Sawatari, E. et al. (2011) International Worm Meeting "Changes in responsivity of olfactory neurons to odor during olfactory adaptation and recovery in Caenorhabditis elegans ."

Teramoto, T., Inoue, A., Sawatari, E., Ishihara, T.

[International Worm Meeting, 2011]

Animals acquire tremendous quantity of information from environment, and process them in their nervous systems. In this process, forgetting is important to prevent excess memory capacity or interference between old and new memories. However the mechanisms of forgetting processes are not elucidated at the molecular and neuronal circuit level. In C. elegans, we found that mutants of P38/JNK pathways regulate forgetting processes for the adaptation to diacetyl. In wild-type animals, the memory for the adaptation to diacetyl is fully recovered within hours, but in the mutants of P38/JNK pathways, the memories were extended to more than one day. The prolonged retention of memory of these mutants was rescued by expressing the wild-type gene product in AWC sensory neurons. In addition, the ceh-36 mutants that have no functional AWC sensory neurons show prolonged retention of memory, suggesting that AWC sensory neurons may secrete molecule inducing forgetting. However, how and where the memory for the adaptation to diacetyl is retained have not been revealed. To examine whether the memory for adaptation to diacetyl is retained in peripheral sensory neurons or interneurons, we analyzed the diacetyl-evoked Ca2+ response in AWA sensory neurons of naive, adapted and recovered animals, by expressing a Ca2+ indicator, YC3.60, in AWA sensory neurons. Ca2+ transient was measured in the PDMS microfluidic device by using same animals for naive, after adaptation and after recovery. In naive animals, diacetyl stimulus induced a Ca2+ increase in the AWA sensory neurons. After continuous perfusion of diacetyl for 1.5 hrs, the Ca2+-responses to the odor were completely diminished, suggesting that the AWA neurons were adapted. Interestingly, after continuous washing out of the odor for 2-4 hrs, odor-evoked increase of Ca2+ was re-emerged. These results suggest that AWA sensory neurons are recoverd to an active state and this time course of the Ca2+ response is very similar to that of the behavioral response to diacetyl. We are currently analyzing P38/JNK mutant to see the neuronal activities during the recovery process from the olfactory adaptation.

Sara Maxwell et al. (2002) European Worm Meeting "Functional analysis of the C. elegans T-box gene family"

Alison Woollard, Roger Pocock, Lizzie Clarke, Sara Maxwell

[European Worm Meeting, 2002]

T-box genes are a group of developmentally important transcription factors united by a common DNA binding domain. T-box genes are present in all metazoan species so far analysed but are absent from yeast. There are 20 T-box genes in C. elegans, more than twice the number found in Drosophila. Many of the C. elegans T-box genes are highly diverged from those found in other species while others have clear orthologues present throughout the metazoan kingdom. One highly conserved T-box gene is mab-9, a member of the tbx20 sub-family1. This was the first C. elegans T-box gene to be identified by mutation and is required for cell fate specification during hindgut and male tail development, and aspects of nervous system function. One other conserved T-box gene has recently been reported to be important for a particular muscle cell fate specification2. We have inactivated the remaining C. elegans T-box genes by RNAi and have found obvious phenotypes only in very few cases. These phenotypes include embryonic lethality, L1 lethality, and a Dpy phenotype with weakly penetrant male tail defects, and will be described in detail. The remaining T-box genes give no obvious phenotype by RNAi. Phylogenetic analysis reveals that several pairs of T-box genes are very similar to eachother and are therefore likely to be the result of recent duplications. This might suggest functional redundancy. Double RNAi experiments have revealed this to be the case with at least two of the T-box gene pairs (see also poster by Pocock et al). Study of the expression patterns of the whole T-box family may suggest other potential redundancy relationships which can be explored by RNAi. Comparison of the C. elegans T-box genes with the set of T-box genes now defined for C. briggsae is being used as a tool for defining potentially important regulatory regions present in orthologous genes.

Hisako Takeshita et al. (2005) International Worm Meeting "Direction of cell polarity is determined by Wnt in C.elegans"

Hisako Takeshita, Hitoshi Sawa, Kota Mizumoto

[International Worm Meeting, 2005]

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, asymmetric divisions of many cells are regulated by Wnt signaling. In particular, asymmetry of the T cell division is regulated by LIN-44/Wnt and the LIN-17/Frizzled receptor. LIN-44/Wnt is expressed in hypodermal cells at the tip of the tail, posterior to the T cell. In lin-44 mutants, the polarity of the T cell division is frequently reversed, while in lin-17 mutants, the division is symmetric. It has been proposed that LIN-44 activates the LIN-17 receptor on the posterior side of the T cell to polarize it. To determine whether LIN-44/Wnt acts as an asymmetric cue that polarizes the T cell, we ectopically expressed LIN-44 in cells anterior to the T cell using the egl-5 promoter (egl-5::LIN-44) to find that polarity reversal phenotype of lin-44 was strongly enhanced. Moreover lin-17 was required for egl-5::LIN-44 to reverse the T-cell polarity. These results suggest that LIN-44 polarize the T cell through the LIN-17 receptor. To further confirm that LIN-44 directly acts on LIN-17 in the T cell, we analyzed the localization of LIN-17::GFP fusion proteins expressed by the seam cell specific promoter (SCM::LIN-17::GFP). This construct rescued defects of lin-17 mutants in the T cell division, indicating that lin-17 functions cell-autonomously in the T cell. We found that LIN-17 localization was localized in the posterior cortex of the T cell in wild type. In lin-44 mutants, the LIN-17 localization was uniform on the cortex. Furthermore, egl-5::LIN-44 reversed the LIN-17 localization to the anterior cortex. These results strongly indicate that LIN-44 directly acts on LIN-17 in the T cell to orient the polarity.

Hryshkevich, Uladzislau et al. (2011) International Worm Meeting "Core promoter T-blocks correlate with gene expression levels in C. elegans."

Yanai, Itai, Hryshkevich, Uladzislau, Hashimshony, Tamar

[International Worm Meeting, 2011]

Core promoters mediate transcription initiation by the integration of diverse regulatory signals encoded in the proximal promoter and enhancers. It has been suggested that genes under simple regulation may have low-complexity permissive promoters. For these genes, the core promoter may serve as the principal regulatory element; however the mechanism by which this occurs is unclear. We report here a periodic poly-thymine motif, we term T-blocks, enriched in occurrences within core promoter forward strands in C. elegans. An increasing number of Tblocks on either strand is associated with increasing nucleosome eviction. Strikingly, only forward strand T-blocks are correlated with expression levels, whereby genes with ³6 T-blocks have five fold higher expression levels than genes with 3 T-blocks. We further demonstrate that differences in T-block numbers between strains predictably affect expression levels of orthologs. Highly expressed genes and genes in operons tend to have a large number of T-blocks, as well as the previously characterized SL1 motif involved in trans-splicing. The presence of T-blocks thus correlates with low nucleosome occupancy and the precision of a trans-splicing motif, suggesting its role at both the DNA and RNA levels. Collectively our results suggest that core promoters may tune gene expression levels through the occurrences of T-blocks, independently of the spatio-temporal regulation mediated by the proximal promoter.

TM Morse et al. (1999) International C. elegans Meeting "Time-series analysis of pirouettes in C. elegans chemotaxis."

TM Morse, JT Pierce, SR Lockery, ML Moravec, TC Ferree, SE Owens, AB Bates

[International C. elegans Meeting, 1999]

Chemotaxis in C. elegans involves a series of abrupt turns (pirouettes) triggered by movement down a gradient of chemical attractant * . Analysis of the time series of concentration change experienced by a worm, together with its pirouette record, suggests a three-stage chemotaxis mechanism in which chemical concentration (C(t)) is differentiated (dC(t)/dt), smoothed (q(t)), and converted into pirouette probability by a nonlinear function (P(q[t])). To test the plausibility of this mechanism, we constructed a computer model in which the smoothing filter and the nonlinearity were estimated from the time series of dC(t)/dt and the pirouette records of real worms. Pirouettes were modeled by sampling randomly, via a Poisson process with probability P(q[t]), from the distribution of direction changes associated with pirouettes in real worms. The average chemotaxis index (time-average of normalized C(t)) of model worms (0.47 +/- 0.05 SD, n = 2000) closely matched the average chemotaxis index of real worms (0.45 +/- 0.04 SD, n = 45), indicating that the three-stage mechanism is quantitatively sufficient to account for C. elegans chemotaxis. To determine the form of the smoothing filter and nonlinearity directly, we have devised a new behavioral assay that subjects unrestrained worms to negative-going impulses in dC/dt as they swim across a sharp border between high and low concentrations of attractant. Preliminary results show that immediately after a border crossing, large impulses make P(t)~= 1, while small impulses make 0 t) >< C. elegans . * Pierce, J.T., and Lockery, S.R., J. Neurosci. (Submitted)

Miller, Raymond R et al. (2009) International Worm Meeting "Examining the interaction of the T-box factor MLS-1 with UNC-37/Groucho in determination of uterine muscle cell fate."

Miller, Raymond R, Crum, Tanya, Okkema, Peter G

[International Worm Meeting, 2009]

T-box family transcription factors play important developmental roles in all multi-cellular animals, including in the development of the limb, heart, and pituitary. However, the mechanism by which these factors function is largely unknown. In many cases, the study of T-box factor function is difficult because of the complex phenotypes T-box factor mutants display, and the difficulty in rescuing loss of function. In Caenorhabditis elegans, the T-box factor muscle lineage specification-1 (MLS-1) is responsible for the decision of precursor cells between vulval and uterine muscle fate. The mls-1 loss of function phenotype is easily examined, and is readily rescued using transgenes. We are using mls-1 to explore the mechanism of T-box factor function. We have found that MLS-1 interacts with the C. elegans Groucho family co-repressor UNC-37 in yeast 2-hybrid experiments, and this interaction is dependent on an MLS-1 engrailed homology 1 (eh1) motif. At low penetrance, unc-37(RNAi) phenocopies the mls-1 loss of function phenotype producing extra vulval muscles, suggesting MLS-1 requires UNC-37. We are currently performing in vivo studies to determine if MLS-1 functions as an UNC-37/Groucho dependent transcriptional repressor. We have found that UNC-37 also interacts with the T-box factor TBX-2 in yeast 2-hybrid assays via a degenerate eh1 motif. eh1 motifs are found in several T-box factors in different species, and we hypothesize interaction with Groucho family co-repressors is a conserved feature of T-box factor function.

Uozumi T et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Live imaging of Ras activity in the olfactory system in C. elegans"

Yamada R, Hirotsu T, Suzuki A, Uozumi T, Ishihara T, Teramoto T

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

The Ras-MAPK (mitogen-activated protein kinase) pathway is a fundamental signaling pathway that regulates many different biological processes. In C. elegans, the Ras-MAPK pathway plays important roles for olfactory perception. Previous immunohistochemical studies have revealed that this pathway is activated within 10 seconds in AWC olfactory neurons by odorant stimuli. However, it is impossible to observe the activation kinetics of the Ras-MAPK pathways by using the immunohistochemical study, because animals are fixed after odorant stimuli. To reveal the spatio-temporal regulation of activation of Ras in living animals, we introduced Raichu-Ras, the FRET-based sensors for the Ras activity, into the AWC olfactory neurons of C. elegans. Using the imaging system we observed that Ras was activated in the olfactory neurons after 2-4 seconds of the application of odorants, and then the Ras was deactivated within 5-6 seconds. These results seem consistent with the previous immunohistochemical studies. Furthermore, we observed that Ras was activated repeatedly by addition of the same odorant, suggesting that the activation of Ras is induced by the increase in concentration of the odorants. In odr-3 or tax-2 mutant animals, the Ras activation was not detected in most individuals. odr-3 and tax-2 genes encode a G&alpha; and a cGMP-gated channel, respectively, which are components of the olfactory signal transduction pathway. These results suggest that Ras is activated downstream of the olfactory signaling pathway in the AWC olfactory neurons. Our new imaging system allows us to understand the function of Ras and may open a new paradigm for the mechanism of olfactory perception by Ras-MAPK pathway.

Kuge, S. et al. (2017) International Worm Meeting "An inverse-type of fluorescent Ca2+ indicator for detecting neuronal inhibition."

Teramoto, T., Matsuda, T., Kuge, S., Ishihara, T., Takeharu, T., Nishihara, T.

[International Worm Meeting, 2017]

Fluorescent Ca2+ indicators have been improved for the last several decades and now are indispensable tools for cell biology, especially neuronal science, because they enable us to monitor neuronal activities. However, these fluorescent Ca2+ indicators including GCaMPs are ideal only for monitoring the activation of neurons. To understand precise functions of the neuronal network, a fluorescent Ca2+ indicator that is suitable for monitoring the neuronal inhibition would be necessary. We previously reported "inverse-pericam", which has a unique property; the fluorescence intensity gets 7-fold dimmer upon Ca2+ binding. We improved "inverse-pericam" by iterative error-prone PCR and got a variant, IP2.0, which shows nearly 20-folds dynamic range in vitro. When IP2.0 was expressed in HeLa cells, the fluorescence of IP2.0 was greatly quenched responding to histamine stimulation, indicating that IP2.0 is useful to detect change of the intracellular concentration of Ca2+. Then we expressed IP2.0 in AWCON neuron of C. elegans and monitored the decrease of Ca2+ concentration during the stimulation of isoamyl alcohol, as well as the increase of Ca2+ concentration after removing isoamyl alcohol. While the fluorescence of GCaMPs has known to be reduced slightly during the stimulation, we observed the increase of fluorescence of IP2.0 during the stimulation. These results suggest that odor stimuli decrease Ca2+ concentration in AWCON neurons. Next, we succeeded in expressing both of IP2.0 and RCaMP2.0 in AWCON neurons to monitor neuronal activation and inhibition simultaneously. Furthermore, we analyzed the neuronal activities of ASEL neurons responding to the concentration of NaCl by expressing IP2.0 and RCaMP2.0. The fluorescece of IP2.0 increased by the decrease of NaCl, indicating that Ca2+ concentration in ASEL neuron is decreased by the decrease of NaCl concentration. These results suggest that IP2.0, which can be used with various red fluorescent Ca2+ probes is suitable for sensitively detecting neuronal activities, even which cannot be sensitively detected by GCaMPs. Now, we are analyzing local Ca2+ dynamics in a whole single neuron by a wide-field imaging system by expressing IP2.0 in AWCON and ASEL neurons and we hope that this analysis makes clear that each sensory neuron has unique properties for informational processing within the neuron. We hope that our imaging system makes it possible to analyze precise Ca2+ dynamics of sequential rapid neuronal activation and inhibition in a whole single neuron and that these results are helpful for understanding the informational processing in the whole neuronal circuit.

Roger Pocock et al. (2003) International Worm Meeting "tbx-8 and tbx-9 are essential for embryonic morphogenesis and act upstream of the posterior patterning gene vab-7"

Roger Pocock, Michael Mitsch, Julie Ahringer, Sara Maxwell, Alison Woollard

[International Worm Meeting, 2003]

T-box genes are a family of transcriptional regulators unified by a conserved T-box DNA binding domain. There are 20 T-box genes in C.elegans, more than in any other species analysed so far but relatively little is known about their function. Using RNA interference, we have inactivated each of the worm T-box genes and have found obvious phenotypes in only a few cases. The remaining T-box genes give no obvious phenotype when inactivated singly by RNAi. Phylogenetic analysis reveals that several pairs of T-box genes are very similar to each other. Therefore, these gene pairs are likely to be the result of recent duplications and may share overlapping functions. Double RNAi experiments have revealed this to be the case with at least one of the T-box gene pairs, tbx-8 and tbx-9, which have been found to have essential, overlapping functions during embryonic morphogenesis. The earliest defect observed is in the process of dorsal hypodermal intercalation, which together with hypodermal ventral enclosure is crucial for embryonic elongation to occur. The dorsal hypodermal migration of cells is aberrant in tbx-8/9 RNAi embryos resulting in defects in elongation and morphogenesis. These embryos also have defects in muscle cell positioning and intestinal development. tbx-8 and tbx-9 are expressed in gut, muscle and hypodermis. Their expression domains largely overlap with both genes being highly expressed in the nuclei of dorsal hypodermal cells undergoing intercalation. tbx-9 single RNAi produces worms which have a bobbed-tail phenotype that is similar to the vab-7 mutant phenotype (vab-7 is an even-skipped homologue required for posterior patterning). This suggests that these genes may be acting in the same pathway. We find that tbx-8 and tbx-9 are both necessary and sufficient for vab-7 expression, and thereby function upstream of vab-7 to activate its expression in particular posterior cells. Furthermore, we find that vab-7 in turn represses another T-box gene, mab-9, in the posterior of embryos. Thus, there are complex interactions between vab-7 and T-box genes in C. elegans. Positive regulation of eve homeobox genes by T-box genes has been reported in other systems yet this is the first demonstration that T-box genes can also be subject to repression by eve. The potential evolutionary association between the role of tbx-8 and tbx-9 in dorsal hypodermal intercalation in the worm and the role of T-box genes in convergent extension movements of cells during morphogenesis of other organisms is an interesting parallel.

Yanping Zhang et al. (2003) International Worm Meeting "tcl-3, a new gene involved in the control of T cell polarity in C. elegans"

Michael A Herman, Yanping Zhang

[International Worm Meeting, 2003]

In C.elegans, as in other animals, the Wnt signaling pathway plays an important role in controlling cell polarities and cell migrations. For example, lin-44/Wnt and lin-17/Frizzled control the asymmetric divisions of T cells in the C. elegans tail. lin-44 and lin-17 also regulate the asymmetric distribution of POP-1/Tcf, a transcriptional regulator of Wnt signaling. In wild type animals, the anterior T cell daughter, T.a, has a higher POP-1 nuclear level than does the posterior daughter, T.p. Mutations in lin-44 cause a reversal of T cell polarity and a corresponding reversal in POP-1 nuclear levels, such that T.a has a lower POP-1 nuclear level and T.p has a higher POP-1 nuclear level. Mutations in lin-17 cause a loss of T cell polarity and equally high POP-1 nuclear levels in both the T.a and T.p cells, suggesting that pop-1 functions after lin-44 and lin-17. We have isolated a mutant defining the new gene, tcl-3(mh32), that controls the asymmetric division of T cell. tcl-3(mh32) complements all known genes that affect T cell polarity, indicating that tcl-3(mh32) defines a new gene. tcl-3(mh32) is a recessive mutation and causes 66.4% (n=64) of T cell divisions to be defective. Examination of T cell descendants indicated that most often four hypodermal cells were generated, suggesting a loss of T cell polarity. Staining with POP-1 antibodies showed that POP-1 nuclear levels were equally high in both T.a and T.p cells in 56 % of tcl-3(mh32) mutants (n=54). This pattern of POP-1 nuclear distribution is similar to that observed in lin-17 mutants, suggesting that tcl-3 also functions before pop-1 in the control of T cell division. Genes that affect T cell polarity, such as lin-44, lin-17, tlp-1 and tcl-2 often also regulate other cell fate specifications. For example, mutations in lin-17 and tcl-2 cause defects in B cell polarity, P11/P12 cell fate specification, gonad development and vulva development. In contrast to these mutants, tcl-3 mutants do not display a B cell polarity defect nor a P11/P12 cell fate defect, indicating that tcl-3 may not be involved in multiple cell fate decisions. We have used snip-SNP mapping to localize tcl-3 to a small interval on the right arm of LGX. We are cloning and further characterizing tcl-3 gene in order to understand its role in the control of the T cell polarity.