-
[
International C. elegans Meeting,
2001]
The hook and hook sensillum, which aid in male mating, are derived from three multipotent hook precursor cells (HPCs), P(9-11).p. Male P(9-11).p cells keep unfused at the L1 stage, and later from the mid-L3 stage each cell exhibits one of the three fates with a invariant pattern of 3*-2*-1* in intact wild type animals. To elucidate the cell interactions among 1*, 2*, and 3* fate cells, following up an experiment of Michael Herman, cell ablations were performed within the P(9-11).p group. The 2* fate in an isolated P10.p is not altered by the disruption of 1*-fated P11.p in the early L3 stage after P10.p migrated to a posterior position close to the cloaca. Within the 2* lineage, ablation of the hook socket cell or its parent cell P10.ppa, but not the hook neurons, has a negative effect on hook structure. To examine whether HOM-C gene
mab-5 has more roles in HPC patterning other than inhibiting the P(9-11).p cell fusion during L1 stage, we use a heat shock-inducible transgenic line muIs9 encoding hs-
mab-5 to manipulate the MAB-5 activity. Overexpression of
mab-5 during mid-L2 to early-L3 stage led to a high percentage of hook abnormalities in adult males, including no hook, misshapen hook, or anterior hook. hs-
mab-5 can block formation of all three hooks in
lin-12(gf) males. Activated LET-23 pathway by
let-23(gf) and
let-60(gf) mutations induces cell division in P9.p. However, additional hook neurons marked by GFP had never been seen. It is not clear if there is any ectopic HPC 2* fate transformation in P9.p or activated LET-23--RAS signaling actually promotes P9.p to adopt a vulval-like fate.
-
[
Worm Breeder's Gazette,
1986]
Much of our understanding of the role of cell-cell communication in C. ent has been derived from studies of equivalence groups, sets of multipotential cells with fates determined by cell interactions. The best characterized is the vulval equivalence group, which consists of six ventral hypodermal cells, P(3-8).p. Each of these cells can express one of three fates, designated 1 , 2 , or 3 , which are determined by an inducing signal from the anchor cell of the gonad. We are interested in extending our knowledge of cell interactions to the cells of the male preanal equivalence group P(9-11) .p, which divide at the same stage and undergo about the same number of rounds of cell divisions as the cells of the vulval equivalence group. The progeny generated by P(9-11).p assume either hypodermal or neuronal cell fates; in particular, the cell P10.papp forms the hypodermal hook, which aids in male mating. The three fates of the cells of the male preanal equivalence group are also known as 1 , 2 and 3 , based upon the hierarchy of replacements seen after cell ablations: P11.p is 1 , P10.p is 2 and P9.p is 3 . In addition, in males, only the cell P9.p has been shown to be tripotential, as are P( 3-8).p in hermaphrodites. We have extended the laser ablation experiments of Sulston and White (Dev. Biol. 78: 577, 1980) to characterize further the control of cell fates within the male preanal equivalence group. First, we have confirmed that P(9-11).p determination occurs during the mid-L2 stage, one stage earlier than the time of P(3-8).p determination. We ablated either P10.p or P11.p at various times after their births in the mid- Ll stage through the early L3 stage and followed the cell lineages of the remaining cells to determine if replacement occurred. Replacement of cell fates was observed if ablations were performed before 20 hours of postembryonic development (mid-L2 stage). However, replacements did not occur if cells were ablated after 20 hours. Second, we have searched for a cell or cells that could be the source of a potential signal analogous to that of the anchor cell. Several cells and sets of cells have been ablated in the Ll stage. Candidates for ablation were selected by their positions relative to P( 9-11).p and/or by sex-specificity. A previous set of ablations performed by John Sulston included: the gonad; B; Y; U; P12.p; K cells; T cells; juvenile preanal ganglion; lumbar ganglion; anal depressor muscle; and tail body muscles (Sulston and White, Dev. Biol. 78: 577, 1980; J. Sulston, personal communication). We have extended and confirmed this set by ablating: the gonad; B; Y; B, U, F and Y; P12; M; VS, V6 and T (both sides); and P(1-12).a. None of these ablations resulted in the loss of 1 or 2 cell fates. Therefore, we have failed to identify a source of a signal. However, it must be kept in mind that we do not know how much of a cell, if any, remains following our laser ablations. It is possible that the remains of a signal-producing cell may still produce a signal. We have attempted to explore further the nature of the intercellular interactions. We tested a model analogous to the current model for vulval development (Sternberg and Horvitz, Cell 44: 761, 1986): a diffusible and spatially graded signal directly determines which of the three distinct fates each cell expresses. According to this model, it should be possible for a Pn.p cell to express a 2 cell fate in the absence of a cell that expresses a 1 cell fate. We have ablated P(10, ll).p in the Ll and observed the fate of P9.p in the L3. In 16/21 animals P9.p migrated posteriorly to occupy a position close to that normally occupied by P10.p. In 14 of those animals P9.p divided once only; in one animal P9.p divided and P9.pp went on to divide another two rounds; and in one animal P9.p expressed a 1 fate (normally expressed by P11.p). Significantly, in no animals was an isolated 2 cell fate observed (the 2 cell fate is normally expressed by P10.p), suggesting that there is no position within a hypothetical gradient that P9.p could assume that would cause it to express the 2 fate. Furthermore, after ablation of P10.p, P9.p efficiently assumed a 2 fate (3/3); after ablation of P11.p, P10.p efficiently assumed a 1 fate (5/5), and in 3/5 of these animals P9.p assumed a 2 fate. In no cases has any cell undergone a 2 fate without another cell assuming a 1 fate, suggesting that the expression of a 1 fate is required for a cell to undergo a 2 fate. Thus, there seems to be at least two differences between the specification of cell fates in the vulval and the preanal equivalence groups: l) the time of determination appears to be one stage earlier for the preanal equivalence group, and 2) there does not appear to be a cell outside the equivalence group producing a graded inducing signal. We currently are considering two alternative models for the specification of P(9-11).p cell fates. Both models assume that a cell with a 1 fate is necessary to induce a neighboring cell to express the 2 fate. In one model, P11.p is determined cell autonomously to express the 1 fate, and only P10.p and P9.p are actually multipotential. In the alternative model, each cell knows anterior from posterior and sends a signal to the cell in front of it, inhibiting that cell from expressing the 1 cell fate. Thus, only the posterior-most cell does not receive the signal and hence expresses the 1 cell fate.
-
[
West Coast Worm Meeting,
2000]
C. elegans P(9-11).p cells in male pre-anal ganglion (PAG) equivalence group have three potential fates, 1, 2, and 3. In wild-type males, P10.p and P11.p adopt the 2 and 1 respectively, divide to generate distinct subsets of cells. P9.p (3 fate), which is undivided, fuses with
hyp7.
osm-6::gfp is specifically expressed in two hook neurons HOA and HOB derived from the 2 lineage. To understand the fate specification in P(9-11).p cells, we constructed a series of strains carrying the
osm-6::gfp marker with mutations in
lin-12,
lin-15 and
mab-5 genes. Instead of normal one pair of GFP expressing cells,
lin-12(gf) mutants showed 2-3 pairs of GFP expression characteristic of the HOA and HOB neurons, indicating a transformation to the 2 fate in P9.p and P11.p cells. In
lin-15(lf) mutants, P9.p could divide and a second pair of GFP expression was observed anteriorly sometimes because of a P9.p P10.p fate transformation. The results suggested that the
lin-12 activity is sufficient to promote the 2 fate and
lin-15, a negative regulator of
let-23 -ras pathway, is required for the 3 fate. The roles of
mab-5 and
let-23/ras are under investigation. A genetic screen for other candidates involved in P(9-11).p fate determination is also in progress.
-
[
Worm Breeder's Gazette,
1978]
The development of the male has been followed to maturity, and the fates of the cells produced by the post-embryonic lineages are now known. Here are a few of the novel points. 1. Sensory rays. The nine pairs of sensory rays are composed of three cells: two neurons and a sheath cell. These sensilla are the only structures in the tail for which there is a clear repeating pattern of cell assignments. 2. Formation of the cloaca and spicules. Daughters of the B cell make up the spicules, each composed of 2 neurons, 4 socket cells and 2 sheath cells. Other cells form the spicule channels and a neuron with striated rootlet that lies over the base of the spicule and probably is a proprioceptor. The elongated shape of the spicules and cloaca is obtained by the dorsal spicule retractor muscles which attach to the dorsal edge of the cloaca and crawl anteriorly. In their absence the cloaca is short and the spicules are crumpled. 3. Rearrangement of the anal depressor muscle. During L4 lethargus, the myofilaments of the depressor muscle detach dorsally and rotate through 90 before reattaching to the gubernaculum. The function of the muscle is thereby completely altered. In the absence of post-embryonic muscle the rearrangement is incomplete: some filaments run in each direction. 4. Regulation. Cell ablations (by laser microbeam) in the young L1 have revealed some capacity for regulation in two areas. (a) Rays are ordinarily made by V5(one), V6(five) and T (three). After ablation of V6, V5 can make most of the rays normally produced by V6. After ablation of V5 and V6, V4 can make one or two rays. Other V cells have not been seen to produce rays, perhaps because they arrive at the tail too late, but V3 was stimulated to go through an extra round of division. (b) Extra neurons, supporting cells and hypodermal cells are added to the preanal ganglion, normally by division of P10.p and P11.p. After ablation of P10.p or P11.p, P9.p can be recruited to this function; the resulting animal is perfect except for the trivial loss of a ventral hypodermal nucleus. No other cell has ever been recruited.
-
[
Worm Breeder's Gazette,
1982]
In the course of investigating the development of the C. elegans male (Develop. Biol., 78, 542-576 and 577-597 (1980)), we made a few random observations on the contribution to mating behaviour of various parts of the body. Since we are not planning to pursue this any further in the near future, here are the data as they stand. Some of these observations will be found also in Jonathan Hodgkin's paper on male mutants (submitted), but are included here for the sake of a coherent account. Microscopy: Mating can be watched at low resolution on plates under a dissecting microscope, and at high resolution on a standard agar mount under Nomarski optics. The latter method is tedious and uncertain, because the males do not perform well under a cover slip. Therefore, individual males were tested by the following procedure, which combines satisfactory mating with moderate resolution. The male was isolated from hermaphrodites for a few hours after the final moult. It was then placed on an NG plate with a very small lawn of bacteria and a single paralysed hermaphrodite (CB369). The male was chased gently into the lawn with a fine hair, and the plate was transferred to a Zeiss microscope preferably having an opalescent sheet instead of a condenser. Use of a low power objective (e.g., Neofluar 6.3 or 10) allowed the various stages of mating to be scored. Subsequently, the male was returned to its own plate, together with four CB369 hermaphrodites, and wild-type progeny were sought after two or three days. Normal Mating Behaviour: The male seems to locate the hermaphrodite more rapidly than would be expected by chance contact, and indeed preliminary evidence for a weak chemotaxis of males towards hermaphrodites has been obtained ( Horvitz and Sulston, unpublished observations). Upon reaching the hermaphrodite, the male frequently crawls forwards beneath it. This behaviour may explain the tendency of males to crawl through small holes in a mesh (Klass, WBG, 3 (1) 9-10 (1977)). As a result, the male's tail comes into contact with the hermaphrodite; he then backs sharply, curving his tail until the ventral side becomes apposed to the hermaphrodite, and continues backing, sliding along the hermaphrodite's body and turning sharply at its ends. When his tail reaches the vulva, he stops abruptly, sometimes after hunting back and forth, and attempts to insert his spicules. The gubernaculum and spicules are moved to a more transverse position, presumably by the action of the gubernacular erector muscles. As soon as the spicules enter the vulva, the intestino-rectal valve is drawn away from the vas deferens and against the dorsal cord by contraction of the sphincter muscle. The valve dividing the seminal vesicle from the vas deferens opens, and sperm begin to flow into the uterus of the hermaphrodite. The first part of the ejaculate consists only of fluid (presumably secretions from the wall of the vas deferens). A good seal is established between the protruded cloaca of the male and the dilated vulva of the hermaphrodite; the spicules, which remain more or less stationary, probably serve as anchors. After ejaculation, the male falls away and gradually retracts his spicules; there is a short refractory period, and the backing response returns gradually in the course of a few minutes. The male also begins the backing behaviour when his tail encounters other smooth objects, including other males and his own body. Solitary males can often be seen moving endlessly backwards with the tail clasping the head. Functional Analysis: Some of the cells involved in the various stages of mating have been identified mainly by laser ablation experiments as follows: Cephalic companions: The cephalic sensilla of the male differ from those of the hermaphrodite in being open to the outside and in being dually innervated (Ward, et. al., J. Comp. Neurol. 160, 313-338 (1975)) . The additional sensory neurons, known as cephalic companions (CEM's) , are therefore suspected of mediating the sex specific chemotaxis of males towards hermaphrodites. Sensory rays: In order to obtain an animal which lacks functional sensory rays, it is only necessary to prevent the development of the structural cells. In each of three very young L4's, either Rn.ap or Rn.app was ablated in every ray group. The resulting adults did not respond to tail contact with a hermaphrodite; however, they repeatedly turned towards it and passed beneath it, as would be expected if the putative chemotactic sense were still operating. Pre-anal ganglion: Loss of the P10.p-type lineage (see Dev. Biol. 78, 581) results in an animal which can back and turn but is unable to locate the vulva within five to ten minutes, however, such individuals usually yield progeny when allowed prolonged access to hermaphrodites ( perhaps by making use of the postcloacal sensilla). This suggests that the hook and its sensillum are used in vulval location, and indeed after ablation of separate components of this sensillum location is erratic though not usually abolished. Ablation of the three ventral hypodermal cells derived from P10 (P10.paaa, P10.paap and P10.papa) does not affect mating. Loss of the P11.p-type lineage, or of P11.pa, leads to very weak mating behaviour in which the various elements cannot be properly scored. This is probably due mainly to the loss of the motor- and inter-neurons derived from P11.pa. These animals sometimes yield progeny eventually. Muscle: Complete loss of male specific muscle, by ablation of the mesoblast (M) in a young L1, has surprisingly little effect upon initial mating behaviour. Such an animal backs, turns and locates the vulva essentially normally. The spicules, of course, do not move, and the shape of the entire cloaca is abnormal. U: Following ablation of rectal cell U (formerly called E) in a young male, the linker cell of the gonad is not destroyed in the usual way. Furthermore, K.a and K' fail to grow anteriorly, and apparently no functional connection is established between the vas deferens and the cloaca. Such an animal mates normally, except that its sperm remain in the vas deferens or leak into the body cavity. Gonad: Complete ablation of the gonad in a young L1 leads to an adult with entirely normal secondary sexual characteristics and normal mating behaviour. [See Figure 1]
-
[
International C. elegans Meeting,
1991]
The relatively complex and sex-specific behavior of male mating is mediated at least in part by the 79 male-specific neurons, most of which reside in the male tail. We have set out to examine the role of these neurons in male mating behavior via the successfully used technique of cell ablation. Since reconstruction data identifying possible neuronal connections is not available for the male, and since cells comprising a common copulatory structure are often generated by a common precursor, we have chosen to follow a lineal approach. That is, male specific post embryonic blast cells which give rise to male copulatory structures andlor neurons are ablated and the resulting phenotype (mating defect), if any, is determined by observation. Given a defect, the progeny of that blast cell are then systematically ablated until the neuron(s) responsible for the phenotype are identified. This approach has been begun for several structures/lineages with the following results: [See Table] We are currently pursuing these and other (nonsensory) male-specific lineages to identify the neurons involved in these and other affected behaviors. For example, using this approach we have determined that of the three neurons generated from the P10.p lineage, the hook sensillum neurons HOA and HOB are required for vulval location while the interneuron PVZ is not. Similarly, using this approach for the B.a lineage we have shown that the sensory/motor neurons SPCL/SPCR are required for spicule insertion while the sensory neurons SPVLISPVR are not.
-
[
Worm Breeder's Gazette,
1982]
In C. elegans the gonad, specifically the anchor cell, induces the ventral hypodermal precursors P(5-7).p to generate the vulva (Kimble, Develop. Biol. 87, 286, 1981). Ablation of any of these vulval precursor cells leads to regulation (Sulston and White, Develop. Biol. 78, 577, 1980). Similar regulation is seen in C. elegans males. We have performed a series of gonadal, P, and Pn.p cell laser ablation experiments in P. redivivus females and males. In P. redivivus, P(5- 8).p generate the vulva; the other Pn.p cells join the hypodermal syncytium. [In C. elegans, P(3,4,8).p divide and their progeny join the syncytium; P(1,2,9-11).p join the syncytium directly.] Our results can be summarized as follows. (1) The P. redivivus gonad is required for female Pn.p divisions and vulval development. The anchor cell (Z4. aaa) is required for the third round of Pn.p divisions. Z4 is required for vulval induction, although a few Pn.p cells can divide in its absence. In males of both species the gonad is not required for Pn.p divisions. (2) Regulation occurs as in C. elegans: the ablation of certain Pn.p cells can result in the replacement of the ablated cell by another Pn.p cell. (3) As in C. elegans, there is a hierarchy of replacements. A higher ranked cell can be replaced by a lower ranked cell but will not replace a lower ranked cell. In C. elegans hermaphrodites, the fate of P6.p is primary (ranks highest in the hierarchy), the fates of P(5,7).p are secondary, and the fates of P(3,4,8).p are tertiary. In P. redivivus females, the fates of P(6,7) .p are primary, the fates of P(5,8).p are secondary, and the fates of P(4,9).p are tertiary. (4) In C. elegans hermaphrodites, P(3-8).p can participate in vulval development and thus define the vulval equivalence group. In P. redivivus females, at least P(4-9).p can participate in vulval development. Thus, the vulval equivalence group differs between the species. (5) In males of both species, P10.p and P11.p generate cells of a pre-anal sensillum (called the hook sensillum in C. elegans); P9.p joins the ventral hypodermal syncytium or, in some C. elegans males generates two daughters that join the syncytium. As in C. elegans, P9.p can replace P10.p. Thus, in P. redivivus, P9.p belongs to both the male and female equivalence groups, indicating that equivalence groups in the two sexes need not be distinct. The differences in vulval regulation between the two species suggest that there are at least four independent levels of control specifying Pn.p fate in vulval development. Different sets of instructions seem to specify: (a) which Pn.p cells can participate in vulval development; (b) which Pn.p cells do participate in vulval development; (c) which Pn.p cells follow primary or secondary fates; and (d) what lineage is generated by cells with the primary, secondary and tertiary fates.
-
[
East Coast Worm Meeting,
1996]
During the development of N2 hermaphrodites, 131 cells commit to and execute the fate of programmed cell death. While several genes have been identified that appear to be required for execution and engulfment by their neighbors, only two genes have been identified that affect the commitment by specific cells to the death process:
ces-1 and
ces-2 (cell death specification 1 ). We are using a genetic screen to identify new ces genes that specifically affect the programmed deaths of cells in the ventral nerve cord. We expect that mutations isolated in the screen will identify genes required for the establishment of cell identity or the execution of the fate of cell death. Of the many programmed deaths available for study, we chose these deaths because they occur during larval development, making them easier to recognize. In addition, because there are multiple deaths in lineally-related cells the screen may be better able to identify weak mutations. To identify new ces genes, we treat
ced-1(
e1735);
ced-2(
e1752) hermaphrodites with EMS and examine the F2 progeny using Nomarski microscopy. In
ced-1;
ced-2 animals engulfment of cell corpses is blocked, and most animals have an easily recognized pattern of three corpses in the anterior ventral cord (descendants of P0, P1 and P2), and seven corpses in the posterior ventral cord (descendants of P9, P10, P11 and P12). Animals with an abnormal number or pattern of cell corpses are selected for further study. To date, 5,400 haploid genomes have been screened and three mutants identified. One has ectopic cell deaths in descendants of P3-P8 in a pattern similar to that of
lin-39( ). In a second mutant strain animals variably have too many or too few deaths in P cell lineages that normally include cell deaths. The third mutant strain has no apparent cell corpses in the ventral cord, but corpses are present in the tail and pharynx. We are continuing to screen for more mutants and are characterizing further those already isolated. 1. Ellis, R.E. & Horvitz, H.R. Development 112, 591-603 (1991). 2. Ellis, H.M. , Ph.D thesis,Genetic control of programmed cell death in the nematode Caenorhabditis elegans (M. I. T., Cambridge, MA, 1985).
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[
C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting,
2008]
We are using C. elegans as a model system to discover new small molecule tools for biological analyses. One obstacle to finding such tools is the large number of xenobiotic defense factors encoded by the worm genome (Lindblom et al., 2006). For example, in a screen of 3,265 small molecules that are bioactive in other systems, we found only 64 (2%) that induce robust phenotypes in the worm (Kwok et al., 2006). We hypothesized that this hit rate is relatively low because most small molecules fail to accumulate in worms. To test this, we developed an HPLC-based assay to measure small molecule accumulation in worm tissue. We surveyed 1,018 compounds from the Spectrum library (Microsource Inc.) for accumulation in whole worms after 6 hours of incubation in 40uM of the small molecules. To ensure confidence in our assignments we established a detection limit of 18uM. Of the 361/1018 compounds that satisfied this criterion, only 25 (6.9%) accumulate in worms. Notably, 2 out of 25 accumulating molecules induce robust phenotype in the worm, compared to 0 out of 336 non-accumulating molecules. We also assayed 25 nematicides obtained from our other small molecule screens, and found that 17 (68%) accumulate. These results support our hypothesis that worms are generally resistant to small molecule accumulation, and show that accumulating small molecules are enriched for bioactivity in C. elegans. Next, the accumulation of an additional 77 compounds from our other screens was assayed, for a complete dataset of 463 small molecules. We used this dataset to build a predictive structure-based model of accumulation. We compiled 4,166 structural descriptors of the small molecules in our dataset, and built a model using a Bayesian Classifier machine learning method. Five-fold cross validation of the model estimates a prediction accuracy of 75.36 +/- 2.04%. We used the model to rank 9,742 compounds of a DIVERSet library (Chembridge Corp.), of which 48 induce robust phenotype in the worm. Encouragingly, 12 of the top-scoring 200 molecules induce phenotype, representing a 12.2-fold enrichment for bioactivity compared to the entire library (
p10). None of the bottom-scoring 200 molecules induce phenotype. These data demonstrate that our model is effective at predicting small molecule accumulation and bioactivity in C. elegans. We hope to use this model to increase our efficiency at identifying new small molecule probes for biological analysis, and to aid the development of potential drug leads using C. elegans as a model.
-
[
West Coast Worm Meeting,
2000]
Cell fusion is a common process in C. elegans. The C. elegans hypodermis consists of several multinucleate syncytia that are generated by the fusion of cells throughout development. The largest syncytium is
hyp7, which spans most of the length of the worm and which contains more than 130 nuclei. We are carrying out two screens to identify mutations affecting cell fusion. First, we screened for mutations that prevent fusion of the Pn.p cells with
hyp7 on the ventral surface of the worm. Mutations identified in this screen affect the decision of these cells to fuse. Second, in order to identify mutations in genes that carry out the cell fusion process, we are screening for mutations that affect fusion of the seam cells that line the lateral surface of the worm. We identified several mutations that affect the pattern of Pn.p cell fusion and have been characterizing two mutations that affect Pn.p cell fusion by altering Hox protein activity. The fusion decision of the 12 Pn.p cells is controlled by two Hox genes,
lin-39 and
mab-5.
lin-39 is expressed in the mid-body [P(3-8).p] and in hermaphrodites prevents fusion of these cells.
mab-5 is expressed more posteriorly [in P(7-11).p] in both sexes, but is not active in hermaphrodite Pn.ps. In
ref-1(
mu220) (REgulator of Fusion) hermaphrodites, P9.p and P10.p fail to fuse with
hyp7. This is due, in part, to inappropriate activation of MAB-5 in
ref-1 hermaphrodites.
ref-1 encodes a gene with two basic-helix-loop-helix DNA binding domains of the hairy/E(spl) family. In males,
lin-39 and
mab-5 each individually prevent Pn.p cell fusion in P(3-6).p and P(9-11).p, respectively. However, in P7.p and P8.p, where both Hox genes are expressed in the same cell, they somehow neutralize one another's activities, so that P7.p and P8.p fuse with
hyp7. In
ref-2(
mu218) males, P7.p and P8.p fail to fuse with
hyp7, perhaps because LIN-39 and MAB-5 fail to cancel each others activities.
ref-2 has been mapped to a 40 kb region on the center of the X chromosome and transformation rescue experiments are in progress. Descendants of the seam cells fuse with
hyp7 at all larval stages and ultimately the seam cells fuse with each other during L4. We are currently using a membrane localized gfp fusion expressed in the seam cells to identify mutants in which seam cell fusions fail to occur.