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19 páginas, 4 figuras. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2022.912319/full#supplementary-material, Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical β-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active β-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo. Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence., This work was supported by the Spanish MICINN grant (PID2019-111225RB-I00) and PROMETEO/2018/055 grant from Generalitat Valenciana to HM, MV, and NF; FPI fellowship BES2016–077156 to IC-B; APOSTD/2018/A/184 and APOSTD2019/058 from Generalitat Valenciana to LG-C and LC-C, respectively

Tesis doctoral, 305 págs, figuras y tablas. Se adjunta índice en fichero aparte, [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 with RFX TFs in the direct regulation of the sensory ciliome. Moreover, our results, together with previous work, show that FKH and RFX TFs act together in the regulation of both motile and sensory cilia, suggesting this regulatory logic could be an ancient trait pre-dating functional sub-specialization of cilia. Finally, we hope our results could help better understand the biological basis of orphan ciliopathies, [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 sinérgica con el conocido regulador maestro de la ciliogénesis DAF19/RFX. En consecuencia, los mutantes para fkh-8 muestran una amplia gama de defectos de comportamiento en una plétora de paradigmas sensoriales, incluyendo la olfacción, la gustación y la mecano-sensación. Así, hemos identificado, por primera vez, un FT que actúa junto con los FTs de la familia RFX en la regulación directa del cilioma sensorial. Además, nuestros resultados, junto con trabajos anteriores, muestran que los FTs FKH y RFX actúan conjuntamente en la regulación de los cilios tanto móviles como sensoriales, lo que sugiere que esta lógica reguladora podría ser un rasgo evolutivo antiguo anterior a la subespecialización funcional de los cilios. Finalmente, esperamos que los resultados de nuestro trabajo ayuden a entender mejor las bases biológicas de las ciliopatías huérfanas, This thesis project has been made possible thanks to a pre-doctoral fellowship from the FPI Programme (BES-2015-072799) conferred by the (now extinct) Spanish Ministry of Economy & Competitivity. The following grants also provided a funding frame throughout the whole research process: “Estudio de los mecanismos transcripcionales que regulan la diferenciación de las neuronas monoaminérgicas y su conservación evolutiva.” SAF2014-56877-R “Dissecting the gene regulatory mechanisms that generate serotonergic neurons and their link to mental disorders.” ERC-St 281920 “Programas de regulación transcripcional asociados a enfermedades genéticas.” SAF2017-84790-R “Regulatory rules and evolution of neuronal gene expression.” ERC-Co 101002203

16 páginas, 2 figuras 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. Our work is funded by H2020 European Research Council (COG-101002203), Ministerio de Ciencia e Innovacion (PID2020-115635RB- I00 and RED2018-102553-T) and Generalitat Valenciana (PROMETEO/2018/055 and PhD fellowship ACIF/2019/079 to E. Sousa)

17 páginas, 6 figuras, 1 tabla. Material suplementario de este artículo online en: https://www.frontiersin.org/articles/10.3389/fcell.2021.770458/full#supplementary-material, 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., This work was supported by the CIDEGenT excellence research program of the Valencian government (CIDEGENT/2019/044) and by the Spanish Ministry of Science, Innovation and Universities (RTI 2018-100872-J-I00) as well as by the Young IBRO Regions Connecting Award to IDP and the ERC Consolidator Grant 101002203 to NF. Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020).

25 páginas, 8 figuras., 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., NF research is funded by European ResearcPRE2018-086h Council (ERC StG 281920; ERC COG 101002203), Spanish Government (SAF2017-84790-R) and Generalitat Valenciana (PROMETEO/2018/055). A.M.T holds the PRE2018-086632 fellowship from Spanish Government and A.J the ACIF/2015/398 fellowship from Generalitat Valenciana. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

16 páginas, 6 figuras, 2 tablas, 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., the Bioinformatics and Biostatistics Unit from Principe Felipe Research Center (CIPF) for providing access to the cluster, cofunded by European Regional Development Funds (FEDER); Funding sources: European Research Council: ERC-StG2011-281920; ERC-Co-2020-101002203; Ministerio de Economía, Industria y Competitividad, Gobierno de España: SAF2017-84790-R; PID2020-115635RB-I00; RED2018-102553-T; Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital, Generalitat Valenciana: PROMETEO/2018/055; ACIF/2019/079.

15 páginas, 8 figuras. Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.186841.supplemental, 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., This work was supported by PhD fellowships from the Ministerio de Educación, Cultura y Deporte (FPU16/02008 to I.R.-B.) and the Ministerio de Economıa y ́Competitividad (BES-2012-053847 to L.R.. and BES-2015-072799 to R.B.-R.); by European Research Council grant (ERC-StG-2011-281920); by Ministerio de Economıa y Competitividad grants (SAF2017-84790-R and SAF2016-75004R) and ́ by the Generalitat Valenciana (PROMETEO/2018/055).

12 páginas, 7 figuras, 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., This work is supported by NICHD (R01HD079682) and Project ALS (A13-0416) to E.O.M. and by NYSTEM pre-doctoral training grant (C026880) to B.A. S.M. is supported by NIGMS (R01GM125722) and the National Science Foundation ABI Innovation Grant No. DBI1564466. Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. M.R. is supported by NYU MSTP (T32GM007308) and Developmental Genetics T32 (T32HD007520) grants. N.F. and M.M.E. are supported by ERC Starting Grant (2011-281920). The authors would like to thank Link Tejavibulya and Apeksha Ashokkumar for their help with molecular biology; Mohammed Khalfan for his help with scRNA-seq analysis. Michael Cammer from the NYU Medical Center Microscopy Core for the ImageJ script used in calcium imaging analysis; and NYU Genomics Core facility. Finally, we would like to thank Steve Small, Nikos Konstantinidis, Pinar Onal, Orly Wapinski, Sevinç Ercan, Chris Rushlow, Claude Desplan and Mazzoni lab members for their helpful suggestions on the manuscript.

Tesis doctoral 296 páginas, figuras y tablas, [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.

36 Pages, 8 Figures, 2 Tables, 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., Ministerio de Economáa y Competitividad SAF2014-56877-R. European Research Council ERC Stg 2011-281920. Generalitat Valenciana ACIF/2013/087. Ministerio de Educación, Cultura y Deporte FPU14/02651. Generalitat Valenciana ACIF/2015/398