ortólogos para AST-1 y CEH-43 (ER81 y DLX2 respectivamente). Nuestro principal interés en esta tesis por tanto, ha sido estudiar si homólogos de CEH-20, el tercer factor conocido en el gusano, participan de manera directa en la especificación dopaminérgica del bulbo en organismos superiores como el ratón. En mamíferos, existen cuatro miembros en la familia PBX: PBX1, El mismo estudio también reveló que la expresión de PBX1a y PBX2 no es exclusiva de las neuronas DA sino que también se expresan en otras células de la capa periglomerular así como en la capa granular (capa más interna) del bulbo olfatorio y otras neuronas del cerebro. Análisis de expresión de la tyrosina hidroxilasa en mutantes condicionales para Pbx1 específicos del linaje dopaminérgico, mutantes nulos para Pbx2 y dobles mutantes, demuestran que Pbx1 pero no Pbx2 es necesario para la diferenciación de las neuronas DA del bulbo, tanto las generadas durante el desarrollo embrionario como en el adulto. Estudios de linaje celular, en el que las neuronas del linaje dopaminérgico quedan permanentemente marcadas con la proteína verde fluorescente demuestran que, en los mutantes para Pbx1, las neuronas están presentes pero no llevan a cabo su correcta diferenciación DA. Por otra parte, también se observó que un pequeño porcentaje de las neuronas del linaje DA en mutantes para Pbx1 expresa ectópicamente Calretinina y/o Calbindina, dos marcadores de otras subpoblaciones de interneuronas del bulbo olfatorio, sugiriendo un cambio de destino celular de DA a otros linajes. Además, hemos determinado que Pbx1 regula la expresión de otros factores de transcripción necesarios para la diferenciación dopaminérgica como MEIS2 y COUP-TF1. Asimismo, los experimentos de electroporación in vivo en ratones neonatos confirman que el fenotipo observado en el bulbo olfatorio no es un efecto secundario derivado de posibles defectos de proliferación sino que es un fenotipo de diferenciación terminal y que puede ser rescatado de forma específica por la isoforma Pbx1a pero no por Pbx1b. Experimentos de sobreexpresión muestran que Pbx1a y en menor medida Pbx1b incrementan la diferenciación a neurona dopaminérgica en las células electroporadas, sugiriendo que en algunos contextos Pbx1 es suficiente para inducir diferenciación DA. Finalmente, hemos detectado que PBX1 se une directamente a las secuen

[EN] Serotonergic transcriptional regulatory logic in Caenorhabditis elegans Neuronal diversity in the nervous system is generated through the activation of multiple unique batteries of terminal differentiation genes, which determine the functional properties of the distinct mature neurons. It is generally accepted that transcription factors (TFs) bind in a combinatorial and cooperative manner to DNA sequences of the genome called enhancers, placing TFs as the main regulators of gene expression. However, how these combinations of TFs identify and activate their target sequences is poorly understood. In this work we use as a paradigm the serotonergic neurons to unravel the regulatory rules that select a cell type-specific transcriptome during terminal differentiation. Serotonergic neurons are present in all eumetazoan groups and are universally defined by their ability to synthesise and release serotonin (5-HT), which is achieved by the expression of the ‘5-HT pathway genes’. Taking advantage of this phylogenetic conservation, we use the simple model organism Caenorhabditis elegans to dissect the transcriptional regulatory logic of serotonergic neurons. C. elegans hermaphrodites have three functionally different serotonergic subclasses: the HSN motorneuron, the ADF sensory neuron and the NSM neurosecretory motorneuron. All three neuron subtypes express the 5-HT pathway genes. Through an in vivo cis-regulatory analysis of these genes we have identified independent cis-regulatory modules (CRM) responsible for their expression in each serotonergic neuron subtype. This modular organisation suggests that different regulatory logics are employed in each neuron subclass to activate its terminal transcriptome. To deepen in our understanding of how cell type-specific transcriptional programmes are implemented we decided to focus the rest of our work on the best characterised serotonergic neuron subtype, the HSN neuron, and carried out an extensive dissection of HSN terminal d, have termed HSN TF collective. This TF code is composed by AST-1 (ETS TF family), UNC-86 (POU TF family), SEM-4 (SPALT TF family), HLH-3 (bHLH TF family), EGL-46 (INSM TF family) and EGL-18 (GATA TF family). The expression of the HSN TF collective is sufficient to induce serotonergic fate in some specific contexts and is required throughout the life of the animal in order to maintain the identity of the HSN neuron. Bioinformatically identified binding site clusters for the six TFs of the HSN TF collective are enriched in known HSN expressed genes compared to a random set of genes. Through in vivo reporter analysis, we demonstrate that this clustering constitutes a regulatory signature that is sufficient for de novo identification of HSN neuron functional enhancers. This regulatory signature contains certain syntactic constrains that further improve the prediction of enhancer expression in the cell. Mouse orthologues of most members of the HSN TF collective are known regulators of the mammalian serotonergic differentiation programme. This homology in both serotonergic regulatory programmes allows for the identification of an additional candidate TF in the worm (PHA-4), orthologue to the mouse FOXA2, and a mouse TF (SALL2), orthologue of the worm SEM-4. Moreover, we prove that mouse orthologues can functionally substitute for their worm counterparts. Finally, Principal Coordinates Analysis suggests that, among C. elegans neurons, the HSN transcriptome most closely resembles that of mouse serotonergic neurons, which reveals deep homology. Our results show that a regulatory signature based on a defined set of TFs is sufficient for enhancer identification using primary DNA sequence. Moreover, our results identify rules governing the transcriptional regulatory code of a critically important neuronal type in two species separated by over 700 million years., [ES] Lógica de regulación transcripcional de las neuronas serotonérgicas en Caenorhabditis elegans La diversidad del sistema nervioso se genera mediante la activación de múltiples baterías únicas de genes efectores, que definen las propiedades funcionales de los diferentes subtipos neuronales. Está bien establecido que los factores de transcripción (FT) se unen de una manera combinatoria y cooperativa a secuencias de ADN presentes en los elementos de regulación en cis del genoma, llamados potenciadores (enhancers en inglés). Esto otorga a los FT un papel central en la regulación de la expresión génica. Sin embargo, no se conocen los mecanismos por los que estas combinaciones de FT identifican y activan sus secuencias diana. En este trabajo se han utilizado las neuronas serotonérgicas como paradigma de investigación de las leyes que regulan la selección del transcriptoma de un tipo neuronal concreto durante la diferenciación terminal. Las neuronas serotonérgicas se encuentran presentes en todos los grupos de eumetazoos y se definen por su habilidad de sintetizar y liberar serotonina (5-HT), lo cual es posible gracias a la expresión de los llamados ‘genes de la vía de la 5-HT’. Aprovechando esta conservación filogenética, hemos utilizado el organismo modelo Caenorhabditis elegans para diseccionar la lógica de regulación transcripcional de las neuronas serotonérgicas. Los hermafroditas C. elegans contienen tres subclases de neuronas serotonérgicas con diferente función: la neurona motora HSN, la neurona secretora ADF y la neurona motora neurosecretora NSM. Mediante un análisis de regulación in vivo de los genes de la vía de la 5-HT, hemos identificado módulos de regulación en cis (MRC) independientes responsables de su expresión en cada uno de los tres subtipos serotonérgicos. Esta organización modular sugiere que cada subclase utiliza una lógica de regulación diferente. Para profundizar en los mecanismos de selección y activación del transcriptoma específico de un tip, bioinformáticos de predicción de sitios de unión para los seis FT del código, muestran que los genes expresados en la neurona HSN están enriquecidos en la presencia de agrupaciones de estos seis sitios de unión, en comparación a un conjunto de genes elegidos al azar. Mediante el análisis de reporteros in vivo, demostramos que esta agrupación constituye una huella reguladora que es suficiente para la identificación de nuevos potenciadores funcionales para la neurona HSN. Además, esta huella reguladora contiene normas sintácticas que mejoran la predicción de potenciadores expresados en la célula. Curiosamente, el programa de diferenciación de las neuronas serotonérgicas en ratón está controlado por FT que son ortólogos a los del nematodo. Esta elevada homología en la regulación nos ha permitido identificar nuevos candidatos a regular las neuronas serotonérgicas del gusano (PHA-4, ortólogo a FOXA2) y del ratón (SALL2, ortólogo a SEM-4). Asimismo, los ortólogos de ratón son capaces de sustituir funcionalmente a los FT equivalentes en gusano. Finalmente, el Análisis de Coordenadas Principales sugiere que, de entre todas las neuronas del gusano, el transcriptoma de la neurona HSN es el que más se asemeja a aquel de las neuronas serotonérgicas de ratón, revelando relaciones de homología profunda. En conclusión, hemos demostrado que la presencia de una huella reguladora basada en un conjunto definido de FT es suficiente para identificar potenciadores, utilizando únicamente la secuencia primaria de ADN. Además, hemos identificado las reglas que gobiernan el código de regulación transcripcional de un tipo neuronal relevante en dos especies separadas hace más de 700 millones de años.

Centenarians not only enjoy an extraordinary aging, but also show a compression of morbidity. Using functional transcriptomic analysis of peripheral blood mononuclear cells (PMBC) we identified 1721 mRNAs differentially expressed by centenarians when compared with septuagenarians and young people. Sub‐network analysis led us to identify Bcl ‐ xL as an important gene up‐regulated in centenarians. It is involved in the control of apoptosis, cellular damage protection and also in modulation of immune response, all associated to healthy aging. Indeed, centenarians display lower plasma cytochrome C levels, higher mitochondrial membrane potential and also less cellular damage accumulation than septuagenarians. Leukocyte chemotaxis and NK cell activity are significantly impaired in septuagenarians compared with young people whereas centenarians maintain them. To further ascertain the functional role of Bcl‐ xL in cellular aging, we found that lymphocytes from septuagenarians transduced with Bcl‐xL display a reduction in senescent‐related markers. Finally, to demonstrate the role of BcL‐xL in longevity at the organism level, C. elegans bearing a gain of function mutation in the BcL‐xL ortholog ced‐9, showed a significant increase in mean and maximal life span. These results show that mRNA expression in centenarians is unique and reveals that BcL‐ xL plays an important role in exceptional aging., Ministerio de Educación y Ciencia (MEC), Red Tematica de investigacion cooperative en envejecimiento y fragilidad (RETICEF) (España), Consejería de Educación, Cultura y Deporte de la Generalitat Valenciana (España), Consejería de Sanidad (Valencia, España), FRAILOMIC-HEALTH, INCLIVA, FEDER, Marie Curie Career, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Centenarians not only enjoy an extraordinary aging, but also show a compression of morbidity. Using functional transcriptomic analysis of peripheral blood mononuclear cells (PMBC) we identified 1721 mRNAs differentially expressed by centenarians when compared with septuagenarians and young people. Sub-network analysis led us to identify Bcl-xL as an important gene up-regulated in centenarians. It is involved in the control of apoptosis, cellular damage protection and also in modulation of immune response, all associated to healthy aging. Indeed, centenarians display lower plasma cytochrome C levels, higher mitochondrial membrane potential and also less cellular damage accumulation than septuagenarians. Leukocyte chemotaxis and NK cell activity are significantly impaired in septuagenarians compared with young people whereas centenarians maintain them. To further ascertain the functional role of Bcl-xL in cellular aging, we found that lymphocytes from septuagenarians transduced with Bcl-xL display a reduction in senescent-related markers. Finally, to demonstrate the role of Bcl-xL in longevity at the organism level, C. elegans bearing a gain of function mutation in the Bcl-xL ortholog ced-9, showed a significant increase in mean and maximal life span. These results show that mRNA expression in centenarians is unique and reveals that Bcl-xL plays an important role in exceptional aging.

Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. We investigate this problem using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, we show that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. We show that the type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features.

Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis elegans hermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs.