Loer CM et al. (1992) Worm Breeder's Gazette "Serotonin and Male Mating Behavior in C. elegans"

Loer CM, Kenyon CJ

[Worm Breeder's Gazette, 1992]

Serotonin antisera stain some neurons specific to the male in C. elegans (The Worm Book, p.377-8). Among these are six cells in the ventral nerve cord that have been tentatively identified as the CP motoneurons derived from P(38).a (G. Garriga, personal communication). These neurons, along with the pair of NSM neurons in the pharynx, are the brightest, most reliably staining serotonin-immunoreactive cells in the worm. EM reconstructions show that CP neurons have neuromuscular junctions with male-specific diagonal muscles, suggesting they may play a role in male mating behavior (The Worm Book, p.121). Mating behavior of the adult male is among the most complex behaviors exhibited by C. elegans. The behavior can be divided into at least five components: recognition, backing, tail curling, vulval location, and copulation (including spicule insertion and sperm transfer); candidate neurons involved in many aspects of this behavior have been identified by ablation (Sulston, WBG 7(2): 22; Liu and Sternberg, Soc. Neurosci. Abstr. 17: 549). When an adult male contacts an hermaphrodite with his tail, he immediately backs along the surface of the hermaphrodite, pressing his tail firmly to the hermaphrodite. When he reaches the end of his intended mate, he makes a tight ventral curl to place his tail in contact with the opposite surface. The male then continues moving his tail along that surface in search of the vulval opening. This behavior may be repeated many times before the vulva is located and copulation is initiated. Application of serotonin to male worms is known to result in a tight ventral curling of the tail (see below), reminiscent of turning exhibited during mating behavior. [See Figure 1] To investigate the role of the CP motoneurons and serotonin in male mating, we assayed the tail curling/turning ability of individual males following ablations or in males containing mutations that alter serotonin expression. Ablation of the mesoblast M eliminates the diagonal muscles of the male, the postsynaptic target of the CP motoneurons. In M-ablated males, application of serotonin no longer caused a tight ventral curling of the tail, although the posterior body was slightly curled ventrally. These males were severely deficient in turning behavior. Ablation of the immediate precursors of CP's (P3-8.aap) also resulted in males that were deficient in turning, although they were not as severely affected as M-ablated males (see graph below). We have also confirmed that the serotonin-containing ventral cord neurons are derived from P(3-8).aap by ablation for example, when P3 .aapand P4 .aapwere ablated and the male subsequently stained with anti-serotonin, the two most anterior cell bodies were missing. We wondered whether worms with altered serotonin expression would show similar defects. cat-4 mutants lack anti-serotonin staining; cat-1 worms have variably decreased serotonin staining (Desai et al, 1988, Nature 336: 638). cat-1 and cat-1 males display decreased mating efficiency (Hodgkin,1983, Genetics 103: 43). cat-4 males were similar to CP-ablated males in their poor turning ability; cat-1 males were variably deficient in turning (see graph below). The mutant ad446 (courtesy of G. Garriga) also lacks serotonin and appears to be similarly defective. Since cat-4 and cat-1 are also deficient in dopamine expression (Sulston et al., 1975, J. Comp. Neurol. 163: 215), we also scored the turning ability of cat-2 mutants, which are dopamine deficient, but wild type for serotonin. These males were only slightly poorer than wild type in turning ability. Although most of the turning defect in cat-4 males may be accounted for by defective CP's, there may also be a sensory deficit at least two ray neurons are stained by serotonin antisera in wild type. Thus, ablation of CP's or reduction of serotonin results in a specific behavioral deficit in male mating behavior. We plan to screen for mutants in turning behavior to obtain additional serotonin-deficient mutations. Mutations that cause loss of serotonin expression may alter genes needed for serotonin synthesis or genes that regulate their expression; these may provide us a handle to understanding the regulation of the serotonergic phenotype. [See Figures 1-4]

Basham SE et al. (1997) Worm Breeder's Gazette "ooc-5 MUTATIONS DISRUPT OOCYTE FORMATION AND DIVISION ORIENTATION"

Rose LS, Basham SE

[Worm Breeder's Gazette, 1997]

Orientation of cell division plays a critical role in the development of both plants and animals. In early C. elegans embryos, two basic patterns of division occur. The AB lineage displays a typical orthogonal pattern of cleavage. In the P lineage, divisions repeatedly occur on the same axis due to a 90 degree rotation of the nuclear-centrosome complex. In the P1 cell, rotation depends on an interaction between astral microtubules and a site on the anterior cortex. We are studying maternal effect lethal mutations that alter spindle orientation in early embryos. In most embryos from animals homozygous for mutations in ooc-5 (for abnormal oocyte formation), the P1 nucleus migrates to the anterior cortex but fails to rotate. This results in P1 dividing transverse instead of along the anterior-posterior axis. As an initial step in understanding the ooc-5 phenotype, we have compared the distribution of cytoskeletal elements in wild-type and ooc-5 mutant embryos. In the P1 cell of wild-type embryos, both actin and CP accumulate in an anterior cortical dot which correlates strongly with nuclear rotation (Waddle et al., 1994, Devel. 120, 2317-2328). The percentage of ooc-5 embryos with a cortical accumulation of CP in the P1 cell is comparable to what we observe in wild-type embryos. These results suggest that the failure of rotation is not due to a lack of CP accumulation. In addition, the microtubule cytoskeleton appears normal in ooc-5 embryos as observed using standard immunofluorescence microscopy. Specifically, astral microtubules are seen projecting toward the cell periphery and appear long enough to interact with the cortex, suggesting that failure of rotation is not caused by microtubules being too short. Early C. elegans embryos are highly polarized. To determine whether ooc-5 mutations disrupt this polarity, we examined ooc-5 embryos for both P granule and PAR-3 localization. In one and two-cell embryos, P granules are localized to the posterior pole of ooc-5 embryos, just as in wild type. In the majority of 4-cell ooc-5 embryos examined however, P granules are seen in two of the four cells. This is presumably due to the failure of nuclear rotation resulting in P1 dividing transverse to the axis of P granule localization. The PAR-3protein is required for embryonic polarity and becomes localized to the anterior periphery of wild-type one-cell embryos (Etemad-Moghadam et al., 1995, Cell 83, 743-752). Our preliminary results show that PAR-3 protein is properly localized to the anterior periphery of one-cell ooc-5 embryos. Together, these observations suggest that altered spindle orientation in ooc-5 embryos is not a result of an overall polarity defect. Mutations in ooc-5 are pleiotropic however, resulting in a germline defect in addition to disrupting spindle orientation in P1. In ooc-5 mutant animals, the germline contains a double row of oocytes that are smaller than wild-type oocytes. Embryos produced by ooc-5 mutants are approximately half the size of a wild-type embryo while their nuclei are approximately 80% wild-type size. This observation raises the possibility that failure of nuclear rotation in these embryos is due to the steric effect of having a near normal size nucleus or microtubule array in a small cell. We have examined other strains that produce small embryos to see if reduced embryo size generally correlates with a failure of nuclear rotation. Homozygous ooc-3 mutants also have a double row of oocytes; the embryos produced are smaller than wild type and nuclear rotation fails to occur in the P1 cell. One interpretation of the ooc-5 and ooc-3 phenotypes is that the reduced distance between centrosomes and the anterior cortex prevents nuclear rotation, for example because microtubules from both asters make stabilizing connections with the anterior cortical site. However, small embryos produced by ceh-18 hermaphrodites can undergo nuclear rotation. In two ceh-18 embryos examined, the distance from the center of the nucleus to the anterior cortex was shorter than in several ooc-5 and ooc-3 embryos. In addition, we have observed several cases in wild-type embryos where the P1 nucleus moves up against the anterior cortex before rotating. These data suggest that lack of rotation in ooc-5 and ooc-3 embryos is not a result of having a shorter distance between the centrosomes and the anterior cortical site. However, we can not rule out other steric effects since ceh-18 and wild-type embryos are larger than ooc-5 embryos in other dimensions. We are beginning a molecular analysis of the ooc-5 gene to gain more insight into its role in germ line formation and spindle orientation.

Hobert O et al. (1999) Worm Breeder's Gazette "The lim-6 LIM homeobox gene regulates GABAergic neuron differentiation, dauer arrest and uterine morphogenesis"

Hobert O, Tessmar K, Ruvkun GB

[Worm Breeder's Gazette, 1999]

lim-6 is the worm ortholog of the mammalian lmx-1 LIM homeobox gene and represents one of seven LIM homeobox genes in C.elegans. A lim-6 reporter gene fusion that contains all exons, introns and 2kB of upstream regulatory regions is exclusively and strongly expressed in the ASG and AWA sensory neurons and in the male CP motorneurons. However, a reporter gene construct that contains a total of 4 kB of upstream sequences including all exons and introns is not expressed in ASG, AWA and CP but is strongly expressed in the GABAeric neurons DVB, AVL, RIS, RME/R, the ASEL chemosensory neuron, the PVT and RIGL/R neurons, in the excretory gland cell and in the developing uterus. This larger GFP reporter gene construct contains all the regulatory regions that are sufficient to rescue the mutant phenotype of lim-6 (see below) and its sites of expression correlate with the sites where lim-6 mutant animals display defective phenotypes. A lim-6 deletion allele, nr2073, was kindly provided by NemaPharm, Inc.; it contains a 1.7 kb deletion which takes out the second LIM domain and the homeodomain and thus presumably represent a loss-of-function allele. lim-6(nr2073) animals are constipated and display strong expulsion defects in the defecation cycle. These defects are presumably due to lim-6 expression and action in AVL and DVB since laser ablation of both neurons causes a similar defect (McIntire et al., 1993) and since we can observe axonal morphology and sprouting defects of the AVL and DVB motorneurons in the lim-6 mutant animals. We also observed that expression of unc-25/GAD, the rate limiting enzyme of GABA synthesis (Jin et al., 1999), is partially dependent on lim-6 gene activity in RIS, AVL and DVB. lim-6 mutant animals display a strong daf-c phenotype in conjunction with daf-7(e1372); this defect can be phenocopied by unc-25(e156), but not by unc-30(e191), arguing that non-D-type GABAergic neurons are involved in regulating dauer arrest and that lim-6 presumably is required for GABA mediated control of dauer arrest. In the developing uterus, lim-6 is transiently expressed in the uterine toroid cells and in the uv2 and uv3 cells. lim-6 mutant animals have a largely reduced brood size and most of their progeny hatches within the parent. In lim-6 mutant animals the uterine toroid cells do not separate and the lumen is clogged. It is intriguing to note the similarities between the expression and mutant phenotypes of the LIM homeobox genes ttx-3, lin-11 and lim-6: All genes are expressed in postmitotic neurons and their expression is maintained throughout adulthood (Hobert et al., 1997, 1998). In each case, the LIM homeobox gene is neither required for the generation of the respective neurons that express the LIM homeobox gene nor for their initial axonal outgrowth. However, in the respective mutant animals these neurons display rather subtle neural sprouting defects, which might be indicative of signaling defects of the respective neuron. Possibly, it is a common theme of LIM homeobox genes to affect synaptic connectivity or synaptic signaling and as a secondary consequence axonal sprouting. References: McIntire et al.(1993) Nature 364, 344-7; Jin et al.(1999) J.Neurosci.19, 539-49; Hobert et al.(1998) J.Neurosci.18, 2084-96; Hobert et al.(1997) Neuron 19, 345-57

Niacaris TR et al. (1998) Worm Breeder's Gazette "Expression patterns of candidate serotonin receptors"

Avery L, Niacaris TR

[Worm Breeder's Gazette, 1998]

Serotonin increases the rate of pharyngeal pumping and egg laying, decreases foraging behavior and is required for proper male mating behavior. Serotonin can influence pharyngeal pumping rate by stimulating MC, a motor neuron which stimulates pharyngeal muscle contraction, or by acting directly on pharyngeal muscle. To determine the receptor or receptors that mediate this response, we have searched the genomic sequence for candidate genes having sequence identity to known vertebrate and invertebrate serotonin receptors. We found seven candidate genes using this method: C02D4.2, C09B7.1, C52B11.3, F01E11.5, F14D12.6, F59C12.2 and M03F4.3. Two of these genes (F59C12.2 and M03F4.3) were previously identified as possible serotonin receptor candidates (Ribeiro and Hamdan IWM97, p278.) We were not able to distinguish octopamine receptors from metabotropic serotonin receptors based on sequence. In addition, there are no sequences in the C. elegans database with significant similarity to the ionotropic serotonin receptor subclass, 5-HT3. We have created GFP fusion constructs of four of the candidate genes (C02D4.2, F01E11.5, F14D12.6 and F59C12.2) using overlap extension PCR. We directly injected these PCR products into worms to determine GFP expression patterns. F01E11.5 and F14D12.6 show limited expression outside the pharynx. The F01E11.5 construct expresses in a few unidentified neurons of the head, while F14D12.6 expression is most pronounced in the spermatheca of the adult. Two of the genes, C02D4.2 and F59C12.2, show pharyngeal expression of the fusion construct. F59C12.2 shows nearly ubiquitous pharyngeal and extrapharyngeal staining. C02D4.2 pharyngeal muscle expression is specific for the metastomal flap muscles, pm1, and the anterior terminal bulb muscles, pm6. The C02D4.2 construct also expresses in muscles of the head, the pharyngeal serotonergic neuron NSM, the ventral nerve cord and unidentified neurons in the nerve ring, head and tail regions. In males, C02D4.2 expresses in dorsal and ventral body muscles of the tail region. The construct also expresses in the male-specific diagonal muscles and serotonergic CP neurons. The expression pattern of C02D4.2 appears to be consistent with several known effects of serotonin. We are currently working to knock out both C02D4.2 and F59C12.2 using PCR detection of EMS-induced deletions of these genes.

Waddle JA et al. (1994) Worm Breeder's Gazette "Acetone Extraction of Early C. elegans Embryos Reveals Persistent, Phalloidin-Stained Rings From Previous Cell Divisions"

Waddle JA, Waterston RH

[Worm Breeder's Gazette, 1994]

Recently, we fortuitously revealed phalloidin-stained, ring-shaped structures shared by sister cells in the early embryo. The detection of these structures requires formaldehyde fixation followed by 100% acetone extraction, a treatment that extracts other phalloidin-binding components including most of the cortical actin cytoskeleton in early nematode embryos. The time of appearance, position, morphology and number of phalloidin-positive rings within embryos suggest these organelles are persistent remnants from prior cell divisions. We refer to these rings as cell division remnants (CDRs). Preliminary characterization of these structures reveals the following: (i) A given embryo always has one fewer CDR than the total number of cells or a number equal to the number of cell divisions. (ii) The CDRs persist through many cell cycles and appear to be shared by all sister cells. (iii) The dimensions and position of the CDRs are similar to the initial size and location of the transient, CP-actin complexes that coincide with the onset of centrosome rotation in the P(1) lineage(1). For example, the CDR from first cleavage is located at a central site in the midfocal plane between AB and P(1) early in the 2-cell stage. Later, nuclear movements in the dividing AB cell push the CDR off-center in a manner which resembles the movement of the transient CP-actin complex during the 2-3 cell transition. (iv) The diameter of CDRs is variable (0.25-2.0 m) and unpredictable. In general, older CDRs are smaller but there is no obvious correlation between ring diameter and the cell cycle. (v) The CDR derived from first cleavage is invariably shared by EMS and ABa at the 4-cell stage, EMS and ABal at the 6-cell stage and MS and ABal at the 8-cell stage. At least some of the above observations suggest that once a CDR is formed, it is not divisible by subsequent cell divisions. Are CDRs involved in cell fate decisions? At the 4-cell stage, a signal from the EMS blastomere is required to instruct the anterior AB daughter to express a fate different from its sister, this interaction requires maternal expression of theglp-1 gene product (2,3). Theglp-1 protein is found in the membrane of both AB-derived cells at the 4-cell stage(4) in agreement with previous experiments which show these two cells have equivalent potential. Paradoxically, the EMS cell contacts both AB-derived cells at the 4-cell stage, yet only the anterior AB daughter expresses the EMS-induced fate change. How an EMS-derived signal is directed to the appropriateGlp-1 expressing AB descendant remains unclear. The invariant "path" of the persistent CDR from first cleavage to the 6-cell stage provides an internal, asymmetrically-positioned structure which could be exploited to distinguish between equivalent sites of contact between AB-derived cells and EMS at the 4 and 6-cell stages. The AB-EMS lineage interactions specify the dorsal-ventral(2) and left-right(5,6) embryonic axes at the 4 and 6-cell stages, respectively. We propose a model whereby the asymmetrically positioned CDR could be exploited to deliver a signal from EMS to specific AB descendants despite apparently equivalent contacts. A CDR model for the specification of the d-v embryonic axis in C. elegans. Fig 1. shows a schematic view of an experiment done by Priess and Thompson(2). At the 2-cell stage, segregation of determinants (shaded region) along the a-p axis in P(1) define the EMS and P(2) "ends" of the cell. Left unperturbed, embryos develop as shown on the left. On the right, the spindle in the AB cell is reoriented by physical manipulation with a blunt micropipette. One consequence of this manipulation is reorientation of the P(1) spindle. We propose that because the centrosome in P(1) is tethered to a CDR from first cleavage (bold circle between cells) the CDR, its associated cortex and determinants, and the anterior centrosome are repositioned as a unit. As a result, the absolute position of the EMS cell changes. At the stage, a signal from EMS is received by the AB daughter that shares the CDR from first cleavage; this interaction defines the d-v axis. A CDR model for the specification of the l-r embryonic axis in C. elegans. Fig. 2 shows a schematic view of an experiment done by Woods(5). EMS and its descendant MS are shaded to emphasize orientation. In all cases, anterior is left and ventral comes out of the page. In unoperated embryos, nuclear movements in the AB cells are skewed such that AB daughters on the left are slightly more anterior than their sisters on the right. If the skew of the AB spindles is reoriented prior to cytokinesis (right column), the l-r axis is switched giving rise to an enantiomeric embryo and adults. Reorientation of the AB cell positions after cytokinesis does not reverse the l-r axis(5). We propose the physical manipulation changes the absolute position of the CDR derived from first cleavage. This change might reroute an EMS-derived signal that makes ABal different from ABar and reverse the l-r axis. A correlation between the position of the CDR and cell fate specification can now be tested. The prediction is that the embryo manipulations described in Figs. 1 and 2 should invariably reposition the CDR upon successful reversal of the embryonic axes. We plan to test the models diagrammed in Figs. 1 and 2 and to investigate the composition of the CDRs using existing antibodies to various cytoskeletal components. REFERENCES: (l) Waddle et al, 1993 Worm Mtg, Abs#2. (2) Priess and Thompson, 1987 (3) Priess et al, 1987 (4) Kimble et al, 1992, CSH Symp Quant Biol 57:401 (5) Wood, 1991 Nature 349:536. (6) Schnabel, 1991, Mech Dev 34:85.

Yandell MD et al. (1996) Worm Breeder's Gazette "MORE EXPRESSION AND OVEREXPRESSION OF A C. ELEGANS TGF-beta HOMOLOGUE, dbl-1"

Wood WB, Yandell MD, Suzuki Y, Ross R

[Worm Breeder's Gazette, 1996]

We have reported previously on the cloning, molecular characterization, expression, and ectopic overexpression of a C. elegans TGF-beta homologue (previously called ceg-1: WBG 13(4):65, 1994; 1995 Meeting Abstracts: 555), which we have now named dbl-1 (dpp-BMP2,4-like). We have continued analysis of dbl-1 expression, using a transgenic him-8 line isolated after coinjection of two dbl-1::GFP constructs containing 4.4 kb of upstream sequence and differing only in the presence or absence of a nuclear localization signal. The expression of both reporters allows us to identify cells both by nuclear position and by cell body and axonal morphology. Fluorescing cells in adults include the anal depressor muscle, neurons of the retro- vesicular ganglion, and VNC motor neurons including those of the pre-anal ganglion and the PDA motor neuron. Male-specific expression is seen in the CP neurons of the VNC, in cells of the dorsal-rectal ganglion, and in two spicule associated neurons of adults, as well as transiently in several still unidentified tail cells of L3-L4 larvae. We have also continued to analyze effects of ectopic dbl-1 overexpression in transgenic lines carrying a dbl-1 cDNA construct (hsp::dbl-1) made with the heat-shock promoter plasmid pPD49.78, which induces well in neuronal and hypodermal cells (A. Fire, personal communication). Since both the dbl-1 expression pattern and existing evidence (Savage et al., 1995 Meeting Abstracts: 56) suggest a role for signaling mediated by a TGF- beta homologue during ray and spicule formation, we examined the effects of dbl-1 overexpression on ray morphogenesis. Heat shocks of hsp::dbl-1 transgenic him-8 L3 males result in a variety of ray transformations, the most common of which (50% penetrance) is transformation of ray 4 to ray 3 with 4-3 fusion, a phenotype also associated with decreased mab-5 activity (Chow and Emmons, Development 120:2579, 1994). Other phenotypes seen at lower frequencies include extra V-rays and multiple-ray fusions. These results suggest a role for dbl-1 in the specification of ray identities. We have refined the genetic map position of dbl-1 on LGV by using PCR to assay for dbl-1 sequences in DNA from inviable embryos homozygous for each of several mapped deficiencies in the region. The results locate dbl-1 in a small region of <.5 map unit (uncovered by nDf32 but not by nDf18) which includes unc-70 and four other genes defined by lethal mutations (Johnsen and Baillie, Genetics 129:735, 1991). To determine whether any of these genes could be dbl-1, we have done DNA coinjection rescue experiments with a rol-6 marker plasmid and cosmid ZC421, which contains the dbl-1 coding region and approximately 20kb of upstream sequence. We have so far injected homozygous unc-70 hermaphrodites as well as heterozygous hermaphrodites from balanced strains carrying lethal mutations representing three of the four essential genes. None of these mutants was rescued. We have also undertaken PCR screening of a high-copy-number Tc1 strain (Andachi and Kohara, 1995 Meeting Abstracts: 96) in attempts to identify nearby Tc1 insertions that would allow generation of a dbl-1 allele.

Seydoux GC et al. (1993) Worm Breeder's Gazette "A lac-Z Fusion Expressed in the C Lineage"

Fire A, Seydoux GC

[Worm Breeder's Gazette, 1993]

During the course of our screen for embryonic expression patterns (WBG 12#4, p20 ),we have isolated a lac-Z fusion that appears to be expressed specifically in the C lineage. This fusion (pGS 15.02) consists of a 3.2 kb fragment of C. elegans genomic DNA fused to the lacZ gene in the pPD21 .28vector. We found that 1 kb of the 3.2 kb is sufficient to drive C specific lac-Z expression (pGS 15.24) and have used three lines carrying an integrated array of pGS15 .24to characterize its expression pattern. To facilitate the identificadon of the lac-Z expressing cells, we have developed a modified method to stain lineaged embryos (thanks to Lois Edgar and Deborah Cowing for their helpful suggestions). Briefly, 2 to 4 cells embryos are transfered in a drop of modified lacZ staining mix (see below) onto a 1.5% agarose pad (also containing modified staining mix) and covered by a coverslip for Nomarski observadon. Embryos are allowed to develop and their lineage is recorded until a desired stage is reached (see WBG 12#2, p 19 for a descripdon of our recording system). At that time, the egg shell and vitelline membrane are disrupted using a laser microbeam to allow the staining mix to enter the embryo. 12 hours later, cells staining for lacZ can be idendfied based on their lineage idendty. In this manner, we found that in the 28 cell-stage embryo the four C-derived blastomeres (Caa, Cap, Cpa, Cpp) all express the fusion in their cytoplasm. (In nonlineaged embryos, we sometimes saw two lacZ-expressing cells in 24 to 26 cellembryos, suggesting that Cp and Ca may occasionally start expressing the fusion towards the end of their cell cycle). The four C-derived blastomeres, as well as their 8 progeny continue to express the fusion during their endre cell-cycle. By the 16 C-cellstage (172 cells), the expression of the fusion weakens but still remains confined to the C lineage. During morphogenesis, no expression of the fusion has been observed in lines carrying the integrated array. Curiously, lines carrying pGS15 .24on an extrachromosomal array continue to express the fusion up to the comma stage. At this dme, expression of the fusion is seen in the nuclei of C-derived hypodermal cells. (We note, however, that expression of the fusion appears somewhat deregulated in non-integrated lines: in the 28 cell-stage embryo, many more cells besides the C-derived blastomeres often express the fusion.) Sequencing pGS15 .24,we have found that it encodes a translational fusion between a novel C. elegans gene and lacZ. We have obtained and sequenced cDNAs corresponding to this gene and found that it has the potential to encode for a 38 kD protein containing an acidic region rich in glutamic acid. No homologies or other features have been noticed as of yet. By Northern analysis, this gene appears to be expressed predominantly in embryos, with no detectable expression in larvae and a low level of expression in adults. Modified Staining Mix: O.O5M NaPi pH 7.5 2mM MgCl2 5mM KFe/5mM KFo 1 ug/ml DAPI 0.004% SDS 5% Sucrose 0.125% X-gal 1% glutaraldehyde

Fred Kippert et al. (2002) Worm Breeder's Gazette "Arrhythmicity of the defecation oscillator in lin-42 mutants"

Fred Kippert, Mark L Blaxter

[Worm Breeder's Gazette, 2002]

Part of the heterochronic gene lin-42 (1) shows significant similarity to the Drosophila and vertebrate per circadian clock genes. This encompasses the PAS domain (and adjacent PAC domain) found in many clock genes. One of most interesting aspect of the Drosophila per gene is that mutations affect not only the circadian clock but also two other timing processes, temperature-dependent development and a temperature-compensated ultradian clock modulating the male's courtship song (2). The defecation oscillator of C. elegans L4 larvae is characterised by a remarkable regularity of the period under a given set of conditions while being highly dependent on temperature and nutritional status. Because of its regularity, it is possible to distinguish between effects on period length and cycle variability in mutants. For example, the period in the clk-1 (qm30) mutant is 50% longer whereas the regularity remains close to that of the wild type. Since defecation is a permanently recurring process, it may be difficult to decide when a mutant is 'arrhythmic'. However, we found that in a presumptive lin-42 null mutant ( mg152 [a gift of Ann Rougvie]), regularity was completely lost, with an increase in the mean coefficient of variation for the 15 cycles of a single L4 from 3.5% to 23.1%. It seems justified to call this arrhythmicity. When we looked at a second lin-42 allele ( n1089 [from the CGC]), we found it to be temperature-sensitive for the oscillator phenotype. At 20degC, period regularity of larvae was close to wild type. At 25degC, the strain became similar to the null mutant. The LIN-42 protein thus appears to play a crucial role in conferring to the defecation oscillator its remarkable regularity. We have recently described a circadian clock in C. elegans (3) and our preliminary investigation indicates that the two alleles behave similarly with respect to the circadian clock, i.e. arrhythmicity in the null mutant and the ts strain at 25degC but close to normal circadian rhythms in the ts strain at 20degC. Like the Drosophila per gene, lin-42 thus appears to be involved in at least 3 different timing processes on different time scales and with different characteristics. Its role in the ultradian and circadian rhythms is now under closer investigation. (1) Jeon M et al (1999) Similarity of the C. elegans developmental timing protein LIN-42 to circadian rhythm proteins. Science 286:1141-1146. (2) Kyriacou CP & Hall JC (1994) Genetic and molecular analysis of Drosophila behavior. Adv. Genet. 31:139-186. (3) Kippert F, Saunders DS & Blaxter ML (2001) C. elegans has a circadian clock. Curr. Biol., in press Strain (30 L4 each) Temperature (degC) Period (sec) Shortest mean (sec) Longest mean (sec) Shortest cycle (sec) Longest cycle (sec) N2 20degC 49.4 46.2 53.5 44 58 N2 25degC 38.2 35.9 42.2 34 44 clk-1 (qm30) 20degC 77.5 70.8 86.5 66 92 lin-42 (n1089) 20degC 52.8 46.7 58.4 43 65 lin-42 (n1089) 25degC 50.5 36.0 62.5 24 91 lin-42 (mg152) 20degC 53.3 43.1 71.4 22 118 Strain (30 L4 each) Temperature (degC) Standard deviation Coefficient of variation (%) Mean of CVs (%) Lowest CV (%) Highest CV (%) N2 20degC 2.2 4.5 3.5 1.7 5.5 N2 25degC 1.9 5.0 3.7 2.8 5.6 clk-1 (qm30) 20degC 5.3 6.8 4.6 2.3 6.1 lin-42 (n1089) 20degC 3.6 6.8 4.2 2.3 7.6 lin-42 (n1089) 25degC 6.7 13.3 14.1 7.2 27.8 lin-42 (mg152) 20degC 7.6 14.2 23.1 10.6 46.2

Wood WB (1990) Worm Breeder's Gazette "Reversal of L-R Asymmetry by Micromanipulation of 6-Cell Embryos: ABal and ABpl are Equivalent to ABar and ABpr, Respectively"

Wood WB

[Worm Breeder's Gazette, 1990]

Both the embryo and the embryonic cell lineage are asymmetric from the 6-cell stage onward, particularly with regard to the ABa lineages ( 1). The general bilateral symmetry of the animal is superimposed on the asymmetric lineage by lineally nonhomologous cells adopting analogous fates at equivalent contra lateral positions during embryogenesis (1), but several asymmetries remain throughout larval development, such as the anterior-posterior positions of the left and right coelomocyte pairs (2) and the anterior-r, posterior-l orientation of the primordial and developing gonad (3). These asymmetries normally have the same handedness in all individuals; in N2 populations grown at 16 C, no animals with reversed gonad handedness were found among about 2500 examined. Handed asymmetry first becomes evident in the embryo between the 4- and 6-cell stages, when skewing of the l-r cleavages of ABa and ABp results in positioning of the al and pl daughters anterior to ar and pr, respectively, with al more ventral than ar [see Wood, WBG 11,#3(May) :73, 1990]. Reversal of handedness was accomplished using methods similar to those of Priess and Thomson (4), by rolling a 4-cell embryo to ventral side (EMS) up with a microneedle and then pressing down and back on the left ventral surface of ABa during AB-cell cleavage. When successful, this procedure reverses the normal skewing of both AB spindles and results in ar and pr being anterior to al and pl, respectively, at completion of the cleavage (see diagram). The embryonic development of a reversed embryo was observed and recorded using the '4D Microscope' developed by J. White. (This consists of a Nomarski microscope equipped with a video camera connected to a computer controlled optical disc recorder and focussing drive motor, which automatically records complete sets of serial optical sections at predetermined intervals and allows subsequent playback of consecutive images from any desired optical section to facilitate lineaging.) Cell assignments in the reversed embryo, based on timing of divisions, relative positions, and lineaging to confirm identities of AB descendants, were entirely consistent with a completely enantiomorphic but otherwise normal cleavage pattern. For example, MS descendants were located on the left side of the early embryo and C descendants on the right; MSa and Ca descendants were to the right of MSp and Cp descendants, respectively, and so on. In the L1 that hatched from this embryo, 14 normally asymmetrically placed nuclei scored were all l-r reversed: Z1, Z2, Z3, and Z4 in the gonad primordium, M, the four cc's, Q2, the exc cell, mu sph, hyp11, and PVR. The adults that developed from other reversed embryos showed reversed handedness for the somatic gonad, placement of coelomocytes, and the positions of neuronal cell bodies relative to processes in the ventral nerve cord. The reversed animals were healthy, moved normally, and were normally fertile. These results indicate that ABal is equivalent to ABar and ABpl to ABpr, and, therefore, that the left-right differences in lineage patterns and cell fates exhibited by descendants of ABa and ABp must be dictated by cell interactions, which differ on the two sides as a consequence of the asymmetric positioning of these cells relative to others in the embryo. The respective cell contacts of the ABal-pl and ABar-pr pairs are still equivalent in the 8-cell embryo despite the asymmetry, and earlier laser ablation experiments (1) provide evidence for cell autonomy of many AB-cell fates subsequent to the 51-cell stage. Therefore, the determinative interactions are most likely to occur between the 15-cell and 51-cell stages. [See Figure 1] Thanks to A. Chisolm, J. Rothman, J. Sulston, and J. White for timely coaching and sage advice.