Fukushige, Tetsunari et al. (2015) International Worm Meeting "Sequential Muscle Cell Fate Determination by Overlapping Functions of Different Transcription Factors in the Descendants of the Anterior Mesoderm Blastomere (MS)."

Fukushige, Tetsunari, Kersey, Rossio, Krause, Micheal

[International Worm Meeting, 2015]

During C.elegans embryonic development, 81 bodywall muscle (BWM) cells are born from four of the six different founder blastomeres (AB, MS, C, D) formed by the first several cell divisions after fertilization. Interestingly, the cascade of factors specifying the BWM fate is different among some of these blastomeres. The Caudal-like transcription factor, PAL-1 combined with a low concentration (or absence) of nuclear POP-1induces muscle cells from posterior blastomeres C and D by directly activating the muscle regulatory factors (MRF: hlh-1/myoD, unc-120/MADS-box and hnd-1/Hand) (1). The regulation of MS BWM fate occurs through a quite different transcriptional cascade, although eventually converging on a similar set of MRFs. Previous studies (2, 3) showed that med-1,2 and tbx-35 genes that are commonly expressed in MS daughter and grand-daughter cells, have overlapping functions to establish the MS cell fate. Furthermore, a ceh-51 mutant by itself does not show any obvious developmental defect in the early MS cell fate specification, but the ceh-51 mutant enhances the mutant phenotype of tbx-35 or MRF gene mutants. We further analyze the temporal and spatial relationship between early MS lineage transcription factors (TBX-35 and CEH-51) and the MRFs for the embryonic muscle cell fate determination. Our results demonstrate a sequential, combinatorial, and variant cascade of transcription factors are used to specify the bodywall muscle cell fate in the MS lineages. These various pathways eventually converge on the activations of hlh-1, defining cells committed to the BWM fate. Understanding this transcriptional cascade has allowed us to manipulate early embryos genetically to produce predominantly MS-like blastomeres that have been profiled for gene expression. We have explored this temporal transcriptional profile to uncover new transcription factors influencing MS muscle cell fates.(1) Fukushige T., et al. Genes Dev., (2006). (2) Maduro MF., et al. Mol. Cell, (2001). (3) Broitman-Maduro G., et al. Development, (2009).

Tetsunari Fukushige et al. (2004) East Coast Worm Meeting "CeMyoD(hlh-1) in embryonic muscle fate determination."

Tetsunari Fukushige, Thomas Brodigan, Michael Krause, Joan McDermott

[East Coast Worm Meeting, 2004]

The MyoD family of basic helix-loop-helix transcription factors regulates skeletal muscle formation and differentiation in mammals. The lone C. elegans member of this family, CeMyoD (encoded by hlh-1 ), is the earliest known factor identified in the body wall muscle cell lineage consistent with an important role in fate specification. However, mutant analysis has shown that CeMyoD activity is not required for body wall muscle formation in C. elegans. The correct number of embryonic muscle cells (81) are formed in hlh-1 loss-of-function mutants and many terminal muscle markers are expressed at apparently normal levels. These mutant animals do have a severe muscle phenotype; hlh-1 mutant muscle functions poorly and results in severe morphogenesis defects and larval lethality. To test if CeMyoD is sufficient for muscle cell fate determination, we generated an integrated transgenic worm carrying a heat-shock promoter:: hlh-1 construct. Ectopically expressed hlh-1 in the early stages of embryogenesis results in widespread conversion of many embryonic cell lineages to muscle as assayed by terminal muscle markers. Markers for other cell fates, such as 3ENB12 (pharyngeal cells), ELT-2 (gut cells) and LIN-26 (hypodermal cells), are not detected in embryos strongly expressing ectopic hlh-1 . These results demonstrate that CeMyoD alone is sufficient to activate the striated muscle cell fate. We have begun to address how the hlh-1 gene is activated during embryogenesis. The early presence of CeMyoD protein in cells restricted to the body wall muscle fate suggests that maternal factors may be regulating hlh-1 expression. This is particularly true in the C and D lineages in which formation of the founder cell is only one or two divisions prior to the onset of CeMyoD accumulation. Using RNAi of maternal components important for early cell determination, we are trying to identify factors that are necessary and sufficient to activate hlh-1 .

Tetsunari Fukushige et al. (2001) International C. elegans Meeting "elt-4, the worm's (and possibly the world's) smallest GATA factor."

Jim McGhee, Helen Tian, Tetsunari Fukushige, Barbara Goszczynski

[International C. elegans Meeting, 2001]

Tetsunari Fukushige et al. (2005) International Worm Meeting "The myogenic potency of HLH-1 reveals wide-spread developmental plasticity in early C. elegans embryos"

Tetsunari Fukushige, Robert Littlejohn, Michael Krause, Thomas M Brodigan

[International Worm Meeting, 2005]

In vertebrates, striated muscle development depends on both the expression of members of the myogenic regulatory factor family (MRFs) and on extrinsic cellular cues, including Wnt signaling. The 81 embryonically born body wall muscle cells in C. elegans are comparable to the striated muscle of vertebrates. These muscle cells all express the gene hlh-1, encoding HLH-1 (CeMyoD) which is the only MRF-related factor in the nematode. However, genetic studies have shown that body wall muscle development occurs in the absence of HLH-1 activity, making the role of this factor in nematode myogenesis unclear. By ectopically expressing hlh-1 in early blastomeres of the C. elegans embryo, we show that CeMyoD is a bona fide MRF that can convert almost all cells to a muscle-like fate, regardless of their lineage of origin. The window during which ectopic HLH-1 can function is surprisingly broad, spanning the first 3 hours of development when cell lineages are normally established and non-muscle cell fate markers begin to be expressed. We have begun to explore the maternal factors controlling zygotic hlh-1 expression. We find that the Caudal-related homeobox factor PAL-1 can activate hlh-1 in blastomeres that either lack POP-1/TCF or that have down-regulated POP-1/TCF in response to Wnt/MAP kinase signaling. The potent myogenic activity of HLH-1 highlights the remarkable developmental plasticity of early C. elegans blastomeres and reveals the evolutionary conservation of MyoD function.

Lei, Haiyan et al. (2009) International Worm Meeting "Defining the embryonic muscle gene regulatory network using chromatin immunoprecipitation and an analysis of potential cis-acting elements."

Fukushige, Tetsunari, Lei, Haiyan, Krause, Michael

[International Worm Meeting, 2009]

Previous work in C. elegans has shown that posterior embryonic body wall muscle lineages are regulated through a genetically defined transcriptional cascade that includes PAL-1/Caudal activation of muscle-specific transcription factors, including HLH-1/MRF and UNC-120/SRF, which together orchestrate specification and differentiation. Using chromatin immunoprecipitation (ChIP) in embryos, we recently demonstrated direct binding of PAL-1 in vivo to an hlh-1 enhancer element (Lei et al., 2009). Through mutational analysis of the evolutionarily conserved sequences within this enhancer, we identify two cis-acting elements and their associated trans-acting factors (PAL-1 and HLH-1) that are critical for the temporal-spatial expression of hlh-1 and proper myogenesis. Our data demonstrates that hlh-1 is indeed a direct target of PAL-1 in the posterior embryonic C. elegans muscle lineages, defining a novel in vivo binding site for this critical developmental regulator. We find that the same enhancer element is also a target of HLH-1 positive auto regulation, underlying (at least in part) the sustained high levels of CeMyoD in bodywall muscle throughout development. In order to further identify the embryonic muscle gene regulatory network, we combined HLH-1 ChIP with whole-genome tiling arrays to identify binding sites throughout the genome. We are currently validating our ChIP-chip data with transgenic reporters. These types of approaches will provide a molecular framework for the gene regulatory network activating the muscle module during embryogenesis.

Konzman, Daniel et al. (2021) International Worm Meeting "The role of O-GlcNAc in fertility of C. elegans males"

Konzman, Daniel, Hanover, John, Krause, Michael, Fukushige, Tetsunari

[International Worm Meeting, 2021]

Mutations in the gene encoding O-GlcNAc transferase lead to embryonic lethality or a severe form of X-linked intellectual disability in humans. This disorder presents with developmental delay, low IQ, and several other developmental defects. To model this disorder, we turned to C. elegans, unique in model organisms in its fully mapped development and in that worms survive without O-GlcNAc. This post-translational protein modification is involved in diverse biological processes including nutrient signaling, signaling, and development. Deletion of the O-GlcNAc transferase gene (ogt-1) results in C. elegans males with reduced ability to successfully mate. This phenotype likely impacts signaling or development and may provide insights into which pathways are involved in this disorder. Compared to wild-type male controls, ogt-1 males have a four-fold reduction in mean offspring, with nearly two thirds failing to produce any offspring at all. Fluorescent sperm tracking assays revealed that ogt-1 males transfer fewer sperm to their mates. We also determined that ogt-1 males show an increased incidence of developmental defects in male tail structures important to mating. Together, these data suggest a defect in mating behavior is the most likely explanation for the phenotype. In support of this, ogt-1 males are less likely than wild-type males to initiate mating when presented with mates. Compared with wild-type males, ogt-1 males are less likely to actively search for mates when isolated from other worms. This indicates these males have an imbalance between mating drive and food-seeking behavior. The genetic amenability of C. elegans allows incorporation of transgenes, which we have exploited to determine the tissue-specific effects of ogt-1 on the mating phenotype. As a positive control, we injected a wild-type copy of ogt-1 into deletion worms and demonstrated it was sufficient to rescue both the lost enzymatic activity and male infertility associated with ogt-1 deletion. Fertility was also rescued by expressing ogt-1 in a hypodermis-specific manner, which further implicates the development of the male copulatory organ, which is largely derived from the hypodermal lineage. Alternatively, the infertility may arise from disruption of signaling pathways within the hypodermis. This study reveals a crucial role for O-GlcNAc in complex developmental and behavioral processes in the C. elegans male. Genetic and molecular studies will further define the developmental and neuronal mechanisms impacted by O-GlcNAc.

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

Gogonea CB et al. (1997) International C. elegans Meeting "EXPRESSION OF THE REPORTER GENE MEC-12::LACZ IN LOCOMOTORY AND CHEMOSENSORY MUTANTS OF C. ELEGANS"

Siddiqui ZK, Siddiqui SS, Gogonea CB

[International C. elegans Meeting, 1997]

Mutations in close to 100 genes affect locomotion. These mutants show uncoordinated locomotion, ranging from slow movement, poor backing, colier, kinkier, and paralyzed phenes (J. Hodgkin, 1997: Appendix in C. elegans II). Previously it was shown that mutants in at least 25 genes harbor defects in the axonal growth and process placement of the six touch receptor neurons (Siddiqui, 1990). A reporter gene mec-12::lacZ has been shown to express in the set of six touch receptor neurons (Fukushige et al., 1997). So far, a variety of cellular defects in the process placement, axonal guidance and axonal growth of the touch cells and the PVR in 15 genes (unc-4, unc-5,unc-30, unc-33, unc-44, unc-51, unc-53, unc-55, unc-61, unc-71, unc-76, unc-83, unc-86, and unc-119) have been observed. Defects include ectopic branching of axons, fore- shortened processes, misplaced cell body positions, extra commissures, and a lack of staining. In addition, we have examined the reporter gene expression in the background of various mechano- sensory mutations (Chalfie and Au, 1989). These results will be summarized. We thank the Caenorhabdtis Genetics Center, and Martin Chalfie for various mutant strains.

Marshall SD et al. (1998) West Coast Worm Meeting "REGULATION OF THE ges-1 GENE FROM THE NEMATODE Caenorhabditis briggsae"

Marshall SD, McGhee JD

[West Coast Worm Meeting, 1998]

The ges-1 gene has been used to investigate how a particular gene can be expressed in only a subset of cells within a complex multicellular organism. The C. elegans ges-1 (Ce-ges-1) gene is only expressed in the E lineage (i.e. gut) of wildtype embryos. Expression is first detected when the gut lineage has only 4-8 cells and the embryo has -150 cells. Previous analysis of the Ce-ges-1 regulatory regions uncovered a gut activator-pharynx/rectum repressor regulatory switch: deletion of a 36 bp fragment containing two tandem WGATAR sites from the upstream Ce-ges-1 promoter switches expression off in the gut and on in the pharynx and rectum (Aamodt et. al., Science, 1991; Egan et al., Dev. Biol. 1995). Experiments performed by Fukushige et al. (Dev. Biol. 1996) have shown that this gut-to-pharynx/rectum switch changes Ce-ges-1 expression from the E lineage into portions of the digestive tract derived from the ABa (anterior pharynx), MS (posterior pharynx), and ABp (rectum) lineages. Further deletions have identified a putative pharynx activator element within 68 bp upstream from the ges-1 initiator methionine (T. Fukushige, unpublished data). A ges-1 homologue from the closely related nematode, C. briggsae, is also expressed in the gut (Kennedy et al., Dev. biol. 1992). Study of the C. briggsae ges-1 (Cb-ges-1) regulatory regions has also revealed a gut to pharynx/rectum regulatory switch, similar to that found for Ce-ges-1 regulation. A tandem pair of WGATA R sites, located ~700 bp upstream from the Cb-ges-1 translational start site, appear to regulate proper Cb-ges-1 gut expression. Additionally, a 90 bp region containing a putative Cb-ges-1 pharynx/rectum activator element is located downstream from the poly(A) signal site. Comparison of the Cb-ges-1 and Ce-ges-1 pharynx activator element does not reveal any obvious sequence or functional conservation between the Cb-ges-1 and Ce-ges-1 pharynx activator elements. However, alignment of the newly finished Cb-ges-1 sequence with Ce-ges-1 reveals a high degree of sequence conservation at the 3' end of both ges-1 genes. Our main conclusion is that the Cb-ges-1 gene, like its Ce-ges-1 counterpart, is capable of switching from expression in the gut to expression in the pharynx and rectum. In both genes, the control of this switch centres on a tandem pair of WGATAR sites. Details of how the expression switch actually work are not yet known but may well differ in the two nematodes.

Jie Yan et al. (2005) International Worm Meeting "Patterning of pho-1 Expression in the C. elegans Intestine"

Tetsunari Fukushige, Jie Yan, Barbara Goszczynski, Jim McGhee

[International Worm Meeting, 2005]

The pho-1 gene encodes a major C. elegans intestinal acid phosphatase, and shows an unexpected maternal lethal effect [1]. PHO-1 activity, as detected by histochemical staining, is present in all the gut cells except the most anterior six cells. Transgenic pho-1::GFP/lacZ reporter constructs are expressed in a similar pattern, indicating that anterior-posterior intestinal patterning is regulated at the level of transcription. We have shown that only one of the three GATA sites in the pho-1 promoter is necessary to activate the reporter gene and pho-1 appears to be a direct target of the ELT-2 GATA factor. We have implicated a zygotic application of the Wnt pathway as important for pho-1 anterior-posterior patterning; our results can be interpreted as if a high concentration of POP-1 represses pho-1 in the intestine anterior. Because there are no obvious POP-1 binding sites in the pho-1 promoter, we suspect that the Wnt pathway regulation is indirect. We are now analyzing the pho-1 promoter in the hope of identifying cis-acting sequences that could be directly involved in pho-1 patterning. pho-1 and its upstream region are quite highly conserved in C. briggsae, and we are using such sequence alignments to identify candidate regulatory sites. [1] Fukushige, et al. (2005) Developmental Biology 279(2):446-61