Flames, Nuria et al. (2009) International Worm Meeting "Regulatory logic of dopaminergic neuron differentiation."

Hobert, Oliver, Flames, Nuria

[International Worm Meeting, 2009]

Vertebrate dopaminergic neurons play a central role in the coordination of movement, attention, memory and reward. In addition, defects in the synthesis or function of the neurotransmitter dopamine underlie several pathological conditions such as Parkinson''s disease, schizophrenia and drug addiction. The dopamine synthesis pathway is extremely well conserved from worms to human. However, the factors that directly activate the expression of the dopamine synthesis pathway genes remain only poorly understood in both organisms. Caenorhabditis elegans hermaphrodite contains eight dopaminergic neurons that are involved in locomotory control and several aspects of neuronal plasticity. These eight neurons fall into 4 distinct classes, each composed of a pair of bilaterally symmetric neurons. All 4 classes display very distinct developmental histories, but nevertheless express the core pathway genes that define the dopaminergic cell fate (dat-1, cat-2, cat-1, cat-4 and bas-1). In this work, we show that in C. elegans, a simple cis-regulatory element controls the expression of all dopamine synthesis pathway genes in all dopaminergic cell types (including also the male dopaminergic neurons). We show that an ETS transcription factor, AST-1, is the trans-acting factor responsible for the activation of the dopaminergic neuron cis-regulatory element. Loss of the ast-1 gene results in loss of specification of all different dopaminergic neuron classes and ectopic expression of ast-1 is sufficient to activate the dopamine production pathway in several additional neurons. We also show that AST-1 is expressed in all the dopaminergic neurons throughout the life of the animal and its function is constantly required to maintain the dopaminergic fate. In conclusion, our studies reveal an astoundingly simple regulatory logic of dopaminergic neuron differentiation.

Nuria Flames et al. (2008) European Worm Meeting "Regulatory logic of dopaminergic cell fate specification"

Oliver Hobert, Nuria Flames

[European Worm Meeting, 2008]

Vertebrate dopaminergic neurons play a central role in the coordination of. movement, attention, memory and reward. In addition, defects in the. synthesis or function of the neurotransmitter dopamine underlie several. pathological conditions such as Parkinson''s disease, schizophrenia and drug. addiction. The dopamine synthesis pathway is extremely well conserved from. worms to human. However, the factors that directly activate the expression. of the dopamine synthesis pathway genes remain only poorly understood in. both organisms.. Caenorhabditis elegans hermaphrodite contains eight dopaminergic neurons. that are involved in locomotory control and several aspects of neuronal. plasticity. These eight neurons fall into 4 distinct classes, each. composed of a pair of bilaterally symmetric neurons. All 4 classes display. very distinct developmental histories, but nevertheless express the core. pathway genes that define the dopaminergic cell fate. In this work, we show. that in C. elegans, a simple cis-regulatory element controls the expression. of all dopamine synthesis pathway genes in all dopaminergic cell types. We. show that an ETS transcription factor, AST-1, is the trans-acting factor. responsible for the activation of the dopaminergic neuron cis-regulatory. element. Loss of the ast-1 gene results in loss of specification of all. different dopaminergic neuron classes and ectopic expression of ast-1 is. sufficient to activate the dopamine production pathway in several. additional neurons. Finally, we show that in mouse the ETS transcription. factor Etv1/ER81 is also necessary for the specification of the olfactory. dopaminergic neurons, suggesting a conserved role for ETS transcription. factors as selector genes for dopaminergic terminal differentiation.. Do not add objects such as pictures, boxes, headers, footers, footnotes,. etc.

Nuria Flames et al. (2007) International Worm Meeting "ETS trancription factors regulate dopaminergic cell fate in C. elegans."

Oliver Hobert, Nuria Flames

[International Worm Meeting, 2007]

Vertebrate dopaminergic (DA) neurons play a central role in the coordination of movement, attention, memory and reward. In addition, their dysfuction is linked to Parkinson''s disease or schizophrenia. In C. elegans, DA neurons are involved in behaviors such as egg-laying, defecation, basal motor activity, response to food sources and habituation to touch (Blakely et al. 2006). C. elegans hermaphrodite contains eight DA neurons, four cephalic cells (CEPs), two anterior deirids (ADEs) and two posterior deirids (PDEs). There is a high conservation at a molecular level in the DA system across phylogeny. Specifically, metabolic pathways for catecholamines appear to be conserved from man to worms. Despite the importance of the DA system, little is know in both mammals and C. elegans about the molecular mechanisms that regulate DA cell fate. In this work, we have undertaken an extensive analysis of the promoter region of several of the genes involved in the DA metabolism to try to uncover a hypothetical common cis regulatory element that would control their expression. We have based our analysis in 6 genes: the dopamine transporter (dat-1) and the tyrosine hydroxylase (cat-2), both exclusive of DA neurons, the vesicular monoamine transporter (cat-1), the GTP cyclohydrolase (cat-4) and the aromatic L-amino acid decarboxylase (bas-1) expressed in DA and serotonergic cells and the dopamine transporter (dop-2) expressed in DA neurons and their target cells. In order to identify the promoter elements responsible for DA expression we performed a promoter dissection analysis of all of these genes. We have found a 25 bp sequence in the dat-1 promoter that is both necessary and sufficient to drive GFP expression specifically in all hermaphrodite DA neurons. Bioinformatics analysis predicted the potential binding of 7 different families of transcription factors. Point mutations in this minimal promoter allowed us to identify the binding site of the ETS transcription factor family as the responsible for DA expression (we have termed this 25bp sequence as the DA motif). All the promoter sequences able to drive DA expression contained predicted DA motifs. Deletion experiments revealed that at least one functional DA motif is necessary for the expression of cat-1, cat-2, cat-4 and bas-1 genes. There are 10 different ETS genes in C. elegans, with available mutants for all of them. A preliminary analysis of the mutants revealed a loss of dat-1::gfp expression in one of them, ast-1 (Schmid et al 2006). We are currently characterizing the role of ast-1 in DA fate as well as the possible role of other ETS genes.

Doitsidou, Maria et al. (2011) International Worm Meeting "A combinatorial code of transcription factors defines the terminal differentiation of dopaminergic neurons."

Doitsidou, Maria, Hobert, Oliver, Felton, Terry, Flames, Nuria

[International Worm Meeting, 2011]

Dopaminergic neurons control various aspects of behaviour and their loss leads to Parkinson's disease. There are 8 dopaminergic (DA) neurons in the C. elegans hermaphrodite that fall into 4 morphological classes. All 4 classes display very distinct developmental histories, but nevertheless express the core pathway genes responsible for the biosynthesis and transport of dopamine. We call these genes the dopamine pathway genes. It was previously shown (Flames, 2009) that ast-1, an ETS transcription factor, is necessary for the expression of all dopamine pathway genes in all dopaminergic cell types. AST-1 exerts its action through binding to the dopaminergic motif, present in the promoters of the dopamine pathway genes. However, neither AST-1 nor its corresponding binding sites are sufficient to activate the dopamine pathway genes. We used a combination of genetic and promoter analysis approaches to identify the transcription factors as well as the corresponding regulatory elements that act in concert with ast-1 for terminal differentiation of DA neurons. Extensive analysis of the dopaminergic motif revealed the presence of functional homeodomain and PBX/Hox binding sites in all DA pathway genes. Automated COPAS forward genetic screens combined with Whole Genome Sequencing, uncovered a homeodomain transcription factor of the Distal-less family, ceh-43, and a Hox domain/MEIS transcription factor, unc-62, that are necessary for terminal differentiation of DA neurons. We find that these transcription factors are required for proper expression of all dopamine pathway genes in all DA neuronal classes. Phenotypic analysis, ectopic expression studies and epistatic relationships between ceh-43, unc-62 and ast-1 will be presented. In addition, candidate approach revealed that a PBX transcription factor, ceh-20, is also involved in terminal differentiation of DA fate. In conclusion, we have uncovered a combinatorial code of transcription factors, as well as the corresponding cis-regulatory elements that control terminal differentiation of dopaminergic neurons.

Doitsidou, Maria et al. (2009) International Worm Meeting "Dopaminergic neuronal cell-fate mutants retrieved through automated screening."

Flames, Nuria, Doitsidou, Maria, Hobert, Oliver

[International Worm Meeting, 2009]

We are interested in dopaminergic neuronal specification and we have utilized an automated method to isolate C. elegans mutants, in which these neurons fail to be appropriately specified. A fluorescence activated sorting mechanism implemented in the COPAS Biosort machine allowed the isolation of mutants with subtle alterations in the cellular specificity of gfp expression(1). This methodology was significantly more efficient than comparable manual screens. We present the various mutants that were isolated from the screens. Some of these mutants are alleles of known transcription factors previously not implicated in dopaminergic cell differentiation, while others define novel genetic loci. Particular focus will be given in the phenotypic characterization of two mutants recently cloned using deep sequencing technology (2)(*), namely a homeobox protein of the Distal-less class and a zing finger transcription factor. (1): Doitsidou et al., Nature Methods, October 2008 (2): Sarin et al., Nature Methods, October 2008 (*): see ''whole genome sequencing for mutant identification'' workshop, and Bigelow H. poster.

Weinberg, Peter J et al. (2013) International Worm Meeting "The SWI/SNF chromatin remodeling complex selectively affects multiple aspects of serotonergic neuron differentiation."

Hobert, Oliver, Flames, Nuria, Weinberg, Peter J, Sawa, Hitoshi, Garriga, Gian

[International Worm Meeting, 2013]

Regulatory programs that control the specification of serotonergic neurons have been investigated by genetic mutant screens in the nematode Caenorhabditis elegans. Loss of a previously uncloned gene, ham-3, affects migration and serotonin antibody staining of the hermaphrodite-specific (HSN) neuron pair. We characterize these defects here in more detail, showing that the defects in serotonin antibody staining are paralleled by a loss of the transcription of all genes involved in serotonin synthesis and transport. This loss is specific to the HSN neuron class as other serotonergic neurons appear to differentiate normally in ham-3 null mutants. Besides failing to migrate appropriately, the HSN neurons also display axon pathfinding defects in ham-3 mutants. However, the HSN neurons are still generated and express a subset of their terminal differentiation features in ham-3 null mutants, demonstrating that ham-3 is a specific regulator of select features of the HSN neurons. We show that ham-3 codes for the C. elegans ortholog of human BAF60, Drosophila Bap60 and yeast Swp73, which are subunits of the yeast SWI/SNF and vertebrate BAF chromatin remodeling complex. We show that the effect of ham-3 on serotonergic fate can be explained by ham-3 regulating the expression of the Spalt/SALL-type Zn finger transcription factor sem-4, a previously identified regulator of serotonin expression in HSN and of the ham-2 Zn transcription factor, a previously identified regulator of HSN migration and axon outgrowth. Our findings provide the first evidence for the involvement of the BAF complex in the acquisition of terminal neuronal identity and constitute genetic proof by germline knockout that a BAF complex component can have cell-type specific roles during development.

Brocal-Ruiz, Rebeca et al. (2021) International Worm Meeting "Forkhead transcription factor FKH-8 is a master regulator of sensory cilia"

Tena, Juan J., Esteve, Ainara, Mora-Martinez, Carlos, Flames, Nuria, Brocal-Ruiz, Rebeca

[International Worm Meeting, 2021]

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 the 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. We have identified FKH-8, a FKH TF, as a terminal selector of the sensory ciliome in C. elegans. fkh-8 expression is restricted to the sixty ciliated sensory neurons of C. elegans, it binds the regulatory regions of the sensory ciliome, it is required for correct ciliome gene expression and cilium morphology and acts synergistically with DAF-19/RFX TF. Accordingly, fkh-8 mutants display a wide range of behavioral defects in a plethora of sensory mediated paradigms, including olfaction, gustation, and mechano-sensation. Importantly, ciliome expression defects in fkh-8 mutants can be rescued by mammalian FoxJ1 but not other FKH TFs. 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, together with previous work, our results 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, our results could help better understand the biological basis of orphan ciliopathies.

Lloret-Fernandez, Carla et al. (2017) International Worm Meeting "Conserved transcriptional regulatory rules define the serotonergic-neuron identity."

Maicas, Miren, Chirivella, Laura, Flames, Nuria, Lloret-Fernandez, Carla, Jimeno, Angela, Weinberg , Peter, Artacho, Alejandro, Mora, Carlos

[International Worm Meeting, 2017]

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 unknown. Here, we identify the cis-regulatory transcriptional code that controls the differentiation of serotonergic (5HT) HSN neurons in C. elegans. Loss of function mutant and cis-regulatory analyses reveal that direct activation of the HSN transcriptome is orchestrated by a collective of six TFs. 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). Bioinformatically identified binding site clusters for these six TFs are enriched in known HSN expressed genes compared to a random set of genes. Through in vivo reporter analysis, we demonstrate that the clustering of TF collective binding sites 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 orthologs of most members of this TF collective are known regulators of mammalian 5HT differentiation programs and 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 5HT neurons, which reveals deep homology. Our results?identify rules governing the transcriptional regulatory code of a critically important neuronal type in two species separated by over 700 million years.

Siehr M et al. (2010) Biology of the C. elegans Male, Madison, WI "DSX-MAB-3 Transcription Factors Specify Multiple Cell Fates within a C. elegans Male Motor Circuit"

Miller R M, Siehr M, Lints R, Koo P K, Bian X, Portman D S

[Biology of the C. elegans Male, Madison, WI, 2010]

DM-domain transcription factors represent one of the few examples of conservation among sexual development programs. In C. elegans, three members of this family, mab-3, mab-23 and dmd-3, have demonstrated roles in male sexual development [1, 2, 3]. Here we show that dmd-3 and mab-23 direct the development of cells in a circuit that controls male posture during mating. This posture, a ventral flexure of the tail, is induced when the sensory rays of the male tail contact a hermaphrodite. mab-23 and dmd-3 mutant males exhibit abnormal posture or are devoid of postural control, respectively. Their postural shortcomings are the result of defects in both sensory and motor cells of the circuit. dmd-3 males have abnormal male-specific muscle morphology and in mab-23 mutants neurotransmitter receptor expression is reduced in these cells. In the sensory rays, neurotransmitter fate patterning among the A-type neurons is abnormal. In wild type males, dopaminergic (DA) fate is restricted to the A neurons of three rays (rays 5, 7 and 9) while the A neurons of other rays are cholinergic. In both mab-23 and dmd-3 mutants almost all A neurons express DA fate and cholinergic fate is absent. Thus, in wild type, mab-23 and dmd-3 suppress inappropriate expression of DA fate and promote establishment of cholinergic fate. The A neurons of rays 5, 7, and 9 are able to adopt DA fate because these cells are preferentially exposed to a TGF-beta (DBL-1) signal which blocks DM-domain gene function. Remarkably, DA fate specification may be controlled by a phylogenetically conserved genetic program. The ETS-domain transcription factor AST-1 promotes establishment of DA fate in neurons common to males and hermaphrodites as well as in the male-specific ray neurons [4]. Our studies suggest that sex-specific patterns of DA fate are established through interaction between this conserved DA fate program and these conserved mediators of the sex determination pathway, a phenomenon that could extend to other animal species. 1. Raymond et al. (1998) Nature. 12: 691-5. 2. Lints and Emmons (2002) Genes Dev. 16: 2390-402. 3. Mason et al. (2008) Development.135: 2373-82. 4. Flames & Hobert (2009) Nature. 16: 885-9.

Siehr, M. et al. (2011) International Worm Meeting "DM-domain genes dmd-3 and mab-23 specify critical cell fate characteristics in the male C. elegans ray sensorimotor circuit."

Portman, D., Siehr, M., Sherlekar, A., Bian, X., Lints, R., Koo, P.

[International Worm Meeting, 2011]

DM-domain (DSX-MAB-3) transcription factors promote gender-specific development in widely divergent organisms. However, precisely how their activity sex-specifically shapes developmental patterning is known for only a few cases. Two DM-domain genes, dmd-3 (1) and mab-23 (2), are expressed in cells of the ray sensorimotor circuit. This circuit induces apposition of the male tail against the hermaphrodite during mating (see Koo et al abstract). There are nine bilateral pairs of ray sensilla on the male tail (numbered 1 to 9) and each contain the sensory endings of two neurons, type A and B. The A-neurons are critical for circuit function and release dopamine and acetylcholine to effect tail posture. We find that dmd-3 and mab-23 are expressed in these neurons and that their loss of function dramatically alters the balance of dopaminergic (DA) and cholinergic (ACh) fate in this population. In wild type males DA fate is confined to three of the nine A-neuron pairs (those of rays 5, 7, and 9). However, in dmd-3 and mab-23 mutants virtually all A-neurons express DA fate; ACh fate (normally expressed by the A-neurons of rays 1 to 4 and 6) is rarely observed. ETS-domain transcription factor gene ast-1 (3) and the C. elegans distal-less homolog dopy-2/ceh-43 (4) establish expression of dopamine biosynthesis genes in all DA neurons in the worm during development. We find that ast-1 and ceh-43 are expressed in all A-neurons and that the ectopic DA fates in the DM-domain gene mutants depend on ast-1/ceh-43 function. The results of genetic analyses suggest a model in which DA fate corresponds to the ground state of A-neurons and is conferred by ast-1/ceh-43. However, in most A-neurons dmd-3/mab-23 suppress ast-1/ceh-43 function and instead promote ACh fate in a ceh-43-dependent manner. The A-neurons of rays 5, 7 and 9 are able to adopt DA fate because DM-domain gene function is blocked by the action of a TGF-beta (DBL-1) signaling pathway, which is specifically activated in the cells of these rays. Mating behavior-, optogenetic- and pharmacological analyses of the DM-domain mutants reveal that these animals are severely defective in mate apposition behavior. These defects can be attributed to mis-specification of A-neuron fates and to defects in the sex-specific patterning of core body wall muscles, which also express dmd-3 and mab-23 during development. 1. Mason et al. (2008) Development.135: 2373-82. 2. Lints & Emmons (2002) Genes Dev. 16: 2390-402. 3. Flames & Hobert (2009) Nature. 16: 885-9. 4. Doitsidou et al. (2008) Nat Methods. 5:869-72.