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First published online 3 August 2006
doi: 10.1242/dev.02507

Development 133, 3329-3340 (2006)
Published by The Company of Biologists 2006
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DIG-1, a novel giant protein, non-autonomously mediates maintenance of nervous system architecture

Claire Y. Bénard1, Alexander Boyanov1, David H. Hall2 and Oliver Hobert1,*

1 Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY 10032, USA.
2 Department of Neuroscience, Center for C. elegans Anatomy, Albert Einstein College of Medicine, Bronx, NY 10461, USA.


Figure 1
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  		Fig. 1. Diagrammatic cross-section of an adult worm showing the position of neuronal cell bodies and axons relative to other structures in C. elegans. The upper panel is a schematic diagram of the complete nervous system of C. elegans (including the position of individual neuronal cell bodies examined in this paper) and the lower panels show cross-sections of two regions in the body examined in this paper, the lateral ganglia in the head region and the VNC.

 

Figure 2
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  		Fig. 2. Axon maintenance defects in sax-8 mutants. (A) Axonal maintenance defect of the two PVQ neurons in sax-8(ky188) mutants. Arrow indicates region of axon flip-over. (B) Quantification of PVQ flip-over defect. (L1: n=40-102; A; n=174-399). `L1' are freshly hatched first larval stage animals. `A' are young adults that have just molted. Error bars indicate standard error of proportion. (C) PVQ flip-over defect of ky188 mutants can be suppressed by paralysis, induced pharmacologically (50 µM levamisol), or genetically, using mutants animals that are paralyzed because of ultrastructural muscle defects [unc-54(e1092) and unc-97(su110)] or neuronal signaling defects [unc-36(e251) and unc-13(e1091)]. The ky188 data are the same as in B. Adult animals were scored (n=122-206). (D,E) Axonal maintenance defects of AVK and RMEV neurons. Animals were scored as young adults only, as these reporters do not allow the visualization these axons in early larvae (n=69-244 for untreated animals). The axonal defects are profoundly suppressed by paralysis using levamisol at 50 µM (n=131, 89, respectively; E). Error bars indicate standard error of proportion.

 

Figure 3
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  		Fig. 3. Cell body maintenance defects in sax-8 mutants. (A) Maintenance defect of amphid chemosensory neurons in sax-8 mutants. Arrows indicate the nerve ring, and arrowhead indicates cell bodies of chemosensory neurons that are anteriorly misplaced. Scale bar: 7 µm. L1 and L4, freshly hatched first and fourth larval stage animals, respectively; A, 3-day-old adult. Error bars indicate standard error of proportion. (B) Quantification of chemosensory neuron displacement. (L1: n=22-85; L4: n=21-53; A: n=93-172). (C,D) Defect in the cell body position of the PVQ neurons. PVQL and PVQR are two bilateral neurons normally placed posterior to the anus. The PVQ cell body can often be found severely misplaced in sax-8 mutants. Notably, the axon still reaches the posterior region and projects normally in all cases. The axon and cell body defects are independent of one another in all alleles, as it is extremely rare that both defects are seen in the same animal (data not shown). E, L1 and A, are early threefold embryos, freshly hatched L1 larvae and young adults, respectively. Error bars indicate the standard error of proportion. (C) Displacement of the PVQ cell body in sax-8 mutants. Arrows indicate the normal position; arrowhead indicates the anteriorly displaced cell body. Scale bars: 5 µm. (D) Quantification of PVQ cell body displacement. (E: n=138-180; L1: n=38-158; A: n=190-438.)

 

Figure 4
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  		Fig. 4. Other cell body position defects in sax-8 mutants. (A) Cell body position defect of the bilateral AVK neurons in sax-8 mutants. Arrows indicate AVK cell bodies. (B) Quantification of the cell body position defect of AVK (L1: n=29-121; A: n=63-94). The position in threefold embryos is already defective (data not shown). L1, freshly hatched first larval stage animals; A, young adults that have just molted. (C) Gonad displacement in freshly hatched L1 larvae. In ky188, ky199 and ky201, the gonad is frequently displaced in the anterior direction, and more rarely in the posterior direction.

 

Figure 5
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  		Fig. 5. Timing of sax-8 action. (A) nu319ts animals grown at 15°C are wild type for chemosensory amphid neuron position (in blue), but are mutant by the fourth larval and adult stages when grown at 25°C (in red). Wild-type animals are fully normal at all three temperatures, and dig-1(nu345), a non-temperature-sensitive mutant displays the same penetrance at all temperatures (data not shown). (B) The adult phenotype of nu139ts animals does not depend on the temperature at which they underwent the development of the nervous system (in embryos and early L1 larvae), but rather on the temperature to which they were exposed as L2, L3, L4 larvae and adults. Animals grown at 15°C up to a given developmental stage, shifted to 25°C and scored as 3-day-old adults are shown in red. Animals grown at 25°C up to a given developmental stage, shifted to 15°C and scored as 3-day-old adults are shown in blue. L1, L2, L3 and L4, first, second, third, and fourth larval stage, respectively; YA, young adult; A, 3-day-old adult. The blue and red broken lines give the penetrance for adult mutants grown continuously at 15°C and 25°C, respectively. Error bars indicate the standard error of proportion. n=50-250 for each data point.

 

Figure 6
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  		Fig. 6. Positional cloning of sax-8. (A) Summary of sax-8 genetic map data. Based on the observed recombination frequencies (as shown), we calculated the position of sax-8 to be at -0.95±0.45. (B) Gene structure of sax-8/dig-1/K07E12.1, taken from www.wormbase.org. The sax-8/dig-1 defects can be rescued with cosmids K07E12 and R05H11 (see Table 1) and with cosmid K07E12 alone (data not shown). (C) Domain structure of DIG-1 and mutant alleles.

 

Figure 7
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  		Fig. 7. dig-1 acts non-autonomously to maintain axon position in the VNC and the lateral ganglion in the head. (A) Expression pattern of dig-1 gfp reporter constructs (see Fig. S2 in the supplementary material for details on reporter constructs and transgenic lines examined). Expression in the gut and in non-neuronal head cells in the commastage embryo (i,ii). Expression in non-neuronal head cells, as well as in body muscles, in late threefold embryos (iii). Expression in the hypodermal syncytium hyp7 in larvae and adults (iv). Strong expression in head muscles and other mesodermal cells, including GLRs, the head mesodermal cell (hmc) (v), and in all the body wall muscles and coelomocytes (vi, vii). The shape of a single body wall muscle cell is outlined in vi. Expression remained strong throughout larval and adult stages. Scale bars: 10 µm. (B) Mosaic analysis. Rescuing cosmids K07E12 and R05H11 were injected at ~15 ng/µl into dig-1(ky188);oyIs14 animals together with myo-3::gfp and F25B3.3::DsRed as lineage markers. One out of 4 of the transgenic lines rescued the PVQ defects and was used for mosaic analysis. Two classes of animals were scored for rescue of axonal defects. `P1-' animals in which the AB cell descendants express F25B3.3::DsRed pan-neuronally, including the ttx-3::gfp-expressing AIY neurons, and myo-3::gfp in a limited number of muscle cells, but not in the body muscle cells. Second, `AB-' animals in which body wall muscle cells, but not enteric muscles, express gfp and in which the neurons do not express F25B3.3::DsRed. (C) Scoring of mosaic, as well as of the transgenic and non-transgenic control animals.

 

Figure 8
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  		Fig. 8. Loss of dig-1 causes defects in basement membrane structure. Transverse section of a region in the head of an adult worm is shown. P, pharynx; D, sensory neuron dendrites; M, muscle. Scale bar: 1 µm. In wild-type animals, thin and `fuzzy' basement membrane material is deposited on tissues that neighbor the axons, such as pharynx and muscle (white arrows). In dig-1 mutants, basement membrane is detached and forms disorganized stacks and whorls (white arrowheads). It is unclear if the whorls of basement membrane material derive from muscle or hypodermis, but they appear thinner and simpler than the pharyngeal basement membrane material. dig-1(ky188) (n=6), dig-1(nu345) (n=2) and dig-1(n1321) (n=1) show similar defects, which are never see in N2 wild-type animals (n=5).

 

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© The Company of Biologists Ltd 2006