Starich TA , Xu J , Skerrett IM , Nicholson BJ , Shaw JE .

GM055437 NIGMS NIH HHS , GM56367 NIGMS NIH HHS , MMF 3183923802 PHS HHS , R01 GM056367 NIGMS NIH HHS , R01 GM055437 NIGMS NIH HHS

	Figure 1. unc-7(e5) wiring defect and unc-7 point mutations. Wiring diagram for coordinated locomotion (adapted from [13,16]) shows chemical (arrows) or electrical (terminated lines) synapses among interneurons (hexagons) and motor neurons (circles) implicated in forward movement (blue), reverse movement (yellow), or movement in both directions (green). Ectopic unc-7(e5) AVA:B motor neuron gap junctions are indicated (red dashed line).
	Figure 2. unc-7 mutations. (A) The unc-7 locus showing the approximate sites of rearrangements within intron 1 resulting in an Unc-7 phenotype. mn382 is a translocation, mn383 is a 6-kb deletion, and mn384 is a 2-kb insertion. (B) unc-7 point mutations and their location in the largest UNC-7 isoform (UNC-7L, 522 amino acids). ATG at M121 is the conserved innexin initiation site and start site of UNC-7SR. The position of GFP in unc-7S::gfp is also indicated. Asterisks indicate stop codons. EL, extracellular loop; TM, transmembrane domain. (The cold sensitive unc-7(hs10) allele was originally used as the basis for defining the gene unc-124 [50], and a heteroallelic interaction with unc-7 was reported [21,50]. Since hs10 is a mutant allele of unc-7, the existence of unc-124 is dubious. See Materials and methods for details.)
	Figure 3. unc-7 transformation rescue. (A) Exon/intron structure of unc-7. Cosmid F56B12 includes presumptive upstream promoter sequences and ends within intron 3. ATG indicates UNC-7L (exon 2), UNC-7S (exon 1S, blue shading), and UNC-7SR (exon 5) start sites. Recombination in vivo between F56B12 and plasmid constructs reconstituted the unc-7 locus. Rescue of forward (F) or reverse (R) locomotion by plasmid constructs, with or without co-injection of F56B12, was determined (isoforms predicted to be expressed are listed in red). Rescue was determined from at least three lines, except (G) (single line generated), and (F) (no rescued lines obtained). (B) Fully rescuing unc-7 min plasmid expressing UNC-7S and UNC-7SR. Red slashes indicate frameshift mutation eliminating UNC-7L expression. (C) unc-7S::gfp construct lacks UNC-7L translational start site and shares 1.4-kb with F56B12. (D) unc-7SΔ1S lacks exon 1S. (E) unc-7-2cDNA6 with exons 2–6 replaced by corresponding cDNA sequence, shares 1 kb of intron 1 overlap with F56B12. (F) unc-7SΔ1S-M121L cannot initiate translation of UNC-7S or UNC-7SR. (G) unc-7S-M121L intiates translation of UNC-7L and UNC-7S with the M121L mutation. (H) Western blot using anti-GFP antibodies detecting UNC-7::GFP isoforms. Predicted sizes of UNC-7L::GFP, -S::GFP, and -SR::GFP are 85, 78, and 72 kDa, respectively. Lane 1, wild type (N2); lanes 2 and 3, unc-7(e5) rescued with unc-7S::gfp + cosmid F56B12, expected to express all isoforms; lane 4, unc-7(e5) rescued with unc-7SΔ1S + F56B12, predicted to express UNC-7L:: and SR::GFP. ND = not determined.
	Figure 4. UNC-7 expression. (A) Wild-type adult stained with affinity-purified anti-UNC-7. Composite of confocal images. (B) Anti-UNC-7 staining of unc-7(e5) mutant; inset shows DAPI stain of nuclei. Anterior to right. (C) Early L1 larva with rescuing unc-7::gfp (plus F56B12) in unc-7(e5), anterior upper right. Right: posterior ventral nerve cord in rescued L3 animal (anterior left). Neurons expressing unc-7::gfp include members of all motor neuron classes (AS9-11, DD5, VD10-11, VA10-11, VB11, DB7, and DA7). (D) Expression of non-rescuing unc-7SΔ::gfp (no cosmid). I1, pharyngeal neuron I1. Anterior to left. Abbreviations: ca, canal-associated process; dnc, dorsal nerve cord; dsl, dorsal sublateral process; nr, nerve ring; ph, pharyngeal nervous system; rvg, retro-vesicular ganglion; ts, tail spike; vnc, ventral nerve cord; vsl, ventral sublateral process.
	Figure 5. Expression patterns of other neuronal innexins. (A) Predicted gene structures of neuronal innexins. Arrows indicate primer binding sites used for PCR-amplified green fluorescent protein (GFP) constructs (see Materials and methods). (B) INX-1::GFP expression (green) in motor neurons; DAPI stained nuclei in red. (C) INX-1::GFP expression (green) in dorsal nerve cord (dnc) and body wall muscles (bwm); dorsal nerve cord is visualized with anti-UNC-33 antibody (red). (D) INX-19::GFP expression in AVA and AVB interneurons. (E) INX-4::GFP expression in sensory neurons and pharyngeal m1 muscle cell. Abbreviations: nr, nerve ring.
	Figure 6. UNC-7 and UNC-9::GFP co-localize. (A) Section of ventral nerve cord (vnc) in a wild-type (N2) animal transformed with UNC-9::GFP. UNC-9::GFP signal enhanced with anti-GFP antibody (green); anti-UNC-7 (red); co-localization (yellow). UNC-9::GFP in body muscle seen in upper half of photo. (B) Co-localization in pharyngeal nerve ring (ph). (C) Co-localization (arrow) in an unc-7(e5) animal rescued for forward locomotion with unc-7S construct (Figure 3C, minus GFP). (D) UNC-9Δ::GFP expression in L1 stage, and (E) in head neurons of L4 stage.
	Figure 7. UNC-7S::GFP expressed in AVB requires UNC-9 expression in B motor neurons for proper localization to AVB:B gap junctions. (A) Diagram of anterior portion of ventral nerve cord (vnc) examined for expression of presumptive AVB:B motor neuron gap junctions. For clarity not all neuronal nuclei are represented; approximate positions of AVA and AVB (purple) posterior to the nerve ring (nr) are indicated. Motor neuron cell bodies lie along the vnc; D class motor neurons send processes dorsally to the dorsal nerve cord (dnc). The retrovesicular ganglion (rvg) is comprised of 20 neurons (including ten motor neurons) on the ventral side; the identity of neurons immediately posterior to the rvg can often be inferred by position, though some variation can occur [37]. (B) unc-7S::gfp construct expressed in unc-7(e5). UNC-7S::GFP expression enhanced with anti-GFP antibody (green); DAPI-stained cell nuclei (red). (C) In unc-4(e120) UNC-7S::GFP additionally localizes to AVB:VA motor neuron gap junctions. (D, E) UNC-7S::GFP localization is more diffuse in unc-9 daf-6 unc-7 (D), and unc-9 single mutants (E). (F) Localization is rescued in unc-9 daf-6 unc-7 by co-expressing UNC-9 in B class motor neurons (Pacr-5::unc-9). (G-I) UNC-7S::GFP expressed in AVB interneurons (Psra-11::unc-7S::gfp) in unc-7(e5) localizes to AVB:B gap junctions (G); localization is lost in unc-9 daf-6 unc-7 animals (H), but is rescued by UNC-9 expression in B motor neurons (Pacr-5::unc-9) (I). (J) UNC-9::GFP expressed in AVB (Punc-7S::unc-9::gfp) in unc-7(e5) animals fails to localize near B motor neuron cell bodies and is diffusely distributed along the ventral nerve cord.
	Figure 8. UNC-9::GFP expessed in B motor neurons requires UNC-7 in AVB for proper localization to AVB:B gap junctions. (A) In wild-type (wt; N2), UNC-9::GFP expressed in B motor neurons (Pacr-5::unc-9::gfp) visualizes puncta in the ventral nerve cord localized near B cell bodies. (B) UNC-9::GFP is expressed more uniformly in the ventral nerve cord in unc-7(e5) animals, and clusters of puncta near B cell bodies are not discernible. (C) In unc-9(fc16), UNC-9::GFP puncta are more widely distributed, but the brightest puncta remain associated with B cell bodies. (D) In unc-9 daf-6 unc-7 animals, Pacr-5::unc-9::gfp is expressed more diffusely in cell bodies (few bright puncta).
	Figure 9. Functional analysis of UNC-7 and UNC-9 in Xenopus oocytes. Left: superimposed traces of junctional currents evoked by 10 mV voltage steps from -100 to +100 mV in one of a pair of Xenopus oocytes injected with cRNAs for the UNC proteins indicated below. Right: graphs of conductance/transjunctional voltage (Gj/Vj) relations for initial conductance (Gj0, open squares) and steady state conductance measured at the end of the 15 s pulse (Gj∞, closed squares) averaged from four to five cell pairs. Gj0 values are normalized to Gj0 at Vj = 0, while Gj∞ values are normalized to Gj0 at the same voltage (Gj∞/Gj0). Error bars represent standard deviation, n = 5. Results shown are for the following pairings, where relative polarity of Vj is defined with respect to the innexins on the right in each pair: (A) UNC-7S/UNC-7S; (B) UNC-7L/UNC-7L; (C) UNC-9/UNC-9; (D) UNC-7S/UNC-9; (E) UNC-7L/UNC-9; (F) UNC-7S/UNC-7L; (G) UNC-7S/UNC-7L+UNC-9, where equal quantities of UNC-7L and UNC-9 cRNA were co-injected into the right cell.
	Figure 10. UNC-9 and UNC-7 fail to heteromerize. (A) Co-expression in interneurons of UNC-7S (red) and UNC-9::GFP (Punc-7S::unc-9::gfp; green) in unc-7(e5) shows little co-localization (yellow). (For clarity DAPI staining is not shown; positions of B motor neuron nuclei are indicated.) UNC-7S does not concentrate in puncta near B cell bodies. (B) Pcex-1::unc-7S::gfp (AVA, AVD interneurons) rescues forward locomotion but does not concentrate in puncta near B cell bodies. (C) Co-expression of Pcex-1::unc-7S (red) and Pcex-1::unc-9::gfp (green) shows little co-localization in an unc-7(e5) background. (D) UNC-7L expressed in interneurons (Punc-7S::unc-7L [M121L]) rescues forward locomotion in unc-7(e5) and localizes to puncta near B motor neuron cell bodies. (E) UNC-7L expressed in B motor neurons (Pacr-5::unc-7L [M121L]) in unc-9 daf-6 unc-7 does not localize to puncta but remains associated with cell bodies (red); co-expressed UNC-9::GFP (Pacr-5::unc-9::gfp; green) is distributed throughout the motor neuron processes.
	Figure 11. Model for composition of heterotypic AVB: B motor neuron gap junction channels.

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