Nuria Flames

How nervous systems balance the generation of robust neuron types with gene expression plasticity mechanisms and how these processes impact cell-type evolution are unknown. Here, we use Caenorhabditis species to study neuron-type robustness, plasticity, and evolution, using VC4 and VC5 cholinergic motoneuron types as models. In Caenorhabditis elegans , we found that epigenetic silencing through histone 3 lysine 9 methylation (H3K9me) is necessary to suppress the expression of the serotonin reuptake gene mod-5/ Sert and a serotonergic phenotype in these cells. In contrast, we observed that VC4 and VC5 neurons in the Angaria group of Caenorhabditis species have evolved an intense serotonergic staining. This phenotype is caused by the emergence of a new enhancer in the mod-5/ Sert locus, which has been recruited to the ancestral neuron-type gene regulatory network. Enhancer transfer from C. angaria is sufficient to impose a constitutive serotonergic fate in C. elegans . Remarkably, acquiring this new trait modulates egg-laying responses to high levels of exogenous serotonin, which can be found in specific nematode environments. Finally, we found that the repression of the serotonergic fate in C. elegans VC4 and VC5 neurons is indeed a plastic trait that can be adjusted in specific environmental growth conditions to elicit egg-laying behaviors similar to those observed in Angaria species. Our work uncovers a dual regulatory paradigm: Epigenetic constraints harmonize neuron-type robustness and plasticity, while enhancer co-option enables evolutionary innovation. This mechanistic plasticity directly links regulatory adaptation to behavioral diversification.

Summary: How nervous systems coordinate the generation of specific neuron types with gene expression plasticity and how these mechanisms impact cell type evolution is unknown. Here we use Caenorhabditis species to study neuron-type robustness, plasticity and evolution, using VC4 and VC5 cholinergic motoneurons as models. In C. elegans , we found that epigenetic silencing through histone 3 lysine 9 methylation (H3K9me) is necessary to suppress the expression of the serotonin reuptake gene mod-5/ Sert and a serotonergic phenotype in these cells. In contrast, we observed that VC4 and VC5 neurons in the Angaria group of species of the Caenorhabditis genus have evolved an intense serotonergic staining. This phenotype is caused by the emergence of a new enhancer in the mod-5/ Sert locus, which has been recruited to the ancestral neuron-type gene regulatory network. Enhancer transfer from C. angaria is sufficient to impose a constitutive serotonergic fate in C. elegans . Remarkably, acquiring this new trait modulates egg-laying responses to high levels of exogenous serotonin, which can be found in specific environments. Finally, we discovered that the repression of the serotonergic fate in C. elegans VC4 and VC5 neurons is indeed a plastic trait that can be adjusted in specific environmental growth conditions to elicit egg-laying behaviours similar to those observed in Angaria species. Our work identifies gene regulatory mechanisms that coordinate the generation of robust neuron-type-specific programs with plastic gene expression responses. These findings identify a gene regulatory framework underlying the evolution of neuron-type-specific features and the emergence of novel behaviours.

Animals rely on their nervous systems to process sensory inputs, integrate these with internal signals, and produce behavioral outputs. This is enabled by the highly specialized morphologies and functions of neurons. Neuronal cells share multiple structural and physiological features, but they also come in a large diversity of types or classes that give the nervous system its broad range of functions and plasticity. This diversity, first recognized over a century ago, spurred classification efforts based on morphology, function, and molecular criteria. Caenorhabditis elegans, with its precisely mapped nervous system at the anatomical level, an extensive molecular description of most of its neurons, and its genetic amenability, has been a prime model for understanding how neurons develop and diversify at a mechanistic level. Here, we review the gene regulatory mechanisms driving neurogenesis and the diversification of neuron classes and subclasses in C. elegans. We discuss our current understanding of the specification of neuronal progenitors and their differentiation in terms of the transcription factors involved and ensuing changes in gene expression and chromatin landscape. The central theme that has emerged is that the identity of a neuron is defined by modules of gene batteries that are under control of parallel yet interconnected regulatory mechanisms. We focus on how, to achieve these terminal identities, cells integrate information along their developmental lineages. Moreover, we discuss how neurons are diversified postembryonically in a time-, genetic sex-, and activity-dependent manner. Finally, we discuss how the understanding of neuronal development can provide insights into the evolution of neuronal diversity.

Cortical neurons are specified during embryonic development but often only acquire their mature properties at relatively late stages of postnatal development. This delay in terminal differentiation is particularly prominent for fast-spiking parvalbumin-expressing (PV+) interneurons, which play critical roles in regulating the function of the cerebral cortex. We found that the maturation of PV+ interneurons is triggered by neuronal activity and mediated by the transcriptional cofactor peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α). Developmental loss of PGC-1α prevents PV+ interneurons from acquiring unique structural, electrophysiological, synaptic, and metabolic features and disrupts their diversification into distinct subtypes. PGC-1α exerts its function as a master regulator of the differentiation of PV+ interneurons by directly controlling gene expression through a transcriptional complex that includes ERRγ and Mef2c. Our results uncover a molecular switch that translates neural activity into a specific transcriptional program promoting the maturation of PV+ interneurons at the appropriate developmental stage.

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.

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.

SUMMARY: Cilia, either motile or non-motile (a.k.a primary or sensory), are complex evolutionary conserved eukaryotic structures composed of hundreds of proteins required for their assembly, structure and function that are collectively known as the ciliome. Ciliome 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 master regulator of the primary ciliome 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 behaviours mediated by sensory cilia. Importantly, we find FKH-8 function can be replaced by mouse FOXJ1 and FOXN4 but not by members of other mouse FKH subfamilies. In conclusion, our results show that RFX and FKH TF families act as master regulators of ciliogenesis also in sensory ciliated cell types and suggest that this regulatory logic could be an ancient trait predating functional cilia sub-specialization.

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.