Reilly, Molly et al. MicroPubl Biol

Hobert, Oliver, Reilly, Molly

[MicroPubl Biol, 2020]

We have previously described the expression patterns of all but one of the 102 homeobox genes present in the C. elegans genome, using either fosmid-based reporter transgenes or CRISPR/Cas9-engineered gfp reporter alleles (Reilly et al., 2020). The last remaining homeobox gene for which we were initially not able to generate a reporter reagent for is ceh-84 (Reilly et al., 2020). As previously recognized by Thomas Brglin (Hench et al., 2015), ceh-84 codes for an unusual homeodomain protein with two divergent homeodomains (Fig.1D). It likely is a tandem duplicate of the ceh-85 gene, which has been classified as a pseudogene (Fig.1B). ceh-84 has no recognizable homologs in other Caenorhabditis species (Hench et al., 2015). Together with its duplicate, ceh-85, localizes to a large cluster of extensively duplicated genes on chromosome II, described by James Thomas (Thomas, 2006). Most of the duplicated genes code for MATH and BTB type proteins (Fig.1A). The entire cluster seems to be C. elegans-specific (Thomas, 2006) and, apart from a number of pseudogenes, it also contains the C. elegans-specific ceh-81, ceh-82 and ceh-83 homeobox genes (Fig.1A).

Reilly, Molly et al. (2019) International Worm Meeting "Do unique homeobox gene codes define all neuron classes of the C. elegans nervous system?"

Hobert, Oliver, Reilly, Molly

[International Worm Meeting, 2019]

A central problem in developmental neuroscience is understanding how the diversity of individual neuron types is specified and maintained. These mechanisms have been probed in C. elegans, where genetic mutant analysis has uncovered a host of transcriptional regulators - called terminal selectors - that control neuron-specific features by binding to regulatory regions of neuron-specific genes to activate their transcription. Despite our progress characterizing these molecular mechanisms, it remains unclear if there are any overarching themes in regulation of terminal nervous system specification. Of the 32 terminal selectors that have been identified, 20 are homeodomain (HD) transcription factors belonging to the homeobox gene family. This is more than 7 times the next leading transcription factor family, which only contribute 3 terminal selectors. Furthermore, those 20 HD terminal selectors are known to control the identity of 86 neuron classes in the C. elegans nervous system. This broad usage of homeobox genes suggests that HD transcription factors may have evolutionarily ancient roles in specifying the terminal identity of an ancestral neuron type and, thus, I hypothesize that a unique combination of HD transcription factors participate in the specification of every neuron class in C. elegans. In my initial analysis of the expression of 64 genomically-tagged homeobox gene reporters, I have found a unique combinatorial expression pattern of homeobox genes for 114 out of 118 neuron classes in the C. elegans hermaphrodite. To determine if they are acting as terminal selectors in each of these neuron classes, I plan to examine the effect of readily available mutant alleles on adoption of neurotransmitter identity. I hypothesize that a global, systematic analysis of homeobox genes will yield a unique combination of homeobox gene regulators for every single neuron type.

Gulez, Burcu et al. (2021) International Worm Meeting "Decoding pharyngeal neuron fate specification"

Reilly, Molly, Gulez, Burcu, Vidal , Berta, Hobert, Oliver

[International Worm Meeting, 2021]

The pharynx has its own nervous system consisting of 14 neuron types that form an independent circuit almost completely isolated from the somatic nervous system. We know that the homeodomain transcription factor (TF) ceh-34 is required to specify all pharyngeal neuron types (see abstract by B. Vidal et al). We hypothesize that there must be other factors operating with ceh-34 in a target gene-dependent manner, forming a combinatorial code that gives each pharyngeal neuron its unique identity. In support of this notion, we found that ceh-34 interacts with two homeobox genes, ttx-3 and unc-86, to affect NSM neuron specification. In order to find additional factors that may collaborate with ceh-34, we are using (1) a candidate gene approach based on homeodomain TF expression, (2) we are performing unbiased forward genetic screens and (3) we are dissecting the cis-regulatory region of terminal effector genes. So far mutant analysis of homeobox genes expressed in the pharynx has shown that ceh-2, ceh-7 and vab-15 affect neurotransmitter identity in specific pharyngeal neurons, making them candidate cofactors for ceh-34. Furthermore, forward genetic screens have yielded four mutants with defects in nlp-8 expression in the I2 neurons. We are in the process of pinpointing the molecular lesion responsible for this mutant phenotype. Finally, dissecting the endogenous unc-17 cis-regulatory region we have identified a conserved motif required for the expression of unc-17 in MC and I1 neurons. We continue to work on these three approaches, which hopefully will lead to a broad understanding of the regulatory code underlying pharyngeal neuron specification.

Varol, Erdem et al. (2021) International Worm Meeting "A computational approach linking neuron-specific gene expression with connectivity"

Barrett, Alec, McWhirter, Rebecca, Vidal, Berta, Tavazoie, Saeed, Hobert, Oliver, Weinreb, Alexis, Miller, David, Xu, Chuan, Taylor, Seth, Paninski, Liam, Yemini, Eviatar, Sestan, Nenad, Basavaraju, Manasa, Litwin-Kumar, Ashok, Cros, Cyril, Reilly, Molly, Santpere, Gabriel, Poff, Abigail, Glenwinkel, Lori, Abrams, Alexander, Hammarlund, Marc, Rafi, Ibnul, Varol, Erdem, Oikonomou, Panos, Cook, Steven

[International Worm Meeting, 2021]

There is strong prior evidence for genetic encoding of synaptogenesis, axon guidance, and synaptic pruning in neural circuits. Despite these foundational observations, the transcriptional codes that drive neural connectivity have not been elucidated. The C. elegans nervous system is a particularly useful model for studying the interplay between genetics and connectivity since its wiring diagram is highly stereotyped and uniquely well-defined by electron microscopy. Furthermore, recent evidence in C. elegans has suggested that a unique combination of transcription factors specifies each of the 118 neuron classes[1]. Motivated by evidence for the stereotypy of neural circuits and for the genetic encoding of neural identity, we introduce a novel statistical technique, termed Network Differential Gene expression analysis (nDGE), to test the hypotheses that neuron-specific gene expression dictates connectivity. Specifically, we test the hypothesis that pre-synaptic neural identity is defined by a "key" gene combination whose post-synaptic targets are determined by a "lock" gene combination. For our approach, we utilize neuron-specific gene expression profiles from the CeNGEN project[2] to investigate transcriptional codes for connectivity in the nerve ring[3]. We hypothesize that the expression of specific cell adhesion molecules (CAM) among synaptically-connected neurons drives synaptic maintenance in the mature nervous system. We posit that CAMs mediating synaptic stability would be more highly expressed in synaptically-connected neurons than in adjacent neurons with membrane contacts but no synapses. Thus, for each neuron, we compare the expression of all possible combinations of pairs of CAMs in the neuron and its synaptic partners relative to the neuron and its non-synaptic adjacent neurons. Two independent comparisons are generated, one for presynaptic neurons and a second result for postsynaptic neurons. Our nDGE analysis reveals that specific combinations of CAMs are correlated with connectivity in different subsets of neurons and thus provides a uniquely comprehensive road map for investigating the genetic blueprint for the nerve ring wiring diagram. Open source software of Network Differential Gene Expression (nDGE) is publicly available at https://github.com/cengenproject/connectivity_analysis along with a vignette showcasing the CAM results. 1. Reilly, M. B., Cros, C., Varol, E., Yemini, E., & Hobert, O. (2020). Unique homeobox codes delineate all the neuron classes of C. elegans. Nature, 584(7822), 595-601. 2. Taylor, S. R., Santpere, G., Weinreb, A., Barrett, A., Reilly, M. B., Xu, C. Varol, E., ... & Miller, D. M. (2020). Molecular topography of an entire nervous system. bioRxiv. 3. Cook, S. J.,... & Emmons, S. W. (2019). Whole-animal connectomes of both Caenorhabditis elegans sexes. Nature, 571(7763), 63-71.

Beitel GJ et al. (1991) International C. elegans Meeting "CLONING AND SEQUENCING OF THE SYNTHETIC MULTIVULVAL GENE lin-9"

Beitel GJ, Horvitz HR

[International C. elegans Meeting, 1991]

A synthetic Multivulva phenotype can result from mutations in two separate genes, while each mutation alone results in an apparently wild-type phenotype (Ferguson and Horvitz, Genetics 123:109-121,1989). Mutations that can cause this synthetic Multivulva (syn Muv) phenotype have been grouped into two classes, A and B, such that a double mutant carrying one mutation in each class will have the syn Muv phenotype. Iin-9(nll2) III is a class B mutation that results in an apparently wild-type phenotype by itself. We previously reported that ZK637 and overlapping cosmids can rescue lin-9 (WBG 11(2 ): p 29,1990). We have now identified an 8 kb subclone with rescuing activity. Using this 8 kb fragment to probe Stuart Kim's cDNA library, six positive plaques representing at least three independent clones were isolated. This 8 kb probe also detects a message of approximately 2.5 kb on Northern blots. The message level appears similar in eggs and mixed-stage populations. We are currently sequencing these cDNAs and looking for evidence that these transcripts do in fact represent the lin-9 gene. This work will be greatly aided by the genomic sequence provided by Molly Craxton, John Sulston and Alan Coulson, as ZK637 was fortuitously chosen as one of the first cosmids to be sequenced in the worm genome sequencing project.

Barrett, Alec et al. (2021) International Worm Meeting "Integrating bulk and single cell transcriptomics for accurate detection of tissue-specific gene expression"

Barrett, Alec, Taylor, Seth R., Weinreb, Alexis, Miller III, David M., Varol, Erdem, Sestan, Nenad, Hobert, Oliver, McWhirter, Rebecca, Li, Mingfeng, Hammarlund, Marc

[International Worm Meeting, 2021]

Advances in RNA-seq for bulk and single cell (sc) approaches have produced increasingly fine dissections of the C. elegans transcriptome. Although both techniques can yield transcriptomes for individual cell types, each comes with strengths and weaknesses. scRNA-Seq affords high resolution, but suffers from dropout, leading to false negatives. Bulk sequencing detects more genes, but suffers from contaminating cell types, resulting in false positives. In this work we integrated these orthogonal approaches to improve the accuracy of both methods. We used bulk samples collected for specific neuron types and sc datasets for all C. elegans neurons and additional non-neuronal cells (1). We used sc data to estimate contamination in each bulk sample, and developed novel methods for removing these gene counts. In one approach we used linear histogram matching to scale sc counts, and subtracted putative contamination using data from non-neuronal clusters. In another approach we used bootstrapping to estimate gene level contributions from target and contaminating tissues in sc data and apply them to bulk counts, providing a bootstrap sample distribution of corrected expression data. We assessed these approaches in two ways: 1) Measuring improvements in calling genes with known expression in all neurons; 2) Examining effects on eliminating genes expressed exclusively in contaminating tissues. We found that our analysis reduced false positives, while maintaining robust true positive detection, thus offering a unique strategy for utilizing complementary bulk and sc RNA-Seq data sets to enhance the accuracy of cell-specific expression profiling data. 1. Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al. Molecular topography of an entire nervous system. bioRxiv. 2020:2020.12.15.422897.

Taylor, Seth R et al. (2021) International Worm Meeting "Molecular topology of an entire nervous system"

Xu, Chuan, Barrett, Alec, Varol, Erdem, Cook, Steven J, Basavaraju, Manasa, Yemini, Eviatar, Hammarlund, Marc, Miller III, David M, Weinreb, Alexis, Abrams, Alexander, Rafi, Ibnul, Santpere, Gabriel, Vidal, Berta, Sestan, Nenad, Oikonomou, Panos, McWhirter, Rebecca, Cros, Cyril, Glenwinkel, Lori, Tavazoie, Saeed, Taylor, Seth R, Hobert, Oliver, Reilly, Molly B, Poff, Abigail

[International Worm Meeting, 2021]

Neurons are the fundamental structural and functional units of the nervous system. Although all neurons share many common features, they also display remarkably diverse morphological and functional characteristics. To uncover the underlying genetic programs that specify individual neuron identities, the CeNGEN consortium produced scRNA-Seq profiles of > 100,000 cells from the L4 stage C. elegans hermaphrodite, including all neuron classes and several non-neuronal cells (e.g., glia, muscle, hypodermis, reproductive tissues). In addition, we identified distinct subclasses for 10 of the 118 anatomically-defined classes. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses unique codes of ~23 neuropeptide genes and ~36 neuropeptide receptors thus pointing to an expansive "wireless" signaling network. To demonstrate the utility of this uniquely comprehensive gene expression catalog, we used computational approaches to identify cis-regulatory elements for neuron-specific gene expression. Because our scRNA-Seq data match the single cell resolution of the wiring diagram, we also sought to correlate expression of cell adhesion proteins with neuron-specific fasciculation and connectivity in the nerve ring. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity and function throughout the C. elegans nervous system. These data are available at cengen.org and can be interrogated with the web application CengenApp at cengen.shinyapps.io/CengenApp.

Zhou, Junxiang et al. (2021) International Worm Meeting "beta-tubulin BEN-1 has a key role in regulating DLK-1 signal transduction"

Jin, Yishi, Sun, Yue, Zhou, Junxiang

[International Worm Meeting, 2021]

The conserved mitogen activated kinase kinase kinase (MAPKKK) DLKs play critical roles in neuronal process growth, synapse formation and axon regeneration. DLKs also act as sensors to mediate neuronal responses to different stresses, including microtubule (MT) disruption. However, it is not clear in which contexts DLKs sense MT stress and which signaling transduction mechanisms control the activity of DLKs to mediate specific functional outcomes. In C. elegans the DLK ortholog DLK-1 is normally expressed in most neurons at low levels. From a forward genetic screen for mutants displaying aberrant DLK-1 levels or localization, we identified a mutant that exhibited aggregated DLK-1 in neuronal soma. Using whole genome sequencing and genetic mapping, we determined that this mutation affected ben-1, one of six beta-tubulin isotypes in C. elegans. Levels of the transcription factor CEBP-1, a downstream target induced by DLK-1, were elevated in neurons of ben-1 mutants. These findings suggest that altering ben-1 function activates dlk-1, resulting in cebp-1 dependent transcription. A ben-1 transcriptional reporter is exclusively expressed in neurons. BEN-1 is known to convey the sensitivity to benomyl1, a microtubule-targeting compound used for killing parasitic nematodes and treating cancers. We found that benomyl treatment also caused neural DLK-1 aggregation, CEBP-1 elevation, as well as neuronal defects such as impaired synaptogenesis. Altogether, our findings suggest that changes in neuronal beta-tubulin BEN-1 containing MT cytoskeleton elicit specific stress responses to activate dlk-1/cebp-1 signaling. 1. Driscoll, M., Dean, E., Reilly, E., Bergholz, E., and Chalfie, M. (1989). Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity. J. Cell Biol. 109, 2993-3003.

Vidal , B. et al. (2019) International Worm Meeting "Decoding pharyngeal neuron fate specification."

Hobert, O., Gulez, B., Vidal , B., Reilly, M.

[International Worm Meeting, 2019]

The pharyngeal nervous system is composed of only 20 neurons belonging to 14 different types, which form a self-contained circuit that is almost independent from the somatic nervous system. This simplicity makes it possible to analyze it in a comprehensive way. Moreover, all pharyngeal neurons directly connect to end organs and can be considered polymodal with sensory-motor characteristics (see abstract by S.J. Cook and S.W.Emmons), a feature that is reminiscent of primitive nervous systems. Thus, understanding how pharyngeal neurons are specified during development might shed light on fundamental aspects of neuronal development. ceh-34, a homeodomain transcription factor of the Six family, is continuously expressed in all pharyngeal neurons and no other neurons outside of the pharynx. Remarkably, we have found that in ceh-34 mutants, pharyngeal neurons are generated, but fail to express a wide array of terminal identity genes, including neurotransmitter pathway genes, indicating that ceh-34 acts as a pharyngeal neuron master regulator ("terminal selector"). Moreover, a conditional AID-based allele demonstrates that ceh-34 is continuously required during the life of the worm to maintain pharyngeal neuron identity. We hypothesize that ceh-34 acts together with other transcription factors to form a combinatorial code that gives each pharyngeal neuron its unique identity. We have found several other homeodomain transcription factors expressed in subsets of pharyngeal neurons (see abstract by M. Reilly and O.Hobert) and we are doing a mutant analysis to test whether they also play a role in the pharyngeal nervous system specification. Moreover, we are performing forward genetic screens to find additional factors in an unbiased way. So far we have identified four mutants that appear to affect I2 neuron identity. We are in the process of pinpointing the causal molecular lesions and further characterizing these new mutants. We hope our efforts will lead to a comprehensive understanding of the regulatory code that dictates pharyngeal neuron development.

Sulston JE et al. (1991) Worm Breeder's Gazette "Genome Sequencing"

Waterston RH, Sulston JE, Metzstein MM, Ainscough R, Shownkeen R, Hawkins TL, Craxton M, Lutterbach B, Green P, Thierry-Mieg J, Wilson RK, Coulson AR, Kozono Y, Staden R, Qui QQ, Du Z, Weigelt LD, Durbin RM

[Worm Breeder's Gazette, 1991]

The Great Adventure has begun! The joint St. Louis Washington University/Cambridge Laboratory of Molecular Biology project to sequence three megabases of contiguous genomic DNA has been funded under the M.R.C. and N.I.H. Human Genome initiatives. The intention is to develop methods and strategies that will allow us to sequence 1Mb of DNA at each site in the final year of the three year project. It will thus act as a pilot project for the eventual sequencing of the entire genome. There are two elements to our general approach. The first is to reduce the redundancy associated with 'shotgun' sequencing schemes by maximizing the use of directed sequencing with synthetic oligonucleotides. The second is to make data retrieval and analysis more efficient by the use of direct read-out fluorescence-detection sequencing machines (both sites have ABI and Pharmacia devices) and sophisticated computer control of day to day data generation and analysis. We are currently addressing a number of problems: which vectors and insert sizes are most efficient for subcloning cosmids? What degree of shotgun redundancy should be obtained before oligo walking? How do the sequencing machines compare? Can double stranded sequencing yield data of good enough quality? (Molly Craxton has developed some excellent double-stranded sequencing protocols, available on request, which work well on cosmid and lambda DNA, in addition to sub-clones). We have begun to sequence in the cluster on chromosome III. The Cambridge group have started on the lin-9/unc-32 cosmid ZK637 and are proceeding rightwards to the edge of the cluster while the St. Louis group have started sequencing around sup-5 on the cosmid ZK370. Obviously, the genome map is essential in allowing us to take such a logical ordered approach. A 3Mb sequence should extend from the right edge of the cluster leftwards beyond mec-14. It is our intention to release sequence data to the community as rapidly as possible, probably within two months following completion ( of cosmid sized pieces). This will be done by incorporation into the databases currently being developed (see articles by R.D. and J. T-M in this issue and Bruce Schatz et al WBG 11/4, and submission to EMBL/Genbank libraries). Initial sequence analysis (predicted coding regions, splice sites, library comparisons etc.) will also be included.