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doi: 10.1242/10.1242/dev.00277

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Identification of spatial and temporal cues that regulate postembryonic expression of axon maintenance factors in the C. elegans ventral nerve cord

Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA

View larger version (44K): [in a new window]   Fig. 1. Features of the C. elegans ventral nerve cord. The VNC in wild-type animals and consequences of microsurgical removal of PVT on VNC architecture are shown. Neurons that have terminated their axon outgrowth (PVQL/R, AVKL/R, RMEV, HSNL/R) are affected by laser removal of PVT or zig-4 mutation, such that their axons flip over the hypodermal ridge (ventral midline) into the opposite fascicle (Aurelio et al., 2002). The upper inset shows gene expression profiles in PVT (see text for references), the lower inset the expression pattern of several LIM homeodomain transcription factors in the tail ganglia of an L1 stage animals (Cassata et al., 2000; Hobert et al., 1998; Hobert et al., 1999b; Sarafi-Reinach et al., 2001; Way and Chalfie, 1989).

View larger version (65K): [in a new window]   Fig. 2. lim-6 and ceh-14 are expressed in the PVT neuron and coordinately affect zig-4::gfp expression. (A) Embryonic expression of lim-6 and ceh-14 reporter gene constructs in PVT (see Materials and Methods for details on expression constructs). Postembryonic expression of lim-6 ceh-14 in PVT has been previously reported (Cassata et al., 2000; Hobert et al., 1999b). (B) Effect of lim-6 ceh-14 on zig-4::gfp (otIs20) expression. The inset shows an enlargement of the tail region with a gfp-fluorescing PVT cell. White star denotes gut autofluorescence. Note that other zig-4::gfp expressing cells are not affected by lim-6 ceh-14. See Fig. 3 for quantitative data on all PVT-expressed zig genes. White arrows points to PVT, which lacks a gfp signal in panel B.

View larger version (38K): [in a new window]   Fig. 3. lim-6 and ceh-14 coordinately affect expression of postembryonically expressed zig genes in the PVT neuron. Individuals with a chromosomally integrated zig::gfp reporter (see Materials and Methods) were crossed into the indicated null mutant backgrounds. Animals were scored at mixed stages. The modest effect of lim-6 on zig-2, zig-3 and zig-4::gfp expression (<30% off) is only manifested after the L3 stage: expression is correctly initiated but fails to be maintained. n=45-500, depending on genotype.

View larger version (46K): [in a new window]   Fig. 4. Generation and fate of the PVT neuron are unaffected in lim-6 ceh-14 double mutants. (A) srq-1::gfp (otIs85) and unc-47::gfp (otIs39) are markers for PVT cell fate. In addition to PVT, srq-1 is expressed in one more tail neuron and several head neurons. The unc-47::gfp constructs labels not only the RMEL/R, RIS, AVL and DVB, as described previously (Eastman et al., 1999), but also the PVT neuron. Expression of the two other PVT cell fate markers used here (B), gpa-2 and pin-2, has been previously documented (Hobert et al., 1999a; Zwaal et al., 1997) and is not shown. (B) Expression of PVT cell fate markers in mutant backgrounds. All alleles shown are molecular nulls. Scoring was carried out in L1 and L2 stage animals. Sample size was 28-187, depending on genotype. Expression of unc47::gfp (otIs39) in PVT is seen only in 15% of post-L3 stage lim-6 ceh-14 animals (n=14), while wild type shows expression in >95% at pre-L3 stages. This stage-dependent downregulation is difficult to interpret, however, because three out of four integrants of the unc47::gfp reporter line juEx60 show no expression in PVT beyond the L3 stage, while only one integrant, otIs39 (tested here), shows expression after the L3 stage. We also noted that in lim-6 ceh-14; otIs39 animals, DVB axon morphology often appears defective.

View larger version (99K): [in a new window]   Fig. 5. Axonal defects in the ventral nerve cord of lim-6 ceh-14 double mutants. (A) VNC defects as assayed with the F25B3.3::gfp (evIs111) marker. Arrows indicate the left and right VNC in wild-type animals. Arrows indicate inappropriate axon flip-overs seen in lim-6 ceh-14 mutants. They are reminiscent of those seen in PVT(—) or zig-4(—) animals (Aurelio et al., 2002). Quantitative data are shown in Table 1. (B) PVQL/R neurons have a wild-type appearance in lim-6 ceh-14 mutant animals. The marker is oyIs14 (see Fig. 8A for oyIs14 in a wild-type background).

View larger version (31K): [in a new window]   Fig. 6. Loss of ceh-14 suppresses the flip-over phenotype of the PVQL and PVQR neurons. PVQL/R was visualized in the individual mutant backgrounds using oyIs14 (sra-6::gfp). All mutant alleles used [lim-6(nr2073), ceh-14(ch3) zig-4 (gk34)] are null alleles. PVT(—) indicates PVT ablation at the early L1 stage. All defects were scored at late larval stages. Numbers within bars indicate sample size.

View larger version (69K): [in a new window]   Fig. 7. The heterochronic gene lin-14 affects zig-4 gene expression but not PVT cell fate. (A) zig-4::gfp (otIs20) expression in PVT (white arrow) is absent in lin-14 mutants. See Table 2 for quantification of defects. The small arrows indicate a pair of non-identified tail neurons that can be occasionally observed to express zig-4::gfp in wild-type animals, but show stronger and more consistent expression in lin-14 mutant animals. (B) PVT adopts its normal fate in lin-14(n179) mutants. 21/21 late L1/L2 stage animals show normal expression of unc-47::gfp (otIs39) in PVT and normal PVT axon morphology (white arrows indicate the cell body and axon of PVT; for wild-type control, see Fig. 4A). All strains were grown at 25°C. PVT cell fate is also unaffected as assessed with a pin-2::gfp (otIs85) marker (data not shown).

View larger version (46K): [in a new window]   Fig. 8. The heterochronic gene lin-14 affects ventral nerve cord structure. (A) Axonal defects in lin-14(n179) mutants visualized with the panneuronal reporter erIs111. (B) Two examples of axonal defects in lin-14(n179) mutants visualized with the PVQL/R reporter oyIs14 (F25B3.3::gfp). See Table 3 for quantification of defects. All strains were grown at 25°C. Arrows indicate inappropriate axon flip-overs; arrowheads indicate correctly positioned left and right VNC axons.

View larger version (35K): [in a new window]   Fig. 9. Misexpression of zig-4 causes PVQL/R axon patterning defects. PVQL/R defects can be observed in animals that misexpress zig-4 under the control of the flp-1 promoter, expressed in the AVKL/R neurons (A), or the myo-3 promoter, expressed in body wall muscles (BWM; B). Animals were scored as adults to reveal maintenance defects (left panels, `Adult') and a subset of those lines were scored as freshly hatched L1 animals to allow scoring for embryonic defects (right panel, `Embryo'). Numbers within bars represent sample size. The broken `control line' in B refers to the average value of the five BWM::control lines that were scored as adults (left). We could not score control lines as freshly hatched larvae (as in A) because of a strong myo-3::gfp background signal. `Control' refers to the respective promoter driving expression of gfp. The kal-1 gene, which codes for a secreted protein (Bülow et al., 2002) was used as an additional control. Control and transgenic lines all contain oyIs14 in the background to allow scoring PVQL/R anatomy.