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Extension of the Caenorhabditis elegans Pharyngeal M1 neuron axon is regulated by multiple mechanisms.

Refai O, Rohs P, Mains PE, Gaudet J - G3 (Bethesda) (2013)

Bottom Line: We found that extension of the M1 pharyngeal axon, which spans the entire length of the pharynx, occurs in two distinct phases.The first proximal phase does not require genes that function in axon extension (unc-34, unc-51, unc-115, and unc-119), whereas the second distal phase does use these genes and is guided in part by the adjacent g1P gland cell projection. unc-34, unc-51, and unc-115 had incompletely penetrant defects and appeared to act in conjunction with the g1P cell for distal outgrowth.One of these mutations appeared to affect the generation or differentiation of the M1 neuron.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

ABSTRACT
The guidance of axons to their correct targets is a critical step in development. The C. elegans pharynx presents an attractive system to study neuronal pathfinding in the context of a developing organ. The worm pharynx contains relatively few cells and cell types, but each cell has a known lineage and stereotyped developmental patterns. We found that extension of the M1 pharyngeal axon, which spans the entire length of the pharynx, occurs in two distinct phases. The first proximal phase does not require genes that function in axon extension (unc-34, unc-51, unc-115, and unc-119), whereas the second distal phase does use these genes and is guided in part by the adjacent g1P gland cell projection. unc-34, unc-51, and unc-115 had incompletely penetrant defects and appeared to act in conjunction with the g1P cell for distal outgrowth. Only unc-119 showed fully penetrant defects for the distal phase. Mutations affecting classical neuronal guidance cues (Netrin, Semaphorin, Slit/Robo, Ephrin) or adhesion molecules (cadherin, IgCAM) had, at best, weak effects on the M1 axon. None of the mutations we tested affected the proximal phase of M1 elongation. In a forward genetic screen, we isolated nine mutations in five genes, three of which are novel, showing defects in M1, including axon overextension, truncation, or ectopic branching. One of these mutations appeared to affect the generation or differentiation of the M1 neuron. We conclude that M1 axon extension is a robust process that is not completely dependent on any single guidance mechanism.

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M1 axon phenotypes (marked with glr-2::gfp) in wild-type (A) and gland-ablated hlh-6::egl-1 (B–D) or in I3 neuron-ablated ceh-2::egl-1 transgenics (E). Arrowhead denotes the M1 cell body and the long arrow in wild-type (A) indicates the nonpharyngeal cells in the nerve ring in these at L4 or young adults. Short arrows highlight M1 defects. (B–D) In 56% of gland-ablated transgenics, the M1 axon is either truncated (B) with abnormal branching in the metacorpus and procorpus (C) or follows an abnormal trajectory in the procorpus (D). (E) In 7% of I3 ablated animals, the M1 axon in the procorpus exhibits an abnormal shape with GFP swelling (arrows), although the axon always extended fully. Scale bar in (A) = 10 µm.
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fig2: M1 axon phenotypes (marked with glr-2::gfp) in wild-type (A) and gland-ablated hlh-6::egl-1 (B–D) or in I3 neuron-ablated ceh-2::egl-1 transgenics (E). Arrowhead denotes the M1 cell body and the long arrow in wild-type (A) indicates the nonpharyngeal cells in the nerve ring in these at L4 or young adults. Short arrows highlight M1 defects. (B–D) In 56% of gland-ablated transgenics, the M1 axon is either truncated (B) with abnormal branching in the metacorpus and procorpus (C) or follows an abnormal trajectory in the procorpus (D). (E) In 7% of I3 ablated animals, the M1 axon in the procorpus exhibits an abnormal shape with GFP swelling (arrows), although the axon always extended fully. Scale bar in (A) = 10 µm.

Mentions: To examine whether the adjacent g1P gland projection is necessary for the M1 axon extension in the procorpus, we genetically ablated the gland cells. The gland-specific hlh-6 promoter (Smit et al. 2008), which is first expressed at the “bean” stage (380 min), was used to drive expression of the pro-apoptotic gene egl-1 (Conradt and Horvitz 1998) in g1P soon after its birth at ∼360 min. We found that the majority of such animals exhibited M1 axon abnormalities within the procorpus. Of gland-ablated worms (n = 140), 35% showed premature axon termination in the procorpus, frequently with extra small branches (Figure 2, B and C and Figure 3). Another 21% extended beyond the procorpus but followed abnormal trajectories (Figure 2D). Notably, the proximal trajectory of the axon from the M1 cell body to the procorpus was normal in all animals. Although other gland cells were killed, none sends projections into the metacorpus or procorpus, so they are unlikely to be involved in M1 guidance. Furthermore, no major structural defects in the other pharyngeal tissues were detected (data not shown).


Extension of the Caenorhabditis elegans Pharyngeal M1 neuron axon is regulated by multiple mechanisms.

Refai O, Rohs P, Mains PE, Gaudet J - G3 (Bethesda) (2013)

M1 axon phenotypes (marked with glr-2::gfp) in wild-type (A) and gland-ablated hlh-6::egl-1 (B–D) or in I3 neuron-ablated ceh-2::egl-1 transgenics (E). Arrowhead denotes the M1 cell body and the long arrow in wild-type (A) indicates the nonpharyngeal cells in the nerve ring in these at L4 or young adults. Short arrows highlight M1 defects. (B–D) In 56% of gland-ablated transgenics, the M1 axon is either truncated (B) with abnormal branching in the metacorpus and procorpus (C) or follows an abnormal trajectory in the procorpus (D). (E) In 7% of I3 ablated animals, the M1 axon in the procorpus exhibits an abnormal shape with GFP swelling (arrows), although the axon always extended fully. Scale bar in (A) = 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815062&req=5

fig2: M1 axon phenotypes (marked with glr-2::gfp) in wild-type (A) and gland-ablated hlh-6::egl-1 (B–D) or in I3 neuron-ablated ceh-2::egl-1 transgenics (E). Arrowhead denotes the M1 cell body and the long arrow in wild-type (A) indicates the nonpharyngeal cells in the nerve ring in these at L4 or young adults. Short arrows highlight M1 defects. (B–D) In 56% of gland-ablated transgenics, the M1 axon is either truncated (B) with abnormal branching in the metacorpus and procorpus (C) or follows an abnormal trajectory in the procorpus (D). (E) In 7% of I3 ablated animals, the M1 axon in the procorpus exhibits an abnormal shape with GFP swelling (arrows), although the axon always extended fully. Scale bar in (A) = 10 µm.
Mentions: To examine whether the adjacent g1P gland projection is necessary for the M1 axon extension in the procorpus, we genetically ablated the gland cells. The gland-specific hlh-6 promoter (Smit et al. 2008), which is first expressed at the “bean” stage (380 min), was used to drive expression of the pro-apoptotic gene egl-1 (Conradt and Horvitz 1998) in g1P soon after its birth at ∼360 min. We found that the majority of such animals exhibited M1 axon abnormalities within the procorpus. Of gland-ablated worms (n = 140), 35% showed premature axon termination in the procorpus, frequently with extra small branches (Figure 2, B and C and Figure 3). Another 21% extended beyond the procorpus but followed abnormal trajectories (Figure 2D). Notably, the proximal trajectory of the axon from the M1 cell body to the procorpus was normal in all animals. Although other gland cells were killed, none sends projections into the metacorpus or procorpus, so they are unlikely to be involved in M1 guidance. Furthermore, no major structural defects in the other pharyngeal tissues were detected (data not shown).

Bottom Line: We found that extension of the M1 pharyngeal axon, which spans the entire length of the pharynx, occurs in two distinct phases.The first proximal phase does not require genes that function in axon extension (unc-34, unc-51, unc-115, and unc-119), whereas the second distal phase does use these genes and is guided in part by the adjacent g1P gland cell projection. unc-34, unc-51, and unc-115 had incompletely penetrant defects and appeared to act in conjunction with the g1P cell for distal outgrowth.One of these mutations appeared to affect the generation or differentiation of the M1 neuron.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

ABSTRACT
The guidance of axons to their correct targets is a critical step in development. The C. elegans pharynx presents an attractive system to study neuronal pathfinding in the context of a developing organ. The worm pharynx contains relatively few cells and cell types, but each cell has a known lineage and stereotyped developmental patterns. We found that extension of the M1 pharyngeal axon, which spans the entire length of the pharynx, occurs in two distinct phases. The first proximal phase does not require genes that function in axon extension (unc-34, unc-51, unc-115, and unc-119), whereas the second distal phase does use these genes and is guided in part by the adjacent g1P gland cell projection. unc-34, unc-51, and unc-115 had incompletely penetrant defects and appeared to act in conjunction with the g1P cell for distal outgrowth. Only unc-119 showed fully penetrant defects for the distal phase. Mutations affecting classical neuronal guidance cues (Netrin, Semaphorin, Slit/Robo, Ephrin) or adhesion molecules (cadherin, IgCAM) had, at best, weak effects on the M1 axon. None of the mutations we tested affected the proximal phase of M1 elongation. In a forward genetic screen, we isolated nine mutations in five genes, three of which are novel, showing defects in M1, including axon overextension, truncation, or ectopic branching. One of these mutations appeared to affect the generation or differentiation of the M1 neuron. We conclude that M1 axon extension is a robust process that is not completely dependent on any single guidance mechanism.

Show MeSH
Related in: MedlinePlus