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- unc.

(C) Co-localization (arrow) in an unc - 7 (e5) animal rescued for forward locomotion with unc - 7S construct (Figure 3C, minus GFP).

One hypothesis for unc - 7 uncoordination is that ectopic AVA: B motor neuron gap junctions interfere with forward locomotion.

It also suggests that in unc - 9 mutants, the native UNC - 7 isoforms expressed in motor neurons (as detected by previous analysis of total UNC - 7 expression) are insufficient to properly localize AVB - expressed UNC - 7S:: GFP (that is, UNC - 7 in motor neurons does not substitute for UNC - 9 in localizing AVB - expressed UNC - 7S).

unc - 93 (e1500) confers a gain - of - function rubberband phenotype that is suppressed by the recessive sup - 10 (mn219) mutation.

For + + / dpy - 8 unc - 124 crosses, 132 wild type and 41 non-Dpy Unc - 124 males were counted, placing dpy - 8 23.7 cM from hs10, and again positioning hs10 very close to unc - 7.

Punc - 7S:: unc - 9:: gfp was expressed in unc - 7 (e5) mutants; UNC - 9:: GFP was broadly but weakly expressed as small puncta throughout the ventral nerve cord (Figure 7J), a pattern reminiscent of UNC - 7S expression in unc - 9 unc - 7 double mutants (Figure 7D).

GFP was seen in puncta near B class motor neuron cell bodies (Figure 7G), although these were generally not as pronounced as with unc - 7S:: gfp.

unc - 124 (hs10) was originally isolated as a cold - sensitive (cs) unc mapping to the left arm of chromosome X between dpy - 8 and unc - 10, at approximately 2.4 cM [50]; unc - 7 maps to the right arm of X at +22 cM.

unc - 9 (fc16) represents a probable null mutation [21].

M121 was mutated to L in unc - 7SΔ1S; this construct (unc - 7SΔ1S - M121L; Figure 3F) plus F56B12 failed to rescue unc - 7, suggesting that UNC - 7L does not have rescuing activity.

When expressed under control of the Punc - 7S promoter, UNC - 7L (Punc - 7S:: unc - 7L [M121L]-RRB- rescued unc - 7 (e5) forward locomotion; though much of the signal remained associated with cell bodies, some puncta were seen localized to AVB: B motor neuron gap junctions (Figure 10D).

unc - 7 (e5) wiring defect and unc - 7 point mutations.

Most unc - 7 genomic constructs were made using subcloned fragments derived from cosmids F09B12 and R07D5.

(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].

First, the genomic region represented by the unc - 9:: gfp construct was shown to fully rescue unc - 9 (fc16) animals, indicating that this region is sufficient to encode a functional unc - 9 product.

(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.

Therefore, the Unc - 7 locomotory defect reflects loss of unc - 7 gap junction channel function, regardless of ectopic gap junction formation, and we sought to determine which gap junctions in the nervous system include UNC - 7 as a component.

However, partial EM reconstruction of an unc - 7 (e5) mutant revealed ectopic gap junctions formed between AVA interneurons that direct backward movement and B class motor neurons involved in forward movement.

unc - 7 transformation rescue.

Although the focus of action of unc - 9 can not be precisely determined from these mosaic experiments, the results are consistent with the interpretation that loss of unc - 9 (+) function from the P1 lineage can be tolerated without apparent effects on locomotion, and that the loss of unc - 9 (+) function from within the AB lineage gives rise to the Unc - 9 phenotype.

Mosaic analysis of unc - 9 followed the same strategy used for unc - 7 [9].

(D) In unc - 9 daf - 6 unc - 7 animals, Pacr - 5:: unc - 9:: gfp is expressed more diffusely in cell bodies (few bright puncta).

Since unc - 7 encodes a gap junction protein, we presumed that the mutant Unc - 7 uncoordinated phenotype reflects a loss of functional gap junction channels.

Additionally, six were identified as Unc - 9 non-Rubberband non-Dyf, giving rise to both wild - type and Unc Rubberband progeny.

It was surprising that re-establishment of AVB: B motor neuron gap junctions was not implicated in the rescue of unc - 7 (e5) forward locomotion by unc - 7S.

An unc - 9:: gfp expression cassette was made by introduction of GFP (afmx) from the partially rescuing unc - 9:: gfp genomic construct (above).

This product was similarly purified in a low - melt agarose gel and used at 1 ng / μl along with 50 ng / μl of the unc - 36 (+) cosmid derivative RIp16 (gift of L Lobel) for microinjection into unc - 36 -LRB--) animals.

The phenotype of unc - 9 daf - 6 unc - 7 mutants was no more severe than unc - 7 alone, consistent with UNC - 7 and UNC - 9 acting in the same process.

(E) UNC - 7L expressed in B motor neurons (Pacr - 5:: unc - 7L [M121L]-RRB- 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.

Although the unc - 7 locus encodes three isoforms that differ in their amino termini, two of these appear functionally equivalent, and the ability of UNC - 7L to contribute to junctions is still uncertain.

Forward and backward movement are both affected, but the forward Unc phenotype is more severe.

unc - 7 mutants are severely uncoordinated.

Of approximately 15,000 animals screened, 2 were identified that met the criteria of being Unc - 9 non-Rubberband Dyf and giving rise to both wild - type and Unc Rubberband progeny.

Therefore, unc - 7 locomotion defects can be rescued by either UNC - 7S or UNC - 7SR, but UNC - 7L can not substitute for this function (although it may have a subtle role or function in another process).

The AVA interneurons in ten unc - 7 (e5) L1 animals were then targeted; none displayed improvement in forward locomotion, supporting the conclusion that ectopic AVA: B gap junctions are not the sole cause of the Unc - 7 phenotype.

Two other strains carrying the unc - 124 (hs10) mutation have been deposited with the C. elegans Genetic Center, and we sequenced the unc - 7 region in HH123 unc - 2 (e55) unc - 124 (hs10) and HH132 dpy - 8 (e130) unc - 124 (hs10); the same unc - 7 mutation was identified in both strains.

(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.