Von Stetina SE MSE. PAR-6, but not E-cadherin and β-integrin, is necessary for epithelial polarization in C. elegans. Developmental Biology [Internet]. 2015;403(1):5-14. Publisher's VersionAbstract

Cell polarity is a fundamental characteristic of epithelial cells. Classical cell biological studies have suggested that establishment and orientation of polarized epithelia depend on outside-in cues that derive from interactions with either neighboring cells or the substratum (Akhtar and Streuli, 2013; Chen and Zhang, 2013; Chung and Andrew, 2008; McNeill et al., 1990; Nejsum and Nelson, 2007; Nelson et al., 2013; Ojakian and Schwimmer, 1994; Wang et al., 1990; Yu et al., 2005). This paradigm has been challenged by examples of epithelia generated in the absence of molecules that mediate cell-cell or cell-matrix interactions, notably E-cadherin and integrins (Baas et al., 2004; Choi et al., 2013; Costa et al., 1998; Harris and Peifer, 2004; Raich et al., 1999; Roote and Zusman, 1995; Vestweber et al., 1985; Williams and Waterston, 1994; Wu et al., 2009). Here we explore an alternative hypothesis, that cadherins and integrins function redundantly to substitute for one another during epithelium formation (Martinez-Rico et al., 2010; Ojakian et al., 2001; Rudkouskaya et al., 2014; Weber et al., 2011). We use C. elegans, which possesses a single E-cadherin (Costa et al., 1998; Hardin et al., 2013; Tepass, 1999) and a single β-integrin (Gettner et al., 1995; Lee et al., 2001), and analyze the arcade cells, which generate an epithelium late in embryogenesis (Portereiko and Mango, 2001; Portereiko et al., 2004), after most maternal factors are depleted. Loss of E-cadherin(HMR-1) in combination with β-integrin(PAT-3) had no impact on the onset or formation of the arcade cell epithelium, nor the epidermis or digestive tract. Moreover, ß-integrin(PAT-3) was not enriched at the basal surface of the arcades, and the candidate PAT-3 binding partner β-laminin(LAM-1) was not detected until after arcade cell polarity was established and exhibited no obvious polarity defect when mutated. Instead, the polarity protein par-6 (Chen and Zhang, 2013; Watts et al., 1996) was required to polarize the arcade cells, and par-6 mutants exhibited mislocalized or absent apical and junctional proteins. We conclude that the arcade cell epithelium polarizes by a PAR-6-mediated pathway that is independent of E-cadherin, β-integrin and β-laminin.

Hsu, H.-T. CBYZLLMH-MA. Recruitment of RNA Polymerase II by the pioneer transcription factor PHA-4. Science [Internet]. 2015;348(6241):1372-1376. Publisher's VersionAbstract
Choi Y MSE. Hunting for Darwin's gemmules and Lamarck's fluid: transgenerational signaling and histone methylation. Biochimica et Biophysica Acta [Internet]. 2014;1839(12):1440-53. Publisher's VersionAbstract

Recent studies have discovered phenotypes induced by a transient treatment or mutation that persist for multiple generations without mutations in DNA. Both invertebrates and vertebrates exhibit such inheritance, and a range of environmental factors can act as a trigger. Now referred to as transgenerational epigenetic inheritance or TEI, this emerging field faces a big challenge-what molecular mechanisms account for inheritance of TEI phenotypes? This review describes examples of TEI and focuses on the possible role of histone methylation and small RNAs in mediating TEI.

Rosains J, Mango SE. Genetic characterization of smg-8 mutants reveals no role in C. elegans nonsense mediated decay. PLoS One. 2012;7(11).Abstract
The nonsense mediated decay (NMD) pathway degrades mRNAs bearing premature translation termination codons. In mammals, SMG-8 has been implicated in the NMD pathway, in part by its association with SMG-1 kinase. Here we use four independent assays to show that C. elegans smg-8 is not required to degrade nonsense-containing mRNAs. We examine the genetic requirement for smg-8 to destabilize the endogenous, natural NMD targets produced by alternative splicing of rpl-7a and rpl-12. We test smg-8 for degradation of the endogenous, NMD target generated by unc-54(r293), which lacks a normal polyadenylation site. We probe the effect of smg-8 on the exogenous NMD target produced by myo-3::GFP, which carries a long 3′ untranslated region that destabilizes mRNAs. None of these known NMD targets is influenced by smg-8 mutations. In addition, smg-8 animals lack classical Smg mutant phenotypes such as a reduced brood size or abnormal vulva. We conclude that smg-8 is unlikely to encode a component critical for NMD.
Mango SE. Ageing: generations of longevity. Nature. 2011;479(7373):302-3.
Meister P, Mango SE, Gasser SM. Locking the genome: nuclear organization and cell fate. Current opinion in genetics & development. 2011;21:167–74.Abstract

The differentiation of pluripotent or totipotent cells into various differentiated cell types is accompanied by a restriction of gene expression patterns, alteration in histone and DNA methylation, and changes in the gross nuclear organization of eu- and heterochromatic domains. Several recent studies have coupled genome-wide mapping of histone modifications with changes in gene expression. Other studies have examined changes in the subnuclear positioning of tissue-specific genes upon transcriptional induction or repression. Here we summarize intriguing correlations of the three phenomena, which suggest that in some cases causal relationships may exist.

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Fakhouri THI, Stevenson J, Chisholm AD, Mango SE. Dynamic chromatin organization during foregut development mediated by the organ selector gene PHA-4/FoxA. PLoS genetics. 2010;6(8).Abstract

Central regulators of cell fate, or selector genes, establish the identity of cells by direct regulation of large cohorts of genes. In Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different sets of target genes at various times and in diverse cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. We have used the Nuclear Spot Assay and GFP reporters to examine PHA-4 interactions with target promoters in living embryos and with single cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen of candidate nuclear factors identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx, but emr-1 did not modulate PHA-4 binding in the intestine. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, may facilitate promoter access and productive transcription. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells, preventing target gene expression in that organ. PHA-4 binding within the pharynx is limited by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.

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Zhong M, Niu W, Lu ZJ, Sarov M, Murray JI, Janette J, Raha D, Sheaffer KL, Lam HYK, Preston E, et al. Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response. PLoS genetics. 2010;6(2):e1000848.Abstract

Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.

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Mango S. Q & A: Susan Mango. Current Biology. 2009;19(7):R276-R277.Abstract
Mango SE. The molecular basis of organ formation: insights from the C. elegans foregut. Annual review of cell and developmental biology [Internet]. 2009;25:597-628. View OnlineAbstract

The digestive tracts of many animals are epithelial tubes with specialized compartments to break down food, remove wastes, combat infection, and signal nutrient availability. C. elegans possesses a linear, epithelial gut tube with foregut, midgut, and hindgut sections. The simple anatomy belies the developmental complexity that is involved in forming the gut from a pool of heterogeneous precursor cells. Here, I focus on the processes that specify cell fates and control morphogenesis within the embryonic foregut (pharynx) and the developmental roles of the pharynx after birth. Maternally donated factors in the pregastrula embryo converge on pha-4, a FoxA transcription factor that specifies organ identity for pharyngeal precursors. Positive feedback loops between PHA-4 and other transcription factors ensure commitment to pharyngeal fate. Binding-site affinity of PHA-4 for its target promoters contributes to the progression of the pharyngeal precursors towards differentiation. During morphogenesis, the pharyngeal precursors form an epithelial tube in a process that is independent of cadherins, catenins, and integrins but requires the kinesin zen-4/MKLP1. After birth, the pharynx and/or pha-4 are involved in repelling pathogens and controlling aging.

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Yuzyuk T, Fakhouri THI, Kiefer J, Mango SE. The polycomb complex protein mes-2/E(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos. Developmental cell. 2009;16(5):699-710.Abstract

We have used expression profiling and in vivo imaging to characterize Caenorhabditis elegans embryos as they transit from a developmentally plastic state to the onset of differentiation. Normally, this transition is accompanied by activation of developmental regulators and differentiation genes, downregulation of early-expressed genes, and large-scale reorganization of chromatin. We find that loss of plasticity and differentiation onset depends on the Polycomb complex protein mes-2/E(Z). mes-2 mutants display prolonged developmental plasticity in response to heterologous developmental regulators. Early-expressed genes remain active, differentiation genes fail to reach wild-type levels, and chromatin retains a decompacted morphology in mes-2 mutants. By contrast, loss of the developmental regulators pha-4/FoxA or end-1/GATA does not prolong plasticity. This study establishes a model by which to analyze developmental plasticity within an intact embryo. mes-2 orchestrates large-scale changes in chromatin organization and gene expression to promote the timely loss of developmental plasticity. Our findings indicate that loss of plasticity can be uncoupled from cell fate specification.

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Sheaffer KL, Updike DL, Mango SE. The Target of Rapamycin pathway antagonizes pha-4/FoxA to control development and aging. Current biology : CB. 2008;18(18):1355-64.Abstract

FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth. We previously identified the AAA+ ATPase homolog ruvb-1 as a potent suppressor of pha-4 mutations.

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Von Stetina SE, Mango SE. Wormnet: a crystal ball for Caenorhabditis elegans. Genome biology. 2008;9(6):226.Abstract

An integrated gene network for Caenorhabditis elegans using data from multiple genome-wide screens encompasses most protein-coding genes and can accurately predict their phenotypes.

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Kiefer JC, a Smith P, Mango SE. PHA-4/FoxA cooperates with TAM-1/TRIM to regulate cell fate restriction in the C. elegans foregut. Developmental biology. 2007;303(2):611-24.Abstract

A key question in development is how pluripotent progenitors are progressively restricted to acquire specific cell fates. Here we investigate how embryonic blastomeres in C. elegans develop into foregut (pharynx) cells in response to the selector gene PHA-4/FoxA. When pha-4 is removed from pharyngeal precursors, they exhibit two alternative responses. Before late-gastrulation (8E stage), these cells lose their pharyngeal identity and acquire an alternative fate such as ectoderm (Specification stage). After the Specification stage, mutant cells develop into aberrant pharyngeal cells (Morphogenesis/Differentiation stage). Two lines of evidence suggest that the Specification stage depends on transcriptional repression of ectodermal genes by pha-4. First, pha-4 exhibits strong synthetic phenotypes with the B class synMuv gene tam-1 (Tandam Array expression Modifier 1) and with a mediator of transcriptional repression, the NuRD complex (NUcleosome Remodeling and histone Deacetylase). Second, pha-4 associates with the promoter of the ectodermal regulator lin-26 and is required to repress lin-26 expression. We propose that restriction of early blastomeres to the pharyngeal fate depends on both repression of ectodermal genes and activation of pharyngeal genes by PHA-4.

Mango SE. The C. elegans pharynx: a model for organogenesis. WormBook : the online review of C. elegans biology [Internet]. 2007:1-26. View OnlineAbstract

The C. elegans foregut (pharynx) has emerged as a powerful system to study organ formation during embryogenesis. Here I review recent advances regarding cell-fate specification and epithelial morphogenesis during pharynx development. Maternally-supplied gene products function prior to gastrulation to establish pluripotent blastomeres. As gastrulation gets under way, pharyngeal precursors become committed to pharyngeal fate in a process that requires PHA-4/FoxA and the Tbox transcription factors TBX-2, TBX-35, TBX-37 and TBX-38. Subsequent waves of gene expression depend on the affinity of PHA-4 for its target promoters, coupled with combinatorial strategies such as feed-forward and positive-feedback loops. During later embryogenesis, pharyngeal precursors undergo reorganization and a mesenchymal-to-epithelial transition to form the linear gut tube. Surprisingly, epithelium formation does not depend on cadherins, catenins or integrins. Rather, the kinesin ZEN-4/MKLP1 and CYK-4/RhoGAP are critical to establish the apical domain during epithelial polarization. Finally, I discuss similarities and differences between the nematode pharynx and the vertebrate heart.

Smith P, Mango SE. Role of T-box gene tbx-2 for anterior foregut muscle development in C. elegans. Developmental biology. 2007;302(1):25-39.Abstract

During organogenesis, pluripotent precursor cells acquire a defined identity such as muscle or nerve. The transition from naïve precursor towards the differentiated state is characterized by sequential waves of gene expression that are determined by regulatory transcription factors. A key question is how transcriptional circuitry dictates the succession of events that accompanies developmental competence, cell fate specification and differentiation. To address this question, we have examined how anterior muscles are established within the Caenorhabditis elegans foregut (pharynx). We find that the T-box transcription factor tbx-2 is essential to form anterior pharyngeal muscles from the ABa blastomere. In the absence of tbx-2 function, ABa-derived cells initiate development normally: they receive glp-1/Notch signaling cues, activate the T-box gene TBX-38 and express the organ selector gene PHA-4/FoxA. However, these cells subsequently arrest development, extinguish PHA-4 and fail to activate PHA-4 target genes. tbx-2 mutant cells do not undergo apoptosis and there is no evidence for adoption of an alternative fate. TBX-2 is expressed in ABa descendants and depends on activation by pha-4 and repression by components of glp-1/Notch signaling. Our analysis suggests that a positive feedback loop between tbx-2 and pha-4 is required for ABa-derived precursors to commit to pharyngeal muscle fate.

Deplancke B, Mukhopadhyay A, Ao W, Elewa AM, a Grove C, Martinez NJ, Sequerra R, Doucette-Stamm L, Reece-Hoyes JS, a Hope I, et al. A gene-centered C. elegans protein-DNA interaction network. Cell. 2006;125(6):1193-205.Abstract

Transcription regulatory networks consist of physical and functional interactions between transcription factors (TFs) and their target genes. The systematic mapping of TF-target gene interactions has been pioneered in unicellular systems, using "TF-centered" methods (e.g., chromatin immunoprecipitation). However, metazoan systems are less amenable to such methods. Here, we used "gene-centered" high-throughput yeast one-hybrid (Y1H) assays to identify 283 interactions between 72 C. elegans digestive tract gene promoters and 117 proteins. The resulting protein-DNA interaction (PDI) network is highly connected and enriched for TFs that are expressed in the digestive tract. We provide functional annotations for approximately 10% of all worm TFs, many of which were previously uncharacterized, and find ten novel putative TFs, illustrating the power of a gene-centered approach. We provide additional in vivo evidence for multiple PDIs and illustrate how the PDI network provides insights into metazoan differential gene expression at a systems level.

Updike DL, Mango SE. Temporal regulation of foregut development by HTZ-1/H2A.Z and PHA-4/FoxA. PLoS genetics. 2006;2(9):e161.Abstract

The histone variant H2A.Z is evolutionarily conserved and plays an essential role in mice, Drosophila, and Tetrahymena. The essential function of H2A.Z is unknown, with some studies suggesting a role in transcriptional repression and others in activation. Here we show that Caenorhabditis elegans HTZ-1/H2A.Z and the remodeling complex MYS-1/ESA1-SSL-1/SWR1 synergize with the FoxA transcription factor PHA-4 to coordinate temporal gene expression during foregut development. We observe dramatic genetic interactions between pha-4 and htz-1, mys-1, and ssl-1. A survey of transcription factors reveals that this interaction is specific, and thus pha-4 is acutely sensitive to reductions in these three proteins. Using a nuclear spot assay to visualize HTZ-1 in living embryos as organogenesis proceeds, we show that HTZ-1 is recruited to foregut promoters at the time of transcriptional onset, and this recruitment requires PHA-4. Loss of htz-1 by RNAi is lethal and leads to delayed expression of a subset of foregut genes. Thus, the effects of PHA-4 on temporal regulation can be explained in part by recruitment of HTZ-1 to target promoters. We suggest PHA-4 and HTZ-1 coordinate temporal gene expression by modulating the chromatin environment.

Jenkins N, Saam JR, Mango SE. CYK-4/GAP provides a localized cue to initiate anteroposterior polarity upon fertilization. Science (New York, N.Y.). 2006;313(5791):1298-301.Abstract

The Caenorhabditis elegans anteroposterior axis is established in response to fertilization by sperm. Here we present evidence that RhoA, the guanine nucleotide-exchange factor ECT-2, and the Rho guanosine triphosphatase-activating protein CYK-4 modulate myosin light-chain activity to create a gradient of actomyosin, which establishes the anterior domain. CYK-4 is enriched within sperm, and paternally donated CYK-4 is required for polarity. These data suggest that CYK-4 provides a molecular link between fertilization and polarity establishment in the one-cell embryo. Orthologs of CYK-4 are expressed in sperm of other species, which suggests that this cue may be evolutionarily conserved.

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Ao W, Gaudet J, Kent JW, Muttumu S, Mango SE. Esearch rticles. 2004;305(September):1743-1746.Abstract