James D McGhee et al. (2005) International Worm Meeting "Efficient specification of the C. elegans endoderm does not require zygotic expression of the med-1 and med-2 genes."

Barbara Goszczynski, James D McGhee

[International Worm Meeting, 2005]

The med-1 and med-2 genes encode a pair of essentially identical GATA factor- related transcription factors that have been proposed to be necessary for specification of the entire C. elegans endoderm (intestine or E lineage) as well as MS-derived mesoderm [1]. med-1 and med-2 are proposed to be the direct downstream targets and the principal effectors of the maternally provided SKN-1 transcription factor; med-1 and med-2 would thus occupy the pivotal interface between maternal and zygotic control of gene expression. The conclusion that med-1 and med-2 are essential for C. elegans endoderm specification was based on a partially-penetrant (~50%) loss of endoderm markers produced by RNA-mediated interference (RNAi) [1]. To determine whether this partial penetrance reflects: (i) inefficient RNAi against early zygotic transcripts; (ii) approximately the expected level of endoderm loss in skn-1 nulls, or; (iii) a further redundancy in the pathway of endoderm specification, we constructed worm strains that segregate embryos lacking both the med-1 gene (because of a gene-specific deletion) and the med-2 gene (using either of two overlapping chromosomal deficiencies). Contrary to expectations, we observe that the large majority (80 - 97 percent) of med-2(-); med-1(-) embryos still express markers of endoderm differentiation. Thus, we conclude that zygotic expression of the med-1 and med-2 genes are not necessary to specify the C. elegans endoderm. Our results could possibly suggest that the C. elegans endoderm can be efficiently specified by a new pathway involving a maternal contribution from the med genes and that (presumably) does not require SKN-1. However, any maternal contribution of the med genes has so far resisted conventional RNAi (maternal injection), under conditions where parallel injections with control dsRNAs (e.g. glp-1, let-413) are highly effective. 1.Maduro, M.F., Meneghini, M.D., Bowerman, B., Broitman-Maduro, G., and Rothman, J.H. (2001). Restriction of mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta homolog is mediated by MED-1 and -2 in C. elegans. Mol Cell 7, 475-485.

Lancaster, Brett et al. (2015) International Worm Meeting "Exploring the relation between transcription factor binding affinity and target gene activation."

McGhee, James, Lancaster, Brett

[International Worm Meeting, 2015]

We are developing a quantitative experimental system to investigate, inside a living animal, how transcription factor binding affinity determines the rate of target gene transcription. The zinc finger transcription factor ELT-2 binds to a 10 base pair (bp) motif with a core TGATAA sequence to regulate the majority (possibly all) of the genes transcribed in the post-specification intestine. The asp-1 gene encodes the major intestinal-specific aspartic protease of the worm and is under direct ELT-2 control; asp-1 transcript levels decrease ~50 fold in elt-2 null mutants and asp-1 has two TGATAA sites in its 1.4kb upstream regulatory promoter (both of which are necessary for transcription). Two reporters were constructed with different single silent mutations in the asp-1 coding sequence, each introducing a novel KpnI site at a unique position. One reporter is under transcriptional control of the wildtype asp-1 promoter, the other is under control of a variant asp-1 promoter in which the sequence of the two ELT-2 binding sites has been altered. Extrachromosomal multicopy transgenic arrays are created by co-injection of the two reporters in equimolar amounts and ensure that each transgene is exposed to the same cellular environment. The total RNA (containing transcripts for both reporters) is purified from a population of transgenic worms and reporter transcripts are reverse transcribed, amplified by PCR, and KpnI digested to reveal a diagnostic band pattern for each reporter following microfluidic electrophoresis. The band intensity is easily quantitated and should reflect in vivo transcript levels. Although TGATAA is the critical sequence on which transcription of asp-1 depends, the four nucleotides flanking each TGATAA (2bp upstream and 2bp downstream) contribute to the total transcriptional activity of the promoter. I show that the asp-1 promoter with TGATAA sites flanked by the nucleotides that appear least frequently in promoters of intestinally enriched or exclusive genes (CATGATAATC) produces 4.3plus_or_minus1.2 fold fewer transcripts than the wildtype promoter (p<0.01). This system provides the means to quantitate the transcriptional potential of any desired sequence, relative to the wildtype control in the same living animals. ELT-2 binding affinity to these sequences will be measured in vitro by competitive electrophoretic mobility shift assays or by fluorescence anisotropy. Also, ELT-2 binding kinetics are being examined in vivo by measuring the diffusion of fluorescently labelled ELT-2 protein at foci in the nucleus containing multiple copies of ELT-2 binding sequences (see poster by Y. Tyulenev et al.).

Lancaster, Brett et al. (2013) International Worm Meeting "A quantitative system to define the role of transcription factor binding affinity in transcriptional activation."

Lancaster, Brett, McGhee, James

[International Worm Meeting, 2013]

We are developing a quantitative experimental system to investigate, inside a living animal, how the affinity of a transcription factor for its cis-acting binding sites influences the rate of target gene transcription. In C. elegans, the zinc finger transcription factor ELT-2 binds to a core TGATAA DNA sequence to regulate the majority (possibly all) of the genes transcribed in the differentiating intestine. Our objective is to quantitatively measure the binding affinity of ELT-2 to a series of binding site variants (e.g. NTGATAAN), and, at the same time, measure the transcriptional output (mRNA) of the target gene with these variants in the promoter, all inside the living worm. The asp-1 gene encodes the major intestinal-specific aspartic protease of the worm, has two TGATAA sites in its 1kb promoter and is under direct ELT-2 control. We have constructed two versions of the asp-1 gene that differ only in the location of a KpnI site introduced by silent mutation. Reporter "A" is regulated by the wildtype asp-1 promoter; reporter "B" is regulated by a mutant reporter in which the wildtype CTGATAAG site has been replaced by, for example, an ATGATAAT site. Equimolar mixtures of the two constructs are introduced into worms (unc-119(-); asp-1(-); elt-4/7(-)) as multicopy transgenic arrays, which guarantees that each transgene, on average, will be exposed to the same cellular environment. RNA isolation followed by RT-PCR, KpnI digestion, and microfluidic gel electrophoresis (Agilent Bioanalyzer) yields distinct and easily quantitated profiles that allow us to estimate the levels of transcript produced by each reporter. Expression of a GFP reporter driven by the asp-1 1kb promoter requires both TGATAA sequences and I have quantitatively replicated this observation using this new system. In the future, binding affinity of purified ELT-2 protein to each binding site variant will be determined by fluorescence anisotropy. I will present our results on the reproducibility and dynamic range of the experimental system, together with possible limitations.

Wiesenfahrt, T. et al. (2017) International Worm Meeting "Coordination of cell shape changes of different cell types during C. elegans elongation."

Mains, Paul, Wiesenfahrt, T., McGhee, James

[International Worm Meeting, 2017]

We are interested in how cells coordinate cell shape changes during embryonic development. The main force during cell shape changes is provided by the actin-myosin network, a component of the cytoskeleton. As a model to analyse how the contractile activity of different cell types is coordinated, we use the elongation of the C. elegans embryo, the process in which the embryo changes from a ball of cells into a worm-like shape. During early elongation a subset of epidermal cells, the lateral cells, contract while the dorso-ventral cells take on a more passive role. We uncovered two parallel pathways that mediate embryonic elongation. Mutations of genes in these pathways can lead to either hypo-contraction (short worms) or hyper-contraction (burst embryos). We suspect that the dominant pathway drives contraction of lateral cells, while the second pathway drives contraction in the more passive dorso-ventral cells. The relative contraction of each set of cells should be altered differently in mutations affecting one or the other pathway. I measure the width of the lateral cells in different mutant backgrounds to test this model. Loss of RGA-2, a Rho GAP that inhibits contraction of dorso-ventral cells, leads to hyper-contraction, whereas loss of RHGF-2 a Rho GEF that triggers lateral contraction, leads to hypo-contraction. In rga-2; rhgf-2 double mutants, which is predicted to switch the contractile properties of the lateral vs. dorso-ventral cells, lateral cells indeed appear wider than in wild type. This implies that the lateral cells are contracting less and dorso-ventral cells are contracting more, even though the worm elongates to the same extent as wild type. In pak-1 mutants, which is predicted to show decreased dorso-ventral contraction, the dorso-ventral cells seem to lose their contractility leading to narrower lateral cells. We are currently using ajm-1::gfp strains to analyse elongation in real time and to see if the changes in lateral cell shape are retained post-embryonically in rga-2; rhgf-2 and pak-1 mutants. Cell shape changes often display a pulsatile behaviour and I tested if this is true for lateral cells during C. elegans elongation, but could find no evidence for pulsatile contractions.

Wiesenfahrt, Tobias et al. (2011) International Worm Meeting "Promoter Analysis of the GATA type transcription factor elt-2."

Berg, Janette, McGhee, James, Wiesenfahrt, Tobias

[International Worm Meeting, 2011]

We have suggested that the GATA type transcription factor ELT-2 is the major regulator of transcription in the C. elegans intestine following endoderm specification, both embryonically and post embryonically. To understand the molecular details of how elt-2 transcription is initiated during embryonic development and is maintained thereafter, we are analyzing the promoter region of elt-2 in C. elegans. Comparison of 5 kb upstream sequences of the elt-2 gene from 4 different Caenorhabditis species revealed three conserved regions (CRI- CRIII). Deletion series (both 3' and 5') as well as analysis of reporter constructs containing different combinations of the CRs suggested that CRI contains the basal promoter and CRIII contains the main enhancer of elt-2. The function of CRII is not yet clear. Rothman and Maduro have shown that the redundant GATA factors END-1 and END-3 are necessary for endoderm specification. Ectopic expression of END-1 can initiate ectopic expression of ELT-2, suggesting that END-1 can activate elt-2 expression in the earliest endoderm lineage. Previous experiments also showed that ELT-2 can bind to its own promoter in vivo. To find potential binding sites for END-1, END-3 and ELT-2, we searched for GATA sites within the CRs of elt-2. We identified 3, 3 and 4 conserved GATA sites within CRI, CRII and CRIII respectively. Band shift assays showed that END-1 and ELT-2 can bind to at least one and all four GATA sites within CRIII in vitro respectively. This suggests that END-1 (END-3 has not yet been tested) can activate elt-2 expression directly. Mutating the GATA sites within CRIII individually and in different combinations, suggested that every CRIII GATA site contributes positively to elt-2 expression. Reporter expression was absent in larvae and adult worms after mutating all GATA sites within CRIII. To test if elt-2 expression relies solely on GATA factors we have engineered a reporter construct consisting of CRIII and CRI in which all GATA sites have been mutated. Results will be presented. To test if ELT-2 can drive intestinal specification and differentiation in the absence of END-1/END-3, we expressed elt-2 under control of the end-1 and end-3 promoters in the end-1/end-3 double mutant (kindly provided by Morris Maduro). Indeed, the end-1p::elt-2 construct is able to rescue the end-1/end-3 double mutant with reasonable penetrance, showing that ELT-2 can drive endoderm specification and further supporting the hypothesis that ELT-2 is involved in the regulation of every gene expressed in the intestine.

Wiesenfahrt, Tobias et al. (2015) International Worm Meeting "ELT-2 can specify, differentiate and maintain the C. elegans intestine in the absence of the endoderm factors END-1,END-3, ELT-4 and ELT-7."

Berg, Janette, Lieb, Jason, Wiesenfahrt, Tobias, Osborne Nishimura, Erin, McGhee, James

[International Worm Meeting, 2015]

We are using the C. elegans intestine to test the flexibility of transcription factor networks. Can we switch factors around within a network and still produce a functional organ? The redundant GATA type transcription factors END-1 and END-3 are expressed from the 1-~8E cell stage and are necessary for endoderm development. Their main function may be to turn on a second pair of GATA type transcription factors, ELT-2 and ELT-7, at the 2-4E cell stage. ELT-2 may be the only necessary transcription factor in the post specification intestine. To determine how elt-2 expression is activated and maintained, we analyzed the 5kb upstream region of elt-2 revealing three conserved regions (CRI-CRIII), containing 3, 3 and 4 conserved GATA sites respectively. 3' and 5' deletion series and analysis of reporter constructs containing the CRs in different combinations show that all CRs contribute positively to elt-2 expression. Only CRIII is able to drive reporter expression by itself from embryos to adults and thus seems to be the most important CR. The conserved GATA sites were the only necessary transcription factor binding sites we were able to identify. Band shift assays showed that END-1, ELT-7 and ELT-2 can bind to all four GATA sites within CRIII in vitro suggesting that END-1 and ELT-7 can activate elt-2 expression directly and that ELT-7 and ELT-2 seem to be directly involved in maintenance of elt-2 expression. CHIP analysis using an ELT-2 monoclonal antibody revealed that ELT-2 binds to all three CRs. To test if ELT-2 can drive intestinal specification, differentiation and maintenance in the absence of END-1/END-3/ELT-4/ELT-7, we expressed elt-2 under control of the end-1 promoter in an end-1/end-3/elt-4/elt-7 quadruple mutant. The end-1p::elt-2 construct is able to rescue the end-1/end-3/elt-4/elt-7 quadruple mutant with >80% penetrance as a multicopy integrated array. The rescued quadruple mutant shows a surprisingly mild phenotype: an embryonic and larval lethality rate of ~3% and ~12% respectively, a reduction of brood size and a slight delay in gastrulation and overall development. An integrated single copy of the end-1p::elt-2 construct is not able to specify the E cell in the absence of end-1/end-3, suggesting that ELT-2 has to be overexpressed to compensate for the loss of end-1/end-3. Our results show that the intestinal transcription factor network is surprisingly flexible: the endoderm differentiation factor ELT-2 can drive endoderm specification by itself and in these experimental conditions is the only GATA type transcription factor necessary for intestinal development.

Kalb, John M. et al. (2009) International Worm Meeting "elt-2 and sbp-1 are the only two transcription factors required in the intestine for viability after endoderm specification."

Nowak, Jessica, Kalb, John M., Larson, Renee A., McGhee, James D.

[International Worm Meeting, 2009]

Our current model is that the GATA-factor ELT-2 is the predominant transcription factor controlling gene expression in the C. elegans intestine following endoderm specification. We are interested if other transcription factors besides ELT-2 have critical roles in the intestine. We have identified roughly 300 different transcription factors in SAGE libraries prepared from FACS-sorted cells of the embryonic intestine. The loss of function of 32 of these transcription factors results in embryonic lethal or larval lethal phenotypes, but their specific requirements in the intestine are difficult to ascertain because these genes may also be expressed in non-intestinal tissues. In order to examine their intestinal function, we used a strain of worms that is RNAi sensitive only in the intestine (OLB11, kindly provided by Olaf Bossinger). OLB11 worms are deficient in RNAi due to a mutation in the rde-1 gene, but have intestinal RNAi rescued by an integrated array containing rde-1 cDNA controlled by the intestinal-specific promoter of the elt-2 gene. Control experiments show the strain behaves as expected: embryos and larvae of injected mothers are completely resistant to RNAi effects of genes expressed exclusively or predominantly outside of the intestine but are sensitive to RNAi against genes expressed exclusively or predominantly in the intestine. Double stranded RNA corresponding to each of the 32 intestinally expressed transcription factors were injected into OLB11 worms and their progeny were scored for viability and growth rates. Intestinal-specific RNAi against only two transcription factors, elt-2 and sbp-1, results in larval lethality, with injected progeny arresting as L1 larvae, matching the RNAi effects of these genes in wild type worms. Intestinal RNAi against four genes, F57B10.1 (let-607), C16A3.4, F23B12.7 and dve-1 causes a slow growth phenotype; however, these worms develop into fertile adults with no apparent defects in the structure of the intestine. Intestinal RNAi against the other 26 transcription factors does not affect viability or growth rates. Currently we are using the OLB11 worms to assess the intestinal function of some of these transcription factors in regulating aging. So far we have determined loss of intestinal skn-1 (RNAi performed by feeding) results in a shortened life span, and we are presently looking at other genes, such as pha-4, in this process.

Wiesenfahrt, Tobias et al. (2017) International Worm Meeting "What regulates the regulators? The C. elegans endoderm, the ELT-2 GATA factor and the chromatin environment."

Duanmu, Jingjie, Flibotte, Stephane, Wiesenfahrt, Tobias, Moerman, Don, Mains, Paul, McGhee, James

[International Worm Meeting, 2017]

We are using the C. elegans intestine to understand the robustness and flexibility of transcription factor networks and how these are influenced by the chromatin environment. The C. elegans endoderm develops under control of a series of partly redundant GATA-type transcription factors: END-3 > END-1 > ELT-7 > ELT-2. ELT-2 is the predominant transcription factor driving intestine differentiation and function, but is not normally involved in endoderm specification. We have recently shown (to our surprise) that ELT-2 can replace all other intestine GATA factors, i.e. can actually specify the intestine, if it is expressed under the end-1 promoter in addition to its own promoter. However, multiple copies of transgenic end-1p::elt-2 are more efficient at specifying the endoderm (88% rescue) than a single copy MosSCI insertion (<1% rescue). In a genetic screen, we mutagenized approximately 14,000 genomes with EMS and identified 17 strains with enhanced ability of a single copy end-1p::elt-2 to specify endoderm. The starting end-1(-) end-3(-) strain, with a single copy insert of end-1p::elt-2, was rescued by an extra-chromosomal array carrying multiple copies of the end-1p::elt-2 together with a heat shock inducible peel-1 construct. Thus we were able to kill all worms that still relied on the extra-chromosomal array for their survival after the EMS treatment. Whole genomic sequences of these independent strains provide a short list of potential candidate genes. Two such candidate genes that were independently mutated in multiple strains have been verified by RNAi or CRISPR: K01G5.9 (4 independent mutations), encoding a Taspase 1 homologue and pqn-82 (2 independent mutations), encoding a homologue of a S. cervisiae SWI/SNF subunit. Taspase appears to be involved in the proteolytic processing of general transcription factors such as TFIIA and trithorax. The SWI/SNF complex appears to be involved in chromatin remodeling and transcriptional regulation. Disrupting the function of these two candidate genes shows a twofold enhancement of ELT-2 levels in the early endoderm relative to the unmutagenized strain, resulting in 50% rescue rate for pqn-82 and 7% rescue rate for K01G5.9. We are currently working on a method to identify the mutated genes in the remaining 11 candidate strains that were hit only once. Thus, this genetic screen could be revealing a set of general chromatin proteins that control the levels of specific transcription factors, such as ELT-2, in the early embryo.

Dalpe, Gratien et al. (2009) International Worm Meeting "PVF-1, a C. elegans homologue of VEGF, functions in male ray positioning."

Tarsitano, Marina, Zhang, Lijia W., Persico, Graziella, Dalpe, Gratien, Culotti, Joseph, McGhee, James D., Zheng, Hong

[International Worm Meeting, 2009]

VEGF is required for both proliferation and chemotaxis of endothelial cells. Although an extensive amount of work describes VEGF''s role in proliferation, its cell migration function is still not understood. In order to understand VEGF''s migration function, we analyzed its single C. elegans homologue PVF-1. We have previously shown that PVF-1 is a secreted protein that has similar biochemical properties to VEGF (Tarsitano et al., 2006). PVF-1 has the ability to bind to VEGFr1 and VEGFr2 and to induce capillary tube formation in HUVEC and neovascularization in chicken embryos. In order to determine the function of PVF-1 in C. elegans we isolated a deletion mutant allele pvf-1 (ev763) that is predicted to be a molecular null. We find that pvf-1 (ev763) mutants do not have any obvious phenotypes. However, pvf-1 mutations enhance the ray displacement phenotype observed in semaphorin-1a/b and plexin-1 mutants (Dalpe et al., 2004; Ginzburg et al., 2002) suggesting PVF-1 has a guidance function in a pathway parallel to Semaphorin-1 mediated signaling. Moreover, using GFP reporter constructs and an antibody raised against PVF-1, we find PVF-1 expression in body wall muscles. Transgenic experiments using a myo-3 promoter driven pvf-1 cDNA rescues the ray positioning phenotype observed in pvf-1;plx-1 double mutants, confirming that body wall muscle expression is sufficient for its function. However, we also obtained similar rescues with constructs using an hsp-16/2 or a lin-44 promoter driving pvf-1 cDNA expression, suggesting that PVF-1 is not an instructive guidance cue for male ray positioning. In the end, we find that a myo-3 promoter driven mouse vegf164 cDNA can perfectly rescue the male ray positioning phenotype of pvf-1;plx-1 double mutants, indicating a functional evolutionary conservation between PVF-1 and mammalian VEGF. All together, the data indicate that PVF-1 is a C. elegans VEGF homologue that functions in male ray cell positioning. Body wall muscle secreted PVF-1 guides ray positioning by functioning in a signaling pathway parallel to the Semaphorin-1/Plexin-1 system although these data do not exclude the possibility that it also functions in the Semaphorin-1 mediated signaling pathway. This study suggests that C. elegans could provide a simpler model system for investigating the role of VEGF in cell migration.

JD Kormish et al. (1999) International C. elegans Meeting "A genetic screen for genes involved in gut development and differentiation"

JD McGhee, JD Kormish

[International C. elegans Meeting, 1999]

To investigate genes potentially involved in gut development and differentiation, we are developing a genetic screen for gut obstructed (gob) mutants. The gut specific elt-2 GATA factor appears necessary for correct gut cell development. elt-2 null mutants arrest as L1s with a blocked intestinal lumen and abnormal brush border (1). When elt-2 null mutants are fed on a mixture of fluorescent beads and Escherichia coli , the beads collect as a large "gob" in the posterior pharynx and anterior gut that can be easily detected under the dissecting microscope. We are now using this gut obstructed phenotype to screen for mutants that should potentially identify genes involved in gut development. Preliminary results of this screen are promising, as several candidates have already been isolated. 1) Fukushige T, Hawkins MG and McGhee JD (1998) Dev Biol 198(2):286-302