Yuan Jiang et al. (2006) Development & Evolution Meeting "CEH-20 regulates the expression of mls-2 in the postembryonic mesoderm"

Yuan Jiang, Jun Liu

[Development & Evolution Meeting, 2006]

mls-2 encodes a HMX homeodomain protein that plays critical roles in the C. elegans postembryonic mesodermal lineage, the M lineage. mls-2 is expressed in the M lineage as well as a few other cell types, such as several pairs of head neurons including ASK and AIM. To uncover mechanisms involved in regulating the expression of mls-2, we generated a series of promoter deletions and identified an M lineage enhancer (position -2221 to -2076) that is required for mls-2 expression in the M lineage. We further identified several essential elements within this M enhancer. Among these elements are two putative Exd/Abd-B binding sites that are conserved in C. briggsae. We have found that mls-2 expression in the M lineage is dependent on the C.elegans Exd/Pbx homolog CEH-20. We are currently testing whether CEH-20 directly binds to the mls-2 promoter and whether CEH-20 regulates mls-2 expression through acting together with the Abd-B class Hox proteins, EGL-5/PHP-3/NOB-1, and the Hth/Meis homolog, UNC-62.

Yong-Uk Lee et al. (2008) ""Inhibition of developmental processes by flavone in Caenorhabditis elegans and its application to the pinewood nematode, Bursaphelenchus xylophilus""

Yoongho Lim, Yong-Uk Lee, Yhong-Hee Shim, Ichiro Kawasaki, Wan-Suk Oh, Young-Ki Paik

[2008]

"Flavone (2-phenyl chromone) is one of the well-known plant flavonoids, but its bioactivity is under explored. When Caenorhabditis elegans or C. briggsae were treated with flavone embryonic and larval lethalities were induced. The larval lethality was pronounced in the earlier larval stages. This anti-nematodal effect was also observed for the pinewood nematode, B. xylophilus. LD50 values were approximately 98 μM and 102 μM for B. xylophilus and C. elegans, respectively. Our results indicate that flavone is an active nematicidal compound that should be further investigated for the purpose of developing a potent anthelmintic drug against B. xylophilus. This study was supported by KRF2006-005-J03401, Forest Science and Technology project (S110707L0501101) and the second BK21 (MOE)"

Anita U Rao et al. (2007) International Worm Meeting "Genetic Dissection of Heme Pathways in C. elegans."

Brian Le, Iqbal Hamza, Anita U Rao

[International Worm Meeting, 2007]

Heme serves as a cofactor for a number of proteins involved in key metabolic processes. In eukaryotes, heme synthesis occurs in the mitochondria by an evolutionarily conserved multi-step pathway. Hemes are hydrophobic and thus insoluble in the aqueous environment of the cell. Moreover, free heme is cytotoxic because of peroxidase activity. We therefore hypothesize that intracellular pathways exist for trafficking of heme from the site of synthesis in the mitochondria to various cellular destinations. However, identification of these heme transport pathways has been difficult because heme synthesis is regulated by multiple effectors and is tightly coordinated with apo-protein synthesis. We have previously shown that C. elegans and related helminths do not make heme albeit requiring exogenous heme for normal metabolic processes. Importantly, C. elegans show a biphasic response for heme; worms are growth-arrested at 1.5 <font face=symbol>m</font>M and at 800 <font face=symbol>m</font>M heme. These results suggest that although worms are obligate heme auxotrophs they are likely to have all the pathways for heme utilization beyond the point of heme synthesis. To identify pathways for heme transport in C. elegans, we exploited their biphasic response to heme by screening for mutants that could survive heme toxicity. We screened 300,000 haploid genomes and isolated 13 mutants at 800 <font face=symbol>m</font>M heme in liquid axenic medium. Based on the mutants growth profile in medium containing low and high heme, we categorized the mutants into three broad phenoclusters: class A, class B and class C. Class A mutants grew robustly under low and high (800 and 1000<font face=symbol>m</font>M) heme, Class B mutants grew exceptionally well under low heme, moderately well at 800 <font face=symbol>m</font>M heme, and not at all at 1000 <font face=symbol>m</font>M heme, and Class C mutants grow moderately well under high heme (800<font face=symbol>m</font>M), but exhibit normal growth under low heme. The mutants were further sub-clustered by using gallium protoporphyrin (GaPP), a toxic heme analog. Complementation analyses revealed that these 13 mutants fall into five complementation groups. Genetic mapping by recombination localized the mutants from each complementation group to chromosome III. We are now producing a high resolution map to define a genetic interval and pinpoint the exact nature of the molecular lesion in these mutants.

Mingfu Wu (2004) Mid-west Worm Meeting "lin(mh56) Controls the Asymmetric B Cell Division and Male Tail Development in C. elegans"

Mingfu Wu

[Mid-west Worm Meeting, 2004]

The B cell is a male-specific blast cell that divides to produce many of the cells that make up the male tail. The B cell divides asymmetrically during the L1 stage. The anterior daughter, B.a, is larger than the posterior daughter, B.p. The polarity of the asymmetric B cell division is controlled by LIN-44/Wnt and LIN-17/Fz, which also regulates the polarities of other cells in tail, including that of the TL and TR cells. However, mutation of some components that function to regulate T cell polarity, such as LIT-1, do not affect B cell polarity, suggesting that the pathways that regulate T and B cell polarities may differ. Recently, we demonstrated that a conserved Planar Cell Polarity-like pathway might regulate B cell polarity (M. Wu and M. Herman, unpublished). In order to identify additional components that affect B cell polarity, a screen for genes that affect B cell first asymmetric cell division was carried out. We screened over 7000 genomes and identified two lin-44 alleles, one mab-9 allele, and three alleles that may define new genes involved in the control of B cell polarity. We have begun characterization of one of these, lin(mh56) . While lin(mh56) mutants do not display T cell polarity defects, the B cell division is defective. Specifically, 30% of lin(mh56) males have B.a and B.p cells of equal size and in five percent B.p was larger than B.a (n=80), which is reversed from what is observed in wild-type males. In addition, lin(mh56) hermaphrodites have a small brood size, are slightly Dumpy and have blunt tails. 90% (n=70) of lin(mh56) males have abnormal tail morphologies with missing, fused or swollen rays. Furthermore, the spicules are crumpled or missing in 53% (n=74) of lin(mh56) males. We used Snip-SNP mapping to localize lin(mh56) to LG III and three factor crosses to further define its location to an interval of several cosmids on the physical map. Rescue experiments are currently underway.

J Liu et al. (1999) International C. elegans Meeting "Hox factors and other components in patterning of embryonic and postembryonic myogenic lineages"

J Liu, AZ Fire

[International C. elegans Meeting, 1999]

Bodywall muscles are derived from four of the embryonic founder cells as well as the postembryonic M blast cell. We are studying patterning of both the embryonic and postembryonic muscles in order to understand how myogenic cell fates are specified during development. As an entry point to study muscle patterning during embryonic development, we characterized enhancer elements from the hlh-1 regulatory region that are able to drive reporter expression in muscle precursors of the MS, D and C lineages. Analyses of these elements suggested regulatory roles of the Hox genes and the hlh-1 gene itself. These analyses also suggested regulatory mechanisms involving unknown bZIP, bHLH and novel transcription factors. We are currently in the process of genetically identifying these factors. The postembryonic M lineage provides an alternative model to study myogenic fate specification. The M lineage gives rise to 14 bodywall muscles, 2 coelomocytes and 16 sex muscles. A number of genes had previously been identified to function in patterning the M lineage, including the Hox gene mab-5 and the bHLH transcription factor twist (1, 2, 3). The role of the Hox factors in patterning the M lineage has been something of a mystery: mab-5 is expressed during the entire M lineage, but mab-5 mutants cause only limited lineage transformation. We will describe a series of experiments indicating a more central role for the Hox genes in activating twist and specifying the M lineage. We found that lin-39 mab-5 double mutants fail to activate twist and completely lack products of the M lineage. Expression (either ectopic or in a normal pattern) of either Hox gene is sufficient to activate twist expression. However, twist activation is not sufficient for specification of the M lineage. Current efforts are directed towards identification of other factors involved in specifying the M lineage. 1. Kenyon, C. (1986), Cell 46: 477-487 2. Harfe B. D., Vaz Gomes A., Kenyon C., Liu J., Krause M., Fire A. (1998) Genes & Dev. 12: 2623-2635 3. A. Corsi, S. Kostas, E. Jorgensen, A. Fire, and M. Krause (poster)

Marisa Foehr et al. (2007) International Worm Meeting "Dorsoventral patterning of the postembryonic mesoderm requires both LIN-12/Notch and TGF-beta signaling."

Marisa Foehr, Jun Liu

[International Worm Meeting, 2007]

The C. elegans post-embryonic mesodermal lineage arises from a single precursor cell, the M mesoblast, which will diversify to generate distinct dorsal and ventral cell types. The dorsal daughter of M gives rise to a subset of body wall muscles and two non-muscle coelomocytes, whereas the ventral daughter of M gives rise to two sex myoblasts in addition to a subset of body wall muscles. Mutations in the C. elegans Schnurri homolog sma-9 cause ventralization of the M lineage. We have previously shown that SMA-9 antagonizes the Sma/Mab TGF-beta signaling pathway to promote dorsal M lineage fates (Foehr et al., 2006). Interestingly, loss-of-function mutations in the Notch receptor homolog lin-12 cause dorsalization of the M lineage (Greenwald et al., 1983), an exact opposite phenotype of sma-9 mutants. We have found that while LIN-12 protein is present in both the dorsal and ventral M lineage cells, the ligands for LIN-12, LAG-2 and APX-1, are asymmetrically localized in cells adjacent to ventral M-derived cells, and they function redundantly in promoting ventral M lineage fates. To investigate how LIN-12/Notch signaling interacts with SMA-9 and the Sma/Mab TGF-beta pathway in regulating M lineage patterning, we generated double and triple mutant combinations among lin-12, sma-9 and dbl-1 (the ligand for the Sma/Mab TGF-beta pathway) and examined their M lineage phenotypes. Our results suggest that the LIN-12/Notch pathway and the Sma/Mab TGF-beta pathway function independently in regulating dorsoventral patterning of the M lineage, with LIN-12/Notch required for ventral M lineage fates, and SMA-9 antagonism of TGF-beta signaling required for dorsal M lineage fates. Our work provides a model for how combined Notch and TGF-beta signaling regulates the developmental potential of two equipotent cells along the dorsoventral axis.

Nirav Amin et al. (2008) Development & Evolution Meeting "Systematic analysis of transcription factors important in C. elegans postembryonic mesodermal development"

Zach Via, Jun Liu, Nirav Amin

[Development & Evolution Meeting, 2008]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on let-381, which encodes a forkhead transcription factor that is essential for C. elegans development. let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between let-381 and these pathways.

Fluet A et al. (1998) West Coast Worm Meeting "AGGREGATION AND TOXICITY OF HUMAN b-AMYLOID PEPTIDE VARIANTS EXPRESSED IN C. ELEGANS"

Fluet A, Johnson CJ, Fay DS, Link CD

[West Coast Worm Meeting, 1998]

We have engineered transgenic C. elegans that express the human b-amyloid peptide, which appears to play a central role in the pathology of Alzheimer's disease. As described previously, these worms, which express the peptide under the control of the unc-54 promoter, are unhealthy, have larval vacuoles, and exhibit a progressive paralysis. To better understand the molecular mechanism of b peptide toxicity, we would like to determine which, if any, of these phenotypes are specific to the expression of b peptide, and which are non-specific effects resulting from overexpression of a foreign peptide. To address this question we have sought to develop non-toxic b peptide variants, which should 'unmask' the non-specific phenotypes. We have specifically attempted to design b peptide variants that are incapable of aggregating into amyloid fibers, which have previously been reported to be directly correlated with b peptide toxicity. Single amino acid changes in the b(1-42) peptide have provided us with two interesting variants that both fail to produce amyloid aggregates: L17P and M35C. Intriguingly, multiple L17P transgenic lines all show the progressive paralysis observed in strains expressing wild-type b peptide. All M35C lines, however, are significantly healthier than the strains expressing wild-type b peptide. While the L17P variant may allow us to differentiate between b peptide's ability to form aggregates and its toxicity, the M35C variant is currently being used to differentiate between specific and non-specific toxicity resulting from b peptide expression. We have also isolated a second non-aggregating version of the b peptide, the single-chain b dimer. Like the M35C variant, this version of the b peptide does not aggregate and animals that express it are healthier than those expressing the wild-type b peptide. Quantitative immunoblot analyses on these strains demonstrate that the differences we see in aggregation of the variants are due to qualitative differences in the peptides and not quantitative differences in their levels of expression. These results also suggest that the progressive paralysis phenotype is specifically due to b peptide toxicity. We are currently using the non- or less-toxic b peptide variants in conjunction with the wild-type b peptide to look for molecular correlates of b peptide toxicity. We have preliminary results from immunoblots of Hsp16 expression that suggest that this heat shock protein is upregulated in response to b amyloid expression, and that this upregulation is significantly reduced in lines expressing the non-aggregating b dimer.

Tian, C. et al. (2009) International Worm Meeting "DRAG-1 is a cell type specific modulator of the Sma/Mab TGF-beta pathway in C. elegans."

Shi, H., Al-Barwani, A., Tian, C., Liu, J., Foehr, M., Sen, D., Lindy, A., Plavskin, Y.

[International Worm Meeting, 2009]

Members of the transforming growth factor b (TGF-b) superfamily play important roles during metazoan development, including cell fate specification, cell proliferation, cell migration and cell death. Mutations in the TGF-b pathway can lead to tumorigenesis and many other somatic and hereditary disorders. In C. elegans, the Sma/Mab TGF-b pathway regulates body size and male tail patterning. Previous studies from our lab have found that the Sma/Mab TGF-b pathway also regulates mesoderm development. In particular, mutations in the C2H2 zinc finger protein SMA-9 cause a dorsal to ventral fate transformation in the postembryonic mesodermal lineage, the M lineage, and this defect can be suppressed by mutations in all the core components of the Sma/Mab TGF-b pathway, suggesting that SMA-9 normally antagonizes the function of Sma/Mab TGF-b signaling in patterning the M lineage (Foehr et al., 2007). In the same sma-9 suppressor screen as described in Foehr et al. (2007), we identified a single locus, recessive sma-9 suppressor mutation drag-1(jj4). Like the Sma/Mab core pathway mutants, jj4 mutants are small and suppress the M lineage defects of sma-9 mutants. However, unlike the Sma/Mab pathway mutants, jj4 mutants do not have male tail defect. Genetic epistasis studies between jj4 and various mutations in the Sma/Mab pathway placed DRAG-1 in the Sma/Mab TGF-b pathway at the level of the ligand-receptor. We mapped and cloned the drag-1 gene and found that drag-1 encodes a putative GPI-anchored protein that is a homolog of the vertebrate BMP co-receptor Dragon. Dragon is a member of the RGM (Repulsive Guidance Molecule) family that is conserved in vertebrates and C. elegans, but not in Drosophila. We found that drag-1 is expressed in the same cells that express sma-6, one of the Sma/Mab TGF-b receptors, and that a functional DRAG-1::GFP fusion is localized outside of the nucleus. Deleting the putative GPI anchor region partially disrupted the function of drag-1, while artificially tethering DRAG-1 to the plasma membrane still allowed DRAG-1 to function. Taken together, our results suggest that DRAG-1 likely functions as a co-receptor in the Sma/Mab TGF-b pathway. We are currently determining whether or not DRAG-1 interacts with the ligand and/or receptors of the Sma/Mab pathway and assaying the functional significance of the interactions in vivo.

Yuan Jiang et al. (2004) East Coast Worm Meeting "MLS-2, an HMX class homeodomain protein essential for mesodermal patterning and cell fate specification"

Jun Kelly Liu, Yuan Jiang

[East Coast Worm Meeting, 2004]

We are interested in understanding mesodermal patterning and fate specification by studying the C. elegans postembryonic mesodermal lineage, the M lineage. The M lineage is derived from a single precursor cell, the M mesoblast, and gives rise to six cell types: striated bodywall muscles (BWMs), nonmuscle coelomocytes (CCs), and four classes of non-striated sex muscles which are descendants of the sex myoblasts (SMs). We are studying the function of the mls-2 (mesodermal lineage specification) gene in M lineage patterning and fate specification. The mls-2(cc615) mutation causes randomization of division planes in the M lineage, and subsequent fate transformation of CCs and BWMs to SMs. In addition, cc615mutants have defects in SM migration and show some larval and adult lethality. We have cloned the wild type mls-2 gene (C39E6.4). mls-2 encodes a homeodomain protein that belongs to the HMX family of homeodomain proteins that are also present in sea urchin, Drosophila and vertebrates., We examined the expression pattern of mls-2 using both functional mls-2::gfp fusion construct and affinity purified anti-MLS-2 antibodies. We found that the MLS-2 protein is localized in nuclei of early M lineage cells and a subset of head neurons. Furthermore, mls-2 expression in the M lineage and the head neurons appears to require distinct cis-acting elements. Overexpression of mls-2 in the early M lineage where mls-2 is normally expressed caused a variety of defects in the M lineage. Forced expression of mls-2 in the later M lineage such as in SMs where mls-2 is not normally expressed resulted in extra rounds of divisions of SMs. This suggests that mls-2 may have multiple roles in the M lineage and that MLS-2 protein level is critical for the correct patterning of the M lineage. The M lineage defects of mls-2(cc615) are very similar to those of mab-5, hlh-1, egl-27 and egl-20 mutants. We are currently carrying out molecular and genetic epistasis experiments to investigate the relationship between mls-2 and those factors.