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Structure and expression of daf-12: A nuclear hormone receptor with three isoforms that are involved in development and aging in Caenorhabditis elegans
Abstract
During Caenorhabditis elegans early larval development environmental conditions promote a cascade of signaling molecules to direct growth to the reproductive adult or to arrest development as a dauer larva. Two parallel chemosensory signal transduction pathways, one of which is transforming growth factor (TGF)-beta-like, converge on the daf-12 gene to regulate dauer formation. A third insulin-like signaling pathway interacts with the daf-12 pathway to regulate both dauer formation and adult longevity. To further understand the role of daf-12 in these processes, we have molecularly characterized this gene. We establish rescue of the mutant dauer defective phenotype with a genomic clone. We show that three transcripts of different lengths, due to differential splicing, are made from the daf-12 gene. The deduced protein isoforms are similar to both DNA- and ligand-binding domains of nuclear hormone receptors. The three daf-12 transcripts are produced throughout development and expression increases during the preparation for and execution of dauer formation. Analysis of various daf mutant strains suggests that the isoform ratios of daf-12 steady-state mRNA are not changed by reduction of function in the TGF-beta and insulin signaling components of the dauer pathway. The daf-12 promoter directs expression of GFP in the pharynx. daf-12 is a C. elegans nuclear hormone receptor with multiple isoforms, is expressed throughout development in distinct cells, and functions under a variety of environmental conditions.
... Furthermore, daf-12 mutant alleles influence the life span of daf-2/InsR mutants in an allele-specific manner. For example, the non-null allele daf-12(m20) [Supplemental Figure 3.1; Snow and Larsen, 2000)] suppresses the extended life span phenotype of daf-2(e1365) [harboring a mutation in the ligand binding domain; ] at all temperatures tested (Gems et al., 1998), whereas it enhances daf-2(e1370) life span extension at high temperatures Gems et al., 1998). In aggregate, these data underscore the need for further investigation into how steroid hormone signaling and DAF-2/InsR signaling interact in life span control. ...
... it is predicted to truncate DAF-12A upstream of the C-terminal ligand binding domain, potentially resulting in a DAF-12A polypeptide that contains an intact zinc finger in the N-terminal DNA binding domain. The DAF-12B isoform, which contains the ligand binding domain but lacks the DNA binding domain, is not affected by m20 Snow and Larsen, 2000)]. The influence of a daf-12 null allele on the dauer-constitutive phenotype of daf-2 mutants has not been explored. ...
... To clarify the epistatic relationship between daf-2 and daf-12, we constructed daf-2;daf-12 double mutants using the daf-12(rh61rh411) null allele [Supplemental Figure 3.1 and Supplemental Table 3.1; heretofore referred to as "daf-12 null"; Snow and Larsen, 2000)] and performed dauer arrest assays at 25°. As expected, both the Class 1 daf-2(e1368) ligand binding domain mutant as well as the Class 2 daf-2(e1370) tyrosine kinase domain mutant had strong dauer-constitutive phenotypes (Supplemental Figure 3.2). ...
FoxO transcription factors were first implicated as regulators of longevity in C. elegans. Reducing insulin/insulin-like growth factor signaling (ILS) extends C. elegans life span in a manner dependent on DAF-16/FoxO. This life span-promoting role of FoxO is potentially conserved, as reducing ILS also enhances mammalian longevity. Additionally, evidence supports roles for FoxO in modulating age-associated diseases in humans, including cancer, osteoporosis, and metabolic dysregulation. Understanding the regulatory mechanisms controlling FoxO activity will supplement our knowledge of age-associated diseases, and possibly also shed light on the biology of aging. In C. elegans, ILS reduction causes an increase in DAF-16/FoxO activity due to its translocation to the nucleus. Nuclear DAF-16/FoxO induces transcriptional programs that promote longevity. We have discovered the EAK pathway, a novel, conserved pathway that inhibits the C. elegans FoxO transcription factor DAF-16. The EAK pathway acts in parallel to PI3K/Akt to inhibit DAF-16/FoxO, independent of controlling its subcellular localization. EAKs do so, at least in part, by promoting the activity of a conserved steroid hormone signaling pathway. This novel mechanism of DAF-16/FoxO inhibition represents a new avenue to pursue to strengthen our understanding of FoxO regulation.
To illuminate the mechanism underlying EAK pathway inhibition of DAF-16/FoxO activity, we performed an unbiased genetic screen to identify suppressors of an eak;akt mutant dauer arrest phenotype (seak mutants). seak genes are predicted to encode molecules that are required to promote dauer arrest by activating DAF-16/FoxO. We have characterized two seak genes: First, we discovered that a conserved Ras GTPase activating protein regulates DAF-16/FoxO activity, likely via the EAK pathway. Second, we have identified a novel function of dosage compensation in C. elegans, discovering that dosage compensation can influence DAF-16/FoxO via the coordinated regulation of ILS pathway genes expressed on the X chromosome. This represents the first elucidation of a post-embryonic function for the dosage compensation complex and suggests that the complex itself might be under the control of signaling pathways that are responsive to the environment. Looking forward, conserved eak and seak genes represent novel therapeutic targets for the modulation of FoxO activity to aid in the fight against diseases of aging.
... Furthermore, daf-12 mutant alleles influence the life span of daf-2/ InsR mutants in an allele-specific manner. For example, the non-null allele daf-12(m20) (see Supporting Information, Figure S1) (Antebi et al. 2000;Snow and Larsen 2000) suppresses the extended life span phenotype of daf-2(e1368) harboring a mutation in the ligand binding domain (Patel et al. 2008) at all temperatures tested (Gems et al. 1998), whereas it enhances daf-2(e1370) life span extension at high temperatures (Gems et al. 1998;Larsen et al. 1995). In aggregate, these data underscore the need for further investigation into how steroid hormone signaling and DAF-2/InsR signaling interact in life span control. ...
... Notably, daf-12(m20) is a nonsense mutation that specifically affects DAF-12A isoforms; it is predicted to truncate DAF-12A upstream of the C-terminal ligand binding domain, potentially resulting in a DAF-12A polypeptide that contains an intact zinc finger in the N-terminal DNA binding domain. The DAF-12B isoform, which contains the ligand binding domain but lacks the DNA binding domain, is not affected by m20 ( Figure S1) (Antebi et al. 2000;Snow and Larsen 2000). The influence of a daf-12 null allele on the dauer-constitutive phenotype of daf-2 mutants has not been explored. ...
... To clarify the epistatic relationship between daf-2 and daf-12, we constructed daf-2;daf-12 double mutants using the daf-12(rh61rh411) null allele ( Figure S1 and Table S1) hereafter referred to as "daf-12 null" (Antebi et al. 2000;Snow and Larsen 2000) and performed dauer arrest assays at 25°. As expected, both the Class 1 daf-2(e1368) ligand binding domain mutant and the Class 2 daf-2(e1370) tyrosine kinase domain mutant had strong dauer-constitutive phenotypes ( Figure S2). ...
Sterol-sensing nuclear receptors and insulin-like growth factor signaling play evolutionarily conserved roles in the control of aging. In the nematode Caenorhabditis elegans, bile-acid-like steroid hormones known as dafachronic acids (DAs) influence longevity by binding to and regulating the activity of the conserved nuclear receptor DAF-12, and the insulin receptor (InsR) ortholog DAF-2 controls life span by inhibiting the FoxO transcription factor DAF-16. How the DA/DAF-12 pathway interacts with DAF-2/InsR signaling to control life span is poorly understood. Here we specifically interrogate the roles of liganded and unliganded DAF-12 in life span control in the context of reduced DAF-2/InsR signaling. In animals with reduced daf-2/InsR activity, mutations that either reduce DA biosynthesis or fully abrogate DAF-12 activity shorten life span, suggesting that liganded DAF-12 promotes longevity. In animals with reduced DAF-2/InsR activity induced by daf-2/InsR RNAi, both liganded and unliganded DAF-12 promote longevity. However, in daf-2/InsR mutants, liganded and unliganded DAF-12 act in opposition to control life span. Thus, multiple DAF-12 activities influence life span in distinct ways in contexts of reduced DAF-2/InsR signaling. Our findings establish new roles for a conserved steroid signaling pathway in life span control and elucidate interactions among DA biosynthetic pathways, DAF-12, and DAF-2/InsR signaling in aging.
... daf-12 mRNA is detected at all developmental stages, but peaks in L2d larvae before dauer formation (Snow & Larsen 2000). Being ubiquitously expressed (Antebi et al 2 0 0 0 ), daf-12 is a candidate for the receipt and transduction of neuron subset-specific signals during development, resulting in an organism-wide developmental decision (Antebi et al 1998;Apfeld & Kenyon 1998;Antebi et al 2000). ...
... daf-12(m20) is a nonsense mutation which has nearly normal gonadal and extragonadal development but is completely Daf-d (Antebi et al 1998(Antebi et al , 2000Snow & Larsen 2000). ...
Males of the nematode Caenorhabditis elegans are longer-lived, more stress resistant and more likely to enter an alternative dispersal stage (dauer) than hermaphrodites. Why might this be? Since these traits are suppressed by insulin-/IGF-l-like signalling (IIS), it seemed possible that IIS levels could be lower in males. Increased male lifespan and stress resistance were dependent upon daf-16, which encodes the transcription factor negatively regulated by IIS, suggesting that IIS is down-regulated in males. Moreover, the male bias to dauer formation was lost in IIS mutants. However, DAF-16::GFP was more localised to the nucleus (where it is active) in hermaphrodites, hence sex differences in DAF-16 activity may occur downstream of IIS. Lifespan is extended by certain uncoordinated (unc) mutations, which disrupt motility by affecting neurons or musculature. Only neuronal unc mutations extended lifespan, suggesting that disruption of neurotransmission and/or neuroendocrine function may be responsible. The 76-dependence of the lifespan increase due to several unc mutations suggests that IIS may be the component affected. Since unc mutations increase lifespan more in males than hermaphrodites, sex differences in IIS/ DAF-16 activity may underlie this effect, at least in part. Hermaphrodite lifespan is regulated by gonadal signalling. Lifespan responses of males to ablation of gonad precursors were reduced relative to hermaphrodites, suggesting reduced lifespan regulation by the male gonad. Moreover, in contrast to hermaphrodites, male lifespan was largely unaffected by mutation of daf-12, which encodes a nuclear hormone receptor that interacts with IIS and is involved with transduction of gonadal signals. Overall, therefore, evidence suggests fundamental sex differences in lifespan regulation. Why might this evolve? Studies of several dioecious and other androdioecious species suggested that increased male lifespan is a general trait among nematodes. Potentially, differential degrees of male rarity and exposure to non-senescent modes of death shape evolution of sex differences in lifespan.
... Expression of GFP reporter constructs from Ce-daf-36 [195] and Ce-daf-12 [193] promoters is down-regulated in dauers, while microarray evidence has shown that Ce-daf-9 and Ce-daf-36 transcripts are up-regulated during dauer recovery [124]. Somewhat contradictorily, Ce-daf-12 transcripts have been shown to be up-regulated during dauer formation [202]. ...
... Curiously, we were unable to identify genes encoding S. stercoralis homologs of Ce-HSD-1 or Ce-DIN-1 in the S. stercoralis draft genome, the S. ratti draft genome, or our de novo assemblies of S. stercoralis transcripts. We also found that the Ss-daf-12 locus encoded a total of seven transcripts encoding three different proteins, with the variability confined to the N-terminus of the predicted protein before the DNAbinding domain, similar to that found in Ce-daf-12 [193,202]. ...
Parasitic nematodes inflict a vast global disease burden in humans as well as animals and plants of agricultural importance; understanding how these worms infect their hosts has significant health and economic implications. In humans, soil-transmitted parasitic nematodes cause hookworm disease and strongyloidiasis, and vector-transmitted parasitic nematodes cause filariasis. The infectious form of the species causing these diseases is a developmentally arrested third-stage larva (L3i). Molecular mechanisms governing L3i developmental arrest and activation within a host have been poorly understood. An analogous developmentally arrested third-stage larva—the dauer larva—forms during stressful environmental conditions in the free-living nematode Caenorhabditis elegans and is controlled by four cellular signaling pathways. The "dauer hypothesis" posits that similar mechanisms regulate dauer and L3i development. The parasitic nematode Strongyloides stercoralis was used to test the dauer hypothesis because its life cycle includes both parasitic and free-living forms. To investigate the role of canonical dauer pathway homologs in regulating L3i arrest and activation, this study utilized transcriptome sequencing (RNAseq), transgenesis, and pharmacological studies. Transcripts encoding cyclic guanosine monophosphate (cGMP) pathway components were coordinately up-regulated in L3i. Application of membrane-permeable 8-bromo-cGMP resulted in activation of L3i and modulation of ligand transcripts in other pathways. In comparison to C. elegans, S. stercoralis has few genes encoding insulin/IGF-1 -like signaling (IIS) ligands, several of which have transcripts modulated during L3i development. Application of the phosphatidylinositol-3 kinase inhibitor, LY294002, prevented L3i activation in host-like conditions. The S. stercoralis transcriptome includes seven homologs of the single C. elegans dauer transforming growth factor β (TGFβ) ligand, three of which are only expressed in L3i. Although the C. elegans nuclear hormone receptor ligand delta7-dafachronic acid (DA) stimulates L3i activation, putative DA biosynthetic genes were not coordinately regulated in L3i development. These data demonstrate that S. stercoralis has homologs for nearly every component in the four canonical dauer pathways, that cGMP signaling may transduce host cues during L3i activation, and that IIS regulates L3i arrest and activation. However, dauer TGFβ signaling appears to function in L3i arrest, an opposite role than in C. elegans, and endogenous DA regulation of L3i development remains largely unexplored.
... Expression of GFP reporter constructs from Ce-daf-36 [128] and Ce-daf-12 [126] promoters is down-regulated in dauers, while microarray evidence has shown that Ce-daf-9 and Ce-daf-36 transcripts are up-regulated during dauer recovery [43]. Somewhat contradictorily, Ce-daf-12 transcripts have been shown to be up-regulated during dauer formation [135]. ...
... Curiously, we were unable to identify genes encoding S. stercoralis homologs of CeHSD-1 or Ce-DIN-1 in the S. stercoralis draft genome, the S. ratti draft genome, or our de novo assemblies of S. stercoralis transcripts. We also found that the Ss-daf-12 locus encoded a total of seven transcripts encoding three different proteins, with the variability confined to the N-terminus of the predicted protein before the DNA-binding domain, similar to that found in Ce-daf-12 [126,135]. ...
The infectious form of many parasitic nematodes, which afflict over one billion people globally, is a developmentally arrested third-stage larva (L3i). The parasitic nematode Strongyloides stercoralis differs from other nematode species that infect humans, in that its life cycle includes both parasitic and free-living forms, which can be leveraged to investigate the mechanisms of L3i arrest and activation. The free-living nematode Caenorhabditis elegans has a similar developmentally arrested larval form, the dauer, whose formation is controlled by four pathways: cyclic GMP (cGMP) signaling, insulin/IGF-1-like signaling (IIS), transforming growth factor β (TGFβ) signaling, and biosynthesis of dafachronic acid (DA) ligands that regulate a nuclear hormone receptor. We hypothesized that homologous pathways are present in S. stercoralis, have similar developmental regulation, and are involved in L3i arrest and activation. To test this, we undertook a deep-sequencing study of the polyadenylated transcriptome, generating over 2.3 billion paired-end reads from seven developmental stages. We constructed developmental expression profiles for S. stercoralis homologs of C. elegans dauer genes identified by BLAST searches of the S. stercoralis genome as well as de novo assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to C. elegans, S. stercoralis has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven S. stercoralis genes encoding homologs of the single C. elegans dauer regulatory TGFβ ligand, three of which are only expressed in L3i. Putative DA biosynthetic genes did not appear to be coordinately regulated in L3i development. Our data suggest that while dauer pathway genes are present in S. stercoralis and may play a role in L3i development, there are significant differences between the two species. Understanding the mechanisms governing L3i development may lead to novel treatment and control strategies.
... 3. DAF-12 is a key coordinator of C. elegans life history C. elegans DAF-12, a homolog of the vertebrate vitamin D receptor (VDR) and the liver X receptor (LXR), plays multiple roles in regulating development and physiology in response to environmental conditions, including major effects on developmental timing, dauer formation, reproductive maturation, metabolism, and lifespan (Antebi et al., 2000;Snow and Larsen, 2000;Gerisch et al., 2001;Gerisch et al., 2007). Thus, DAF-12 appears to be a hormonal master coordinator of C. elegans life history (Fielenbach and Antebi, 2008;Magner and Antebi, 2008). ...
... In Drosophila, EcR signaling is similarly modulated by co-activators (taiman; Bai et al., 2000) or co-repressors (SMRTER or Alien; Tsai et al., 1999;Dressel et al., 1999), and it would be interesting to examine the effects of such cofactors on lifespan regulation. Moreover, both DAF-12 and EcR have several receptor isoforms (Bender et al., 1997;Antebi et al., 2000;Mouillet et al., 2001), and it is possible that distinct isoforms might have different effects on lifespan (Snow and Larsen, 2000). ...
In the last two decades it has become clear that hormones and gene mutations in endocrine signaling pathways can exert major effects on lifespan and related life history traits in worms, flies, mice, and other organisms. While most of this research has focused on insulin/insulin-like growth factor-1 signaling, a peptide hormone pathway, recent work has shown that also lipophilic hormones play an important role in modulating lifespan and other life history traits. Here we review how steroid hormones, a particular group of lipophilic hormones, affect life history traits in the nematode worm (Caenorhabditis elegans) and the fruit fly (Drosophila melanogaster), with a particular focus on longevity. Interestingly, a comparison suggests that parallel endocrine principles might be at work in worms and flies in these species and that steroid hormones interact with the gonad to affect lifespan.
... To this end, we fractionated whole animal lysates by sucrose density gradient ultracentrifugation to analyse the polyribosome association of endogenous let-7 targets in wild-type and let-7(n2853) mutant C. elegans at the L3 developmental stage, when let-7 activity is low, and at the late L4 stage, when let-7 activity is high ( Figure 1A; Supplementary Figure S1). As the two let-7 targets daf-12 and lin-41 Grosshans et al, 2005) are expressed at very low levels in L4 stage larvae (Snow and Larsen, 2000;Bagga et al, 2005 and this study, below), we used reverse transcription-quantitative PCR (RT-qPCR) to quantify them. It is to be noted that all experiments were performed using random hexamer oligonucleotides to prime RT, to include even mRNA, the poly(A) tail of which might be short due to the action of the miRNA (Eulalio et al, 2008a;Filipowicz et al, 2008). ...
... For daf-12 mRNA, its low abundance prevents detection by northern blotting even in unfractionated, total RNA without prior selection of polyadenylated mRNA (Snow and Larsen, 2000). Therefore, to confirm that our RT-qPCR assay similarly measures the levels of full-length daf-12 mRNA, we tested a second set of primers, and obtained comparable results as expected (Supplementary Figure S5A). ...
MicroRNAs (miRNAs) repress target genes through a poorly defined antisense mechanism. Cell-free and cell-based assays have supported the idea that miRNAs repress their target mRNAs by blocking initiation of translation, whereas studies in animal models argued against this possibility. We examined endogenous targets of the let-7 miRNA, an important regulator of stem cell fates. We report that let-7 represses translation initiation in Caenorhabditis elegans, demonstrating this mode of action for the first time in an organism. Unexpectedly, although the lin-4 miRNA was previously reported to repress its targets at a step downstream of translation initiation, we also observe repression of translation initiation for this miRNA. This repressive mechanism, which frequently but not always coincides with transcript degradation, requires the GW182 proteins AIN-1 and AIN-2, and acts on several mRNAs targeted by different miRNAs. Our analysis of an expanded set of endogenous miRNA targets therefore indicates widespread repression of translation initiation under physiological conditions and establishes C. elegans as a genetic system for dissection of the underlying mechanisms.
... Four DAF-12 protein isoforms have been reported (Antebi et al. 2000;Snow and Larsen 2000). Two (A1 and A3) encompass all 17 DAF-12 exons and differ by 16 amino acids in exon 12. ...
... These two isoforms are truncated within exon 3 before the Zn-finger DNA-binding domain (DBD) by the daf-12(m583) mutation and are unlikely to have activity in this mutant. A third reported isoform (A2), which includes all of the DAF-12 protein sequence, including the DBD, Hinge, and the ligand-binding domain (LBD), but excluding the N-terminal hypervariable region, begins within exon 3 and is unaffected by the m583 mutation ( Snow and Larsen 2000). Since daf-12(m583) is dauer defective as well as male leaving defective, the A2 isoform alone apparently cannot supply daf-12 activity for either of these phenotypes. ...
Coordination of animal behavior with reproductive status is often achieved through elaboration of hormones by the gonad. In the nematode Caenorhabditis elegans, adult males explore their environment to locate mates. Mate searching is regulated by presence of mates, nutritional status, and a signal from the gonad. Here we show that the gonadal signal acts via the nuclear receptor DAF-12, a protein known to regulate several C. elegans life-history traits. DAF-12 has both activational and organizational functions to stimulate exploratory behavior and acts downstream of the gonadal signal, outside of the gonad. DAF-12 acts upstream of sensory input from mating partners and physiological signals indicating nutritional status. Mate searching was rescued in germ-line ablated animals, but not if both germ line and somatic gonad were ablated, by a precursor of the DAF-12 ligand, dafachronic acid (DA). The results are interpreted to suggest that the germ line produces a DA precursor that is converted to DA outside of the germ line, possibly in the somatic gonad. As it does in other pathways in which it functions, in regulation of male mate searching behavior DAF-12 acts at a choice point between alternatives favoring reproduction (mate searching) vs. survival (remaining on food).
... It is hypothesized that these two pathways cooperate so that multiple signal inputs can be used to inform dauer and lifespan decisions (Shaw et al., 2007). Some of this signal integration may occur through DAF-12/nuclear hormone receptor (Antebi et al., 2000;Snow and Larsen, 2000). The DAF-7 pathway functions epistatically with IIS in regulating nictation behavior (Lee et al., 2017). ...
The Transforming Growth Factor-β (TGF-β) superfamily of signaling molecules plays critical roles in development, differentiation, homeostasis, and disease. Due to the conservation of these ligands and their signaling pathways, genetic studies in invertebrate systems including the nematode Caenorhabditis elegans have been instrumental in identifying signaling mechanisms. C. elegans is also a premier organism for research in longevity and healthy aging. Here we summarize current knowledge on the roles of TGF-β signaling in aging and immunity.
... To determine whether a cell-autonomous dauer decision could also be sufficient to induce dendrite growth arrest, we turned to the DAF-12 NHR, which binds DIN-1 to promote dauer entry [30,33,34]. As a type II nuclear receptor, DAF-12 is thought to bind DNA constitutively, repressing transcription in the absence of its hormone ligand in the dauer-promoting, adverse-environment state and activating transcription when ligand is bound in the RD-promoting, favorable-environment state [35,36] (Fig 2B). Whereas daf-12(o) null mutants cannot enter dauer and exhibit a shortened adult lifespan in RD, the previously identified gain-offunction allele daf-12(r273) promotes constitutive dauer entry and extends adult lifespan in RD [34,[36][37][38] (Fig 2B). ...
Neurons have a lifespan that parallels that of the organism and are largely irreplaceable. Their unusually long lifespan predisposes neurons to neurodegenerative disease. We sought to identify physiological mechanisms that delay neuron aging in Caenorhabditis elegans by asking how neuron morphological aging is arrested in the long-lived, alternate organismal state, the dauer diapause. We find that a hormone signaling pathway, the abnormal DAuer Formation (DAF) 12 nuclear hormone receptor (NHR) pathway, functions cell-intrinsically in the dauer diapause to arrest neuron morphological aging, and that same pathway can be cell-autonomously manipulated during normal organismal aging to delay neuron morphological aging. This delayed aging is mediated by suppressing constitutive endocytosis, which alters the subcellular localization of the actin regulator T cell lymphoma Invasion And Metastasis 1 (TIAM-1), thereby decreasing age-dependent neurite growth. Intriguingly, we show that suppressed endocytosis appears to be a general feature of cells in diapause, suggestive that this may be a mechanism to halt the growth and other age-related programs supported by most endosome recycling.
... We used both the whole C. elegans daf-12 gene, and the heavily conserved DNA-binding domain [27,53,54] for tblastn/blastp searching the available Strongyloides spp. genomes [34]. ...
The gene daf-12 has long shown to be involved in the dauer pathway in Caenorhabditis elegans (C. elegans). Due to the similarities of the dauer larvae of C. elegans and infective larvae of certain parasitic nematodes such as Strongyloides spp., this gene has also been suspected to be involved in the development of infective larvae. Previous research has shown that the application of dafachronic acid, the steroid hormone ligand of DAF-12 in C. elegans, affects the development of infective larvae and metabolism in Strongyloides. However, a lack of tools for either forward or reverse genetics within Strongyloides has limited studies of gene function within these important parasites. After determining whether Strongyloides had the requisite proteins for RNAi, we developed and report here the first successful RNAi by soaking protocol for Strongyloides ratti (S. ratti) and use this protocol to study the functions of daf-12 within S. ratti. Suppression of daf-12 in S. ratti severely impairs the formation of infective larvae of the direct cycle and redirects development towards the non-infective (non-dauer) free-living life cycle. Further, daf-12(RNAi) S. ratti produce slightly but significantly fewer offspring and these offspring are developmentally delayed or incapable of completing their development to infective larvae (L3i). Whilst the successful daf-12(RNAi) L3i are still able to infect a new host, the resulting infection is less productive and shorter lived. Further, daf-12 knockdown affects metabolism in S. ratti resulting in a shift from aerobic towards anaerobic fat metabolism. Finally, daf-12(RNAi) S. ratti have reduced tolerance of temperature stress.
... This functional domain can be found in growth HRs or NHRs. The NHR consists of two parts, a DNA binding domain and an LBD [14]; the growth HR features one more domain, identified as a transactivation domain [15]. The LBD represents the C-terminal domain, responsible for the ligand binding step and the induction of downstream HR signaling. ...
Previously, we found that the expression of several genes, including HR, varied in Drosophila melanogaster after white spot syndrome virus (WSSV) infection. In this present study, we further investigated the role of HR in Kuruma shrimp, Marsupenaeus japonicus and determined its anti-apoptosis and anti-inflammation role in the innate immune system. We successfully identified a partial sequence (866 bp in length) of the M. japonicus hormone receptor ligand binding domain (mjHR_LBD/mjHR). The 5' end of mjHR was successfully obtained; the open reading frame (ORF) ran from 33 to 701 bp, and encoded a protein containing 222 amino acids. mjHR belonged to the ligand binding domain of hormone receptors, was most likely part of a nuclear hormone receptor, and shared a close evolutionary relationship with other arthropods, such as insects. mjHR was expressed predominantly in immunity tissues such as gills, hemolymph and the hepatopancreas. WSSV infection could cause the down-regulation of mjHR, while infection with Vibrio alginolyticus could cause significant up-regulation of mjHR. The expression of mjHR was knocked down by dsRNA expressed by an engineered LITMUS 38i-HR plasmid. Virus and bacteria challenge experiment showed that the mortality of WSSV-infected shrimps was elevated in the absence of HR while the mortality of shrimps infected with V. alginolyticus was slightly reduced. Phenoloxidase (PO) activity, phagocytosis and apoptosis were promoted, while superoxide dismutase (SOD) activity was impaired, indicating that mjHR functions in an anti-apoptosis and anti-inflammation manner to prevent shrimp death caused by an over-load of immunity responses. Differences between mjHR expression and mortality change after WSSV or V. alginolyticus infection indicated that there was a different strategy for viruses or bacteria when confronted with the innate immune system.
... To control the posthatching decision to enter the dauer phase, Daf-7 signals through two TGF-β family type I and type II receptors, encoded by daf-1 and daf-4, respectively (Georgi et al. 1990;Estevez et al. 1993). Daf-7-induced signals then converge on the expression of daf-12 (Thomas et al. 1993) and act as a signaling mechanism that transmits environmental information to directly control the larval and reproductive development of C. elegans Ren et al. 1996;Antebi et al. 1998;Snow and Larsen 2000). ...
The transforming growth factor β (TGF-β) family has a profound impact on the reproductive function of various organisms. In this review, we discuss how highly conserved members of the TGF-β family influence the reproductive function across several species. We briefly discuss how TGF-β-related proteins balance germ-cell proliferation and differentiation as well as dauer entry and exit in Caenorhabditis elegans. In Drosophila melanogaster, TGF-β-related proteins maintain germ stem-cell identity and eggshell patterning. We then provide an in-depth analysis of landmark studies performed using transgenic mouse models and discuss how these data have uncovered basic developmental aspects of male and female reproductive development. In particular, we discuss the roles of the various TGF-β family ligands and receptors in primordial germ-cell development, sexual differentiation, and gonadal cell development. We also discuss how mutant mouse studies showed the contribution of TGF-β family signaling to embryonic and postnatal testis and ovarian development. We conclude the review by describing data obtained from human studies, which highlight the importance of the TGF-β family in normal female reproductive function during pregnancy and in various gynecologic pathologies.
... Hr96 encodes a nuclear receptor represented by an orthologous group of three C. elegans genes: daf-12, nhr-8, and nhr-48 [8]. NHR-8 is implicated in xenobiotic stress [9], while DAF-12 functions in dauer arrest, lipid metabolism, insulin signaling, and longevity [10][11][12]. ...
Supporting results and additional explanatory text.
... Consistent with this interpretation, dbl-1/TGF-β is expressed in neurons while its receptor and downstream SMADs are expressed broadly [40,41,43,62,63]. Likewise, daf-36 is expressed in the intestine, daf-9/CYP450 is expressed in the epidermis and XXX cells, and daf-12/NHR is expressed broadly [29,32,34,64]. We speculate that DAF-16/FOXO functions in several of these tissues to inhibit these genes directly or indirectly. ...
Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This "L1 arrest" (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.
... Noteworthy at this point is that the locus daf-12 is very complex. DAF-12 is a nuclear hormone receptor that is required for regulating multiple stage-specific aspects of nematode development (Antebi et al., 2000;Snow and Larsen, 2000). It encods multiple alternatively spliced isoforms and thereby is capable of mutating to daf-d, daf-c and heterochronic phenotypes independently (Savage-Dunn, 2005). ...
Die mittlere Lebenserwartung des Menschen ist über die letzten 200 Jahre kontinuierlich gestiegen. Da Langlebigkeit ohne Gesundheit wenig Wert besitzt, ist es ein zentrales Anliegen, das Auftreten altersbedingter Krankheiten zu mindern. Besonders pflanzliche Phytochemikalien, im speziellen Polyphenole (PPs), sollen erheblich an der Gesundheitsförderung mitwirken. Die exakten Mechanismen jedoch, welche die Wirkvielfalt erklären könnten, sind nicht im Detail bekannt. Diese Fragen können nur durch in vivo Studien an Modelorganismen beantwortet werden, die sowohl die Lebensdauer, sowie physiologische und genetische Parameter einschließen. In dieser Studie wurden drei PPs mit lebensverlängernden Eigenschaften in C. elegans identifiziert: Quercetin (Q), Kaffeesäure (CA) und Rosmarinsäure (RA). Für alle drei PPs wurden hormetische Konzentration-Wirkungs-Kurven gefunden, dennoch war die Hormetin-typische Aktivierung einer Stressantwort (gemessen als Geneexpressions-Level von Hitzeschock-Proteinen) auf Q und RA beschränkt. Eine Umverteilung von Ressourcen nach dem Prinzip der „Disposable Soma Theorie“ konnte anhand von Abweichungen in der Größe, verändertem Lipid-Metabolismus und verzögerter Reproduktion (bei gleichbleibender Anzahl der Nachkommen), für alle drei PPs gezeigt werden. Während direkte CR-Effekte ausgeschlossen wurden, ist dies nicht möglich für durch CA und RA ausgelöste indirekte CR-Effekte, da beide die Lebensspanne von sir-2.1 Mutanten nicht verlängern konnten. Alle drei PPs verlängerten die Lebensspanne von mev-1 Mutanten, jedoch wurde eine erhöhte TAC in vivo und eine reduzierte oxidative Schädigung, nur durch Q- und CA- Gabe erreicht. Die genetischen Wirkwege der PPs wurden durch Lebensdauer- und Thermotoleranztests mit in alters-relevanten Genen mutierten Nematoden definiert. Die gesundheitsfördernden Eigenschaften von CA und RA konnten so osr-1, sek-1, sir-2.1 and unc-43, sowie daf-16 im Falle von CA, zugeschrieben werden. Die Mechanismen von Q wurden in größerem Umfang, durch die Integration von durchgeführten Lebensdauertests und Microarray-Studien einerseits und einer umfassenden Meta-Analyse von veröffentlichten, alters-relevanten Genexpressions-Profilen andererseits, analysiert. Q wirkt vermutlich durch ein komplexes Zusammenspiel von konservierten genetischen Signalwegen, im Speziellen dem Insulin-ähnlichen (ILS), TGF-beta, p38 MAPK, CAMKII und möglicherweise auch über eine von der Keimbahn und somatischen Gonade ausgehenden Signalwirkung. Zusammenfassend lässt sich sagen, dass sowohl in vivo antioxidative und prooxidative Eigenschaften, die Modulation auf Genebene, sowie eine Umverteilung von Ressourcen zu gewissen Teilen (abhängig vom PP) zur Lebensverlängerung beitragen.
... Groups NR1J and NR1I cluster in the tree (Fig 1). No members of the NR1I group, including vitamin D receptor (VDR) or pregnane X receptors (PXR), were identified in B. glabrata and L. gigantea; however NR1J group in protostomes shares similarity with vertebrate NR1I group [110] and there is evidence that both NR1I/NR1J groups share a common ancestor [24]. NR1I receptors are considered as natural sensors and are involved in xenobiotic metabolism in vertebrates [111]; however, other studies have indicated that NR1J members might regulate xenobiotic responses in D. melanogaster and C. elegans [112,113]. ...
Nuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates , however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates , such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail en-docrine systems are fundamentally different.
... Finally, outputs of these three pathways are collated by a nuclear hormone receptor, DAF-12, which is considered the master regulator for entry into dauer development (Inoue and Thomas, 2000;Snow and Larsen, 2000), for which the ligands are dafachronic acids, sterols produced by cytochrome p450 DAF-9 (Jia et al., 2002;Motola et al., 2006). Therefore under the dauer hypothesis, one would expect both the molecular and cellular control of dauer entry to be conserved in parasitic nematodes. ...
How any complex trait has evolved is a fascinating question, yet the evolution of parasitism among the nematodes is arguably one of the most arresting. How did free-living nematodes cross that seemingly insurmountable evolutionary chasm between soil dwelling and survival inside another organism? Which of the many finely honed responses to the varied and harsh environments of free-living nematodes provided the material upon which natural selection could act? Although several complementary theories explain this phenomenon, I will focus on the dauer hypothesis. The dauer hypothesis posits that the arrested third-stage dauer larvae of free-living nematodes such as Caenorhabditis elegans are, due to their manyphysiological similarities with infective third-stage larvae of parasitic nematodes, a pre-adaptation to parasitism. If so, then a logical extension of this hypothesis is that the molecular pathways which control entry into and recovery from dauer formation by free-living nematodes in response to environmental cues have been co-opted to control the processes of infective larval arrest and activation in parasitic nematodes. The molecular machinery that controls dauer entry and exit is present in a wide range of parasitic nematodes. However, the developmental outputs of the different pathways are both conserved and divergent, not only between populations of C. elegans or between C. elegans and parasitic nematodes but also between different species of parasitic nematodes. Thus the picture that emerges is more nuanced than originally predicted and may provide insights into the evolution of such an interesting and complex trait.
... Gerontology 2004;50:265–290 277 ways are synergistically modulated by post-receptor signaling of daf-1/daf-4 [Ogg et al., 1997; Snow and Larsen, 2000]. The daf-1/daf-4 receptors are proposed to bind a TGFß-type protein coded for by daf-7 [Estevez et al., 1993]. ...
A mechanistic understanding of aging has yet to be described; this paper puts forth a new theory that has the potential to explain aging in all sexually reproductive life forms. The theory also puts forth a new definition of aging – any change in an organism over time. This definition includes not only the changes associated with the loss of function (i.e. senescence, the commonly accepted definition of aging), but also the changes associated with the gain of function (growth and development). Using this definition, the rate of aging would be synonymous with the rate of change. The rate of change/aging is most rapid during the fetal period when organisms develop from a single cell at conception to a multicellular organism at birth. Therefore, ‘fetal aging’ would be determined by factors regulating the rate of mitogenesis, differentiation, and cell death. We suggest that these factors also are responsible for regulating aging throughout life. Thus, whatever controls mitogenesis, differentiation and cell death must also control aging. Since life-extending modalities consistently affect reproduction, and reproductive hormones are known to regulate mitogenesis and differentiation, we propose that aging is primarily regulated by the hormones that control reproduction (hence, the Reproductive-Cell Cycle Theory of Aging). In mammals, reproduction is controlled by the hypothalamic-pituitary-gonadal (HPG) axis hormones. Longevity inducing interventions, including caloric restriction, decrease fertility by suppressing HPG axis hormones and HPG hormones are known to affect signaling through the well-documented longevity regulating GH/IGF-1/PI3K/Akt/Forkhead pathway. This is exemplified by genetic alterations in Caenorhabditis elegans where homologues of the HPG axis pathways, as well as the daf-2 and daf-9 pathways, all converge on daf-16, the homologue of human Forkhead that functions in the regulation of cell cycle events. In summary, we propose that the hormones that regulate reproduction act in an antagonistic pleiotrophic manner to control aging via cell cycle signaling; promoting growth and development early in life in order to achieve reproduction, but later in life, in a futile attempt to maintain reproduction, become dysregulated and drive senescence.
... Only six NHRs have predicted or confirmed targets and binding sites. With the exception of daf-12, which is expressed in most tissues, including the pharynx (Snow and Larsen, 2000), none of the well-characterized NHRs are expressed in the gland cells (Gissendanner et al., 2004;Miyabayashi et al., 1999). However, in our search for new gland-specific genes described earlier, we identified two uncharacterized NHR genes, nhr-48 and nhr-60 (Supplementary Table 1) that had reported gland expression (Gissendanner et al., 2004). ...
The Caenorhabditis elegans pharyngeal glands represent one of five cell types in the pharynx. We have previously shown that the bHLH transcription factor, HLH-6, is required for gland development and for expression of many, but not all, gland genes (Smit et al., 2008). Here, we have identified additional gland-expressed genes and find that transcriptional regulatory inputs other than HLH-6 are necessary for their regulation. We demonstrate that at least two hlh-6 independent gland genes, nas-12 and Y8A9A.2, require a cis-acting motif (HRL3- Hlh-6 Regulatory eLement 3), previously described based on its requirement for hlh-6 expression (Ghai and Gaudet, 2008). We also show that expression of the gland-expressed genes, ZK596.1, scl-3, wrt-3, and Y76B12C.3, rely on cis-elements and trans-acting factor(s) other than HLH-6 and HRL3. In addition, we show that negative regulatory mechanisms are employed to refine the spatial expression of some genes, resulting in expression in only a subset of the five gland cells. We show that one of these genes, Y8A9A.2, is negatively regulated by the NHR transcription factor encoded by nhr-48, which represses Y8A9A.2 expression in the g1A cells. We also show that another gene expressed in the reciprocal subset of gland cells, phat-5, is negatively regulated in the g1P and g2 cells by an unknown factor acting through a conserved cis-element in the phat-5 promoter. Overall, this work reveals levels of regulation of gene expression in a single cell type beyond that previously known, and suggests mechanisms by which the different gland sub-types are distinguished.
... We have not identified factors responsible for choosing the alternative splice acceptor sites in C. elegans males, however the existence of male-specific alternative splicing for unc-55 could serve as a starting point for further exploration of this phenomenon. Tissue, developmental stage and male-specific alternative splicing have now been reported for a number of genes in C. elegans (this study; Snow and Larsen, 2000;Zahler, 2005 for a summary). ...
In Caenorhabditis elegans, uncoordinated (unc)-55 encodes a nuclear hormone receptor that is necessary for coordinated movement and male mating. An unc-55 reporter gene revealed a sexually dimorphic pattern: early in post-embryonic motor neurons in both sexes; and later in a subset of male-specific cells that included an interneuron and eight muscle cells. A behavioral analysis coupled with RNA interference (RNAi) revealed that males require UNC-55 to execute copulatory motor programs. Two mRNA isoforms (unc-55a and unc-55b) were detected throughout post-embryonic development in males, whereas only one, unc-55a, was detected in hermaphrodites. In unc-55 mutant males isoform a rescued the locomotion and mating defect, whereas isoform b rescued the mating defect only. Isoform b represents the first report of male-specific splicing in C. elegans. In addition, isoform b extended the number of days that transgenic unc-55 mutant males mated when compared to males rescued with isoform a, suggesting an anabolic role for the nuclear hormone receptor. The male-specific expression and splicing is part of a regulatory hierarchy that includes two key genes, male abnormal (mab)-5 and mab-9, required for the generation and differentiation of male-specific cells. We suggest that UNC-55 acts as an interface between genes involved in male tail pattern formation and those responsible for function.
... Gerontology 2004;50:265–290 277 ways are synergistically modulated by post-receptor signaling of daf-1/daf-4 [Ogg et al., 1997; Snow and Larsen, 2000]. The daf-1/daf-4 receptors are proposed to bind a TGFß-type protein coded for by daf-7 [Estevez et al., 1993]. ...
The Reproductive-Cell Cycle Theory posits that the hormones that regulate reproduction act in an antagonistic pleiotrophic manner to control aging via cell cycle signaling; promoting growth and development early in life in order to achieve reproduction, but later in life, in a futile attempt to maintain reproduction, become dysregulated and drive senescence. Since reproduction is the most important function of an organism from the perspective of the survival of the species, if reproductive-cell cycle signaling factors determine the rate of growth, determine the rate of development, determine the rate of reproduction, and determine the rate of senescence, then by definition they determine the rate of aging and thus lifespan. The theory is able to explain: 1) the simultaneous regulation of the rate of aging and reproduction as evidenced by the fact that environmental conditions and experimental interventions known to extend longevity are associated with decreased reproductive-cell cycle signaling factors, thereby slowing aging and preserving fertility in a hostile reproductive environment; 2) two phenomena that are closely related to species lifespan-the rate of growth and development and the ultimate size of the animal; 3). the apparent paradox that size is directly proportional to lifespan and inversely proportional to fertility between species but vice versa within a species; 4). how differing rates of reproduction between species is associated with differences in their lifespan; 5). why we develop aging-related diseases; and 6). an evolutionarily credible reason for why and how aging occurs-these hormones act in an antagonistic pleiotrophic manner via cell cycle signaling; promoting growth and development early in life in order to achieve reproduction, but later in life, in a futile attempt to maintain reproduction, become dysregulated and drive senescence (dyosis). In essence, the Reproductive-Cell Cycle Theory can explain aging in all sexually reproductive life forms.
... These findings led to the model that dafachronic acid extends the lifespan of germline-deficient animals simply by activating a lifespan extending activity of DAF-12/ NHR. The m20 allele is predicted to eliminate the function of two isoforms of DAF-12/NHR while leaving a third isoform intact [16,17]. Therefore, it was interesting to ask how animals carrying a putative null DAF-12/NHR allele would respond to germline ablation. ...
In Caenorhabditis elegans and Drosophila melanogaster, removing the germline precursor cells increases lifespan. In worms, and possibly also in flies, this lifespan extension requires the presence of somatic reproductive tissues. How the somatic gonad signals other tissues to increase lifespan is not known. The lifespan increase triggered by loss of the germ cells is known to require sterol hormone signaling, as reducing the activity of the nuclear hormone receptor DAF-12, or genes required for synthesis of the DAF-12 ligand dafachronic acid, prevents germline loss from extending lifespan. In addition to sterol signaling, the FOXO transcription factor DAF-16 is required to extend lifespan in animals that lack germ cells. DAF-12/NHR is known to assist with the nuclear accumulation of DAF-16/FOXO in these animals, yet we find that loss of DAF-12/NHR has little or no effect on the expression of at least some DAF-16/FOXO target genes. In this study, we show that the DAF-12-sterol signaling pathway has a second function to activate a distinct set of genes and extend lifespan in response to the somatic reproductive tissues. When germline-deficient animals lacking somatic reproductive tissues are given dafachronic acid, their expression of DAF-12/NHR-dependent target genes is restored and their lifespan is increased. Together, our findings indicate that in C. elegans lacking germ cells, the somatic reproductive tissues promote longevity via steroid hormone signaling to DAF-12.
... In Caenorhabditis elegans, stressful conditions such as overcrowding, high temperature or starvation translate into pheromonal cues eventually processed through signalling pathways including insulin/IGF, TGFb-like and guanylyl cyclase pathways. As a result, the concentration of peculiar steroidal hormones (D4-dafachronic acids and D7-dafachronic acids) decreases, and the transition to dauer larva is released (Antebi et al. 1998Antebi et al. , 2000 Snow & Larsen 2000; Motola et al. 2006; Giroux & Corey 2007). These hormones are required for dauer larva development in C. elegans (Vowels & Thomas 1992). ...
Metazoan life cycles can be complex in different ways. A number of diverse phenotypes and reproductive events can sequentially occur along the cycle, and at certain stages a variety of developmental and reproductive options can be available to the animal, the choice among which depends on a combination of organismal and environmental conditions.
We hypothesize that a diversity of phenotypes arranged in developmental sequence throughout an animal's life cycle may have evolved by genetic assimilation of alternative phenotypes originally triggered by environmental cues.
This is supported by similarities between the developmental mechanisms mediating phenotype change and alternative phenotype determination during ontogeny and the common ecological condition that favour both forms of phenotypic variation.
The comparison of transcription profiles from different developmental stages throughout a complex life cycle with those from alternative phenotypes in closely related polyphenic animals is expected to offer critical evidence upon which to evaluate our hypothesis.
... However, recent studies revealed that DHR96 might play a role in xenobiotic stress responses in D. melanogaster [37]. Additionally, DAF-12, a NR1J member in C. elegans has been implicated in dia- pause [38]. Receptor gene identification and accompanying phylogenetic analyses revealed that D. pulex possesses a unique group of nuclear receptors consisting of three genes that share sequence similarities with HR96 and to a lesser extent VDR. ...
The nuclear receptor superfamily currently consists of seven gene subfamilies that encompass over 80 distinct receptor proteins. These transcription factors typically share a common five-domain structure with a highly conserved DNA-binding domain. Some nuclear receptors are ubiquitous among the metazoans, while others are unique to specific phylogenetic groups. Crustaceans represent the second largest group of arthropods with insects being the largest. However, relative to insects, little is known about the nuclear receptors of crustaceans. The aim of this study was to identify putative nuclear receptors from the first assembled genome of a crustacean Daphnia pulex http://wFleaBase.org. Nuclear receptor expression was evaluated and receptors were subjected to phylogenetic analyses to gain insight into evolution and function.
Twenty-five putative nuclear receptors were identified in D. pulex based on the presence of a conserved DNA-binding domain. All of the nuclear receptor protein sequences contain a highly homologous DNA-binding domain and a less conserved ligand-binding domain with the exception of the NR0A group. These receptors lack a ligand-binding domain. Phylogenetic analysis revealed the presence of all seven receptor subfamilies. The D. pulex genome contains several nuclear receptors that have vertebrate orthologs. However, several nuclear receptor members that are represented in vertebrates are absent from D. pulex. Notable absences include receptors of the 1C group (peroxisome proliferators-activated receptors), the 3A group (estrogen receptor), and the 3C group (androgen, progestogen, mineralcorticoid, and glucocorticoid receptors). The D. pulex genome also contains nuclear receptor orthologs that are present in insects and nematodes but not vertebrates, including putative nuclear receptors within the NR0A group. A novel group of receptors, designated HR97, was identified in D. pulex that groups with the HR96/CeNHR8/48/DAF12 clade, but forms its own sub-clade. Gene products were detected in adult female D. pulex for 21 of the 25 receptors.
Nuclear receptors are ancient proteins with highly conserved DNA-binding domains. The DNA-binding domains of the nuclear receptors of D. pulex contain the same degree of conservation that is typically found within nuclear receptors of other species. Most of the receptors identified in D. pulex have orthologs within the vertebrate and invertebrate lineages examined with the exception of the novel HR97 group and the Dappu-HR10 and potentially the Dappu-HR11 receptors found in D. pulex. These groups of receptors may harbour functions that are intrinsic to crustacean physiology.
... Mutations in lin-4 cause reiteration of the L1 fate at subsequent larval stages, while mutations in lin-14 lead to precocious adoption of the L2 stage fate at the L1 larval stage and premature terminal differentiation of seam cells, one stage earlier than wild type (Chalfie et al., 1981;Ambros and Horvitz, 1984). Multiple heterochronic genes, including lin-28 (encoding a cold shock domain protein), daf-12 (encoding a nuclear hormone receptor), lin-46 (encoding a homolog of the scaffolding protein gephyrin) and the let-7 family miRNAs (mir-48, mir-84 and mir-241), may converge on the regulation of the C. elegans Hunchback homolog, hbl-1, to control the succession from L2 to L3 (Moss et al., 1997;Antebi et al., 1998Antebi et al., , 2000Snow and Larsen, 2000;Pepper et al., 2004;Abbott et al., 2005;Li et al., 2005;Moss, 2007;Bethke et al., 2009). In lin-28 mutants, L2-stage events are skipped and the larval/adult switch takes place at the L3 stage (Ambros and Horvitz, 1984). ...
The temporal sequence of cell division and differentiation is explicitly controlled for succession and synchrony of developmental events. In this study we describe how the Caenorhabditis elegans gene sel-7 specifies the L3 stage-specific fate of seam cells, which adopt temporal specificities at each of four larval stages. Loss of function of sel-7 causes reiteration of the L2 stage fate at the L3 stage. sel-7 is involved in regulating the temporal expression pattern of hbl-1, which is a key factor in specifying the L2/L3 progression. We also show that sel-7 functions redundantly with other retarded heterochronic genes, including lin-46, daf-12 and the let-7 family miRNAs, in preventing adoption of the L2 fate at later stages. Expression of sel-7 in seam cells is temporally regulated through an evolutionarily conserved regulatory element located in intron 4 of sel-7. We further demonstrate that reiteration of the L2 proliferative seam cell division at the L3 stage in sel-7 mutants requires activity of the transcriptional mediator complex. Our study reveals that sel-7 functions as a novel heterochronic gene in controlling temporal cell identities and also demonstrates a role of the transcriptional mediator complex in integrating temporal information to specify seam cell division patterns in C. elegans.
... In C. elegans, pheromonal cues that indicate overcrowding, high temperature or starvation are processed through several signaling pathways including insulin/IGF, TGFβ-like, and guanylyl cyclase pathways (Fig. 1A)45678151617181920. This results in the decrease of a class of steroidal hormones, Δ4-DAfachronic acid and Δ7-DAfachronic acid (DAs) and shifts the nuclear hormone receptor DAF-12 from its ligand-bound form to a ligand-free form, which specifies the dauer fate2122232425. DAF-12 is strictly required for C. elegans dauer development [26] and its systemic expression suggests a role in the specification of dauer fate at the level of individual tissues [24]. Despite the wealth of knowledge of dauer formation in C. elegans, the extent to which this paradigm applies to other nematodes is not comprehensively investigated. ...
Under harsh environmental conditions, Caenorhabditis elegans larvae undergo arrest and form dauer larvae that can attach to other animals to facilitate dispersal. It has been argued that this phenomenon, called phoresy, represents an intermediate step toward parasitism. Indeed, parasitic nematodes invade their hosts as infective larvae, a stage that shows striking morphological similarities to dauer larvae. Although the molecular regulation of dauer entry in C. elegans involves insulin and TGF-beta signaling, studies of TGF-beta orthologs in parasitic nematodes didn't provide evidence for a common origin of dauer and infective larvae. To identify conserved regulators between Caenorhabditis and parasitic nematodes, we used an evolutionary approach involving Pristionchus pacificus as an intermediate. We show by mutational and pharmacological analysis that Pristionchus and Caenorhabditis share the dafachronic acid-DAF-12 system as the core endocrine module for dauer formation. One dafachronic acid, Delta7-DA, has a conserved role in the mammalian parasite Strongyloides papillosus by controlling entry into the infective stage. Application of Delta7-DA blocks formation of infective larvae and results in free-living animals. Conservation of this small molecule ligand represents a fundamental link between dauer and infective larvae and might provide a general strategy for nematode parasitism.
... A group of lipophylic, steroid-like molecules also modulate longevity. These molecules require the nuclear hormone receptor DAF-12 for their effects (Antebi et al., 1998Antebi et al., , 2000 Snow and Larsen, 2000). Three of the steroid-like molecules are bile acids (Held et al., 2006; Motola et al., 2006), and a fourth is the steroid precursor pregnenolone (Broue et al., 2007). ...
In recent years, there has been significant growth in our understanding of the regulation of longevity. The most notable change is the identification and detailed description of a number of molecular pathways modulating the rate of aging. A good portion of this new data has come from studies using the genetic model organism Caenorhabditis elegans. In this review, we provide an overview of physiological systems that are involved in the modulation of aging in C. elegans, then discuss the known endocrine signaling systems that are likely to couple these systems together. Finally, we present a working model describing how aging may be regulated as a coordinated system, communicating through endocrine signals.
... Finally, larvae with a mutation in the daf-12 gene are Daf-d, and these mutations suppress the Daf-c defect of daf-7 and interact with particular alleles of daf-2 (23-27). The daf-12 gene shows homology to nuclear hormone receptors (42,43). In wild-type larvae under growth conditions, the inferred order of gene function places the TGF-β-like daf-7 and the guanylyl cyclase daf-11 pathways both functioning to inactivate the daf-12 gene, thereby preventing dauer formation. ...
Protein damage that accumulates during aging can be mitigated by a repair methyltransferase, the l-isoaspartyl-O-methyltransferase. In Caenorhabditis elegans, the pcm-1 gene encodes this enzyme. In response to pheromone, we show that pcm-1 mutants form fewer dauer larvae with reduced survival due to loss of the methyltransferase activity. Mutations in daf-2, an insulin/insulin-like growth factor-1-like receptor, and daf-7, a transforming growth factor-β–like ligand, modulate pcm-1 dauer defects. Additionally, daf-2 and daf-7 mutant dauer larvae live significantly longer than wild type. Although dauer larvae are resistant to many environmental stressors,
a proportionately larger decrease in dauer larvae life spans occurred at 25°C compared to 20°C than in adult life span. At
25°C, mutation of the daf-7 or pcm-1 genes does not change adult life span, whereas mutation of the daf-2 gene and overexpression of PCM-1 increases adult life span. Thus, there are both overlapping and distinct mechanisms that
specify dauer and adult longevity.
... Among them, DAF-12/NHR is perhaps best understood for its various roles in dauer formation, fat metabolism, developmental timing, gonadal maturation, and longevity. DAF-12/NHR is most closely related to the vertebrate vitamin D and LXR receptors (Antebi et al. 2000; Snow and Larsen 2000), but regulates reproductive maturation analogous to the estrogen receptor. Genetic epistasis experiments reveal that it works at the end of the dauer pathways, downstream from cGMP, TGF-, and IIS pathways (Fig. 2; Riddle et al. 1981; Vowels and Thomas 1992; Thomas et al. 1993). ...
Because life is often unpredictable, dynamic, and complex, all animals have evolved remarkable abilities to cope with changes in their external environment and internal physiology. This regulatory plasticity leads to shifts in behavior and metabolism, as well as to changes in development, growth, and reproduction, which is thought to improve the chances of survival and reproductive success. In favorable environments, the nematode Caenorhabditis elegans develops rapidly to reproductive maturity, but in adverse environments, animals arrest at the dauer diapause, a long-lived stress resistant stage. A molecular and genetic analysis of dauer formation has revealed key insights into how sensory and dietary cues are coupled to conserved endocrine pathways, including insulin/IGF, TGF-beta, serotonergic, and steroid hormone signal transduction, which govern the choice between reproduction and survival. These and other pathways reveal a molecular basis for metazoan plasticity in response to extrinsic and intrinsic signals.
... One candidate for a negative regulator of lin-28 is the product of the daf-12 gene. daf-12 encodes a nuclear hormone receptor that has multiple roles in C. elegans development (Antebi et al., 1998(Antebi et al., , 2000Snow and Larson, 2000). Antebi et al. (1998) showed that certain alleles of daf-12 cause a retarded phenotype. ...
The heterochronic gene lin-28 of the nematode Caenorhabditis elegans controls the relative timing of diverse developmental events during the animal's larval stages. lin-28 is stage-specifically regulated by two genetic circuits: negatively by the 22-nt RNA lin-4 and positively by the heterochronic gene lin-14. Here, we show that lin-28 is repressed during normal development by a mechanism that acts on its mRNA after translation initiation. We provide evidence that lin-14 inhibits a negative regulation that is independent of the lin-4 RNA and involves the gene daf-12, which encodes a nuclear hormone receptor. The lin-4-independent repression does not affect the initiation of translation on the lin-28 mRNA, and like the lin-4-mediated repression, acts through the gene's 3'-untranslated region. In addition, we find that lin-4 is not sufficient to cause repression of lin-28 if the lin-4-independent circuit is inhibited. Therefore, the lin-4-independent circuit likely contributes substantially to the down-regulation of lin-28 that occurs during normal development. The role of lin-4 may be to initiate or potentiate the lin-4-independent circuit. We speculate that a parallel lin-4-independent regulatory mechanism regulates the expression of lin-14.
... Multiple genes that regulate the process have been identified and ordered by epistasis analysis into three intersecting pathways (Riddle and Albert, 1997 ). daf-12 functions at the intersection of these pathways as a critical regulator of dauer development (Antebi et al., 2000; Snow and Larsen, 2000 ). daf-12 also functions in multiple processes during nondauer postembryonic development, including temporal fate and gonad development, to provide coordinated regulation of distinct stage-specific developmental programs in a range of cell types (Antebi et al., 1998). ...
The estrogen receptors are members of a large family of transcription factors, which include receptors for steroid hormones, retinoids, vitamin D and thyroid hormone, as well as several orphan receptors with yet undiscovered ligands. The process by which small lipophilic molecules signal in target tissues is complex and involves nuclear receptor associations with a multitude of coregulator proteins. These proteins participate in and recruit many enzymatic and structural activities that allow modulation of chromatin structure to facilitate stimulation or repression of gene expression. This review will present an overview of those regulatory factors that interact with the estrogen receptors and participate in the transmission of the estrogenic signal.
... We have shown that daf-12 regulates the timing of let-7 expression: mutations in daf-12 that result in retarded heterochronic phenotypes cause retarded expression of let-7 and let-7::gfp. A DAF-12::GFP fusion revealed high expression in the hypodermal seam cells (Antebi et al., 2000), and both the DAF-12::GFP fusion protein and daf-12 mRNA are expressed throughout development (Antebi et al., 2000;Snow and Larsen, 2000), placing DAF-12 in the right place at the right time. One possibility is that DAF-12 regulation of let-7 could be direct with DAF-12 filling the role of the TREB and binding to the TRE to activate the transcription of let-7. ...
MicroRNAs (miRNAs) are a large family of small regulatory RNAs that are poorly understood. The let-7 miRNA regulates the timing of the developmental switch from larval to adult cell fates during Caenorhabditis elegans development. Expression of let-7 RNA is temporally regulated, with robust expression in the fourth larval and adult stages. Here, we show that, like let-7 RNA, a transcriptional fusion of the let-7 promoter to gfp is temporally regulated, indicating that let-7 is transcriptionally controlled. Temporal upregulation of let-7 transcription requires an enhancer element, the temporal regulatory element (TRE), situated about 1200 base pairs upstream of the start of the mature let-7 RNA. The TRE is both necessary and sufficient for this temporal upregulation. A TRE binding factor (TREB) is able to bind to the TRE, and a 22-base pair inverted repeat within the TRE is necessary and sufficient for this binding. We also find that the nuclear hormone receptor DAF-12 and the RNA binding protein LIN-28 are both required for the correct timing of let-7 RNA and let-7::gfp expression. We speculate that these heterochronic genes regulate let-7 expression through its TRE.
... The C-terminal domains of the human proteins in this family and DAF-16 have been shown to have similar functional properties despite very limited sequence identity (Nasrin et al., 2000; Lee et al., 2001), suggesting that such properties as conformation and hydropathy may play more important roles in the function of this domain than sequence identity. In C. elegans, daf-12 encodes a member of the steroid nuclear receptor family of transcription factors, and daf-12 interacts with the insulin-like branch of the dauer pathway to regulate dauer development (Riddle and Albert, 1997; Snow and Larsen, 2000). A proposed S. stercoralis ortholog of daf-12 has been identified (Siddiqui et al., 2000). ...
A forkhead transcription factor gene, fktf-1, which we propose to be orthologous to the Caenorhabditis elegans dauer-regulatory gene daf-16 has been discovered in the parasitic nematode Strongyloides stercoralis. Genomic and cDNA sequences from both species predict alternately spliced a and b message isoforms. In contrast to C. elegans, where two a isoforms, daf-16a1 and daf-16a2, are found, a single fktf-1a isoform is found in S. stercoralis. Five of the 10 introns found in the C. elegans gene are found in the proposed S. stercoralis ortholog. Functional motifs common to DAF-16 and several mammalian forkhead transcription factors are conserved in FKTF-1. These include the forkhead DNA binding domain, four Akt/protein kinase B phosphorylation sites and a C-terminal domain that may associate with factors such as the steroid receptor coactivator and other factors necessary for transcriptional regulation. An N-terminal serine-rich domain found in DAF-16A is greatly expanded in FKTF-1A. This domain is missing in DAF-16B, FKTF-1B and all mammalian orthologs. FKTF-1 shows the closest phylogenetic relationship to DAF-16 among all known mammalian and nematode forkhead transcription factors. Like its proposed Caenorhabditis ortholog, the fktf-1 message is expressed at all stages of the life cycle examined thus far. Discovery of fktf-1 indicates the presence of an insulin-like signalling pathway in S. stercoralis similar to that known to regulate dauer development in C. elegans. This pathway is a likely candidate to control infective larval arrest and reactivation as well as regulation of the switch between parasitic and free-living development in the parasite.
Food is an ultimate regulator of animal reproduction. Because of its invariant cell lineage, ease of synchronized culture, and powerful genetics, the nematode Caenorhabditis elegans (C. elegans) has served as an excellent model system for delineating the genetic pathways that mediate the nutritional regulation of germline development. C. elegans possesses multiple nutritional checkpoints during post-embryonic development that temporally arrest developmental programs in the germline and somatic stem/progenitor cells. The insulin/insulin-like growth factor (IGF) signaling (IIS) pathway and other factors such as adenosine monophosphate (AMP)-activated kinase (AMPK) and mechanistic (or mammalian) Target of Rapamycin Complex 1 (mTORC1) constitute the signaling network dedicated to regulating developmental quiescence of the germline. Furthermore, additional nutrient-responsive pathways adjust the size of the germline stem/progenitor cell pool by altering the balance between self-renewal and differentiation, as well as the balance between cellular survival and death. These findings illustrate the molecular mechanisms that coordinate germline development with the dietary environment by altering the behavior of its stem/progenitor cells.
In the last decade of the 20th century one model organism has received more attention from gerontologists than any other, namely the free-living soil nematode Caenorhabditis elegans (C. elegans). We owe much of our general view of the genetic determination of lifespan to this organism. C. elegans became a popular organism for the analysis of complex biological problems following the pioneering work of Sidney Brenner and Sir John Sulston who determined the developmental cellular fate map [1]. It is also noted for the early work of Robert Horvitz and colleagues who used the cell lineage map to begin a genetic dissection of programmed cell death [2].
The lifespan of metazoans can be extended by environmental conditions: caloric restriction (CR) in a wide range of organisms (1), low temperature in some poikilothermic animals (2), and low oxygen concentrations in the nematode Caenorhabditis elegans (3). The mechanisms by which each condition slows the aging rate have not yet been fully elucidated. Function of CR has been postulated as hormonal changes, altered gene expression, lowered metabolic rate, and a reduced generation rate of mitochondrial reactive oxygen species (ROS). Lifespan could also be lengthened by environmental perturbations. Hormesis is a phenomenon occurring when agents that are harmful at high doses or over long periods, actually produce beneficial effects, such as lifespan extension, when used at low doses or over short periods. C. elegans shows lifespanextension hormesis when exposed to low doses of radiation (4) or short-term heat (5), hyperoxia (6), or hyperbaric oxygen (7). These treatments are associated with adaptive resistance to lethal thermal or oxidative stress, and the gene expression of stress-defense proteins (5,6,8).
Lipopolysaccharide (LPS) is the main surface constituent of Gram-negative bacteria. Lipid A, the hydrophobic moiety, outer monolayer of the outer cell membrane forms the major component of LPS. Immunogenic Lipid A is recognized by the innate immune system through the TLR 4/MD-2 complex. Pseudomonas aeruginosa PAO1, a Gram-negative bacterium is known to cause nosocomial infection and known for its adaptation to adverse environmental conditions. Pseudomonas aeruginosa can infect a broad host spectrum including Caenorhabditis elegans, a simple free living soil nematode. Here, we reveal that PAO1 modifies its Lipid A during the host interaction with C. elegans. The penta-acylated form of Lipid A was identified by using matrix assisted laser desorption ionization–time of flight analysis and the β-(1,6)-linked disaccharide of glucosamine with phosphate groups, 2 and 2′ amide linked fatty acid chain and 3 and 3′ ester linked fatty acids were investigated for the modification using the non destructive 1H NMR, spin–lattice (T
1) relaxation measurement, differential scanning calorimetry. T
1 relaxation measurements showed that the 2 and 2′ amide linked fatty acid chain, –CH in the glucosamine disaccharide of PAO1 lipid A, in an exposed host had a different spin lattice relaxation time compared to an unexposed host and the findings were reconfirmed using in vitro human corneal epithelial cells cell lines. Furthermore, scanning electron microscope and confocal laser scanning microscopy analysis revealed that the P. aeruginosa PAO1 biofilm formation was disturbed in the exposed host condition. The daf-12, daf-16, tol-1, pmk-1, ins-7 and ilys3 immune genes of C. elegans were examined with live bacterial and isolated lipid moiety infection and the expression was found to be highly specific. Overall, the present study revealed that PAO1 modified its 2 and 2′ amide linked fatty acid chain in the lipid A of PAO1 LPS during the exposed host condition.
Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA was increased 100-fold through carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap hybrid mass spectrometer outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer. The HR-SIM method has a limit of quantification of as low as 1 pg of DA, which enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms DA increases from 0.04 ± 0.02 ng DA/mg protein in the L1 larval stage to 1.21 ± 0.67 ng DA/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, three genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-β, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.
Transforming Growth Factor-β (TGF-β) superfamily ligands regulate many aspects of cell identity, function, and survival in multicellular animals. Genes encoding five TGF-β family members are present in the genome of C. elegans. Two of the ligands, DBL-1 and DAF-7, signal through a canonical receptor-Smad signaling pathway; while a third ligand, UNC-129, interacts with a noncanonical signaling pathway. No function has yet been associated with the remaining two ligands. Here we summarize these signaling pathways and their biological functions.
Recent self-forgiveness research raises thorny issues: Are people who claim to forgive themselves merely excusing their offenses? Might it be adaptive for people to feel distress after they hurt others? This study asked 138 undergraduates to reflect on times when they offended others. Feelings of self-condemnation were associated with maladjustment, as shown in prior studies. However, participants reported more prosocial responses (repentance and a sense of being humbled) if they accepted responsibility, experienced remorse, and found that reducing negative feelings required effort. Egotism was associated with reluctance to accept responsibility, whereas a more shame-prone, neurotic pattern was associated with self-condemnation. To avoid confounding self-forgiveness with excusing, future studies should include responsibility ratings and should also distinguish between remorse and self-condemnation.
The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.
Daumone is one of the three purified and artificially synthesized components of the Caenorhabditis elegans dauer pheromone. It affects the major signal transduction pathways known to discriminate between developmental arrest at the dauer stage and growth to the adult [the transforming growth factor beta (TGF-beta) and daf-2/IGF1R pathways], just as natural pheromone extracts do. Transcription of daf-7/TGF-beta is reduced in pre-dauer larvae, and nuclear localization of the DAF-16/FOXO transcription factor is increased in embryos and L1 larvae exposed to synthetic daumone. However, daumone does not require the cilia in the amphidial neurons to produce these effects nor does it require the Galpha protein GPA-3 to induce dauer entry, although GPA-3 is required for dauer induction by natural dauer pheromone extracts. Synthetic daumone has physiological effects that have not been observed with natural pheromone. It is toxic at the concentrations required for bioassay and is lethal to mutants with defective cuticles. The molecular and physiological effects of daumone and natural dauer pheromone are only partially overlapping.
With the characterization of the Smads 5 years ago, it became possible to trace the TGFbeta signal transduction pathway from the plasma membrane to the nucleus. Since that time, many Smad interaction partners, cofactors and target genes have been identified using a variety of experimental approaches and model systems. Understanding how these partners generate tissue specificity and crosstalk between pathways is an ongoing pursuit for the field of TGFbeta signal transduction. The nematode Caenorhabditis elegans provides a simple, genetically tractable model organism in which to address this goal. This review will examine progress towards the identification of cellular and molecular targets of TGFbeta-related signaling in C. elegans.
The cell division and differentiation events that occur during the development of the nematode Caenorhabditis elegans are nearly identical between different individuals, a feature that distinguishes this organism from larger and more complex metazoans, such as humans and Drosophila. In view of this discrepancy, it might be expected that the regulation of cell growth, division and differentiation in C. elegans would involve mechanisms separate from those utilized in larger animals. However, the results of recent genetic, molecular and cellular studies indicate that C. elegans employs an arsenal of developmental regulatory mechanisms quite similar to those wielded by its arthropod and vertebrate relatives. Thus, the nematode system is providing both novel and complementary insights into the general problem of how growth and patterning events are integrated in development. This review offers a general perspective on the regulation of cell division and growth in C. elegans, emphasizing recent studies of these crucial aspects of development.
It is difficult to exaggerate the progress that has been made in biogerontology over the last 15 years. As with all scientific revolutions, a few experiments in a small number of laboratories have changed the way in which we think about and design experiments. As a result of these experiments, there is much evidence to suggest that a rudimentary understanding of some of the processes that cause aging will be available in the next decade. One particular area of progress is the molecular genetics of lifespan. Although one may draw some distinctions between chronological lifespan and normal aging, extended lifespan remains one of the best indicators that an intervention in an aging process has been made. The isolation of a long-lived variant of a laboratory invertebrate is now essentially a trivial project but the information obtained from this approach is proving invaluable. As with most other biological problems, the most important experimental developments are coming from studying simple organisms in a reductionist fashion.
The dauer larva of the nematode Caenorhabditis elegans is a good model system for investigating the regulation of developmental fates by environmental cues. Here we show that SDF-9, a protein tyrosine phosphatase-like molecule, is involved in the regulation of dauer larva formation. The dauer larva of sdf-9 mutants is different from a normal dauer larva but resembles the dauer-like larva of daf-9 and daf-12 dauer-constitutive mutants. Like these mutants, the dauer-constitutive phenotypes of sdf-9 mutants were greatly enhanced by cholesterol deprivation. Epistasis analyses, together with the relationship between sdf-9 mutations and daf-9 expression, suggested that SDF-9 increases the activity of DAF-9 or helps the execution of the DAF-9 function. SDF-9 was expressed in two head cells in which DAF-9 is expressed. By their position and by genetic mosaic experiments, we identified these cells as XXXL/R cells, which are known as embryonic hypodermal cells and whose function at later stages is unknown. Killing of the sdf-9-expressing cells in the wild-type first-stage larva induced formation of the dauer-like larva. Since this study on SDF-9 and former studies on DAF-9 showed that the functions of these proteins are related to those of steroids, XXXL/R cells seem to play a key role in the metabolism or function of a steroid hormone(s) that acts in dauer regulation.
It has been established that an intricate program of gene expression controls progression through the different stages in development. The equally complex biological phenomenon known as aging is genetically determined and environmentally modulated. This review focuses on the genetic component of aging, with a special emphasis on differential gene expression. At least two genetic pathways regulating organism longevity act by modifying gene expression. Many genes are also subjected to age-dependent transcriptional regulation. Some age-related gene expression changes are prevented by caloric restriction, the most robust intervention that slows down the aging process. Manipulating the expression of some age-regulated genes can extend an organism's life span. Remarkably, the activity of many transcription regulatory elements is linked to physiological age as opposed to chronological age, indicating that orderly and tightly controlled regulatory pathways are active during aging.
Several genes involved in the determination of life span have been identified by mutation in the free-living soil nematode Caenorhabditis elegans. One of the key pathways studied in the context of life span is the DAF-2 pathway. The daf-2 gene is homologous to the insulin and insulin-like growth factor 1 receptor families. A downstream gene, daf-16, encodes a protein that is homologous to the forkhead transcription factor. A study by McElwee, Bubb, and Thomas, published in the current issue of Aging Cell, used genome-scale gene expression analysis to search for genes that are differentially expressed between long-lived daf-2(e1370) and short-lived daf-16(m27);daf-2(e1370) animals. In doing so, they identified candidate direct and indirect targets of DAF-16. In this Perspective, I discuss the results of this study.
Several investigators have generated long-lived nematode worms (Caenorhabditis elegans) in the past decade by mutation of genes in the organism in order to study the genetics of aging and longevity. Dozens of longevity assurance genes (LAG) that dramatically increase the longevity of this organism have been identified. All long-lived mutants of C. elegans are also resistant to environmental stress, such as high temperature, reactive oxygen species (ROS), and ultraviolet irradiation. Double mutations of some LAGs further extended life span up to 400%, providing more insight into cellular mechanisms that put limits on the life span of organisms. With the availability of the LAG mutants and the combined DNA microarray and RNAi technology, the understanding of actual biochemical processes that determine life span is within reach: the downstream signal transduction pathway may regulate life span by up-regulating pro-longevity genes such as those that encode antioxidant enzymes and/or stress-response proteins, and down-regulating specific life-shortening genes. Furthermore, longevity could be modified through chemical manipulation. Results from these studies further support the free radical theory of aging, suggest that the molecular mechanism of aging process may be shared in all organisms, and provide insight for therapeutic intervention in age-related diseases.
The central nervous system (CNS) represents the organ with the highest structural and functional complexity. Accordingly, uncovering the mechanisms leading to cell diversity, patterning and connectivity in the CNS is one of the major challenges in developmental biology. The developing CNS of the fruitfly Drosophila melanogaster is an ideal model system to study these processes. Several principle questions regarding neurogenesis (like stem cell formation, cell fate specification, axonal pathfinding) have been addressed in Drosophila by focusing on the relatively simply structured truncal parts of the nervous system. However, information processing (e.g. vision, olfaction), behavior, learning and memory require highly specialized structures, which are located in the brain. Owing to much higher complexity and hidden segmental organisation our understanding of brain development is still quite rudimentary. Considerable advances have been made recently in bringing the resolution of brain structures to the level of individual cells and their lineages, which significantly facilitates investigations into the mechanisms controlling brain development.
This book provides an overview of some major facets of recent research on Drosophila brain development. The individual chapters were written by experts in each field. V. Hartenstein et al. survey the generic cell types that make up the developing brain and describe the morphogenesis of neural lineages and their relationship to neuropile compartments in the larval brain. Recent findings on anterioposterior regionalization and on dorsoventral patterning in the embryonic brain are reviewed in the chapters by R. Lichtneckert and H. Reichert and by R. Urbach and G. Technau, respectively. Both processes show striking parallels between Drosophila and mouse. Photoactivated gene expression as a means for tracing cell fate through embryonic brain development is demonstrated in J. Minden´s chapter. At present, the best characterized neural network on the developmental, structural, and functional level is the chemosensory system, to which three chapters are devoted: R. Stocker´s chapter covers the design of the larval chemosensory system and shows that it prefigures the adult system. V. Rodrigues and T. Hummel summarize recent findings on the specification and connectivity development of the adult olfactory receptor neurons. P. Laissue and B. Vosshall review the molecular biology, neuroanatomy and function of the adult olfactory system. A further focus of research is the visual system, with the optic lobes comprising about half of the adult fly brain. The genetic and cellular principles which direct the assembly of the optic lobes are highlighted in the chapter by K. Fischbach and P. Hiesinger. The central brain harbors distinct neuropils like the central complex and the mushroom bodies, as well as “diffused neuropiles” which lack clearly demarcated structures. K. Ito and T. Awasaki review the organization of the adult central brain and show how its complex architecture evolves from clonally related neural circuits.
This book will be helpful to those who want to study brain development in the fly. As knowledge extracted from the Drosophila model has often proven to be of more general relevance, comparative aspects are included in most chapters. Therefore, this book should also be useful for researchers working on brain development in other organisms and on brain evolution, as well as for instructors and advanced students in the field of developmental neurobiology.
In mammals, insulin signalling regulates glucose transport together with the expression and activity of various metabolic enzymes. In the nematode Caenorhabditis elegans, a related pathway regulates metabolism, development and longevity. Wild-type animals enter the developmentally arrested dauer stage in response to high levels of a secreted pheromone, accumulating large amounts of fat in their intestines and hypodermis. Mutants in DAF-2 (a homologue of the mammalian insulin receptor) and AGE-1 (a homologue of the catalytic subunit of mammalian phosphatidylinositol 3-OH kinase) arrest development at the dauer stage. Moreover, animals bearing weak or temperature-sensitive mutations in daf-2 and age-1 can develop reproductively, but nevertheless show increased energy storage and longevity. Here we show that null mutations in daf-16 suppress the effects of mutations in daf-2 or age-1; lack of daf-16 bypasses the need for this insulin receptor-like signalling pathway. The principal role of DAF-2/AGE-1 signalling is thus to antagonize DAF-16. daf-16 is widely expressed and encodes three members of the Fork head family of transcription factors. The DAF-2 pathway acts synergistically with the pathway activated by a nematode TGF-β-type signal, DAF-7, suggesting that DAF-16 cooperates with nematode SMAD proteins in regulating the transcription of key metabolic and developmental control genes. The probable human ortholognes of DAF-16, FKHR and AFX, may also act downstream of insulin signalling and cooperate with TGF-β effectors in mediating metabolic regulation. These genes may be dysregulated in diabetes.
The venom of the black widow spider (BWSV) uniquely contains a family of high molecular weight proteins that cause uncontrolled vesicle release in synapses. Two membrane receptors for BWSV have been identified, one of these being latrophilin/CIRL (LPH), a member of the G-protein coupled receptor superfamily of cell-signalling receptors and the other being neurexin. In mammals, LPH and neurexin have been shown to bind BWSV, but their function is unclear. We established C.elegans as a model system for studying the effects of BWSV by microinjection of venom into wild-type (N2) C.elegans, which showed that the venom had an acute lethal effect over a million-fold range of concentrations. BWSV treated with SDS (0.1%) or heat before injection reduced the kill rate in N2 C.elegans to zero, this suggests that the active component of the venom is a protein. FPLC of BWSV demonstrated that the active component of BWSV toxic to C.elegans resembles epsilon- latroinsectotoxin. Identification of a homologue of the latrophilin gene in C.elegans, BO457.1, induced a functional knockout of the latrophilin gene by RNA interference (RNAi). The knockout was examined for a change in phenotype, which occurred in RNAi treated worms, compared to N2, and was extensively characterised. LPH knockout C.elegans were completely resistant to the lethal effects of BWSV over the same concentration range as that used in the N2 worms, whereas RNAi of CYP37A1, BO286.2 and neurexin 1alpha homologue has no effect on BWSV toxicity. We have shown that a C.elegans latrophilin homologue mediates the toxic effects of black widow spider venom in the nematode and identified a high molecular weight latrotoxin that kills C.elegans. Additionally, the data provide evidence for an important role of LPH in nerve cell function.
Dauer larva formation in Caenorhabditis elegans is controlled by chemosensory cells that respond to environmental cues. Genetic interactions among mutations in 23 genes that affect dauer larva formation were investigated. Mutations in seven genes that cause constitutive dauer formation, and mutations in 16 genes that either block dauer formation or result in the formation of abnormal dauers, were analyzed. Double mutants between dauer-constitutive and dauer-defective mutations were constructed and characterized for their capacity to form dauer larvae. Many of the genes could be interpreted to lie in a simple linear epistasis pathway. Three genes, daf-16, daf-18 and daf-20, may affect downstream steps in a branched part of the pathway. Three other genes, daf-2, daf-3 and daf-5, displayed partial or complex epistasis interactions that were difficult to interpret as part of a simple linear pathway. Dauer-defective mutations in nine genes cause structurally defective chemosensory cilia, thereby blocking chemosensation. Mutations in all nine of these genes appear to fall at a single step in the epistasis pathway. Dauer-constitutive mutations in one gene, daf-11, were strongly suppressed for dauer formation by mutations in the nine cilium-structure genes. Mutations in the other six dauer-constitutive genes caused dauer formation despite the absence of functional chemosensory endings. These results suggest that daf-11 is directly involved in chemosensory transduction essential for dauer formation, while the other Daf-c genes play roles downstream of the chemosensory step.
We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.
The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.
We have isolated cosmid clones containing the chromosomal chicken progesterone receptor gene. The gene consists of eight exons and is approximately 38 kilobases long. Individual exons correlate well with conserved functional domains of the receptor molecule. Alternative polyadenylylation in the second intron results in a putative non-hormone-binding protein. The cap site of the gene is heterogeneous over at least 14 base pairs and lies in a very G + C-rich region. The promoter lacks "TATA" and "CAAT" boxes, but CCGCCC motifs exist in the surrounding region.
The specificity of the cyclic AMP-dependent protein kinase was examined using two series of dodecapeptides as substrates. One series consisted of peptides of the general sequence (Gly)x-Arg-Arg-(Gly)y-Ala-Ser-Leu-Gly in which x + y = 6. The other series consisted of peptides of the sequence (Gly)x-Lys-Arg-(Gly)y-Ala-Ser-Leu-Gly in which x + y was again equal to 6. The peptides Gly-Gly-Gly-Gly-Gly-Gly-Gly-Arg-Arg-Ser-Leu-Gly and Gly-Gly-Gly-Gly-Gly-Gly-Gly-Lys-Arg-Ser-Leu-Gly were also examined. In the series in which the adjacent arginines were located various distances from the serine, the substrate for which the enzyme clearly exhibited optimal kinetic constants contained one amino acid residue between the basic residues and serine. Direct binding studies of N alpha-[3H]acetyl peptides to catalytic subunit of cyclic AMP-dependent protein kinase revealed a correlation between binding affinity and the ability to serve as substrate for the enzyme. In the second series in which the adjacent basic amino acids were Lys-Arg, optimal kinetic constants were again obtained when these residues were separated from serine by a single amino acid. This latter result was surprising in view of phosphorylation site sequences in the known physiologically significant protein substrates for the kinase, since those containing Lys-Arg all contain two amino acids between these residues and serine.
The substrate specificity of cGMP-dependent protein kinase has been investigated by examining the ability of the enzyme to phosphorylate a series of synthetic peptides that correspond to the amino acid sequence at its site of autophosphorylation. The undecapeptide Ile53-Gly-Pro-Arg-Thr-Thr58-Arg-Ala-Gln-Gly-Ile63 which corresponds to the sequence around threonine-58 in cGMP-dependent protein kinase (Takio, K., Smith, S.B., Walsh, K.A., Krebs, E.G., and Titani, K. (1983) J. Biol. Chem. 258, 5531-5536) was synthesized and tested as a substrate for that enzyme. It was phosphorylated to the extent of 1.0 mol of phosphate/mol of peptide. Analysis of the products of Edman degradation of the phosphopeptide indicated that only threonine-58 was phosphorylated, as is the case for the autophosphorylation reaction in the native enzyme. The peptide was phosphorylated by cGMP-dependent protein kinase with a Km value of 578 +/- 25 microM and a Vmax of 0.069 +/- 0.003 mumol/min/mg of enzyme. This low Vmax value is consistent with the relatively slow rate of the autophosphorylation reaction. An analog peptide that contained serine in place of threonine-58 was also phosphorylated to 1.0 mol of phosphate/mol of peptide. That phosphopeptide contained only phosphoserine. The serine-containing analog peptide had a Km value similar to that of the parent peptide but was phosphorylated with a 70-fold higher Vmax value. Substitution of arginine-56 in the parent peptide by an alanine residue resulted in a peptide that was essentially not a substrate. Substitution of arginine-59, COOH-terminal to the phosphorylatable threonine, yielded a peptide with a Vmax similar to that of the parent peptide but a Km value of almost 22,000 microM. These results indicate that serine is a better phosphate-accepting residue than is threonine and that both arginine residues around the site of autophosphorylation are important specificity determinants for the cGMP-dependent protein kinase.
Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.
The nematode Caenorhabditis elegans responds to conditions of overcrowding and limited food by arresting development as a dauer larva. Genetic analysis of mutations that alter dauer larva formation (daf mutations) is presented along with an updated genetic pathway for dauer vs. nondauer development. Mutations in the daf-2 and daf-23 genes double adult life span, whereas mutations in four other dauer-constitutive genes positioned in a separate branch of this pathway (daf-1, daf-4, daf-7 and daf-8) do not. The increased life spans are suppressed completely by a daf-16 mutation and partially in a daf-2; daf-18 double mutant. A genetic pathway for determination of adult life span is presented based on the same strains and growth conditions used to characterize Daf phenotypes. Both dauer larva formation and adult life span are affected in daf-2; daf-12 double mutants in an allele-specific manner. Mutations in daf-12 do not extend adult life span, but certain combinations of daf-2 and daf-12 mutant alleles nearly quadruple it. This synergistic effect, which does not equivalently extend the fertile period, is the largest genetic extension of life span yet observed in a metazoan.
The Caenorhabditis elegans dauer larva is specialized for dispersal without growth and is formed under conditions of overcrowding and limited food.
The daf-7 gene, required for transducing environmental cues that support continuous development with plentiful food, encodes a transforming
growth factor-β (TGF-β) superfamily member. A daf-7 reporter construct is expressed in the ASI chemosensory neurons. Dauer-inducing pheromone inhibits daf-7 expression and promotes dauer formation, whereas food reactivates daf-7 expression and promotes recovery from the dauer state. When the food/pheromone ratio is high, the level of daf-7 mRNA peaks during the L1 larval stage, when commitment to non-dauer development is made.
A C. elegans neurosecretory signaling system regulates whether animals enter the reproductive life cycle or arrest development at the
long-lived dauer diapause stage. daf-2, a key gene in the genetic pathway that mediates this endocrine signaling, encodes an insulin receptor family member. Decreases
in DAF-2 signaling induce metabolic and developmental changes, as in mammalian metabolic control by the insulin receptor.
Decreased DAF-2 signaling also causes an increase in life-span. Life-span regulation by insulin-like metabolic control is
analogous to mammalian longevity enhancement induced by caloric restriction, suggesting a general link between metabolism,
diapause, and longevity.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
The functions of the ecdysone-induced DHR3 and E75B orphan nuclear receptors in the early stages of Drosophila metamorphosis
were investigated. DHR3 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis.
It also induces βFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal
pulse of ecdysone. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming
a complex with DHR3 on the βFTZF1 promoter, thereby providing a timing mechanism for βFTZF1 induction that is dependent on the disappearance of E75B.
During the course of normal embryonic and post-embryonic development, 131 cells in a Caenorhabditis elegans hermaphrodite undergo programmed cell death. Loss of function mutations in either of the genes ced-3 or ced-4 abolish cell deaths, enabling these `undead' cells to survive and be incorporated into the adult with no obvious deleterious consequences. Ultrastructural reconstructions have shown that undead cells exhibit many differentiated characteristics. Most of the reconstructed cells appeared to be neurons with all the characteristic features associated with such cells, such as processes, synaptic vesicles and presynaptic specializations. However, clear morphological differences were seen among the undead neurons, suggesting a diversity of cell type. One of the reconstructed cells was a rectal epithelial cell, which had displaced its lineal sister that normally functions in this role. Removal of the ability to undergo programmed cell death by mutation therefore reveals a diversity of cryptic differentiated states that are acquired by cells that normally are destined to die.
The nuclear hormone receptors comprise a super-family of ligand-modulated transcription factors that regulate homeostasis, reproduction, development, and differentiation. Three amino acids within the zinc finger DNA binding motif determine target gene specificity. Groups of receptors exist with similar DNA binding specificity. A complex carboxy terminal region mediates ligand binding, dimerization, and hormone-relieved transcriptional inactivation. We summarize the current understanding of these phenomena and suggest a novel model that structurally and functionally links these events. This “regulatory zipper model” may explain the mechanism by which ligand activates nuclear hormone receptors.
1,25-Dihydroxy vitamin D3[1,25(OH)2D3] an active form of vitamin D, has roles in many biological phenomena such as calcium homeostasis and bone formation1−3, which are thought to be mediated by the 1,25(OH)2D3 receptor (VDR), a member of the nuclear hormone receptor superfamily4−6. However, the molecular basis for the actions of 1,25(OH)2D3 in bone formation, its role during development and VDR genetic polymorphisms for predicting bone mineral density7 are uncertain. To investigate the functional role of VDR, we generated mice deficient in VDR by gene targeting. We report here that in VDR null mutant mice, no defects in development and growth were observed before weaning, irrespective of reduced expression of vitamin D target genes. After weaning, however, mutants failed to thrive, with appearance of alopoecia, hypocalcaemia and infertility, and bone formation was severely impaired as a typical feature of vitamin D−dependent rickets type II (refs 8,9). Unlike humans with this disease, most of the null mutant mice died within 15 weeks after birth, and uterine hypoplasia with impaired folliculogenesis was found in female reproductive organs. These defects, such as alopoecia and uterine hypoplasia, were not observed in vitamin D−deficient animals. The findings establish a critical role for VDR in growth, bone formation and female reproduction in the post-weaning stage.
A cDNA encoding a nuclear hormone receptor of the steroid/thyroid receptor superfamily was obtained from third-stage larvae(L3) of the parasitic roundworm Strongyloides stercoralis. A recombinant clone was isolated via screening of an S. stercoralis cDNA library with a polymerase chain reaction (PCR)-generated probe. The insert of 2583 bp contained the complete coding
sequence of the receptor homologue. The conceptually translated amino acid sequence of this open reading frame encodes a 753-amino-acid-residue
protein with an apparent molecular weight of 83.6 kDa and a predicted isoelectric point (pI) of 8.52. The coding sequence
is 69% AT and the noncoding sequence is 72% AT, reflecting a characteristic A/T codon bias of S. stercoralis. In this report the amino acid sequence of the S. stercoralis nuclear hormone receptor of the steroid/thyroid receptor superfamily is compared with that of nuclear hormones of Caenorhabditis elegans, human orphan nuclear receptors, and insect ecdysone receptors. The potential role of steroids in the induction of hyperinfection
syndrome is also discussed.
The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms. There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly conserved genes provides evidence for a regional organization of the chromosomes.
The ancestry of the cells in the hermaphrodite and male gonadal somatic structures of C. elegans has been traced from the two gonadal somatic progenitor cells (Z1 and Z4) that are present in the newly hatched larvae of both sexes. The lineages of Z1 and Z4 are essentially invariant. In hermaphrodites, they give rise to a symmetrical group of structures consisting of 143 cells, and in males, they give rise to an asymmetrical group of structures consisting of 56 cells. The male gonad can be distinguished from the hermaphrodite gonad soon after the first division of Z1 and Z4. However, the development of Z1 and Z4 in hermaphrodites shares several features in common with their development in males suggesting that the two programs are controlled by similar mechanisms. In the hermaphrodite lineage, a variability in the positions of two cells is correlated with a variability in the lineages of four cells. This variability suggests that cell-cell interaction may play a more significant role in organisms that develop by invariant lineages than has hitherto been considered. None of the somatic structures (e.g., uterus, spermatheca, vas deferens) develops as a clone of a single cell. Instead, cells that arise early in the Z1–Z4 lineage generally contribute descendants to more than one structure, and individual structures consist of descendants of more than one lineage.
The number of nongonadal nuclei in the free-living soil nematode Caenorhabditis elegans increases from about 550 in the newly hatched larva to about 810 in the mature hermaphrodite and to about 970 in the mature male. The pattern of cell divisions which leads to this increase is essentially invariant among individuals; rigidly determined cell lineages generate a fixed number of progeny cells of strictly specified fates. These lineages range in length from one to eight sequential divisions and lead to significant developmental changes in the neuronal, muscular, hypodermal, and digestive systems. Frequently, several blast cells follow the same asymmetric program of divisions; lineally equivalent progeny of such cells generally differentiate into functionally equivalent cells. We have determined these cell lineages by direct observation of the divisions, migrations, and deaths of individual cells in living nematodes. Many of the cell lineages are involved in sexual maturation. At hatching, the hermaphrodite and male are almost identical morphologically; by the adult stage, gross anatomical differences are obvious. Some of these sexual differences arise from blast cells whose division patterns are initially identical in the male and in the hermaphrodite but later diverge. In the hermaphrodite, these cells produce structures used in egg-laying and mating, whereas, in the male, they produce morphologically different structures which function before and during copulation. In addition, development of the male involves a number of lineages derived from cells which do not divide in the hermaphrodite. Similar postembryonic developmental events occur in other nematode species.
In the postembryological development of the free-living nematode Caenorhabditis elegans, a morphologically recognizable, nongrowing stage, called the dauerlarva, may arise. Using synchronous populations and following growth and molting, it has been shown that the dauerlarva is formed by a facultative, reversible arrest at a specific point in the life cycle, the second of four cuticle molts, in response to external conditions.At each molt a normal animal passes through “lethargus,” a stage in which feeding and locomotion are transiently arrested. In the dauerlarva stage, feeding is arrested indefinitely and locomotion is markedly reduced. A simple quantitative assay, based on the exceptional resistance of dauerlarvae to sodium dodecyl sulfate (SDS), has been developed to study dauerlarva formation and its reversal. The SDS resistance of dauerlarvae requires both non-feeding and an especially impermeable cuticle. Dauerlarva formation can be efficiently induced by limiting the concentration of bacteria (the food supply), but not by complete starvation. Quantitative recovery to normal development can be induced by transfer to fresh medium with excess bacteria. Simpler stimuli can elicit recovery at slower rates, the principal factors besides nutrition being nutrition being optimal ionic and osmotic conditions and a noninhibitory concentration of animals. There are identifiable stages in recovery, beginning with a resumption of feeding. The cuticle, ultrastructurally very different from normal cuticle, is shed at the next molt, after which development appears normal. A temperature-sensitive mutant, which forms dauerlarvae at high temperature despite the presence of abundant food, is described, and the use of dauerlarvae for further mutant isolation is discussed.
Under conditions unfavorable to growth, the nematode Caenorhabditis elegans enters a developmentally arrested stage, the dauer larva. We have examined gene expression in the dauer larva and during recovery from the dauer stage. Run-on transcription assays with isolated nuclei reveal a depression of general RNA polymerase II transcription to 11-17% of that in other stages. Transcription of individual gene families (including actin, collagen, hsp70, and histone) is similarly depressed relative to actively growing stages. Dauer larvae are, however, capable of being induced for heat shock messages, indicating that they are competent to initiate and elongate transcripts. For most genes surveyed, reduced transcription in dauer larvae correlates with a decrease in message abundance. Hsp70 mRNA, however, is transcribed at lower rates but accumulates at levels comparable to those in other stages. Interestingly, dauer larvae are 15-fold enriched in a mRNA for a C. elegans hsp90 gene. Hsp90 mRNA accumulation is regulated at least in part by differential stability. Dauer larvae thus appear to have a unique pattern of gene expression. Upon placement in food, dauer larvae reenter the developmental pathway as late-stage larvae. Dauer recovery is accompanied by a temporally regulated sequence of gene expression. At least four distinct patterns of gene expression can be distinguished during exit from the dauer stage. Steady-state levels of hsp70 and polyubiquitin mRNA rise sharply within 75 min of recovery before declining by the fourth hour. Actin and histone mRNAs increase steadily following 2-4 hr of recovery, whereas myosin mRNA increases after 10 hr. In contrast, hsp90 mRNA declines sharply within the first 75 min of recovery. Changes in mRNA populations during dauer formation and exit may be physiologically relevant.
This chapter presents experimental approaches to elucidate the precise molecular mechanism by which steroid receptors regulate the initiation of target gene transcription and the role of the hormonal ligand in this process. For determining the direct actions of receptor on DNA transcription, a cell-free—reconstituted—transcription system is required. In such a system, the concentration of receptor, ligand, general transcription factors, and target genes could be manipulated. In the initial experiments, the effect of purified cPR (50–80% pure) or a purified cPR derivative expressed in Escherichia coli are measured on transcription of templates that lacked or contained progesterone response elements (PREs). To examine the mechanism by which progesterone receptor interacts with general transcription factors to stimulate RNA synthesis in vitro, it was investigated whether the receptor participates in the formation of a stable preinitiation—rapid start—complex of transcription factors. In this investigation described in the chapter, in an attempt to investigate the mechanism by which regulatory proteins such as steroid hormone receptors interact with core promoters to enhance transcription, the cell-free transcription system was used.
Point mutagenesis of the nuclear targeting sequence of nucleoplasmin has identified two interdependent basic domains. These are separated by 10 intervening "spacer" amino acids that tolerate point mutations and some insertions. Amino acids in both basic domains are required for nuclear targeting, and the transport defect of a mutation in one domain is amplified by a simultaneous mutation in the other. Therefore, these basic domains are interdependent. A strikingly similar motif of two clusters of basic residues is seen in the nuclear targeting sequence of Xenopus N1. It is also conserved in the related nucleolar protein NO38. Several other short sequences known to be necessary for nuclear targeting fall within a similar motif.
The dauer larva is a developmentally arrested, non-feeding dispersal stage normally formed in response to overcrowding and limited food. The daf-1 gene specifies an intermediate step in a hierarchy of genes thought to specify a pathway for neural transduction of environmental cues. Mutations in daf-1 result in constitutive formation of dauer larvae even in abundant food. This gene has been cloned by Tc1-transposon tagging, and it appears to encode a new class of serine/threonine kinase. A daf-1 probe detects a 2.5 kb mRNA of low abundance, and the DNA sequence indicates that the gene encodes a 669 amino acid protein, with a putative transmembrane domain and a C-terminal protein kinase domain most closely related to the cytosolic, raf proto-oncogene family. Hence, the daf-1 product appears to be a cell-surface receptor required for transduction of environmental signals into an appropriate developmental response.
The nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that regulate homeostasis, reproduction, development, and differentiation. Three amino acids within the zinc finger DNA binding motif determine target gene specificity. Groups of receptors exist with similar DNA binding specificity. A complex carboxy terminal region mediates ligand binding, dimerization, and hormone-relieved transcriptional inactivation. We summarize the current understanding of these phenomena and suggest a novel model that structurally and functionally links these events. This "regulatory zipper model" may explain the mechanism by which ligand activates nuclear hormone receptors.
During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).
The cDNA for the 1,25-dihydroxyvitamin D3 receptor has been isolated by screening a rat kidney lambda gt11 expression library with a mixture of three previously identified anti-receptor monoclonal antibodies. Protein produced from this cDNA reacts with each of the three monoclonal antibodies but does not react with an antibody specific for the porcine receptor or an irrelevant antibody. In addition, the expressed protein specifically binds 1,25-dihydroxyvitamin D3 but does not bind estradiol, progesterone, testosterone, cortisol, or aldosterone. The nucleotide sequence for this cDNA has been determined and used to deduce the amino acid sequence of the protein. This amino acid sequence contains a region that matches perfectly the partial amino acid sequence determined for the receptor isolated from porcine intestine. This amino acid sequence also closely matches the reported amino acid sequence of the putative DNA-binding finger of the avian 1,25-dihydroxyvitamin D3 receptor.
Receptors for retinoic acid, vitamin D3 and the steroid and thyroid hormones belong to a family of ligand-activated enhancer-binding factors which are composed of a number of functional domains required for ligand and DNA binding, nuclear translocation, dimerization and trans-activation of transcription. Ligand binding, which may promote dissociation of the receptor from a heat shock protein, results in the binding of the ligand-receptor complex as a dimer to its cognate palindromic responsive element. The mechanism by which the DNA-bound receptor activates transcription is unknown but appears to involve more than one trans-activating domain.
Analyses of steroid receptors are important for understanding molecular details of transcriptional control, as well as providing
insight as to how an individual transacting factor contributes to cell identity and function. These studies have led to the
identification of a superfamily of regulatory proteins that include receptors for thyroid hormone and the vertebrate morphogen
retinoic acid. Although animals employ complex and often distinct ways to control their physiology and development, the discovery
of receptor-related molecules in a wide range of species suggests that mechanisms underlying morphogenesis and homeostasis
may be more ubiquitous than previously expected.
Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
The dauer larva of Caenorhabditis elegans is a developmentally arrested stage induced by starvation or overcrowding. Mutant genes controlling the ability to form dauer larvae interact in a way which allows them to be ordered in a pathway. Mutant phenotypes suggest that the pathway corresponds to neural processing of environmental stimuli.
We have designed a rapid cloning and screening strategy to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, we isolated three Drosophila genes, designated DHR38, DHR78, and DHR96. All three genes are expressed throughout third-instar larval and prepupal development. DHR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. DHR78 and DHR96 are orphan receptor genes. DHR78 is induced by 20-hydroxyecdysone (20E) in cultured larval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats. DHR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20E receptor can bind to each of the sequences recognized by DHR78 and DHR96, indicating that these proteins may compete with the receptor for binding to a common set of target sequences.
Excerpt
The C. elegans genome project is part of a larger effort to understand how the information encoded in its DN A specifies the biology of this small nematode worm (Brenner 1974; Wood et al. 1988). It was selected for study because it is a highly tractable experimental system. Its short generation time (3 1/2 days), large brood size (300), and its modes of reproduction (self-fertilizing hermaphrodite and cross-fertilizing males) have aided genetic analysis. More than 950 genes have already been identified through mutation (Edgley and Riddle 1990; J. Hodgkin and R. Durbin, pers. comm.). Its small size (∼1 mm), defined number of cells (959 somatic nuclei), and transparent body and eggshell have permitted the elucidation of the cell lineage (Sulston et al. 1983) and the reconstruction of the entire nervous system at the level of the synapse (White et al. 1986). The relatively small genome has facilitated molecular investigations, which...
Under conditions of high population density and low food, Caenorhabditis elegans forms an alternative third larval stage, called the dauer stage, which is resistant to desiccation and harsh environments. Genetic analysis of some dauer constitutive (Daf-c) and dauer defective (Daf-d) mutants has revealed a complex pathway that is likely to function in particular neurons and/or responding tissues. Here we analyze the genetic interactions between three genes which comprise a branch of the dauer formation pathway that acts in parallel to or downstream of the other branches of the pathway, the Daf-c genes daf-2 and daf-23 and the Daf-d gene daf-16. Unlike mutations in other Daf-c genes, mutations in both daf-2 and daf-23 cause non-conditional arrest at the dauer stage. Our epistasis analysis suggests that daf-2 and daf-23 are functioning at a similar point in the dauer pathway. First, mutations in daf-2 and daf-23 are epistatic to mutations in the same set of Daf-d genes. Second, daf-2 and daf-23 mutants are suppressed by mutations in daf-16. Mutations in daf-16 do not suppress any of the other Daf-c mutants as efficiently as they suppress daf-2 and daf-23 mutants. Third, double mutants between either daf-2 or daf-23 and several other daf-d mutants exhibit an unusual interaction. Based on these results, we present a model for the function of daf-2, daf-23 and daf-16 in dauer formation.
In D. melanogaster a pulse of the steroid hormone ecdysone triggers the larval-to-adult metamorphosis, a complex process in which this hormone induces imaginal tissues to generate adult structures and larval tissues to degenerate. We show that the EcR gene encodes three ecdysone receptor isoforms (EcR-A, EcR-B1, and EcR-B2) that have common DNA- and hormone-binding domains but different N-terminal regions. We have used isoform-specific monoclonal antibodies to show that at the onset of metamorphosis different ecdysone target tissues express different isoform combinations in a manner consistent with the proposition that the different metamorphic responses of these tissues require different combinations of the EcR isoforms. We have also determined temporal developmental profiles of the EcR isoforms and their mRNAs in whole animals, showing that different isoforms predominate at different developmental stages that are marked by a pulse of ecdysone.
The bone morphogenetic protein (BMP) family is a conserved group of signalling molecules within the transforming growth factor-beta (TGF-beta) superfamily. This group, including the Drosophila decapentaplegic (dpp) protein and the mammalian BMPs, mediates cellular interactions and tissue differentiation during development. Here we show that a homologue of human BMPs controls a developmental switch in the life cycle of the free-living soil nematode Caenorhabditis elegans. Starvation and overcrowding induce C. elegans to form a developmentally arrested, third-stage dauer larva. The daf-4 gene, which acts to inhibit dauer larva formation and promote growth, encodes a receptor protein kinase similar to the daf-1, activin and TGF-beta receptor serine/threonine kinases. When expressed in monkey COS cells, the daf-4 receptor binds human BMP-2 and BMP-4. The daf-4 receptor is the first to be identified for any growth factor in the BMP family.
SHP is an orphan member of the nuclear hormone receptor superfamily that contains the dimerization and ligand-binding domain
found in other family members but lacks the conserved DNA binding domain. In the yeast two-hybrid system, SHP interacted with
several conventional and orphan members of the receptor superfamily, including retinoid receptors, the thyroid hormone receptor,
and the orphan receptor MB67. SHP also interacted directly with these receptors in vitro. In mammalian cells, SHP specifically
inhibited transactivation by the superfamily members with which it interacted. These results suggest that SHP functions as
a negative regulator of receptor-dependent signaling pathways.
A pheromone-induced neurosecretory pathway in Caenorhabditis elegans triggers developmental arrest and an increase in longevity at the dauer diapause stage. The gene age-1 is required for non-dauer development and normal senescence. age-1 encodes a homologue of mammalian phosphatidylinositol-3-OH kinase (PI(3)K) catalytic subunits. Lack of both maternal and zygotic age-1 activity causes dauer formation, whereas animals with maternal but not zygotic age-1 activity develop as non-dauers that live more than twice as long as normal. These data suggest that phosphatidylinositol signalling mediated by AGE-1 protein controls lifespan and the dauer diapause decision.
Formation of the C. elegans dauer larva is repressed by the chemosensory neurons ADF, ASI, and ASG. Mutant analysis has defined two parallel genetic pathways that control dauer formation. By killing neurons in these mutants, we show that mutations in one of these genetic pathways disrupt dauer repression by ADF, ASI, and ASG. One gene in this pathway is daf-7, which encodes a TGFbeta-related protein. We find that daf-7::GFP fusions are expressed specifically in ASI and that expression is regulated by dauer-inducing sensory stimuli. We also show that a different chemosensory neuron, ASJ, functions in parallel to these neurons to induce dauer formation. Mutations in the second genetic pathway activate dauer formation in an ASJ-dependent manner. Thus, the genetic redundancy in this process is reflected at the neuronal level.
The functions of the ecdysone-induced DHR3 and E75B orphan nuclear receptors in the early stages of Drosophila metamorphosis were investigated. DHR3 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis. It also induces betaFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming a complex with DHR3 on the betaFTZF1 promoter, thereby providing a timing mechanism for betaFTZF1 induction that is dependent on the disappearance of E75B.