1 2 3 Resource 4 5 6 A cellular and regulatory map of the cholinergic nervous system 7 of C.elegans 8 9 10 Laura Pereira1,2*, Paschalis Kratsios1,2¶, Esther Serrano-Saiz1,2¶, Hila Sheftel3, Avi Mayo 3, 11 David H. Hall4, John G. White5, Brigitte LeBoeuf6, L. Rene Garcia2,6, Uri Alon 3and Oliver 12 Hobert1,2* 13 14 1Department of Biological Sciences, Department of Biochemistry and Molecular Biophysics, 15 Columbia University, New York, USA 16 2 Howard Hughes Medical Institute 17 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 18 4Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 19 5 MRC Laboratory of Molecular Biology, Cambridge, England 20 6 Department of Biology, Texas A&M University, College Station, Texas, USA 21 ¶ these authors contributed equally to this work 22 * correspondence: [email protected], [email protected] 23 ABSTRACT 24 25 Nervous system maps are of critical importance for understanding how nervous 26 systemsdevelop and function. We systematically map here all cholinergic neuron types in the 27 male and hermaphrodite C.elegans nervous system. We find that acetylcholine is the most 28 broadly used neurotransmitter and we analyze its usage relative to other neurotransmitters 29 within the context of the entire connectome and within specific network motifs embedded in 30 the connectome. We reveal several dynamic aspects of cholinergic neurotransmitter identity, 31 including a sexually dimorphic glutamatergic to cholinergic neurotransmitter switch in a sex- 32 shared interneuron. An expression pattern analysis of ACh-gated anion channels furthermore 33 suggests that ACh may also operate very broadly as an inhibitory neurotransmitter. As a first 34 application of this comprehensive neurotransmitter map, we identify transcriptional control 35 mechanisms that control cholinergic neurotransmitter identity and cholinergic circuit 36 assembly. 37 38 39 INTRODUCTION 40 Nervous system maps that describe a wide range of distinct structural and molecular 41 parameters are essential for an understanding of nervous system development and function. 42 Tremendous efforts have been and are being made to map connectomes(Bargmann and 43 Marder, 2013; Plaza et al., 2014). Connectomes now exist for small anatomic regions of 44 mouse and fly brains(Helmstaedter et al., 2013; Kasthuri et al., 2015; Takemura et al., 2013), 45 but the only complete, system-wide connectome remains that of the nematode C. elegans, 46 both in its hermaphroditic and male form(Albertson and Thomson, 1976; Jarrell et al., 2012; 47 White et al., 1986). However, these anatomical maps are incomplete without the elucidation 48 of chemical maps that describe the synaptically released neurotransmitters through which 49 anatomically connected neurons communicate with one another.But even in C. elegans, let 50 alone other organisms, there have so far been only limited efforts to precisely map 51 neurotransmitter identities on a system-wide level with single neuron resolution. In C.elegans, 52 a combination of direct staining methods and expression analysis of neurotransmitter-specific 53 enzymes and transporters have defined the likely complete complement of GABAergic, 54 glutamatergic and aminergic neurotransmitter systems. Specifically, out of the 118 55 anatomically distinct neuron classes in the hermaphrodite (amounting to a total of 302 56 neurons), six classes (26 neurons) are GABAergic(McIntire et al., 1993), 38 are glutamatergic 57 (78 neurons) and 13 are aminergic (i.e. serotonergic, dopaminergic, etc.). 58 The last remaining neurotransmitter system – the cholinergic system – has not been 59 completely mapped. Antibody staining against the vesicular acetylcholine (ACh) transporter, 60 VAChT (encoded by unc-17) and the ACh-synthesizing choline acetyltransferase ChAT 61 (encoded by cha-1) revealed the cholinergic identity of a number of neurons in the nervous 62 system (Alfonso et al., 1993; Duerr et al., 2008). However, due to the synaptic localization of 63 the VAChT and ChAT proteins, expression could only be unambiguously assigned to about 64 one dozen neuron classes, mostly in the ventral nerve cord and a few isolated head and tail 65 neurons (seeTable 1for a summary of previous studies on cholinergic neuron identity). The 66 authors of these previous studies explicitly noted that many additional VAChT/ChAT- 67 expressing neuron classes await identification (Duerr et al., 2008).Ensuing studies using 68 reporter genes that capture cis-regulatory elements of parts of the unc-17/VAChTlocus 69 identified the cholinergic identity of a few additional neuron classes (Table1), but the extent to 70 which ACh is used in the nervous system has remained unclear.In the male nervous system, 71 composed of 23 additional neuron classes, neurotransmitter identities are even less well 72 defined (not just ACh, but other systems as well).Here we map the usage of ACh in both the 73 hermaphrodite and male nervous systems. We show that ACh is the most broadly used 74 neurotransmitter in the C.elegans nervous system, employed by more than half of all 75 neurons. 76 The tremendous benefits ofa neurotransmitter map include the ability to precisely 77 dissect and understand neuronal circuit function. For example, knowledge of the cholinergic 78 identity of theAIY interneuron (Altun-Gultekin et al., 2001)helped to define thetwo distinct 79 behavioral outputs of AIY, one controlled via an ACh-mediated activation of the RIB 80 interneuron and another controlled byACh-mediated inhibition of the AIZ interneuron, via an 81 ACh-gated chloride channel(Li et al., 2014). The cholinergic neurotransmitter map presented 82 here will provide a resource to further functionally dissect circuit function in the C.elegans 83 nervous system. 84 Since neurotransmitter identity represents a key feature of a neuron, the knowledge of 85 the cholinergic identityprovides aresource for studying how a neuron adopts its specific fate 86 during development. For example, the assignment of glutamatergic identity to a host of 87 distinct C. elegans neurons has enabled us to define phylogenetically conserved regulatory 88 features of glutamatergic neuron differentiation (Serrano-Saiz et al., 2013). Moreover, the 89 long known cholinergic identity of ventral cord motor neurons provided an entry point to 90 studyhow their terminal differentiation is controlled(Kratsios et al., 2015; Kratsios et al., 2011). 91 Previous studies describing the mechanism of cholinergic identity control have pointed to a 92 modular control system in which neuron-type specific combinations of transcription factors 93 turn on cholinergic pathway genes(Altun-Gultekin et al., 2001; Kratsios et al., 2011; Zhang et 94 al., 2014). Since previous studies only examined a relatively small number of neurons, the 95 problem of cholinergic identity control has not yet encompassed a circuit level analysis. 96 Through a genetic screen and a candidate gene approach we reveal common themes in the 97 form of circuit-associated transcription factors that control the identity of all neurons within 98 defined circuits or circuit-associated network motifs. Taken together, we anticipate that 99 neurotransmitter maps like those provided here represent an invaluable resource for the 100 C.elegans community that will serve as a high-resolution starting point for various types 101 ofbehavioral and developmental analyses. 102 103 104 RESULTS AND DISCUSSION 105 106 Defining cholinergic neurons 107 Cholinergic neurotransmitter identity is defined by the expression of the enzyme 108 choline acetyltransferase (ChAT; encoded by cha-1 in C. elegans)and the vesicular ACh 109 transporter (VAChT; encoded by unc-17in C.elegans); see Figure 1A for a description of the 110 cholinergic pathway genes. Coexpression of these two genes is ensured via theirorganization 111 into an operon-like structure called the cholinergic locus(Figure 1B). This operon-like 112 organization is conserved from invertebrates to vertebrates(Eiden, 1998). Other possible 113 diagnostic features of cholinergic neurons often used in vertebrates are the expression of the 114 enzyme that breaksdownACh, acetylcholinesterase (AChE/ace; four genes in C.elegans; 115 (Arpagaus et al., 1998)) and the reuptake transporter of the breakdown product choline (ChT; 116 encoded by cho-1in C.elegans (Okuda et al., 2000) ). Whether these genes are expressed in 117 all cholinergic neurons and/or restricted to all cholinergic neurons is, however, unclear. 118 To define cholinergic neuron types, we generated transgenic lines expressing fosmid 119 based reporters for the unc-17 andcha-1locus, the cho-1 locusand several acegenes(Figure 120 1B). Fosmids contain 30-40 kb genomic sequences, including genes upstream and 121 downstream of the gene of interest and usually contain all cis-regulatory information involved 122 in regulating expression of a specific gene. Differently colored fluorescent proteins were used 123 to assess the relative overlap of these genes to one another(Figure 1C-E). The fosmid lines 124 that monitor cho-1 and ace-1/-2expression are nuclear localized reporters, in which the 125 fluorescent tag is separated from the respective genomic locus by an SL2 trans-splicing 126 event and targeted to the nucleus (see Material and Methods). The fosmid line for the unc-17 127 locus is, in contrast, a direct fusion of gfp to the unc-17 gene, thereby revealing the 128 subcellular localization of unc-17. 129 Multiple lines for each reporter