Synapse formation is a process tightly controlled in space and time. How gene regulatory mechanisms specify spatial and temporal aspects of synapse formation is not well understood. In the nematode Caenorhabditis elegans, two subtypes of the D-type inhibitory motor neuron (MN) classes, the dorsal D (DD) and ventral D (VD) neurons, extend axons along both the dorsal and ventral nerve cords1. The embryonically generated DD motor neurons initially innervate ventral muscles in the first (L1) larval stage and receive their synaptic input from cholinergic motor neurons in the dorsal cord. They rewire by the end of the L1 moult to innervate dorsal muscles and to be innervated by newly formed ventral cholinergic motor neurons1. VD motor neurons develop after the L1 moult; they take over the innervation of ventral muscles and receive their synaptic input from dorsal cholinergic motor neurons. We show here that the spatiotemporal control of synaptic wiring of the D-type neurons is controlled by an intersectional transcriptional strategy in which the UNC-30 Pitx-type homeodomain transcription factor acts together, in embryonic and early larval stages, with the temporally controlled LIN-14 transcription factor to prevent premature synapse rewiring of the DD motor neurons and, together with the UNC-55 nuclear hormone receptor, to prevent aberrant VD synaptic wiring in later larval and adult stages. A key effector of this intersectional transcription factor combination is a novel synaptic organizer molecule, the single immunoglobulin domain protein OIG-1. OIG-1 is perisynaptically localized along the synaptic outputs of the D-type motor neurons in a temporally controlled manner and is required for appropriate selection of both pre- and post-synaptic partners.
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We thank the TransgeneOme project, the CGC, J. L. Bessereau, M. Gendrel, S. Y. Kerk for reagents, Q. Chen for microinjection, E. Southgate and N. Thomson for electron microscopy analyses, S. Brenner for first noting the neural defects of unc-30(e191), E. Yemini for assistance with worm tracking, and D. Miller, S. He, I. Greenwald and members of the Hobert laboratory for comments on this manuscript. This work was funded by the National Institutes of Health (R01NS039996-05 and R01NS050266-03), the Howard Hughes Medical Institute and the UK Medical Research Council.
The authors declare no competing financial interests.
Extended data figures and tables
a, Reconstructions of a VD4 and a DD2 MN from an unc-30(e191) animal compared to the same neurons in a wild-type animal. Cell bodies (large black dots) are all situated in the ventral cord. Processes emanate anteriorly (upwards on plots) from the cell body and run along the ventral cord. Lateral branches leave the ventral cord (V) and run round to the dorsal cord (D) as a circumferential commissure (broken horizontal process in the plots). Commissures from unc-30 mutant type-D neurons are situated in the same regions as those of their wild-type counterparts. However, the cell bodies of DD neurons are often displaced anteriorly in the mutants, with the consequence that DD neurons have shorter processes in the ventral cord (C). Processes in the dorsal cord run anteriorly in mutant animals, whereas they branch with the main branch running posteriorly in wild-type animals. Neuromuscular junctions (NMJs) in unc-30 mutants are made predominantly in the dorsal cord by both the DDs and VDs, whereas in the wild type, only DD neurons innervate dorsal muscles. The synaptic inputs to the DD and VD neurons in mutant animals (inward pointing arrows for chemical synapses and ‘T’ for gap junctions) are generally abnormal. The reconstructed DD2 neuron received synapses from several unidentified processes on the ventral side (depicted with a ‘?’). These processes do not belong to VA or VB neurons, the normal pre-synaptic partners of DD, as all the local VA and VB neurons were identified. From the location and synaptic behaviour of these processes, it is probable that they belong to interneurons which span the length of the cord and do not usually innervate D-type neurons. An asterisk (‘*’) indicates that a synapse has multiple post-synaptic elements. A total of six VDs and three DDs were reconstructed. Each reconstruction covered around 2,000 electron microscopy sections, corresponding to a length of 100 µm along the body of the animal. The unc-30(e191) mutation does not affect MN cell body position or the synaptic behaviour of the DA/DB neurons, except in regard to their synapses to D-type neurones; this made it possible to unambiguously identify D type neurones from their positions and by eliminating other identified classes of MNs. Electron microscopy and reconstructions of micrographs of serial sections were performed as described in ref. 31. b, Electron micrographs. The processes of DD and VD neurons normally run subjacent to the bounding basal lamina of the ventral cord immediately dorsal to the axons of the VA and VB neurons (a). NMJs are made through the basal lamina onto muscle arms (M). In unc-30(e191) animals, the axons of the DD and VD neurons wander round the cord and do not run in defined locations; the configuration shown in b is typical but not stereotyped. Very few NMJs are made in the ventral cord by the DD or VD neurons in unc-30 mutants; those that are made look rather small (c). Atypical synapses (d) are often made onto DD or VD processes from neurons such as the touch receptor neuron AVM (6). It is probable that these synapses are not normally found as the processes of AVM, and DD or VD do not normally run alongside each other in wild-type animals. Both the DD and VD MNs make NMJs to dorsal muscles in the dorsal cord of unc-30 mutants, whereas in wild-type animals, only DD neurons do so and VD neurons innervate ventral muscles in the ventral cord. e, f, NMJs made by VD (e) and DD (f) neurons in the dorsal cord of an unc-30(e191) mutant. Scale bars, 1 µm.
a, Presynaptic marker RAB-3 ectopically localizes to the dorsal nerve cord (DNC; marked in red) in lin-14 mutant animals. RAB-3–GFP puncta (from otEx5663, unc-30p::gfp::rab-3) localize mostly to the ventral nerve cord (VNC; marked in red) in wild-type L1 animals (left). Ectopic RAB-3–GFP puncta localize mostly to the dorsal nerve cord in 95% of lin-14 L1 animals (right, scored in progeny from lin-14 null animals carrying a lin-14 rescue array23). Ventral and dorsal nerve cords are indicated by red dotted lines. L1 animals were obtained by hypochlorite-treating gravid adult animals and letting embryos hatch and arrest in M9 for 16–18 h. n > 20 for each strain scored. b, RAB-3 ectopically localizes to the dorsal nerve cord in unc-55 L4 mutant animals. RAB-3–GFP puncta localizes to both the ventral (VNC) and dorsal (DNC) nerve cord in 100% of wild-type L4 animals (left). RAB-3–GFP puncta localize mostly to the dorsal nerve cord in 100% of unc-55 L4 animals (right). Ventral and dorsal nerve cords are indicated by red dotted lines. Signals between the nerve cords are autofluorescence from the gut. n > 20 for each strain scored. c, RAB-3 ectopically localizes to the dorsal nerve cord in oig-1 mutants. RAB-3 normally localizes to the ventral nerve cord (VNC, marked in red) in wild-type L1 animals (top left). Ectopic RAB-3–GFP puncta localize to the dorsal nerve cord in 55% of oig-1 L1 animals (top right, compared to 20% of wild-type animals). L1 animals were obtained by hypochlorite-treating gravid adult animals and letting embryos hatch and arrest in M9 for 16–18 h. n > 20 for each strain scored. In wild-type L4 animals, more RAB-3–GFP puncta are localized in the VNC than in the DNC of the animal (bottom, black dots). Conversely, in oig-1 mutants, more RAB-3–GFP puncta are localized in the DNC than in the VNC (bottom, red dots). **P < 0.01, n = 20 for each strain, original magnification, ×630.
a, Expression of unc-30 is not affected by loss of lin-14 or unc-55. A 2.4 kb unc-30 promoter gfp fusion reporter is expressed in the DD MNs (green circles) in wild-type L1 animals; this expression is not affected in lin-14(−) mutant animals (scored in progeny from lin-14 null mothers carrying a lin-14 rescue array23). An unc-30 fosmid-based reporter, kindly provided by the TransgeneOme project, is expressed in the DD (green circles) and VD (blue squares) MNs (DD4 to VD10 shown) in wild-type L4 animals; this expression is not affected in unc-55(e1170) L4 animals. n > 20 for each genotype. b, Expression of a lin-14 fosmid-based reporter construct32 is unaffected by loss of unc-30. lin-14 is expressed in the DA, DB, and DD MNs in the VNC at the L1 stage (average number of VNC cells = 15); this expression is not affected in unc-30(e191) mutant L1s (average number of VNC cells = 15); n > 20 for each genotype. Loss of unc-30 also does not affect unc-55 expression, as shown by ref. 11. Original magnification, ×630.
Extended Data Figure 4 Deletion of a putative UNC-30 binding site results in loss of oig-1 expression in the D-type neurons.
Regions of the oig-1 promoter were fused to gfp to analyse expression. (+) indicates robust expression of the reporter construct in the specified cell type, whereas (−) indicates loss of expression in the specified cell type. Twenty worms at both the L1 and L4 stage were scored for each line. Expression of a 1 kb promoter reporter (prom 1) recapitulates expression of the oig-1fosmid::gfp reporter in the D-type MNs (see Fig. 2). This region contained 3 elements that exactly match the UNC-30 consensus binding site (TAATC, purple box14,) and multiple others that are a partial match to the UNC-30 binding site (magenta and blue boxes). Further deletion of this prom 1 defined a minimal 125 bp element that is sufficient to drive oig-1 expression in the D-type MNs (prom 6). This element contains two sites that partially match the UNC-30 binding sequence. Deletion of the AAATC site in the context of the 1 kb promoter (prom 9) has no effect on oig-1 expression in the D-type MNs. Deletion of the TAAAC site in the context of the 1 kb promoter reporter (prom 10) results in complete loss of oig-1 expression specifically in the D-type MNs. We noted that some of the smaller transcriptional reporter lines show extended expression of gfp in the DD motor neurons. Since DD rewiring is delayed upon partial removal of the homeobox gene irx-1 (ref. 13), it is possible that in these reporters, potential IRX-1 binding sites are deleted. We have not pursued the effect of irx-1 on oig-1 expression as the lethality associated with complete loss of irx-1 function complicates an analysis of irx-1 null mutant phenotypes in D-type motor neurons.
a, oig-1 mutants display locomotory defects. Locomotion of L4 animals was analysed with tracking assays28. The graphs on the left side of each panel correspond to assays comparing wild-type, oig-1 mutant, and oig-1; unc-30p::oig-1 animals. The graphs on the right side of each panel correspond to assays comparing wild-type, oig-1 mutant, and oig-1; oig-1fosmid::gfp animals. Twenty animals (each dot on a plot) were tracked for each genotype for both comparisons. Mean and Q values are indicated. Note that in a previously published analysis of a large panel of available mutants, the same set of locomotory defects that we describe here for oig-1 mutants were found to be affected in unc-55 mutants28, albeit in a stronger manner than oig-1 mutants. Also note that the very strong locomotory defects unc-30 defects are qualitatively very different from oig-1 mutants, but this is to be expected as unc-30 mutants do not only show the synaptic defects that we describe here, but also lack the neurotransmitter GABA (ref. 3), thereby disabling any neuromuscular signalling. Top, The midbody speed of oig-1 mutant animals is significantly lower than that of wild-type animals. This defect is partially rescued (statistically different from oig-1 mutants but also from wild-type animals) by expressing unc-30p::oig-1 in oig-1 animals (left graph). The lower midbody speed of oig-1 mutants is completely rescued (statistically different from oig-1 mutants but not from wild-type animals) by expressing the oig-1fosmid::gfp in oig-1 mutants. Middle, oig-1 mutants exhibit more dwelling than wild-type L4 animals. This defect is partially rescued (statistically different from oig-1 mutants but also from wild-type animals) by expressing unc-30p::oig-1 in oig-1 animals (left graph). The increased dwelling of oig-1 mutants is completely rescued (statistically different from oig-1 mutants but from not wild-type animals) by expressing the oig-1fosmid::gfp in oig-1 mutants. Bottom, oig-1 mutants exhibit an increased path curvature compared to wild-type animals. This defect is not rescued by expressing unc-30p::oig-1 in oig-1 animals (left graph). The increased path curvature of oig-1 mutants is completely rescued (statistically different from oig-1 mutants but not from wild-type animals) by expressing the oig-1fosmid::gfp in oig-1 mutants. b, Aldicarb-sensitivity defects in oig-1 mutants. oig-1 mutant young adult animals (red squares), which display aberrant GABAergic synapses in both the ventral and dorsal cord, show hypersensitivity to aldicarb-induced paralysis compared to wild type (black triangles). Expression of oig-1fosmid::gfp from a multicopy transgenic array (green circles) does not only rescue the oig-1 mutant phenotype, but even results in a slight hyposensitivity to aldicarb. Worms (blinded for genotype) were tested every 15 min for paralysis by touching the head and tail three times each. n = 20 for each strain, repeated 3 times. c, Expression of oig-1 in the D-type neurons rescues ectopic DD synapses in the dorsal nerve cord. At the L1 stage when only the DD MNs are present, SNB-1–GFP (from juIs1-unc-25p::snb-1::gfp) localizes to the ventral nerve cord (VNC) in wild-type animals (top left). Ectopic SNB-1–GFP puncta localize to the dorsal nerve cord (DNC) of oig-1 mutant L1s (top right). This phenotype is rescued by expressing oig-1 in the D-type MNs (unc-30p::oig-1, otEx4955, bottom left), but not by expressing oig-1 in the neighbouring cholinergic MNs (unc-3p::oig-1, otEx4942, bottom right). Original magnification, ×630; white boxes indicate the dorsal nerve cord.
Cholinergic innervation to the D-type MNs is visualized with an unc-47p::acr-12::gfp reporter transgene, maintained in an acr-12(ok367) mutant background17. a, ACR-12 puncta localization is affected by loss of lin-14. In wild-type L1 animals, ACR-12 puncta are observed only in the DD neurons in the dorsal nerve cord (DNC) (left). In lin-14 mutant L1 animals (scored in progeny from lin-14 null animals carrying a lin-14 rescue array23), ACR-12 puncta are detected in the ventral nerve cord (VNC) of the DD MNs. Quantification of this data are represented in the graph. Some dorsal puncta in the DD MNs were still observed in 83% of the lin-14 mutant L1s that had puncta in the ventral nerve cord. L1 animals were obtained by hypochlorite-treating gravid adult animals and letting embryos hatch and arrest in M9 for 16–18 h. n > 20 for each strain scored, **P < 0.01. b, ACR-12 puncta localization is affected by loss of unc-55. In wild-type L4 animals, ACR-12 puncta are observed in both the ventral (VNC) and dorsal (DNC) nerve cords (left). In unc-55 mutant L4 animals, ACR-12 puncta are observed mostly in the ventral nerve cord of unc-55 mutants. Ventral and dorsal nerve cords are marked by red dotted lines. Quantification of this data are represented in the graph. n > 20 for each strain, **P < 0.01, original magnification, ×630.
At the L1 stage when only the DD MNs are present, ectopic SNB-1–GFP puncta (from juIs1-unc-25p::SNB-1::GFP) localize to the dorsal nerve cord (DNC) of oig-1 mutant L1s (red bar) but not in wild-type animal (black bar) (see Fig. 3a). Based on an alignment with the hidden Markov model (HMM) Ig domain (top), a highly conserved residue (W75) and a nonconserved residue (E64) in the OIG-1 Ig domain were mutated in the context of an unc-30p::oig-1 transgene that is able to rescue the L1 ectopic synapse defects (see Fig. 3a). The unc-30p::oig-1E64A transgenes (otEx6212, otEx6213) were still able to rescue the synaptic defects of oig-1 L1 animals (green bars), whereas the unc-30p::oig-1W75A transgenes (otEx6214,otEx6215) had no rescue ability (blue bars). L1 animals were obtained by hypochlorite-treating gravid adult animals and letting embryos hatch and arrest in M9 for 16–18 h. n > 20 for each strain scored, **P < 0.01, *P < 0.05.
OIG-1–GFP (from oig-1fosmid::gfp) localizes in a punctate manner along axons in the nerve ring (blue arrow) and along a pair of neurons in the pharynx, tentatively identified as the M2 MNs (red arrows point to cell body and process). These neurons form synapses onto pharyngeal muscles along their processes, and these processes also show punctate localization of OIG-1. Original magnification, ×630.
OIG-1–GFP (from oig-1fosmid::gfp) localizes to the ventral nerve cord (VNC) of wild-type L1 animals (left). In sad-1 mutants (middle), OIG-1–GFP is ectopically localized to the dorsal side (DNC) of L1 animals. In strd-1 mutants, OIG-1–GFP is ectopically localized to the dorsal side of L1 animals. Quantification of the data are shown in graph. n > 20 for each strain scored, **P < 0.01, original magnification, ×630.
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Howell, K., White, J. & Hobert, O. Spatiotemporal control of a novel synaptic organizer molecule. Nature 523, 83–87 (2015). https://doi.org/10.1038/nature14545
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