Jarriault et al. 10.1073/pnas.0712159105. |
Fig. 3. Ultrastructural characteristics of P12.pa. (A) Electron micrograph of the rectal area of an L4 hermaphrodite showing the B and the P12.pa cells wrapped around the rectum, both displaying a train-rail shape. The cell contour of the P12.pa cell is in a red dotted line, whereas the cell contour of the B cell is in a black dotted line. Boxed is the area shown at a higher magnification in B. Note the similarity between the pictures here and in Fig. 2A. (B) Blow-up of the boxed area in A, illustrating an adherens junction (arrowhead) between the apical membranes of P12.pa and B. An asterisk indicates the rectal slit.
Fig. 4. Marker expression in Y and PDA. (A) The epithelial marker CHE-14::GFP is expressed in Y. (Right) Fluorescent photomicrograph of Y expressing CHE-14::GFP in a newly hatched L1 animal (40 of 40 early L1s expressed che-14 in Y). (Left) The corresponding Nomarski photomicrograph. CHE-14 can be detected at the apical side of the Y cell, bordering the rectal slit (white arrow), and some faint fluorescence can be seen in Y cytoplasm; note that both sides of the rectal slit are marked with CHE-14, although only one is shown in focus; arrowhead, Y nucleus. (B) The epithelial markers DLG-1 and AJM-1 are expressed in Y. Fluorescent photomicrographs of the subapical part of the Y cell (white arrowheads) expressing both DLG-1 (green, Left) and AJM-1 (red, Center) in the same L1 animal (overlay in yellow, Right; 41 of 41 and 27 of 27 early L1s expressed dlg-1 or ajm-1 in Y respectively); thin arrow, rectal slit. (C) PDA is a neuron in the L3 stage. Photomicrograph of the PDA motor neuron cell body (arrowhead) and axonal process (thick arrow) in a cog-1::gfp L3 larva. PDA's axon is first directed toward the posterior of the animal on its ventral side, then it runs round to the dorsal side through a right-handed commissure past the rectum (thick arrow) and goes toward the anterior part of the worm in the dorsal cord. In cog-1::gfp or ace-3/4::gfp animals, PDA's process (thick arrow) stops just posterior to the pharynx (asterisk). The anus is indicated by a thin arrow.
Fig. 5. Schematic representation of phenotypes. (A) Partial lineage of P11 and P12 during the L1 stage in a wild-type animal. P11.p does not divide further and displays a characteristic epithelial morphology. P12.p divides at the end of the L1 stage to gives rise to P12.pa, which will replace Y in the rectum and to P12.pp, which dies. (B) The rectal area at the L1 and L3 stages in wild-type animals or in mutant backgrounds as indicated. A thin arrow indicates the anus. In L3 and older wild-type animals, three epithelial cells can be found in the rectal and preanal area: P11.p, P12.pa, and U cells. In bar-1, Y migration is blocked but transdifferentiation occurs; in lin-15, the extra P12.pa cell fails to transdifferentiate; egl-5 and sem-4, Y transdifferentiation is blocked; egl- 38 and mab-9, "extra Y" cells remain rectal and fail to transdifferentiate; lin-12(d), the extra Y transdifferentiates as PDA. See Results in main text and Tables 2 and 3 for data. The supernumerary Y-like cell is indicated as Y*. For simplicity, K, K', and the axonal projections of PDA and DA9 are not drawn.
Table 4. Heterochronic genes affecting early larval stages do not affect PDA formation
Genotype | No PDA, % (n) |
lin-4(e912) | 0 (46) |
lin-14(n179)* | 1 (98) |
lin-28(n719) | 0 (39) |
The lin-4, lin-14, and lin-28 heterochronic genes control the timing of many L1 and L2 stage-specific events (1). No PDA, percentage of animals with no PDA. L4 hermaphrodites were scored for the presence of a PDA motor neuron by using syIs63 [cog-1::gfp] or fpIs1 [ace-3/4::gfp]. Both the appearance and the position of PDA were wild type. (n) represents the total number of animal scored. In addition, the timing of the Y-to-PDA transdifferentiation appears to be independent of these genes, as precocious PDA formation was not evident in the lin-14(n179) precocious mutant: 0 of 28 L1 animals and 1 of 17 late L2 had differentiated PDA neurons.
*Grown and scored at 25°C.
1. Rougvie AE (2001) Control of developmental timing in animals. Nat Rev Genet 2:690-701.
Table 5. Summary of markers expression
A. Expression of markers in Y and PDA | |||
Transgene | Marks | Y | PDA |
CHE-14::GFP | Apical | 40 of 40 | 0 of 40 |
AJM-1::GFP | CeJ | 41 of 41 | 0 of 30 |
DLG-1::GFP | CeJ | 27 of 27 | 0 of 48 |
egl-5::gfp * | Rectal | 56 of 56 | 0 of 56 |
egl-26::gfp * | Y, B | 36 of 39 | 0 of 34 |
unc-119::gfp | Neurons | 0 of 26 | All |
F25B3.3::gfp | Neurons | 0 of 32 | All |
cog-1::gfp | PDA | 0 of 30 | 59 of 59 |
ace-3/4::gfp | PDA | 0 of 45 | 48 of 51 |
Transgenes are fully described in SI Text. CeJ, C. elegans junctions (1). Expression in Y was scored in the L1 stage; expression in PDA was scored in the L3 and L4 stages.
*Complete profile of expression in rectal cells is shown below.
B. Expression of egl-5::gfp and egl-26::gfp in the rectal cells | |||||||||
Reporter | Stage | n | Y, % | P12.pa, % | B, % | U, % | F, % | K, % | K', % |
egl-5 | L1 | 56 | 100 | - | 100 | 100 | 92 | 100 | 100 |
L4† | 40 | 0 | 0 | 100 | 100 | 100 | 100 | 100 | |
egl-26 | L1 | 39 | 92 | - | 100 | 25 | 56 | nd | nd |
L4† | 34 | 0 | 97 | 100 | 0 | 0 | nd | nd | |
n
, number of newly hatched L1s and L3 or older hermaphrodites scored.†
L3 and older animals, designated here as L4, all show the same pattern. The percentage of animals with GFP in a given cell is shown. egl-5::gfp is expressed in Y in L1 larvae and B, U, F, K, and K' throughout development, but not in P12.pa. egl-26::gfp is expressed in Y and B in L1 larvae and in P12.pa and B in older larvae.1. Michaux G, Legouis R, Labouesse M (2001) Epithelial biology: lessons from Caenorhabditis elegans. Gene 277:83-100.
Table 6. Engulfment of prospective neurons does not underlie the Y-to-PDA transformation
Genotype | Apoptosis phenotype | Persistent Y, % (n) | Normal PDA, % (n) |
ced-3(n717)^ | No cell death | 0 (65) | 100 (61) |
ced-3(n1286) | No cell death | nd | 100 (29) |
ced-10(n3246) | No engulfment | 0 (16) | 100 (31) |
ced-1(n1735) | No engulfment | 0 (27) | nd |
ced-12(k149) | No engulfment | 0 (34) | nd |
In cell death mutants, the "undead" cells often take on the differentiated characteristics of their sisters or lineal homologs (1). In the tail, just anterior to the rectum of hermaphrodites, there are several dying prospective neurons: in the L1 stage, the sisters of the P12.aaa and P11.aaa neuroblasts - as well as the sister of P12.pa - and in the L2 stage, the sisters of the VA11 and VA12 neurons. L4 hermaphrodites were scored for the persistence of Y as an epithelial cell in the anterior rectum by Nomarski optics or for the presence of a PDA motor neuron by using syIs63 [cog-1::gfp] or fpIs1 [ace-3/4::gfp]. Both the appearance and the position of PDA were wild type. (n), total number of animal scored. ˆ, P12.pp death was 50% penetrant (33 of 65).
1. White JG, Southgate E, Thomson JN (1991) On the nature of the undead cells in the nematode Caenorhabditis elegans. Phil Tans R Soc London Ser B 331:263-271.
Table 7. Y-to-PDA transdifferentiation occurs in mutants with no P12 cell
Genotype | 2 P11.p, % (n) | Mutant Y, % (n) | 2 cells in rectum, % (n') | 1 PDA, % (n) |
bar-1(ga80) | 94 (87) | 77 (87) | 81 (82) | 98 (50) |
let-60(sy93) | 27 (92) | 10 (92) | 36 (25) | nd |
lin-44(n1792) | 24 (50) | 10 (50) | 41 (12) | nd |
Reduced activity of either the Wnt or EGF pathways causes P12 to adopt the fate of P11, resulting in a characteristic "2 P11.p" phenotype (1). L4 hermaphrodites were scored for the presence of an additional cell in the anterior rectum; (n), total number of animal scored; % mutant Y, percentage of worms where Y did not migrate; % 2 cells in rectum, percentage of animals with 2 cells in the rectum (i.e., Y did not migrate) among those mutants that displayed a 2 P11.p mutant phenotype (n'); % 1 PDA, percentage of the animals that had one wild-type-looking PDA neuron expressing cog-1::gfp, irrespective of its location. In addition, ablation of the "extra P11" cell in newly hatched bar-1(0) L1 larvae did not rescue the Y migration (0 of 8 wild-type); two cells were always found but the ventral one at Y's original place had a neuronal morphology suggestive of differentiation into PDA.
1. Jiang LI, Sternberg PW (1998) Interactions of EGF, Wnt and HOM-C genes specify the P12 neuroectoblast fate in C. elegans. Development 125:2337-2347.
SI Text
Markers for Y.
mcIs47 [dlg-1::gfp; rol-6(d)], a gift of Michel Labouesse (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale U596, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7104, Illkirch, France and Université Louis Pasteur, Strasbourg, France), expresses a GFP-tagged adherens junction marker in epithelial cells; mcEx242 [che-14::gfp; rol-6(d) (1)], a gift of Michel Labouesse, expresses a GFP-tagged apical membrane marker in epithelial cells; jcIs1 [ajm-1::gfp; rol-6(d) (2)] expresses a GFP-tagged adherens junction marker in epithelial cells; mcEx [lin-26::gfp; rol-6(d)], a gift of Michel Labouesse, expresses gfp in all rectal cells and P12.pa; bxIs7 [egl-5::gfp; (3)], a gift of Scott Emmons (Albert Einstein College of Medicine, New York, NY); we found that gfp is expressed in all rectal cells in transgenic L1 larvae, including Y, and then in all rectal cells except for P12.pa in transgenic L4 and older animals; kuIs36 [pegl-26::gfp; unc-119(+) (4)] a transcriptional fusion gift of Wendy Hanna-Rose (Pennsylvania State University, University Park, PA); egl-26 is expressed in cells forming tubes, including Y, B and P12.pa; kuIs34 [psem-4::gfp; unc-119(+) (5)], a gift of Wendy Hanna-Rose; sem-4 has been reported to be expressed among other cells in the rectal B and F cells as well as in cells in the preanal ganglion (5). We extended these observations by scoring a sem-4:: gfp reporter in the rectal area and found that sem-4 is expressed in all rectal cells in L1s hermaphrodites (i.e., Y, U, B, F, K, and K') and in all rectal cells, including P12.pa, in L3 or older worms.Markers for PDA.
fpIs1 [pace-3/4::gfp; pha-1(+) (this study)]; a transcriptional fusion; syIs63 [cog-1::cog-1::gfp; unc-119(+) (6)], a translational fusion; we identified cog-1, which had been described to be expressed in unidentified cells in the preanal ganglion of late larvae (6), as a useful PDA marker; arEx627 [pexp-1::GFP; dpy-20(+) (this study)], a transcriptional fusion.Other markers.
ccIs4251 [pmyo-3::NgfplacZ; pmyo-3::Mtgfp; dpy-20(+), (7)], a gift of Mike Krause (National Institutes of Health, Bethesda, MD). myo-3 is expressed in muscles and we confirmed it is not expressed in Y (0/35 L1 animals); edIs6 [unc-119::gfp; rol-6(d) (8)], a panneuronal marker; evIs111 [F25B3.3::gfp; dpy-20(+) (9)], a panneuronal marker; enIs7 [pced-1::ced-1::gfp; unc-76(+) (10)], a gift of Zheng Zhou (Baylor College of Medicine, Houston, TX); ced-1 is expressed in engulfing cells.Identification of Molecular Markers for PDA.
PDA is easily confused with two other neurons in the preanal ganglion (PAG), named DA8 and DA9 (11, 12). However, they may be distinguished by using two criteria: time of appearance (DA8 and DA9 are present in early L1 larvae; the transdifferentiation of Y into PDA occurs later) and axon trajectory [DA8 sends its process through a left-hand commissure, PDA and DA9 through a right-hand commissure (11, 12)]. A transgene was scored positive for expression in PDA if all of the following conditions were met: the GFP-positive cell in the PAG is not seen in L1s; the axonal process of the GFP-positive neuron is as expected (commisure and trajectory): the GFP-positive neuron sends its axon toward the dorsal cord through a right-handed commissure; the position of the cell body in the PAG is just anterior to the U cell in young L3 hermaphrodites. In addition, when possible, the transgenic worms were lineaged to follow Y's transformation into PDA in those strains, and/or Y was killed by using a laser beam in a newly hatched L1 bearing a transgene of interest and the absence of the PDA neuron was analyzed in L4 animals. By using these criteria, only three genes were found or confirmed to be expressed in PDA: ace-3/4, exp-1, and cog-1. A number of markers previously thought to be expressed in PDA were reclassified [wrk-1, dop-2, ksv-1, dbl-1].Electron Microscopy.
Two newly hatched N2 L1 hermaphrodites (0-1 h after hatching) and one L4 hermaphrodite were cryofixed by high-pressure freezing as described (13). Freeze substitution was processed in the Leica AFS for 60 h at -90°C and then for 6 h at -30°C in 1% osmium tetroxyde, 0.5% uranyl acetate, 0.5% glutaraldehyde, and 2% H2O in pure acetone. Samples were then rinsed and infiltrated with graded concentrations of an epoxy resin (Epon812; Fluka). The final steps of the infiltration were done at room temperature, when the epon concentration was >70%. Ten microns of serial ultrathin sections (50-70 nm) of the rectal area for 2 L1 and 1 L4 hermaphrodites were collected and contrasted in lead citrate and uranyl acetate before imaging with a SiS Megaview 3 CCD camera mounted on a FEI Morgagni TEM operated at 70 kV.Correlating the Timing of the Y-to-PDA Transdifferentiation to the Development of the Somatic Gonad.
To be able to follow precisely the Y-to-PDA transformation, we correlated the temporal sequence of these events to the development of the gonad, focusing on the somatic gonad precursors divisions (Fig. 1B). This was then used as a mean to precisely stage the various wild-type and mutant animals used in this study.The gonad of a newly hatched L1 hermaphrodite is made of four cells: two germ-line precursors, named Z2 and Z3, and two somatic precursors, named Z1 and Z4, which undergo three rounds of divisions in a stereotyped pattern (14, 15). Three hermaphrodites were lineaged from the beginning of the L1 stage to the L3 stage as described (14), after the timing of divisions of Z1 and Z4 and of their descendants and the aspect and migration of Y or P12.pa. Our observations were then confirmed by picking L1 and L2 animals at various stages and by checking that our correlations held up. Briefly, Y appears epithelial and a part of the rectum until the P12.pa cell is born, which correlates with the Z1 and Z4 somatic precursors having undergone at least one round of division. When the somatic precursors have undergone two rounds of divisions, Y is found in between U and P12.pa, next to the rectum. Then, Y starts its migration as the nucleus appears to squeeze out of the rectum. This phase correlated with loss of expression of the epithelial markers (data not shown). By the time the somatic gonadal precursors have undergone three rounds of divisions, Y has almost reached its final position, and when an AC cell can be identified and the somatic primordium cells have rearranged (15), the PDA neuron has been formed, and PDA markers are expressed.
T Shift Experiments.
To assess whether lin-12 activity was needed after Y's birth and specification, we used the temperature-sensitive allele lin-12(n676n930ts) (16). L1 larvae were scored for the presence of a Y cell in the anterior rectum by Nomarski optics, and L3 and older larvae were scored for the presence of a PDA motor neuron by checking expression of the cog-1::gfp marker. When grown at 15°C, 15 of 24 lin-12(n676n930) L1s have a Y cell in the anterior rectum, and 33 of 50 L3s or older have a PDA neuron made. By comparison, no Y cell is found in L1 grown at 25°C (17), and 0 of 35 lin-12(n676n930) L3 or older grown at 25°C have a PDA neuron made. In a first set of experiments, lin-12(n676n930) embryos laid at 15°C were transferred to 25°C. The embryos used corresponded to stages ranging from just before Y's birth to past the 3-fold stage [≈253-578 min of development after the first cleavage at 20°C; the Y cell is born ≈290 min after the first cleavage at 20°C in the wild-type embryo (18)]. Two hours 50 min later, larvae that had already hatched were removed. After an additional 4 h 10 min, half of the L1 found on the plate were scored for the presence of a Y cell, whereas the other half was transferred to a new plate at 25°C and scored when the worms reached the L3 stage or older for the presence of the PDA neuron. In a second set of experiments, we sought to restrict the developmental time window of the embryos analyzed and transferred to 25°C embryos grown at 15°C that had reached anywhere between the lima bean stage to the 1 [1/2] fold stage [≈320-417 min after the first cleavage at 20°C].1. Michaux G, Gansmuller A, Hindelang C, Labouesse M (2000) CHE-14, a protein with a sterol-sensing domain, is required for apical sorting in C. elegans ectodermal epithelial cells. Curr Biol 10:1098-1107.
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