Stefanakis, Nikolaos et al. (2021) International Worm Meeting "The development and functions of GLR glia."

Shaham, Shai, Stefanakis, Nikolaos

[International Worm Meeting, 2021]

C. elegans glia can broadly be divided into three classes: 46 sensory-neuron associated glia (sheath and socket cells), four nerve-ring wrapping glia (CEPsh cells), and six GLR (Glia Like cells of the Nerve Ring) glia. While roles of C. elegans sensory organ glia and nerve-ring wrapping glia have been characterized, little is known about the development and functions of GLR glia. The GLR cell bodies lie in a six-fold symmetry just posterior to the nerve ring and extend anterior leaf-like processes that form a seal around the pharynx, isolating the nerve ring from the body cavity. These processes lie in close proximity to the muscle arm plate in the nerve ring, where neuromuscular junctions (NMJ) form with head motor neurons. Based on their morphological characteristics, position, and gene and neurotransmitter expression patterns, GLR roles in NMJ development, activity and motor behavior are likely. To aid our studies and to understand the molecular basis of the development and functions of GLR glia we first performed GLR-transcriptome analysis. In order to identify genes controlling GLR fate specification and differentiation we are following a candidate gene approach in which mutants and RNAi for transcription factor genes enriched in the GLR transcriptome are tested for defects in GLR gene expression and morphology. We have so far discovered two transcription factors that are important regulators of GLR cell fate specification and differentiation. Apart from exposing regulators of GLR development, newly made conditional mutants in these genes will also be used to inform us on possible functions of these cells in NMJ control. To directly examine GLR functions in NMJ, we have generated a stable transgenic line in which GLR glia are ablated at the L1 stage. GLR-ablated animals display various motor defects including reduced speed, extended locomotory pausing and increased reversal frequency. A significant overrepresentation for genes related to NMJ synaptic transmission among the most highly enriched GLR-genes further supports our hypothesis that GLR glia could have important functions at the NMJ. We are currently following a candidate gene approach and cell specific rescue experiments to study the involvement of these genes in the observed motor-behavior defects. Progress in these studies will be presented.

Stefanakis, Nikolaos et al. (2011) International Worm Meeting "Regulatory logic of pan-neuronal gene expression in C. elegans."

Hobert, Oliver, Carrera, Ines, Stefanakis, Nikolaos

[International Worm Meeting, 2011]

The adult C. elegans nervous system is remarkable diverse consisting of 302 neurons grouped in 118 anatomical classes. The morphological and functional diversity of mature differentiated neurons of each class is reflected in their different molecular composition. The expression of different sets of genes gives each neuron its "neuron-type specific" identity. However, all neurons in a nervous system have common morphological characteristics namely cellular projections and synapses. The molecular correlates to those common features are encoded by pan-neuronal genes. While a lot of information is available about how specific neuronal types are generated, the regulatory programs that govern pan-neuronal gene expression are poorly understood. In this study our main goal is to dissect the cis-regulatory logic of pan-neuronal gene expression and to identify the trans-acting factors responsible for its execution. To set the basis of our analysis we have first constructed a reporter for the pan-neuronal synaptic gene rab-3. We have extensively studied the expression pattern of this transgene, which appears to be expressed in almost all the neurons of an adult hermaphrodite worm. We then defined a pan-neuronal gene battery consisting of synaptic proteins and neuronal-specialized cytoskeleton components that are broadly expressed in the nervous system. We study the spatial and temporal expression pattern of each of these genes by tagging their locus with a nuclear localized YFP using fosmid recombineering, and comparing the overlap of expression between the rab-3 reporter and these fosmid reporters. We are currently dissecting the cis-regulatory architecture of each member of our list by generating NLS-TagRFP deletion constructs of their promoter regions. Although we are verifying that most of the genes under study are expressed throughout the nervous system of the adult worm in a similar spatial and temporal pattern, deletion analysis of their promoters shows that there might not be a common regulatory logic among them. While for many genes a piece-meal manner of regulation is suggested, there are other genes where only a small region of the promoter is still sufficient to drive expression in most neurons. Progress on this analysis will be presented.

Stefanakis, Nikolaos et al. (2015) International Worm Meeting "Redundant modular control of pan-neuronal gene expression in C. elegans."

Hobert, Oliver, Carrera, Ines, Stefanakis, Nikolaos, Leyva-Diaz, Eduardo

[International Worm Meeting, 2015]

While neuronal cell types in a nervous system display an astounding degree of diversity in their molecular composition, all neuron types share a panel of common features defined by the expression of pan-neuronal genes. Though a great deal is known about how the expression of neuron-type specific identity features is controlled in the nervous system, there is a striking dearth of insight into how pan-neuronal gene expression programs are controlled. We define here a set of 23 conserved genes which are expressed throughout the entire nervous system of C. elegans, most of them encoding for genes involved in synaptic vesicle biology and neuropeptide processing. Through an extensive analysis of the cis-regulatory control region of this pan-neuronal gene battery, we address several distinct scenarios for how pan-neuronal gene expression could be achieved. We found that pan-neuronal gene expression is controlled through a surprisingly complex array of redundantly acting cis-regulatory modules that direct expression to broad, overlapping domains throughout the nervous system. These redundantly acting cis-regulatory modules are responsive to a host of distinct trans-acting factors. We found that terminal selector factors and HOX genes as some examples of these factors, however the identity of most of them remains unknown. We are currently conducting multiple genetic screens to identify these trans-acting factors that bind to those elements and control pan-neuronal gene expression. Neuronal gene expression programs therefore fall into two fundamentally distinct classes. Neuron type-specific genes are generally controlled by discrete and non-redundantly acting regulatory inputs, while pan-neuronal gene expression is controlled by diverse, redundant molecular mechanisms. These two fundamentally distinct strategies of controlling gene expression in the nervous system may be a reflection of the evolutionary history of neuronal cell types.

Carrera, Ines et al. (2013) International Worm Meeting "Regulatory logic of pan-neuronal gene expression."

Stefanakis, Nikolaos, Hobert, Oliver, Carrera, Ines

[International Worm Meeting, 2013]

Cell fate decisions in the vertebrate nervous system are particularly complex as the nervous system is composed of a remarkably heterogeneous assemblage of cell types. Although a lot is known about how specific transcription factors, or Terminal Selectors (TS), specify different neuronal types by coregulating neuron-type specific terminal differentiation genes, much less is understood about the regulatory programs that control the expression of those neuronal features shared by every neuron, pan-neuronal features. Addressing this question is key to understanding how neuronal fate is determined. In this work, we are dissecting the cis-regulatory logic of broadly expressed neuronal genes and also identifying those trans-acting factors that regulate them. Promoter bashing analysis of cis-regulatory control elements of pan-neuronal genes shows a piecemeal regulation of gene expression in different neuronal types as well as redundant elements. We find that TS are also able to regulate expression of isolated cis-regulatory modules of some pan-neuronal genes, although in TS mutants full promoters of these genes are not affected. We are currently conducting genetic screens to identify these redundant transcription factors. Analysis of the temporal and spatial expression of broadly expressed neuronal genes by fosmid reporters show expression in the nervous system as well as in other tissues. Progress towards the understanding of how pan-neuronal gene expression is regulated will be presented.

Leyva-Diaz, Eduardo et al. (2017) International Worm Meeting "pals-22, a member of an expanded C. elegans gene family, is involved in repetitive transgene silencing."

Leyva-Diaz, Eduardo, Hobert, Oliver, Stefanakis, Nikolaos, Carrera, Ines

[International Worm Meeting, 2017]

Repetitive DNA sequences are subject to chromatin-based gene silencing in various animal species. Under specific circumstances repetitive DNA sequences can escape such silencing. For example, while exogenously added, extrachromosomal DNA sequences that are stably inherited in multicopy, repetitive arrays in the nematode C. elegans are often silenced in the germline, such silencing often does not occur in the soma. This indicates that somatic cells may utilize factors that prevent repetitive DNA silencing. Such "anti-silencing" factors have been revealed through genetic screens that identified mutant loci in which repetitive transgenic arrays are aberrantly silenced in the soma (Fischer et al. 2008, Grishok et al. 2005, Hsieh et al. 1999, Knight and Bass 2002). We describe here a novel mutant locus, pals-22 (for protein containing ALS2CR12 domain) with a transgene silencing phenotype. We find that pals-22 is a member of a large family of divergent genes, defined by the presence of an ALS2CR12 domain (39 members, including 2 pseudogenes). While family members are highly divergent they show striking patterns of genomic clustering. The family expansion appears C. elegans specific and has not occurred to the same extent in other nematode species (e.g. C. briggsae only codes for 6 pals genes). A PALS-22::GFP fusion protein that is fully functional in rescue assays displays a cytoplasmic subcellular localization, which rule out any direct chromatin-mediated silencing mechanism. This fusion protein is expressed across different tissues: the nervous system (pan-neuronally), muscle (pharyngeal, vulval, anal and body wall muscle), gut and seam cells. Lastly, the transgene silencing phenotype of pals-22 depends on the biogenesis of small RNAs, since silencing is abolished in the RNAi defective mutant rde-4, suggesting that pals-22 might regulate RNAi dependent silencing in the cytoplasm of neurons and other tissues.

Leyva-Diaz, Eduardo et al. (2017) International Worm Meeting "Identification of transcription factors regulating pan-neuronal gene expression."

Stefanakis, Nikolaos, Hobert, Oliver, Leyva-Diaz, Eduardo, Carrera, Ines

[International Worm Meeting, 2017]

While neuronal cell types in a nervous system display an astounding degree of diversity in their molecular composition, all neuron types share a panel of common features defined by the expression of pan-neuronal genes. Though a great deal is known about how the expression of neuron-type specific identity features is controlled in the nervous system, there is a lack of knowledge into which factors are controlling pan-neuronal gene expression programs. Through an extensive analysis of the cis-regulatory control region of a battery of pan-neuronal genes, we addressed several distinct scenarios for how pan-neuronal gene expression could be achieved. We found that pan-neuronal gene expression is controlled through a surprisingly complex array of redundantly acting cis-regulatory modules that direct expression to broad, overlapping domains through the nervous system. These redundantly acting cis-regulatory modules are responsive to a host of distinct trans-acting factors. We found that terminal selector factors and HOX genes as some examples of these factors, however the identity of most of them remains unknown. We are currently conducting multiple genetic screens to identify these trans-acting factors that bind to those cis-regulatory elements and control pan-neuronal gene expression. Our first mutants are presently being cloned and tested for involvement in the regulation of different pan-neuronal genes. In addition, we are performing a small-scale cis-regulatory analysis of a conserved vertebrate pan-neuronal gene to test whether this redundant modular model is conserved in vertebrates. Identifying factors involved in pan-neuronal gene regulation will provide insights into how the fully differentiated neuronal fate is developed, and might reveal key regulators of neuronal identity maintenance during normal development and neurodegeneration.