spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Knobel, K. M.
Right arrow Articles by Bastiani, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Knobel, K. M.
Right arrow Articles by Bastiani, M. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

UNC-119 suppresses axon branching in C. elegans

Karla M. Knobel{ddagger}, Warren S. Davis, Erik M. Jorgensen* and Michael J. Bastiani

Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, USA
{ddagger} Present address: School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705-2222, USA



View larger version (107K):

[in a new window]
  		Fig. 1. unc-119(ed3) mutants have branched motorneuron axons. (A) Confocal micrograph of a wild-type young adult (38 hours after hatching) expressing GFP in GABA neurons. Anterior is to the right; posterior to the left. GFP was expressed in 19 D-type motorneuron cell bodies located along the tightly fasciculated ventral nerve cord (open arrow) and their commissures (arrowhead). The dorsal nerve cord was contiguous along the length of the animal (arrow). (B) Confocal micrograph of an unc-119(ed3) young adult expressing GFP in GABA neurons. There were branches in many of the axons that reached the dorsal cord (arrowheads), and large gaps in the dorsal nerve cord (solid arrow). The ventral nerve cord was defasciculated (open arrow). (C) GABA motor neuron structure was abnormal in unc-119(ed3) mutants scored at 38 hours after hatching. All wild-type axons were normal (left, n=21 worms, 273 axons). Although some unc-119(ed3) axons were normal (left, 27% ±2.8, n=9 worms, 144 axons), many reached the dorsal midline but failed to extend normally along the dorsal nerve cord (middle left, 30% ±3.9), many axons were branched (middle right, 31% ±6.0), and some axons failed to reach the dorsal nerve cord (right, 12.3% ±3.0). These differences from wild-type are significant (asterisk *, P<0.0001, t-test; error bars represent standard error of the mean). (D) Confocal micrograph of unc-119(ed3) axons branching at the dorsal body wall muscle. Brightfield illumination revealed the border between muscle and intestine (dotted line). Axons branched at the muscle (arrow) and at the lateral nerve cord (arrowhead). The dorsal nerve cord is marked by an open arrow. Scale bars (A,B) 25 µm, (C) 10 µm.

 


View larger version (80K):

[in a new window]
  		Fig. 2. UNC-119 is expressed in neurons. (A) GFP was expressed in the GABA motorneuron cell bodies (arrows) and axons (**) in a wild-type worm expressing a Punc-47::GFP construct. (B) In the same wild-type individual, anti-UNC-119 antibodies labeled multiple cell bodies (arrowhead and arrows) and axons in the left (*) and right (**) ventral nerve cords. (C) GFP was also expressed in the GABA motorneuron commissures of wild-type worm carrying a Punc-47::GFP construct (right ventral nerve cord: **, axon: arrow). (D) Anti-UNC-119 antibodies labeled the axons in the right ventral nerve cord (**), the commissures seen in (C, arrow), and the lateral nerve cord (arrowhead). Scale bar (A-D) 5 µm.

 


View larger version (73K):

[in a new window]
  		Fig. 3. UNC-119 functions cell-autonomously to suppress axon branching. (A) Expression of a GFP-tagged UNC-119 protein in the GABA motor neurons of an unc-119(ed3) mutant restored GABA motor axon structure (arrowheads). Note that there were no gaps in the dorsal nerve cord (arrow). (B) GFP expression in the CAN lateral cord neurons (arrowhead) of wild-type animals. The lateral cord axons were not branched. (C) The CAN axons of unc-119(ed3) mutants were branched (arrowhead). (D) Expression of a GFP-tagged UNC-119 protein in the GABA neurons of unc-119(ed3) mutants (left arrow) failed to rescue the branching of the CAN axons (arrowhead). (E) 100% of D-type motor neuron axons in wild-type worms were morphologically normal (black, n=16 worms, 96 axons). Only 27% ±2.8 of VD and DD axons were normal in unc-119(ed3) mutants (gray, n=9 worms, 144 axons). Expression of a GFP-tagged UNC-119 protein in the GABA neurons of unc-119(ed3) mutants restored the morphology of the DD and VD axons (white, 85% ±7.3, n=10 worms, 95 axons). (F) 100% of wild-type worms scored had unbranched lateral nerve cord CAN axons (black, n=12 worms). All of unc-119(ed3) mutants scored had branched CAN axons (gray, n=10 worms). Expression of a GFP-tagged UNC-119 protein in the GABA neurons of unc-119(ed3) mutants failed to rescue the branched morphology of the CAN axons (white, 8% n=13 worms). Scale bars (A-D) 10 µm.

 


View larger version (54K):

[in a new window]
  		Fig. 4. DD axon morphology changes over time in unc-119(ed3) mutants. (A) Confocal micrograph of an unc-119(ed3) L1 larvae expressing GFP in the GABA motor neurons. The DD axons (arrows) bifurcated normally at the dorsal nerve cord (arrowhead). (B) The DD neurons in an adult unc-119(ed3) mutant were branched (arrows). There were large gaps in the dorsal nerve cord (arrowhead). (C,D). The number of branched DD axons increased over time in unc-119(ed3) mutants. (C) At 1 hour after hatching 93% ±3.2 of wild-type axons were normal (left, n=12 worms, 71 axons). Most unc-119(ed3) DD axons were also normal at this stage (left, 58% ±8.1, n=15 worms, 79 axons). Some unc-119(ed3) axons reached the dorsal nerve cord but failed to extend normally along the dorsal midline (middle left, 28% ±7.2), which was also true for some wild-type axons at this time (7% ±3.2). Only a small fraction of mutant DD axons were visibly branched (middle right, 7% ±4.0), or failed to reach the dorsal nerve cord (right, 7% ±6.7). (D) At 48 hours after hatching, all wild-type axons were morphologically normal (left, n=16 worms, 96 axons). However, the fraction of normal axons in unc-119(ed3) mutants was significantly reduced (left, 25% ±8.1, n=8 worms, 40 axons). The fraction of axons that failed to extend along the dorsal midline did not change significantly (middle left, 20% ±3.8). The number of branched axons in unc-119(ed3) mutants increased eightfold (middle right, 55% ±5.0). The percentage of axons that failed to reach the dorsal nerve cord did change significantly (0%, right). Significance between time points is indicated by an asterisk (*, P<0.0001, t-test). Error bars represent the standard error of the mean. Scale bars (A,B) 10 µm.

 


View larger version (87K):

[in a new window]
  		Fig. 5. VD growth cone migration is normal in unc-119(ed3) mutants. (A) Time-lapse images of a migrating VD growth cone in a wild-type animal expressing GFP in the GABA motor neurons. At time 0:00, the lateral growth cone was round as it migrated circumferentially across the epidermis. When it contacted the dorsal body wall muscle, the growth cone formed an anvil-shaped structure (arrowhead, 0:24). A single finger extended between the muscle and epidermal cells toward the dorsal nerve cord (solid arrow, 0:56). Eventually this finger contacted the dorsal nerve cord where a new growth cone formed (arrowhead 1:36). The original growth cone located at the ventral side of the body wall muscle was collapsing (open arrowhead). (B) Schematic of wild-type growth cone imaged in A. The dorsal nerve cord is indicated by a dotted line. (C) Time-lapse images of a migrating VD growth cone in an unc-119(ed3) mutant expressing GFP in the GABA motor neurons. At 0:00, the lateral growth cone was round. When the growth cone contacted the dorsal body wall muscle (0:25, arrowhead) it formed an anvil-shaped structure. The growth cone sent fingers dorsally (solid arrows, 0:35) that contacted the dorsal nerve cord. At 1:05 the finger reached the dorsal nerve cord where a new growth cone formed. The ventral growth cone (open arrowhead) collapsed. (D) Schematic of unc-119(ed3) growth cone imaged in C. The dorsal nerve cord is indicated by a dotted line. Scale bars (A,C) 5 µm.

 


View larger version (56K):

[in a new window]
  		Fig. 6. unc-119(ed3) DD axon branches are produced by secondary growth cones. (A) Sprouting from DD axons increased with time. Lateral sprouts (black dots) extending from DD axons were observed before 15 hours in unc-119(ed3) mutants expressing GFP in the GABA motor neurons. Sprout activity peaked at 15 hours post-hatching (33%) and was nearly absent by 30 hours after hatching. Growth cones extending from DD axons (open squares) increased in number after 10 hours, by 20 hours 44% of DD axons scored had supernumerary growth cones. Axon branches (+) became visible 30 hours after hatching. VD growth cone migration occurred between 15 and 30 hours after hatching in unc-119(ed3) mutants (gray field). During this analysis we did not observe sprouts or axonal growth cones in wild-type animals at any time (n=7). (B) Axonal growth cones produced supernumerary branches in unc-119(ed3) mutants expressing GFP in the GABA motor neurons. Time-lapse analysis of an unc-119(ed3) axon showed an axonal growth cone (arrow, 0:00) extending multiple branches towards the dorsal nerve cord (arrow, 0:24 to 1:02). One of these branches projected to the dorsal nerve cord (arrow, 0:46). Note that the axon extended along the dorsal midline retracted (compare arrowheads in 0:00 and 1:02). Below is a schematic of the growth cone. Scale bar is 5 µm.

 


View larger version (31K):

[in a new window]
  		Fig. 7. UNC-119 is required throughout life. (A) Expression of UNC-119 prior to DD axon migration initially rescued the morphology of the DD axons (L1 and L2) relative to non-heat-shocked siblings (-HS). As the animals age, axon morphology declined (AD, adult). unc-119(ed3)mutants carrying the [Phsp::UNC-119] (+HS) constructs were heat shocked prior to DD axon migration (0-5 hours after fertilization). (B) Expression of UNC-119 after axon migration maintained the morphology of the DD neurons. unc-119(ed3) animals carrying the [Phsp::UNC-119] constructs were heat shocked after DD axon migration was completed (10-15 hours after fertilization). (C) Expression of UNC-119 during the L1 larval stage restored axon morphology. unc-119(ed3) carrying the [Phsp::UNC-119] arrays were heat shocked during the L1 larval stage (0-5 hours after hatching). (D) Expression of UNC-119 in the L3 larval stage prevented the decline of axon morphology. unc-119(ed3) animals carrying the [Phsp::UNC-119] arrays were heat shocked in L3 larval stage (36 hours after hatching). The asterisks indicate significant difference compared to the non-heat-shocked controls as determined by an unpaired t-test: *** P<0.0001, ** P< 0.001, *P<0.05.

 


View larger version (92K):

[in a new window]
  		Fig. 8. Synapses are formed in unc-119(ed3) mutants. (A) Dorsal view of a wild-type adult animal expressing GFP-tagged synaptobrevin in the DD neurons located in the dorsal nerve cord. DD synapses (arrow) were uniformly distributed along the dorsal nerve cord. (B) Dorsal (DD) synapses in unc-119(ed3) young adults were present but discontinuous because of large gaps in the dorsal nerve cord. (C) Ventral view of a wild-type adult expressing GFP-tagged synaptobrevin in the VD synapses of the ventral nerve cord. VD synapses were located along the ventral nerve cord of wild-type worms (arrow). (D) The distribution of VD synapses in the ventral nerve cord of unc-119 adults was abnormal. A lateral branch that formed synapses is indicated by an arrowhead. Scale bars 10 µm.

 


View larger version (246K):

[in a new window]
  		Fig. 9. Synapse ultrastructure is normal in unc-119 mutants. Presynaptic specializations are indicated. (A) A GABA neuromuscular junction in the ventral nerve cord of a wild-type adult. (B) A GABA neuromuscular junction in the ventral nerve cord of an unc-119(e2498) adult. (C) An acetylcholine neuromuscular junction in the ventral nerve cord of a wild-type adult. (D) An acetylcholine neuromuscular junction in the ventral nerve cord of an unc-119(e2498) adult. (E) The number of synaptic vesicles per active zone was similar in wild type (black, 262±31.8 vesicles per active zone), unc-119(ed3)(gray, 246±33.0), and unc-119(e2498)(white, 207±39.0). These numbers are not significantly different. Error bars represent standard deviation. Scale bar 10 nm.

 


View larger version (85K):

[in a new window]
  		Fig. 10. Synapse localization is disrupted in unc-119(ed3) mutants. (A) Ventral view of a newly hatched wild-type L1 larvae expressing GFP-tagged synaptobrevin in DD neurons. Presynaptic varicosities are indicated by an arrow. (B) Ventral view of a newly hatched unc-119(ed3) L1 larvae expressing GFP-tagged synaptobrevin in DD neurons (arrow). (C) There were no synaptic varicosities visible in the dorsal nerve cord of wild-type L1 larvae (arrow). (D) Presynaptic varicosities were seen in the dorsal nerve cord of unc-119(ed3) mutants (arrow). (E) Dorsal nerve cord varicosities were observed in 52% of unc-119(ed3) DD3 axons (wild-type worms: 0% n=21 (black); unc-119: n=23 (gray)). Scale bars (A-D) 5 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2001