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.

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.

Elvis Huarcaya Najarro et al. (2006) Development & Evolution Meeting "Mutations in tcl-1 cause defects in T cell polarity"

Elvis Huarcaya Najarro, Krista Williams, Michael Herman, Jennifer Griffiths

[Development & Evolution Meeting, 2006]

In C. elegans, the anteroposterior polarities of a pair of cells in the tail, called TL and TR, are controlled by a WNT signal encoded by lin-44. Mutations in lin-44 cause the reversal of T cell polarity. Mutations in lin-17, which encodes a Frizzled-like protein, cause the loss of T cell polarity, suggesting that LIN-17 is a receptor of the LIN-44 WNT signal. Other Wnt pathway components that have been shown to function in the control of T cell polarity include LIT-1/NLK, WRM-1/beta-catenin, SYS-1/beta-catenin and POP-1/TCF. As in many other anterior-posterior asymmetric cell divisions, the nuclear level of POP-1 is higher in the anterior T cell daughter, T.a, than it is in the posterior daughter, T.p. We have identified several mutations that define new genes involved in the control of T cell polarity, including tcl-1. The tcl-1 mutation causes the loss of neural cell fates in the T cell lineage and is incompletely penetrant. Genetic tests suggest that our tcl-1 allele might cause a weak novel function. Analysis of T cell lineages revealed that T.p often fails to divide in tcl-1 hermaphrodites. We also have determined that tcl-1 affects distribution of GFP::POP-1 to the T cell daughters, thus TCL-1 functions early in the control of T cell polarity. tcl-1 mutants also appear to have an additional defect affecting either the neurons or the supporting cells of the sensory phasmid in the tail. We have rescued tcl-1 with cosmid F55A12, on which there are 10 candidate predicted genes. A genomic fragment that contained both F55A12.2 and ppk-1 rescued tcl-1 mutants, whereas a fragment containing only ppk-1 did not. Furthermore, F55A12.2 is only 712 bp downstream of ppk-1 and is trans-spliced to SL2, suggesting that these two genes are in an operon. Although, it is not yet clear which corresponds to tcl-1 and how it might function in T cell asymmetry, ppk-1 encodes a type I phosphatidylinosital phosphate kinase. These enzymes phosphorylate phosphatidylinositol 4-phosphate to generate phosphatidylinositol 4,5 bisphosphate (PI4, 5P2) that can modulate many cellular behaviors, including cell proliferation. Establishing a role for ppk-1 in cellular asymmetry would be an important finding and could link cell signaling to cell polarity.

X Zhao et al. (1999) International C. elegans Meeting "Identification and Analysis of Three New Mutations Involved in the Control of Cell Polarity During C. elegans Development"

TM Ratliff, MA Herman, X Zhao

[International C. elegans Meeting, 1999]

We are interested in how cell polarity is controlled during metazoan development. Our approach is to identify and study genes involved in the control of cell polarity by identifying mutations that disrupt the polarities of individual cells. Mutations in three genes: lin-44, lin-17 and egl-27 have previously been found to affect the polarity of certain cells in the tail called TL and TR. lin-44 encodes a WNT signaling protein that is made by the skin cells at the tip of the developing tail and functions to specify the polarity of the more anterior T cells. lin-17 encodes a putative WNT receptor and egl-27 encodes a protein similar to a metastasis-associated factor, MTA1, and has been shown to be required within the T cells for normal polarity. We screened for additional mutants that affect T cell polarity to identify new genes that may interact with the genes known to control T cell polarity. We screened approximately 45,000 haploid genomes and isolated three new mutants: mh15, mh17 and mh21. A fourth mutation, mn593, was isolated in a preliminary screen of this type (see abstract by K. Williams and M. A. Herman). All three mutations are recessive and have been mapped to distinct intervals on LG IV. Phenotypic analyses indicate that mh15 and mh21 mutants display more severe T cell defects than do mh17 mutants. In addition, all three mutations cause males to have abnormal tail morphologies. Some mh15 and mh17 male tails appear to have fused rays. The T cell division pattern is defective in these mutants. Cell lineage analysis of mh15 indicates that several T cell daughters fail to divide and cell fates are not properly specified. These observations are consistent with a defect in T cell polarity. We are further analyzing the phenotypes of all three mutants and attempting to clone these genes by microinjection of relevant cosmid and YAC clones.

Xiaojun Zhao et al. (2002) Mid-west Worm Meeting "tcl-2 encodes a novel protein that acts synergistically with Wnt signaling pathways in C. elegans"

Michael A Herman, Hitoshi Sawa, Xiaojun Zhao

[Mid-west Worm Meeting, 2002]

During the development of multicellular organisms asymmetric cell divisions are required to increase the cell numbers and generate diverse cell types. In C. elegans, as in other animals, the conserved Wnt signaling pathway controls asymmetric cell divisions and cell fate specifications. For example, in the C. elegans tail, lin-44/Wnt and lin-17/frizzled control the asymmetric division of the TL and TR cells (collectively referred to as T cells). In wild-type animals, POP-1, a TCF-4/LEF-1 homolog, is distributed asymmetrically to the nuclei of the T cell daughters: the anterior daughter, T.a, has a higher level of POP-1 than does the posterior daughter, T.p. Mutations in lin-44cause a reversal of T cell polarity and a corresponding reversal in the level of POP-1: T.a has a lower level of POP-1 and T.p. has a higher level. Mutations in lin-17 cause a loss of T cell polarity and equally high level of POP-1 in both T.a and T.p. This suggests that pop-1 is downstream of lin-44 and lin-17. We have isolated several mutations defining the new genes that interact with lin-44 and lin-17 in the control of asymmetric division of the T cell. One of these is tcl-2 (T cell lineage defective). Cell lineage analysis has revealed that tcl-2 mutations cause defects in the pattern and asymmetry of the T cell divisions. Furthermore, both T.a and T.p had equally high levels of POP-1 in tcl-2 mutants, as we observed in lin-17 mutants, suggesting that tcl-2 also functions before pop-1 in the control of the T cell division. We cloned tcl-2 by transformation rescue and found sequence changes in four sequenced tcl-2 mutant alleles. No related proteins have been found in a variety of database searches, suggesting that tcl-2 encodes a novel protein. A tcl-2::gfptranslational fusion construct rescued the T cell division defect and was weakly expressed in the nuclei of the T cells and certain T cell descendants. In addition, we found that tcl-2mutants also have defects in the development of gonadal axes and the specification of the P11/P12 cell fate, both of which involve the Wnt signaling pathway. Double mutant analyses revealed that tcl-2 acts synergistically with lin-44 and lin-17 in the control of gonad development and P11/P12 cell fate specification. A role for LIN-44 in the development of the gonadal axes was unexpected, as neither lin-44 nor any other C. elegans Wnt gene has been shown to be involved in gonad development. Our data suggest that tcl-2 may either act in a parallel pathway or to modulate the Wnt signaling pathway in the control of the asymmetric T cell divisions, gonad development and P11/P12 fate specification.