Cardin, Derrick L. et al. (2013) International Worm Meeting "Activity and functional interactions of the leucine-rich protein PAN-1 during larval development."

Gissendanner, Chris R., Cardin, Derrick L.

[International Worm Meeting, 2013]

PAN-1 is a novel nematode-specific leucine-rich (LRR) protein that exhibits complexity in both structure and function. The pan-1 gene encodes three protein isoforms: a predicted type I transmembrane protein with extracellular LRRs and two cytoplasmic proteins, one with a smaller number of LRRs and one lacking LRRs. PAN-1 participates in multiple developmental processes. It associates with P-granules in the germline (Gao et al, 2012) and is also required for larval development. For the latter, pan-1 is necessary for early larval development and the L4 to adult transition. pan-1 promotes growth of the germline, somatic gonad, and vulva during later larval stages and is required for progression of the L4 to adult molting cycle. To better understand the function of PAN-1 during larval development we have initiated a series of studies to address the cellular activities of this protein. Expression of the transmembrane isoform of PAN-1 lacking a cytoplasmic domain is dominant-negative, indicating that PAN-1 may dimerize and transduce an intracellular signal. Using these dominant-negative effects as an assay, we are performing conditional expression of different cytoplasmic domain-deleted constructs to identify critical regions of the intracellular domain. We are also characterizing interactions between pan-1 and other genes. We have found that lin-29 loss of function suppresses the molting progression and vulva development phenotypes in pan-1 RNAi animals. We are further characterizing this interaction to determine how pan-1 intersects with lin-29 in molting regulation. In addition, loss of function of lron-12, which encodes a secreted LRR protein, enhances the pan-1 RNAi phenotype. lron-12 mutants exhibit a larval growth phenotype indicating that pan-1 and lron-12 function together to regulate larval development. We propose that PAN-1 participates in a novel nematode-specific signaling pathway regulating life cycle progression.

Ge Gao et al. (2008) Development & Evolution Meeting "A P-granule Associated Novel Protein And Its Relationship To The C.elegans GLHs"

Ge Gao, Karen Bennett

[Development & Evolution Meeting, 2008]

P-granules are germline-specific protein and RNA complexes found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the P-granules and are necessary for fertility in C. elegans. PAN-1, a P-granule Associated Novel protein, was identified as a GLH-binding partner by yeast two hybrid assays. This binding was also confirmed by pull down assays after in vitro co-expression of His-tagged PAN-1 and GST-tagged GLH-1 in baculovirus. When pan-1 is eliminated by RNA interference (RNAi), C. elegans larvae arrest between the L1 and L2 stages and can survive eight days at 20oC (equal to several generations in wild type worms). After backcrossing to eliminate potential non-specific mutations, a pan-1(gk142) deletion strain exhibits the same "forever-young" phenotype as seen by RNAi. mRNA analysis and protein expression studies show that PAN-1 is not germline specific but is germline enhanced. To further study PAN-1 germline function, we made use of the rrf-1 strain. rrf-1 encodes an RNA directed RNA polymerase required for RNAi in C. elegans somatic tissue. By doing pan-1 RNAi into the rrf-1 strain, the pan-1 larval arrest phenotype is rescued and PAN-1 protein is expressed in the somatic tissue while it is eliminated in the germline. Use of pan-1(RNAi) in the rrf-1 strain gives us the ability to study pan-1 germline function. Our data show that GLH-1 protein levels are somewhat down regulated in the rrf-1 strain after pan-1(RNAi) compared to the rrf-1 strain alone, suggesting that PAN-1 could function as a regulatory protein that affects C. elegans germline development. pan-1(RNAi) into the rrf-1 strain also results in fewer progeny compared to the rrf-1 stain alone or to wild type worms. Experiments are ongoing to investigate whether PAN-1 co localizes with P-granule proteins such as PGL-1 and GLH-1. If PAN-1 regulates the GLHs, we predict it might form a regulatory complex with two other GLH binding partners, CSN-5 and KGB-1.

Li, Meng et al. (2019) International Worm Meeting "PAN-1 interacts with MYRF to regulate synaptic rewiring in DDs."

Xia, Shili, Qi, Yingchuan, Li, Meng, Shao, Dongyu

[International Worm Meeting, 2019]

In C. elegans, six GABAergic neurons (named DD neurons) rewire during development by reversing their axonal and dendritic domains without other obvious morphological transitions. This unique event provides an attractive system in which we can identify the molecular requirements of circuit remodeling. We have previously identified the conserved gene myrf-1 and its paralog myrf-2 as key positive regulators of DD neuron rewiring. Using immunoprecipitation of GFP::MYRF-1 from C. elegans tissues followed by quantitative mass spectrometry analyses, we have identified PAN-1 (P-granule Associated Novel protein), a novel leucine rich repeat (LRR) domain-containing protein, as an interactor of MYRF in vivo. pan-1 null mutants exhibit blocked synaptic rewiring in DD neurons. The membrane-associated isoform of PAN-1B rescues synaptic rewiring cell-autonomously. Over-expression of MYRF restores the rewiring defect of pan-1(0) mutants, indicating that PAN-1 acts upstream of MYRF.

Ge Gao et al. (2004) Mid-west Worm Meeting "Loss of pan-1 causes a Peter Pan-like phenotype in C. elegans"

Ge Gao, Karen Bennett

[Mid-west Worm Meeting, 2004]

P-granules are complexes of proteins and RNA found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the germline specific P-granules, which are necessary for fertility in C. elegans . PAN-1, a P-granule associated novel protein, was identified as a GLH-binding protein in yeast two hybrid assays. PAN-1 contains some conserved amino acids of N-terminal F-box motifs, as well as sixteen leucine-rich repeats and a weak FOG-2 homology (FTH) motif, each found in F-box proteins. F-box proteins, in the SCF (SKP-1, Cullin, F-box) complex, utilize ubiquitin-mediated substrate degradation. When pan-1 is eliminated by RNA interference (RNAi), the larvae arrest between the L1 and L2 stages and can survive eight days at 20 0 C. A pan-1(gk142) deletion strain exhibits the same 'forever-young' phenotype. mRNA analysis and protein expression show that PAN-1 is not germline specific but is germline enhanced. Experiments are ongoing to separate potential germline and somatic functions of PAN-1. If PAN-1 belongs to the family of F-box proteins, it may be implicated in regulating GLH protein levels, as are two other GLH binding proteins, CSN-5 and KGB-1.

Ge Gao et al. (2006) Development & Evolution Meeting "Loss Of pan-1 Causes a Peter Pan-like Phenotype in C. elegans"

Karen Bennett, Ge Gao

[Development & Evolution Meeting, 2006]

P-granules are complexes of proteins and RNA found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the germline specific P-granules, which are necessary for fertility in C. elegans. PAN-1, a P-granule Associated Novel protein, was identified as a GLH-binding protein in yeast two hybrid assays. PAN-1 contains some conserved amino acids of N-terminal F-box motifs, as well as sixteen leucine-rich repeats and a weak FOG-2 homology (FTH) motif, each found in F-box proteins. F-box proteins, in the SCF (SKP-1, Cullin, F-box) complex, utilize ubiquitin-mediated substrate degradation. When pan-1 is eliminated by RNA interference (RNAi), C. elegans larvae arrest between the L1 and L2 stages and can survive eight days at 20ºC, which is equal to several generations in wild type worms. After backcrossing to eliminate potential non-specific mutations, a pan-1(gk142) deletion strain exhibits the same "forever-young" phenotype as seen by RNAi. mRNA analysis and protein expression studies show that PAN-1 is not germline specific but is germline enhanced. Experiments are ongoing to confirm the PAN-1 binding to GLHs in the baculovirus system, as well as potential germline functions of PAN-1. If PAN-1 belongs to the very large (>400) family of C. elegans F-box proteins, it may be implicated in regulating GLH protein levels, as are two other GLH binding proteins, CSN-5 and KGB-1.

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.

Ilya Ruvinsky et al. (2005) International Worm Meeting "A cis-regulatory motif that allows identification of pan-neuronal genes in C. elegans"

Gary Ruvkun, Uwe Ohler, Christopher Burge, Ilya Ruvinsky

[International Worm Meeting, 2005]

Pan-neuronal genes are expressed in most or all types of neurons and thus define generic neuronal features and provide a window into the developmental origin of the nervous system. We computationally predicted a ten-nucleotide cis-regulatory motif shared by many pan-neuronal genes and demonstrated that it is required for broad neuronal expression. Our results suggest that global and cell-type specific controls act in concert to establish pan-neuronal gene expression. Using the newly discovered motif and genome-level gene expression data, we systematically identified a set of 234 candidate pan-neuronal genes, the largest collection of such genes in any organism. The known involvement of many of these genes in neurogenesis and physiology of the nervous system supports the utility of this set for future targeted analyses.

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.

Katherine L Berry et al. (2001) International C. elegans Meeting "Neuronal Defects Elicited by Ectopic Expression of C. elegans Kallmann Protein"

Oliver Hobert, Hannes E Buelow, Katherine L Berry

[International C. elegans Meeting, 2001]

The Kallmann Syndrome gene, KAL-1 , codes for a cell surface protein containing a protease inhibitor domain and three FNIII repeats. Mutations in this gene cause defects in axon targeting in the olfactory system of humans. The C. elegans ortholog, Cekal-1 , shows high structural homology to human KAL-1 and is expressed in a subset of C. elegans neurons as well as in the excretory canal and uterine lumen (1). To investigate potential effects of Cekal-1 on the nervous system of C. elegans, we ectopically expressed the gene under a variety of neuron specific promoters. Ectopic expression of CeKAL-1 in either the AFD thermosensory neuron or the AIY interneuron causes a cell-autonomous, highly penetrant, protein-specific neurite sprouting defect (1). A modifier screen of the sprouting defect in AIY yielded seven modifier mutants in five complementation groups; these mutants either suppressed or qualitatively enhanced the sprouting phenotype and only modified neurites generated by CeKAL-1 (1). We are currently testing whether the modifier mutants of the AIY sprouting defect are also capable of modifying the AFD sprouting defect. To determine the extent of cellular responsiveness to ectopic CeKAL-1, we expressed CeKAL-1 pan-neurally using the promoter of unc-119 , a novel pan-neural gene. We obtained four integrated lines and investigated these for neuronal and non-neuronal defects. The most striking non-neuronal defect exhibited is the vab (variably abnormal) phenotype, suggesting defective ventral enclosure of the hypodermis during embryogenesis. Consistent with this, we see embryonic expression of CeKAL-1 at the time of ventral enclosure in cells that appear to be neuroblasts. To investigate for neuronal defects in the lines in which CeKAL-1 is pan-neurally expressed, we systematically constructed doubles with a variety of neuron-specific GFP reporters. In the pan-neural CeKal-1 lines, we observe a highly penetrant (50%) severe short stop phenotype in the AIY interneuron, a phenotype notably distinct from the sprouting phenotype that results from cell-autonomous expression of CeKAL-1 in AIY. A double mutant expressing CeKAL-1 both in AIY and pan-neurally shows both the short stop and sprouting phenotypes, indicating that pan-neural expression of CeKAL-1 does not interfere with its cell-autonomous effect in this case. In the pan-neural CeKAL-1 lines we also observe, in addition to several low penetrance (less than 10%) axon pathfinding defects, a striking and moderately penetrant (15-25%) axon pathfinding defect in the amphid sensory neurons. Whereas the axons of these neurons normally enter the nerve ring through the amphid commissure, in the pan-neural CeKAL-1 lines, DiI filling reveals altered pathfinding: These axons, as a fascicle, sometimes wander in any direction or more frequently enter the nerve ring directly via a lateral route. We note that this phenotype is unlikely to result from a general defect in dorsal to ventral pathfinding since dorsally to ventrally directed processes of the touch neurons show wild type routing. Visualization of this lateral entry phenotype using a GFP reporter specific for AFD reveals that the axons of AFDL and R appear to successfully meet in the nerve ring even when one or both partner takes a lateral entry route. We also see that this axon pathfinding defect is frequently associated with ectopic neurites originating at the neuronal cell bodies. We note that in sax-3(ky200) and unc-6(ev400) mutants the amphid commisure axons also take a lateral route into the nerve ring (2), and we are investigating whether the pan-neural CeKAL-1 lateral entry phenotype may be dependent on ROBO or netrin. 1. Bulow, H., Berry, K., Zhu, J., and Hobert, O. Worm Breeders Gazette16(4): 28 (Oct. 1, 2000) 2. Zallen, J., Kirch, S., and Bargmann, C. (1999) Development , 126:3679-3692.