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.

Schulze, Jens et al. (2011) International Worm Meeting "Evolution of embryonic development in nematodes."

Schulze, Jens, Schierenberg, Einhard

[International Worm Meeting, 2011]

Nematodes are well suited for a comparative study of early embryogenesis. Analyzing development of a single model system like Caenorhabditis elegans does not shed any light on the degree of evolutionary modifications within the taxon Nematoda. For better understanding evolution of development among nematodes including the identification of plesiomorphic and apomorphic characters, we compared early embryogenesis of representatives from all 12 nematode clades (phylogeny after Holterman et al., 2006; Mol. Biol. Evol. 23:1792-1800). Our data reveal that embryogenesis is unexpectedly variable with floating transitions that can be interpreted as frozen images of evolutionary change. Particularly, members of clade 1 and 2 differ massively from the standard C. elegans. Nevertheless, some basic developmental similarities appear to be common among all nematodes, e.g. the general existence of at least partial cell lineages and the influence of Polarity Organizing Centers (POCs). These POCs are required for generating a linear sequence of cells along the a-p axis which constitute the ventral midline. Depending on phylogenetic position fewer or more cells of this midline divide into left and right daughters this way establishing bilateral symmetry within individual lineages. Our studies reveal the stepwise emergence of founder cells and dramatic fate shifts during evolution. The comparison between embryogenesis of the basal nematode Tobrilus stefanskii and the tardigrade Hypsibius dujardini (Gabriel et al., 2007; Dev. Biol. 312, 545-59) with respect to the early cell division pattern revealed surprising similarities between these two. This may be considered as embryological support for the Ecdysozoa hypothesis.

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.