Nuclear receptors in nematode development: Natural experiments made by a phylum

BBAGRM-00768; No. of pages: 14; 4C: 2, 3, 9 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Review

Nuclear receptors in nematode development: Natural experiments made by a phylum☆,☆☆ Marta Kostrouchova a,⁎, Zdenek Kostrouch b a b

Laboratory of Molecular Biology and Genetics, Charles University in Prague, Prague, Czech Republic Laboratory of Molecular Pathology, Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic

a r t i c l e

i n f o

Article history: Received 5 February 2014 Received in revised form 21 June 2014 Accepted 23 June 2014 Available online xxxx Keywords: Nuclear receptor Nematode Caenorhabditis elegans Development Evolution

a b s t r a c t The development of complex multicellular organisms is dependent on regulatory decisions that are necessary for the establishment of specific differentiation and metabolic cellular states. Nuclear receptors (NRs) form a large family of transcription factors that play critical roles in the regulation of development and metabolism of Metazoa. Based on their DNA binding and ligand binding domains, NRs are divided into eight NR subfamilies from which representatives of six subfamilies are present in both deuterostomes and protostomes indicating their early evolutionary origin. In some nematode species, especially in Caenorhabditis, the family of NRs expanded to a large number of genes strikingly exceeding the number of NR genes in vertebrates or insects. Nematode NRs, including the multiplied Caenorhabditis genes, show clear relation to vertebrate and insect homologues belonging to six of the eight main NR subfamilies. This review summarizes advances in research of nematode NRs and their developmental functions. Nematode NRs can reveal evolutionarily conserved mechanisms that regulate specific developmental and metabolic processes as well as new regulatory adaptations. They represent the results of a large number of natural experiments with structural and functional potential of NRs for the evolution of the phylum. The conserved and divergent character of nematode NRs adds a new dimension to our understanding of the general biology of regulation by NRs. This article is part of a Special Issue entitled: Nuclear receptors in animal development. © 2014 Elsevier B.V. All rights reserved.

Abbreviations: AR (NR3C4), androgen receptor; CAR (NR1I3), constitutive androstane receptor; COUP-TF (NR2F), chicken ovalbumin upstream promoter transcription factor; DAF-12, (abnormal) dauer formation (NR)-12; DAX-1 (NR0B1), dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; DBD, DNA binding domain; (D)HR-3 (HR46, NR1F4), (Drosophila) hormone receptor-3, hormone receptor-like in 46; DPR-1, dauer pheromone responsive (NR)-1; E75, -78, ecdysone-induced protein 75, -78; EAR2 (NR2F6), V-erbA-related protein 2; EcR (NR1H1), ecdysone receptor; ER (NR3A1), estrogen receptor; ERR (NR3B4), estrogen related receptor; FAX-1, (defective) fasciculations of axons phenotype (NR)-1; FXR (NR1H4), farnesoid X receptor; GCNF, germ cell nuclear factor; GFP, green fluorescence protein; GR (NR3C1), glucocorticoid receptor; HNF4 (NR2A1), hepatocyte nuclear factor-4; LBD, ligand binding domain; LRH1 (NR5A2), liver receptor homologue-1; LXR (NR1H), liver X receptor; MR (NR3C2), mineralocorticoid receptor; NGFIB (NUR77, NR4A1), nerve growth factor IB; NHR(s), nuclear hormone receptor(s); NR(s), nuclear receptor(s); NOR1 (NR4A3), neuron-derived orphan receptor-1; NURR1 (NR4A2), nuclear receptor related-1; ODR-7, odorant response abnormal (NR)-7; PNR (NR2E3), photoreceptor cell-specific nuclear receptor; PPAR (NR1C), peroxisome proliferator-activated receptor; PR (NR3C3), progesterone receptor; PXR (NR1I2), pregnane X receptor; RAR (NR1B), retinoic acid receptor; RNAi, RNA interference; ROS, reactive oxygen species; ROR (NR1F), retinoid-related orphan receptor; RXR (NR2B), retinoid X receptor; SEX-1, signal element on X-1; SF-1 (NR5A1), steroidogenic factor-1; SHP (NR0B2), small heterodimer partner (NR); SVP (NR2F3), seven-up (NR); TLL (NR2E2), tailless (NR); TLX (NR2E1), vertebrate homologue of the Drosophilla tailless gene (NR); TR2, −4 (NR2C1, −2), testicular receptor 2, −4; TR (THR, NR1A), thyroid hormone receptor; UNC, uncoordinated (NR); USP (NR2B4), ultraspiracle protein; VDR (NR1I1), vitamin D receptor ☆ This article is part of a Special Issue entitled: Nuclear receptors in animal development. ☆☆ Authors thank an anonymous reviewer of a grant application for valuable comments and inspiration that was used in the title. ⁎ Corresponding author. E-mail address: [email protected] (M. Kostrouchova).

1. Introduction 1.1. Overall characteristics of NRs in nematodes Nuclear receptors (NRs) are very characteristic transcription factors found only in metazoan species. NRs are critically important for the regulation of metabolism, development and reproduction. Their molecular structure includes two highly conserved domains. The DNA binding domain (DBD) which interacts with response elements in promoters of regulated genes and the ligand binding domain (LBD), which in the case of proteins with known ligands binds and is regulated by smallmolecule ligands. The DBD consists of two protein loops (called zinc fingers), each contain a zinc ion coordinating tetrahedrically four cysteine residues. The number of amino acid residues forming the molecular signature is also conserved across the animal phyla and has in an absolute majority of cases the formula Cys-X2-Cys-X13-Cys-X2-CysX15-Cys-X5-Cys-X9-Cys-X2-Cys [1–4]. There are exceptions to this rule. Genome sequencing projects and focused studies aimed at cloning NRs from different species identified homologues of classical NRs with slightly variable length of the sequence separating the two zinc fingers. The sequence separating the two zinc fingers contains in most NRs 15 amino acid residues. In some NRs, such as in the vertebrate thyroid hormone receptors, it contains 17 amino acid residues. Less frequently there is a variable number of amino acids in sequences forming the

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Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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feature of their regulatory and evolutionary potential. This is strongly supported by the multiplication of NR2A members in Caenorhabditis [17,18]. The number of NRs varies between Rhabditida species reaching 289 in C. elegans, 232 in C. briggsae and 256 in C. remanei [19]. Accumulated data from genome sequencing projects and functional studies indicate that some NRs are highly conserved and are likely to keep orthologous functions in different phyla. In the majority of other NRs, it is possible to detect a structural and functional relationship to a particular NR subfamily but not to a particular receptor within this subfamily. Of the 289 NRs (denominated as nhr−, daf-12, fax-1, sex-1, unc-55, odr-7, dpr-1) found in the C. elegans genome WormBase WS241 [20] only 15 to 20 are considered conserved [21–23] based on higher sequence similarity and functional studies (Fig. 2). The other 269 NRs show variable relation to HNF4 and are more related to other NRs found only in nematodes than to vertebrate or insect homologues [18,22,24]. Their sequence conservation and their proven expression indicates that the majority of them are functional genes but their exact function or relation to particular NRs from other phyla based on mutual comparison of sequence may be difficult or misleading (as pointed out by Laudet and others [25]). In C. elegans, the conserved NRs have been intensively studied. NRs have been shown to have critical roles in the regulation of nematode development and metabolism (reviewed below). Functional data not only revealed new facts concerning particular nematode developmental pathways but also shed light on the relation of nematode NRs to their closest vertebrate and insect homologues. They further support the evolutionary links between particular members of NR subfamilies in distant phyla. Several NRs that are classified as non-conserved (but still related to NR2A–HNF4) were shown to have critical functions in nematodes. Despite the diversity of the primary sequence an absolute majority of these seemingly non-conserved NRs is still highly conserved and consistently new studies are revealing important developmental and metabolic roles for these multiplied nematode specific NRs. Detailed knowledge about their functions is likely to contribute to the general

Insecta

Drosophila

Arthropoda

Protostomes

loops between the coordinating cysteines. Exceptionally, NRs with two DBD domains are found in some species, e.g. in Platyhelminthes Schistosoma mansoni [5], Schmidtea mediterranea, Dugesia japonica, in the mollusk Lottia gigantea, and in the arthropod Daphnia pulex [6]. Another exception is represented by TLX, whose DBD differs from other NRs and forms a longer signature with additional cysteines [7]. TLX emerged very likely early in the evolution of metazoans since this NR is found in Cnidaria [8] and the unusual DBD is conserved in many metazoan species whose genomes have been sequenced. The LBD of NRs is also very characteristic at the structural level. It consists of 11 to 12 helices that form a highly conserved secondary structure despite the quite high variability in the primary sequence of various NRs [9]. Based on the sequence similarities of both DBD and LBD, it was possible to detect homologous NRs in several species of vertebrates, insects and nematodes and comparison of NRs from more or less distant species allowed subclassification of NRs into eight main subfamilies [10]. It is surprising that this division, which was based mostly on vertebrate and insect NRs [10] is very consistent with the evolutionary scheme that can be built from the large number of NRs now identified in sequenced genomes of distant taxa [8,11–13]. This most likely reflects the inherent properties for their regulatory and evolutionary potential. Comparison of nematode, vertebrate and insect NRs indicates that the six main subfamilies of NRs were already evolved in animal species existing before the split of deuterostomes and protostomes [14,15] (Fig. 1). The number of NRs, however, differs in-between species not only in the total number of NR genes in the genomes but also in the number of NRs within certain NR subfamilies, especially in NR1 through NR4. Contrary to that, subfamilies NR5 and NR6 contain in sequenced genomes only one representative of each. Species as distant as those of Cnidaria and vertebrates show examples of multiplication and subspeciation of NRs within certain NR subfamilies. In certain phyla, the multiplication of NRs can be attributed to whole genome duplications (WGD), such as in vertebrates which experienced WGD twice [16]. Radiation and multiplication of NRs within subfamilies, however, cannot be simply explained by WGD and the duplication of NRs may be considered a primordial

Ecdysozoa

2

Lophotrochozoa Homo NR1-NR6

NR1/NR4, NR2, NR3, NR6 (NR2A, B, C/D, E(5x), F (5x))

Gnathostomes Cephalochordata – Tunicata

Deuterostomes

Nematoda

Cnidaria Porifera

Fig. 1. Schematic representation of nuclear receptors found in sequenced genomes in relation to deuterostome and protostome evolution. The phylogenetic tree is derived from [227]. The occurrence of representative NR subfamilies indicates the early origin of founders of NRs (based on [8,10,14]). Numbers in brackets indicate the number of NRs in selected species.

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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NEMATODES (Caenorhabditis elegans)

NR subfamilies NR3

NHR-6 (NGFIB-like)

NR4 NR1

SEX-1/NHR-24 (RevErbA-like,RAR-like) NHR-85 (RevErbA-like) NHR-23/ CHR3 (HR3-like) NHR-48,-8, DAF-12(VDR-, PXR-, CAR-

VERTEBRATES (Homo sapiens) α,β (NR3A1-2) A - ERα,β B - ERRα,β, α,β, γ (NR3B1-3) C - GR, MR, PR, AR (NR3C1-4) A - NGFIB (NR4A1) NURR1 (NR4A2) NOR1 (NR4A3) A - TRα,β α,β (NR1A1-2) B - RARα,β, α,β, γ (NR1B1-3) C - PPARα,β/ α,β/ δ,, γ (NR1C1-3) D - RevErbAα,β α,β (NR1D1-2) F - RORα,β, α,β, γ (NR1F1-3) H - LXRα,β α,β, FXR (NR1H3,2,4) I - VDR, PXR, CAR (NR1I1-3)

-like)

X - (NRs with 2 DBDs)

INSECTS (Drosophila melanogaster) ERR (NR3B4) HR38 (NR4A4, NGFIB-like)

E75 (NR1D3, RevErbA-like), E78 (RevErbA-like) HR3 (NR1F4) ECR (NR1H1) HR96 (NR1J; VDR-, PXR-, CAR- like)

NHR-25 (NR5A, FTZ-F1-like)

NR5

A - SF1/FTZ1 (NR5A1) LRH1 (NR5A2)

FTZ-F1 (HR39, NR5A, SF1-like)

NHR-91 (GCNF-like)

NR6

A - GCNF (NR6A1)

HR4 (GCNF-like)

Aa - NHR-31, -60, -80, -114 Ab - NHR-14, -40, -49, 64 Ac – supNRs with unknown functions (∼260)

NR2

A - HNF4 α, (β), γ (NR2A1-2)

HNF-4 (NR2A4)

B - RXRα, α, β, γ (NR2B1-3)

USP (RXR-like)

NHR-41 (NR2D1, TR2/4-like)

C - TR2, -4 (NR2C1-2)

HR78 (NR2D1, TR2/4-like)

NHR-67 (Tll/Tlx-like) FAX-1/NHR-29 (PNR-like)

E - TLX, PNR (NR2A1, 3)

TLL (NR2E2), HR51 (NR2E3, PNR-like), DSF (NR2E4, PNR-like), HR83 (NR2E5), NR2E6

UNC-55 (COUP-TF-like)

F - COUP-TFI, -II, EAR2 (NR2F1,2,6)

SVP (NR2F3, COUP-TF-like)

(NR0)

3

B - DAX1, SHP (NR0B1-2)

Fig. 2. Relation of highly conserved nematode NRs to vertebrate and insect NR subfamilies. The scheme shows the likely relationship of nematode NRs to the NR classification of vertebrate and insect NRs. Five subfamilies of NRs have close homologues in the C. elegans genome. NR3 whose origin is set before the split of diploblasts, deuterostomes and protostomes has no known homologue in the C. elegans genome.

biology of the regulation by NRs (e.g. NHR-2, NHR-31, NHR-40, NHR-60, NHR-80, NHR-114 [21,26–31]). In some cases, functional data indicate that several multiplied NRs that are classified as non-conserved relate to one vertebrate NR to the extent that it is possible to view them as diversified orthologues that underwent subspeciation as it is in the case of HNF4 homologues NHR-31 [29], NHR-69 [32], NHR-80 [33], and NHR-114 [31]. C. elegans NRs are scattered over all chromosomes and are oriented in both directions. Positional clustering can be viewed well using clustering analysis of chromosomes that contain a high number of NRs. Genes that belong to this category of NRs are predominantly localized on chromosome V. They are however also present in high numbers on chromosome IV and X and some representatives are found on all chromosomes (WormBase WS241) [18,21,34]. Supplementary Table 1 lists the presumable orthologues in the C. elegans genome. Although the most ancient subfamily of NRs is undoubtedly the NR2 subfamily and its members are among the most conserved between many phyla (e.g. diploblasts, vertebrates and mollusks), in nematodes the conservation is less apparent and one major founder, RXR, which is highly conserved between diploblastic species, vertebrates and mollusks, is missing in Caenorhabditis elegans. It is however found in several other nematode species [35,36]. Most C. elegans NRs are more or less related to HNF4. This group expanded in Rhabditidae to a large number of genes that are showing signs of ongoing duplications [17,37–39]. Despite the fact that many HNF4 related NRs are dispensable under laboratory conditions, an increasing number of targeted studies is revealing their conserved or newly acquired functionality.

2. Nematode NRs in relation to NR classification and evolution Even though the classification of NRs was originally proposed based on a limited number of representatives known mostly from vertebrates and insects, it proves to be helpful for the understanding of the regulatory potentials and evolutionary links of NRs in general. The classification was based on the presence and sequence similarities of conserved domains, but it is consistent with the regulatory potential of NRs [14, 40], since these domains dictate the core of the regulatory mechanisms. Most NRs are expressed in different cell and tissue types and have multiple roles in the regulation of metabolism and development in most if not all metazoan species including those that have a relatively small number of NRs, such as insects or Cnidaria. Genes that are multiplied within the NR families show signs of neofunctionalization and subfunctionalization that are employed in the establishment of new regulatory levels [41–43]. This scenario is, however, very likely the driving force for the evolution of regulation by NRs in general as well as in nematodes. 2.1. NR2 — the subfamily of founders of metazoan NRs expanded in nematodes Genome sequencing of diploblastic species indicates with high probability that the NR2 subfamily is the subfamily of founders of metazoan NRs [11,44,45]. Although it is still not clear if the original receptor was related more to HNF4, RXR or COUP-TF, or another member of this subfamily which has disappeared, these three NRs are present in the genome of Trichoplax adhaerens and in cnidarian species [8]. They are

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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also found in all metazoan phyla whose reference genomes are known. Despite the extent of conservation of NR2 members in other phyla, conservation of RXR, HNF4 and COUP-TF in nematodes is much smaller. In search for genes that evolved from members of the NR2 subfamily it seems that RXR was lost from the genomes of the Rhabditidae, while the orthologues of other members of the NR2 subfamily can be found. In C. elegans, the closest homologue of COUP-TF and Seven-up is UNC-55. NHR-67, FAX-1, NHR-239 and NHR-111 are related to TLX/TLL. NHR41 is related to testicular receptor TR2/4 and the rest of NRs (~269) is related to HNF4α. They are however not orthologues in the sense of two way sequence comparison. On the contrary, these genes are diversified in their sequence in both DNA binding and ligand binding domains and are classified as supplementary NRs (supNRs) [18]. 2.1.1. NR2A–HNF4 like HNF4α is a member of the NR2 subfamily, which is highly conserved between diploblasts, vertebrates and insects. It is likely to be the most ancient NR in the evolution of Metazoa as it is found in Porifera [44, 46], Placozoa [11], Cnidaria [8] and all metazoan species studied to date [12]. The human genome contains two HNF4 genes, alpha and gamma. Both HNF4 members in vertebrates as well as in insects are predominantly important for the development of the gastrointestinal system and for metabolism. Targeted disruption (knockout) of HNF4α results in defects of embryonic development, homeostasis and metabolism, impairment of the liver and the biliary and central nervous system [47]. HNF4γ was shown to constitutively bind fatty acids. It is expressed in the gastrointestinal system (duodenum, jejunum, ileum, and colon), in the liver, and with lower levels in the gall bladder, kidney, skin, central nervous system and endocrine system (adrenal gland, thyroid and pancreas). Its targeted disruption results in defects in behavior, homeostasis and metabolism, growth and development [48]. In Xenopus, HNF4β is expressed in the liver, kidney, stomach, intestine, lung, ovary and testis and regulates oogenesis and embryogenesis [49]. In insects, similarly as in vertebrates, HNF4 regulates development of the gut and reproductive organs [50]. From the 269 C. elegans NRs related to HNF4, only some were studied in detail: NHR-49, NHR-31, NHR-64, NHR-14, NHR-80, NHR-40, and NHR-60. Several possible evolutionary pressures that may have led to the multiplication of these NRs were suggested. It is intriguing to speculate that the multiplied NRs evolved under the pressure of the environment and may be involved in response to xenobiotics. It is possible that the loss of heterodimerizing NR, RXR, participated in the evolutionary pressure for conservation of the multiplied NRs. RXRs, however, do not heterodimerize with members of the NR2 subfamily, but NR1 and NR4 subfamily [51]. The pressure for heterodimerization is documented by the newly evolved heterodimerization that is independent of RXR as is the case of NHR-49 within the NR2 subfamily [33]. It therefore also seems possible that many multiplied nematode NR2 members arose by a mechanism that specializes HNF4 functions (and in this respect the highly conserved HNF4 homologues are supporting orthologous as well as new, paralogous functions). It will be discussed later that several nematode NRs evolved to support functions that are dependent on members of other NR subfamilies in non-nematode phyla. The members of the NR2A group in C. elegans that have been characterized to date can be divided to members with features of functional conservation with HNF4 in other phyla and members that adopted functions supported by other NRs. For the purpose of this review, the first group of functional orthologues have been named NR2A-a, the second NR2A-b and the remaining NRs that are still not sufficiently characterized NR2A-c. The NR2A-a group includes NHR-80, NHR-114, NHR-31, and NHR60, while the NR2A-b group includes NHR-49, NHR-40, NHR-64, and NHR-14. The NR2A-c group contains approximately 260 NRs whose functions were still not sufficiently studied. It can be expected that

based on results of future studies many NRs from group NR2A-c will move to one of the first two groups. 2.1.1.1. NR2A-a: HNF4-related genes with functions similar to HNF4 in vertebrates and insects 2.1.1.1.1. NHR-80. NHR-80 is a representative member of the group of HNF4-related genes that are not only similar to HNF4 in sequence but also regulate functions dependent on HNF4 in other species. In this respect NHR-80 is a structural and functional orthologue of HNF4. NHR-80 is expressed in the intestine and regulates expression of the delta-9 desaturases (FAT-5, FAT-6, and FAT-7), thus affecting fatty acid metabolism. It is also involved in a mechanism that connects signals from the germline with longevity. Depletion of germ cells increases expression of NHR-80 in intestinal cells and this further extends the life span in animals with an ablated germline that already have an extended life span. Germline ablation causes the NHR-80 dependent up-regulation of the stearoyl-CoA desaturase, fat-6, that produces oleic acid from stearic acid [26,30]. NHR-80 can heterodimerize with NHR-49 [33,52] and form a regulatory network with NHR-49, NHR-66 and NHR-13 that regulate fatty acid metabolism and mitochondrial morphology and function. NHR-49 mutant animals have a shortened lifespan. This is caused by NHR-80 and NHR-13 dependent up regulation of fatty acid desaturases and in result, nhr-49 mutant animals have significantly altered mitochondrial morphology and function. These phenotypes are also found in NHR-66 and NHR-80 mediated activities [33,52]. In C. elegans, prevention of germline stem cell proliferation results in a 60% extension of lifespan, termed “gonadal longevity”. This gonadal longevity relies on the transcriptional activities of the steroid nuclear receptor DAF-12 and other factors (DAF-16-homologue of vertebrate FOXO transcription factor, PHA-4-homologue of the FOXA transcription factor and NHR-80). These transcription factors form a network which has an impact on the regulation of fatty acid lipolysis, autophagy, stress resistance and on other processes, involved in homeostasis and life span [53]. NHR-80 is also involved in the regulation of longevity in germline ablated animals through interaction with NDG-4, which is likely to act as a transmembrane acyltransferase. nhr-80 loss of function leads to increased stress resistance. Reduction of function of NDG-4 increases the lifespan up to five times on the background of simultaneous inhibition of insulin/IGF-1 signaling. The NDG-4 dependent effect on increased longevity in germline ablated animals requires NHR-80, illustrating the complexity of NHR-80 functional connections in the integration of signals from the germline to metabolism [54]. 2.1.1.1.2. NHR-114. NHR-114 is highly related to HNF4 and has been shown to be critical for the mechanism that protects germline stem cells from adverse effects of dietary metabolites. NHR-114 loss of function on certain bacterial diets causes accumulation of cell division defects, which decrease fertility up to sterility. This mechanism requires tryptophan, an essential amino acid. However for the effect of tryptophan to take place, live bacteria are required, suggesting that bacterial tryptophan metabolism, not the uptake of tryptophan itself, suppresses the sterility caused by NHR-114 loss of function. This mechanism may be evolutionarily conserved considering the conserved expression pattern of HNF4 (alpha and gamma in intestine) and the proven role of HNF4 α in glucose metabolism. Regulation of life span and fertility through detoxification mechanisms and an effect on germline cells is likely to be evolutionarily conserved [31]. 2.1.1.1.3. NHR-31. NHR-31 should also be considered a HNF4 homologue. Its activity is required for proper development and function of the excretory cell. This function is mediated through the control of the expression of genes encoding subunits of the vacuolar ATPase. The transgene of nhr-31-promoter fused with gfp is expressed at high levels in the excretory cell from embryogenesis to adulthood. Its expression is also detected in the intestine and other cells localized in the distal part of the body [29].

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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Interestingly, this function in excretory organs is likely to be orthologous with HNF4α since HNF4α was shown to orchestrate the expression of genes critical for the regulation of a set of cell proliferation genes in kidney cells [55]. 2.1.1.1.4. NHR-60. NHR-60 is a member of the NR2A group which has overlapping features with HNF4. It is related in sequence and its expression follows the pattern found in vertebrate and insect HNF4 homologues. It is however expressed ubiquitously from one cell embryo to adulthood, as well as in the germline. In seam cells, NHR-60 is downstream of NHR-23 [28]. NHR-60 function projects to fundamental processes of embryonic and larval development. Its inhibition causes developmental arrest with incomplete ventral closure and severe defects in morphogenesis that include defective elongation and severe defects of development of seam cells during larval development. The function of NHR-60 is dependent on the C-terminal domain since the expression of a transgene lacking the presumable AF2 domain induces similar phenotypes as nhr-60 or nhr-23 inhibition by RNAi indicating that part of the NHR-23 regulatory function is mediated by NHR-60 [28]. 2.1.1.2. NR2A-b: HNF4 related genes that evolved to regulate functions similar to functions regulated in vertebrates and insects by NRs other than HNF4 2.1.1.2.1. NHR-49. NHR-49 is a regulator of fat metabolism and life span in C. elegans. The deletion of NHR-49 gave rise to worms with elevated fat content and shortened life span. Using a quantitative RT-PCR screen, it has been found that NHR-49 is important for two distinct aspects of lipid metabolism, for fatty acid desaturation and fatty acid beta-oxidation [56]. Transcriptional profiles in an nhr-49(nr2041) deletion strain (compared to N2 wild-type worms) using oligonucleotide microarrays identified sphingolipid processing and lipid remodeling genes as targets of NHR-49 [33]. Candidate NHR-49 co-factors identified by yeast two-hybrid screens include NHR-13, NHR-22, NHR-66, NHR-105 and MDT-15 [33,56–58]. NHR-49 interacts functionally or physically (hetorodimerizes) with other members of the NR2A group; with NHR-66 in regulation of sphingolipid and lipid remodeling genes, with NHR-80 in regulation of genes involved in fatty acid desaturation and NHR-13 in regulation of genes involved in the desaturase pathway. A direct physical interaction between NHR-49 and NHR-13 was not detected. NHR-49 is involved in the regulation of processes that project to mitochondrial morphology and function and this phenotype is also dependent on NHR-66 and NHR-80 mediated activities [33]. It seems likely that a tight functional requirement for co-regulation of target genes may represent the evolutionary pressure for the establishment of heterodimerization and NHR-49 may be an example of such evolution of dimerization between NRs. By sequence similarity NHR-49 resembles most mammalian HNF4, but its metabolic functions make it a PPAR (NR1C) functional homologue. Correspondingly, lipid lowering fibrates promoted C. elegans longevity in an NHR-49 dependent manner possibly by promoting mitochondrial hormesis (a favorable biological response in mitochondria to low exposures to ROS). It has been postulated that due to stimulation of fatty acid oxidation, ROS formation increases, which in turn acts as a signal to increase stress response, antioxidant defense and increase expression of superoxide dismutase. Increased expression of superoxide dismutase, which is a major factor in antioxidative defense, may be the key factor in NHR-49-dependent effect on longevity [59]. 2.1.1.2.2. NHR-40. NHR-40 is expressed in pharyngeal, body wall, and sex muscle cells as well as in a subset of neurons. The down regulation of nhr-40 by RNAi, or a mutant with a deletion in an intron, results in late embryonic and early larval arrest with defects in elongation and morphogenesis. The nhr-40 loss of function phenotype includes irregular development of body wall muscle cells and impaired movement and coordination resembling neuromuscular affection [27]. Its loss of function leads to impaired expression of muscle-related proteins on the proteome level which is aggravated by low temperature or food restriction

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suggesting a role of NHR-40 in connecting the metabolic state and muscle development [60]. 2.1.1.2.3. NHR-64. NHR-64 is broadly expressed and is detected in anterior and posterior neurons, the ventral nerve cord, the pharynx, gut, and hypodermis [61]. NHR-64 was shown, together with NHR-49 and NHR-80, to be critical for the expression of stearoyl-CoA desaturase and regulation of balanced composition of fatty acids genes [26,56]. Its inactivation by RNAi suppresses low fat stores in stearoyl-CoA desaturase-deficient fat-6; fat-7 double mutants and in sterol regulatory element binding protein (SREBP) sbp-1 mutants. In consequence, it also improves the growth rate of the fat-6; fat-7and sbp-1 mutant strains. Illustrating the need for special conditions that are likely to be necessary to uncover regulatory functions of many nematode NRs, inhibition by nhr-64 RNAi in wild type animals leads only to small changes in fatty acid composition. On the mRNA level, inhibition of nhr-64 by RNAi leads to an altered expression of 14 studied metabolic genes (six decreased and eight increased expression). Among them, the acetyl-CoA synthetase gene (acs-2) and an acyl-CoA oxidase gene (F08A8.4) homologous to human peroxisomal acyl-CoA oxidase-1 had decreased respectively whereas acetyl-CoA carboxylase and de-novo synthetized monomethyl branched chain fatty acids had increased. It has been suggested that NHR-64 is likely to be critical in promoting fatty acid oxidation in mitochondria and peroxisomes and is a potent regulator of fat storage in C. elegans [62]. 2.1.1.2.4. NHR-69. NHR-69 is strongly expressed in the gut and hypodermis during all larval stages and weakly in the uterus (in the late L4 larvae and in adults). It is also detected in the rectal epithelia and posterior pharynx. The nhr-69::gfp transgene regulated by the nhr-69 promoter was detected in the nucleus of the E8 intestinal precursor cells in developing embryos and later throughout larval development until adulthood. In adults, it is expressed in the ASI neurons, in tail neurons, and in the hypodermis. It localizes exclusively to the nuclei [32]. NHR-69 associates with a Smad protein DAF-8 in vivo and in vitro. Double mutants of daf-8; nhr-69 result in a defective neuropeptide secretion and phenotypes of reduced insulin signaling (increased expression of sod-3 and gst-10 and a longer life span). Expression of exp-2, encoding a voltage-gated potassium channel, is synergistically increased in daf-8 and nhr-69 double mutants (but not in single mutants). EXP-2 represses the secretion of the insulin-like peptide DAF-28 in ASI neurons. Reversely, exp-2 mutation shortens the extended life span of daf-8; nhr-69 double mutants putting EXP-2 downstream of DAF-8 and NHR-69. Over-expression of NHR-69 in DAF-28-secreting ASI neurons leads to a hypoglycemic phenotype that is rescued by exogenous glucose. This indicates that NHR-69 together with DAF-8/R-Smad inhibit transcription of exp-2 to promote DAF-28 secretion. NHR-69 thus exemplifies a physiological crosstalk between TGF-β and HNF4αlike signaling in C. elegans [32]. Although NHR-69 is in sequence analysis related to HNF-4, it was suggested to act as a receptor of estrogenic compounds [63] and testosterone [64]. Since HNF4 is now considered to be the liganded founder of NRs that were conserved during metazoan evolution, it is possible that binding of steroid molecules by nematode NHF4-related NRs may be derived from such HNF4 ancestral function [12]. 2.1.1.2.5. NHR-14. NHR-14 is an HNF4 homologue which was proposed to be a C. elegans NR binding estrogen. Selected transcriptional effects of estradiol were found to be linked to NHR-14 [63]. The expression of vitelogenins, which responds to estrogens, has been found to be independent of NHR-14 suggesting that several HNF4 homologues may bind estrogenic molecules and functionally respond to them [65]. 2.1.1.2.6. NR2A-c: HNF4 related genes with unknown functions in nematodes. Many of the remaining NR2A members were studied on the expression level and on the basis of RNAi. Their functional expression indicated that these NR2A group members are likely to be functional genes but their functions are not obvious in RNAi screens under laboratory conditions. Several studies have proved regulated expression of NRs that fall into this category [38,61]. Signs of recent duplications

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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and limited conservation between species of Rhabditidae can be documented indicating the ongoing evolution of genes that are related to HNF4. Several clusters of recently duplicated genes can be found, especially on chromosome V [18]. Ongoing expressional diversification of recently multiplied NR genes was documented on a NR cluster on chromosome V [38]. 2.1.2. NR2B–RXR like RXR is a highly conserved member of the NR2 subfamily present in most Metazoan phyla from Placozoa [11], Cnidaria [44,66], Arthropoda [67,68], mollusks [69] and all deuterostome species studied to date [12,70–74]. The unique position of RXR in NRs is given by its evolutionarily conserved ability to heterodimerize with other NRs. In vertebrates, heterodimerization partners include members of the NR1 subfamily [NR1C1-3 (PPARα,β/δ,γ), NR1B1-3 (RARα,β,γ), NRH1-2 (LXRα,β), NR1A1-2 (TRα,β), NR1I1-2 (VDR and PXR), NR1H4 (FXR)] and NR4 subfamily [NR4A1-2 (Nur77 and Nurr1)] ([75,76]). Specific functions of RXR homodimers are also found in vertebrates and were shown to have both transactivating [77,78] as well as transrepressing regulatory functions [79]. Arthropod and mollusk RXR homologues (Ultraspiracle, USP in insects), dimerize with an invertebrate member of the NR1 subfamily, an Ecdysone receptor (EcR) that binds and is activated by the insect steroid hormone Ecdysone [80,81]. The heterodimerization of RXR is an evolutionarily old property of RXR conserved between arthropods and vertebrates [82,83]. Specific functions of RXR homodimers are also found in vertebrates and shown to have both transactivating [77,78] as well as transrepressing regulatory functions [79] (reviewed in [84]). Vertebrate RXRs are widely expressed [85]. RXRβ is found in most cells, while RXRα and RXRγ are expressed in certain tissues as in the intestine, liver, kidney, epidermis and in muscles and the brain. The high affinity ligands of RXRs were identified. The high affinity ligand is a stereoisomer of retinoic acid (RA), 9-cis RA [86]. However, other ligands were proposed as endogenous ligands although with lower affinity (Reviewed in [87]). In invertebrates, RXR homologue Usp regulates together with its heterodimerizing partner EcR critical developmental processes including insect metamorphosis, reproduction and behavior. Invertebrate RXRs show variable affinities to small-molecule ligands. High affinity binding was shown in the case of RXR in a cubomedusan jellyfish Tripedalia cystophora [66]. Insect and crustacean RXRs were shown to bind terpenes such as juvenile hormone and methyl farnesoate which are likely endogenous ligands [88]. Specific binding of 9-cis RA to RXR together with its transactivating functions and the natural occurrence of 9-cis RA in embryos was found in an evolutionarily ancient insect Locusta migratoria [89]. RXR from a freshwater mollusk Biomphalaria glabrata also specifically binds 9-cis RA and cis-4, 7, 10, 13, 16, 19docosahexaenoic acid with transactivating effects on gene transcription [69]. 2.1.2.1. Nematode RXR — evolutionary experiments with an ancient regulatory cascade. Although an RXR homologue seems to be absent in Rhabditidae, it is found in several species of other nematode families. An RXR homologue is found in the genome of Pristonchus pacificus [36]. The genomes of the parasitic nematode Dirofilaria immitis [35] and of Brugia malayi both encode RXR and EcR [21,90,91] supporting the presence of RXR in basal nematode species. RXR is found in the genome of the parasitic nematode Pristionchus pacificus together with its dimerizing partner from the NR1 subfamily EcR [92]. P. pacificus was recently developed as a laboratory model organism [93,94]. The expression of P. pacificus RXR, Ppa-pnhr-1, is observed in all postembryonic stages in an oscillating pattern that correlates with molting. The highest expression is in the midintermolt period and lowest during molting. The expression of the EcR homologue, Ppa-pnhr-2, is stable. Thus, RXR-EcR regulation is likely to be at least partially conserved in some nematode species [36]. The

presence of RXR-EcR regulation in nematode species supports the classification of nematodes as Ecdysozoa. Keeping with this, RXR homologues from Filaria show similar conserved functionality. Brugia malayi RXR, Bma-RXR is expressed in L1 larvae and in adults. Bma-RXR is able to dimerize with Bma-EcR and the LBD of Bma-EcR was capable of transducing an ecdysteroid signal in a mammalian cell system [35,90]. RXR homologues are also found in Onchocerca volvulus Ov-RXR [90] and in Dirofilaria immitis Di-RXR-1. Di-RXR-1 was shown to be able to functionally interact with Drosophila EcR [35]. 2.1.3. NR2C/D–TR2/4 2.1.3.1. NHR-41. NHR-41 (NR2D1) is highly similar to the Drosophila DHR78 (NR2D1), which is essential for the onset of metamorphosis, and to the mammalian TR2 and TR4 receptors (NR2C1-2) that are widely expressed and act as negative regulators of several NRs [95,96]. NHR41 plays a role in the formation of SDS-resistant dauer larvae. Using two different reporter fusions, the expression of nhr-41α was detected in hypodermal-seam cells, excretory cell, and rectal epithelia, while the expression of nhr-41β was detected in gut, chemosensory neurons, and in head and tail hypodermis. nhr-41 mRNAs are expressed in all larval stages at precise intervals relative to molting; mRNA produced by the upstream promoter sequence is detected at the time larval molts occur, while mRNA produced by the promoter sequence in the fourth intron is detected at variable levels during larval development with distinct peaks of expression at the beginning of the intermolt [61]. This suggests that the regulation of molting by NR2D1 receptors is conserved between nematodes and insects [97]. 2.1.4. NR2E–TLX/TLL The NR2E group contains two NRs in vertebrates (TLX and PNR) [7,98] and four to six NRs in insects (including TLL, HR51, DSF, HR83) [13]. Members of this group can be found in Cnidaria [8], flatworms [99] and in most if not all metazoan phyla except for the Placozoa. TLX regulates development of neural tissues and retina [7,100]. PNR is expressed in photoreceptors and regulates their development [98,101]. Mutations in the PNR gene caused defects in the number and type of photoreceptor cells in the retina, enhanced S-cone sensitivity, retinal degeneration, and other retinal defects, suggesting that it may function in the specification of neuron identity [102]. In insects, this group is enlarged to six genes [103]. Both in insects and vertebrates, TLX/TLL specify terminal cell fate during embryogenesis and neural stem cell maintenance. They are constitutive transcriptional repressors [104]. In nematodes, this group includes four NRs FAX-1, NHR-67, NHR111, and NHR-239. 2.1.4.1. FAX-1. FAX-1 functions in the specification of neuron identity. It regulates the path-finding of axons that extend along the ventral nerve cord and axons in the nerve ring and is required for normal neurotransmitter expression [105–107]. FAX-1 protein accumulates in the nuclei of 18 neurons (AVA, AVB, and AVE interneuron pairs) that coordinate body movements. Mutations in fax-1 cause axon path-finding defects and a loss of FMRFamide-like (Phe-Met-Arg-Phe-NH2-like) neurotransmitter expression in the bilaterally-paired AVK interneurons which result in locomotion defects [105,106]. fax-1 and unc-42, a paired-homeodomain protein, specify cell identity in AVA and AVE interneurons by complementary regulation of target effector genes. The genes downstream of fax-1 include presynaptic products, such as the neurotransmitter precursor genes flp-1, ncs-1, and opt-3, and postsynaptic products, such as those encoded by the nmr-1, nmr-2 genes [107]. The LBD of FAX-1 diverged from vertebrate and Drosophila orthologues. The FAX-1 DBD is conserved in 83% of amino acid residues when compared to the human nuclear receptor PNR (NR2E3) [98,101,106].

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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2.1.4.2. NHR-67. The nhr-67 gene encodes the nematode orthologue of the tailless nuclear receptor (TLL) gene. nhr-67 is expressed in a subset of head neurons. Inhibition of nhr-67 (by RNAi) results in slow-growing worms with cuticle-shedding defects, egg-laying defects, and a protruding vulva (Pvl), suggesting that NHR-67 plays multiple roles in development [61]. nhr-67 is also expressed in multiple vulval cells and interacts with three transcriptional regulators — Nkx6-type homeobox gene cog-1, lin-11 (LIM homeodomain transcription factor), and egl-38 (homologue of Pax2/5/8) and forms composite expression patterns with downstream genes. It is important for cell fusion events during morphogenesis of the vulva [108,109]. NHR-67 regulates the migration of the male linker cell (as a cellautonomous, stage-specific regulator of timing in linker cell migration programs during L3 and L4 stage) [110]. The linker cell in C. elegans males undergoes a stereotyped migration that guides gonad organogenesis. It occurs with precise timing. NHR-67 is required for this event [110]. Whole transcriptome expression analysis of a linker cell inhibited for NHR-67 by RNAi identified 22–25% of 10,000 analyzed genes as upor down-regulated (20-fold) [111]. The involvement of genes identified as NHR-67 dependent in linker cell migration was confirmed in 22% (45 of 204) of genes by functional analysis [111]. NHR-67 acts at distinct steps to determine the identity and subsequent left/right (L/R) asymmetric subtype diversification of a class of gustatory ASE neurons. Additionally, it also regulates the expression of a sensory neuron-type-specific selector gene, che-1, which encodes a zinc-finger transcription factor [112]. NHR-67 regulates development of cells that form the hermaphrodite uterus [113]. This is dependent on coordinated signaling between the uterine anchor cell (AC) and the ventral uterine cell (VU). NHR-67 shows a dynamic pattern of expression in AC and VU cells. It functions upstream of the lin-12/Notch receptor in the VU lineages, upstream of the lag-2/Delta signal in the AC, and in a pathway that includes hlh-2/ daughterless and egl-43/Evi1 transcription factors [113]. FAX-1 and NHR-67 have overlapping DNA binding preferences. This is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 in their DNA-binding domains. This amino acid position is part of the “P box” and plays a critical role in binding site specificity. FAX-1 binds a much larger repertoire of half-sites than NHR-67. While NHR-67 binds both monomeric and dimeric sites, FAX-1 binds only dimeric sites. These binding specificities are conserved between NR2E receptors in nematodes and vertebrates suggesting evolutionarily conserved regulatory mechanisms [114]. These interspecies structure–function relationships were tested using genetic and genomic means. The NR2E group includes two additional members, NHR-239 which is most related to insect HR83 and NHR-111 which appears to be a recently evolved paralogue of FAX-1. NHR-239 in C. elegans and C. briggsae have truncated and highly diverged LBDs. nhr-111 is broadly expressed, while nhr-239 is expressed only in a small subset of neurons. FAX-1 is indispensable for normal movement. Functional analysis of the FAX-1 LBD in an in vivo assay revealed that it is not required for at least some developmental functions since the movement phenotype can be rescued by highly divergent LBDs of C. elegans NHR-111 and NHR-67, LBD of C. briggsae FAX-1 or by a transgene lacking the LBD. This suggests that the relatively high level of sequence divergence for Caenorhabditis elegans LBDs reflects in the case of the NR2E group members the relaxed selection of the primary sequence as opposed to a divergent positive selection [115]. This concept may be more general and may be applicable to NR2E members in other phyla as well as to some HNF4 related genes. 2.1.5. NR2F–COUP-TF-like COUP-TF, although present in the genomes of animals that lack a real nervous system seems to be evolutionarily related to the evolution of neurons and the nervous system. Vertebrate COUP-TFs are broadly, but not ubiquitously expressed in multiple tissues throughout embryonic development and are

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indispensable for proper organogenesis. In vertebrates COUP-TFs have critical roles in the differentiation of specialized mature cells from common stem/progenitors (reviewed in [116]). Similarly as in vertebrates, the insect SVP is involved in the regulation of neuronal development and cell specification [117,118]. 2.1.5.1. UNC-55. UNC-55 is expressed in neurons (in 2 pairs of neurons in the nerve ring, 13 pairs of motor neurons in the ventral nerve cord, and in a pair of cells in the pre-anal ganglion). The expression of unc-55 in the ventral nerve cord is temporally restricted in L2 and L3 larval stages and is located in ventral D (VD) motor neurons, but not in dorsal D (DD) motor neurons. The dorsal D GABAergic and ventral D GABAergic motor neurons are involved in a cross-inhibitory network, which is responsible for the coordinated movement of animals. Specific changes in synaptic location occur in dorsal D neurons during early larval development. Initially ventral D synapses are relocated to the dorsal side. The ventral D motor neurons are not remodeling synapses, because this function is inhibited by UNC-55 [119,120]. unc-55 mutants have asymmetric locomotion when moving backward, due to changes of the synaptic pattern of the ventral D motor neurons. They adopted the pattern from the dorsal D motor neurons [119]. The ectopic expression of unc-55 in the dorsal D motor neurons causes them to adopt ventral D motor neuron features [121]. UNC-55 is also necessary for male mating. Two mRNA isoforms (unc-55a and unc-55b) were detected during post-embryonic development in males, whereas only one, unc-55a, was detected in hermaphrodites [120]. In unc-55 mutant males the isoform unc-55a rescued both the locomotion and mating defects, whereas isoform unc-55b rescued the mating defect only. UNC-55 functions as a link between the generation of male-specific lineages and the genes, which are involved in activating the programs necessary for copulation [122]. The function of UNC-55 downstream synaptic remodeling genes was studied using a combination of a cell-specific profiling and RNAi. One of the targets was the Iroquois-like homeodomain protein, IRX-1. It functions as a key regulator of remodeling in dorsal D neurons [123]. Thus, keeping with the important position of COUP-TF during the evolution of Metazoa, UNC-55 is a conserved nematode orthologue of COUP-TF/SVP. 2.2. NR1 — conserved and evolving mechanisms of members of the NR1 subfamily regulate nematode development The NR1 subfamily is a very ancient subfamily of NRs which originated before the separation of deuterostomes and protostomes [15]. The NR1 subfamily includes 20 members in humans (Fig. 2) that are divided into seven classes including (A) thyroid hormone receptors α and β (TRα, β) (NR1A1-2), (B) retinoic acid receptors α, β and γ (RARα, β, γ) (NR1B1-3), (C) peroxisome proliferators activated receptors α/β, δ and γ (PPARα/β, δ, γ) (NR1C1-3), (D) RevErbA α and β (NR1D1-2), (F) −ROR α, β and γ (NR1F1-3), (H) −LXR α and β, and FXR (NR1H3,2,4), (I) VDR, PXR, and CAR (NR1I1-3). Platyhelminths NR1 subfamily members that contain 2 DBDs are classified as NR1X [5,6]. These NRs are critical regulators of development, metabolism and developmental timing and have a role in cancer as shown for TRs (reviewed in [124,125]), RARs (reviewed in [126,127]), VDR (reviewed in [128]), and PPARs (reviewed in [129]). Insect NR1 members include E75 (NR1D3), E78 (NR1E1), HR3 (NR1F4), EcR (NR1H1) and HR96 (NR1J1). Insect members of this subfamily are involved in complex regulatory networks that are important for a wide range of developmental and metabolic processes including embryogenesis, metamorphosis, reproduction, and homeostasis (reviewed in [130]). There are six C. elegans NRs that show relation to the NR1 subfamily members: SEX-1 and NHR-85 show limited homology to NR1 members, probably to RevErbA (NR1D) or RARs (NR1B); NHR-23 is a close homologue of ROR in vertebrates and HR3 in insects (NR1F); DAF-12, NHR-48

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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and NHR-8 show strongest similarity with VDR and LXR (NR1I) and HR96 in Drosophila (NR1J). Homologues of NR1H (EcR) are not present in sequenced genomes of Rhabditidae, they are, however found in Pristionchus pacificus, Haemonchus contortus and in Filaria. 2.2.1. NR1B–NR1D homologues SEX-1 and NHR-85 2.2.1.1. SEX-1. SEX-1, originally cloned as CNR14, shows high sequence similarity to vertebrate RevErbA and RARs [3]. It is expressed ubiquitously from oogenesis to mid embryogenesis with maximum expression detected at the onset of gastrulation (28–100 cell stage), and later gradually decreases to the stage of 550 cells when it becomes undetectable by antibody staining [131]. SEX-1 has been shown to associate with xol-1 promoter and to function as a very potent inhibitor of xol-1 expression. This subsequently affects sex determination through the mechanism of X-chromosome dosage compensation [131]. SEX-1 inhibits xol-1 expression in cooperation with ONECUT homeodomain protein CEH-39 which is necessary together with SEX-1 for complete repression of xol-1 [132–134]. SEX-1 dependent repression of xol-1 is complemented by a second regulatory step at the RNA level through an RNA binding protein FOX-1 which has an additive effect in lethality caused by SEX-1 inactivation in XX-hermaphrodite animals [132–134]. The mechanistic function of SEX-1, that is direct inhibition of transcription puts SEX-1 as a functional homologue of RevErbA, which is also a transcriptional repressor based on its lack of the AF-2 domain. However, SEX-1 has a structurally recognizable AF-2 domain and keeping with its possible function as an activator, it was found to act during sex determination at multiple steps also downstream of XOL-1. SEX-1 functions synergistically with sdc genes at the level of the dosage compensation complex (DCC). It was speculated that this function may control the activity of some sdc genes or the stability of the SDC protein complex, or it could function with undefined genes in the sexdetermination pathway at the same regulatory step as the sdc genes or could function independently [132]. If such independent function of SEX-1 is based on its activation function-2 motif has to be determined. It can be anticipated that SEX-1, as a functional homologue of RevErbA, had to arise from a member of the NR1 subfamily that contained the AF-2 domain, that was later lost during evolution of vertebrate RevErbA [15]. RevErbA transmits its repressive function through the interaction with nuclear receptor corepressor NCoR [135,136]. C. elegans NCoR is critical for the development of the gonad through two mechanisms that are both mechanistically transcription inhibition but one is acting by inhibiting the expression of regulatory RNAs [137]. It is therefore possible that both steps in SEX-1 dependent sex determination mechanistically repress transcription. A role for siRNA in chromosome dosage compensation and selective recognition of X-chromatin was found in Drosophila [138]. 2.2.1.2. NHR-85. NHR-85 encodes a nuclear receptor of the NR1 subfamily that is highly similar to the insect E75 nuclear receptor that regulates molting and metamorphosis, and human RevErbA (NR1D1), which is involved in the regulation of the circadian rhythm. Based on the fusion transgenes carrying the nhr-85 promoter fused to gfp, its expression was detected in the vulva, the hypodermis, and in specialized epithelia of the rectum and excretory duct. While nhr-85 RNAi by injection did not produce phenotypes in the N2 background, 15% of F1 adult progeny of injected rrf-3(pk1426) hermaphrodites were egg-laying defective (Egl phenotype) [61]. 2.2.2. NR1F NHR-23 is a close homologue of vertebrate RORs and insect HR3 2.2.2.1. NHR-23. NHR-23 (CHR3) (NR1F4) is related to RAR/RXR/TR NRs by its P box sequence and is the closest nematode homologue of ROR/ RZRα in vertebrates and DHR3 in Drosophila melanogaster [3,139,140].

It is a critical regulator of C. elegans larval development. Its expression is high in inter-molting phases but decreases in molts. Inhibition of nhr-23 by RNAi results in larval arrests and severe defects of molting in all four larval stages [140]. Its expression is regulated by miRNAs let-7 and mir-87 [141]. Downstream of NHR-23 are collagen genes, enzymes and genes likely to regulate development such as the hedgehog related genes (Hh-r) and several NRs [140,142,143]. NHR-23 forms a regulatory cascade with NHR-60. In seam cells, nhr-60 expression is dependent on NHR-23. Another member of the NR family, NHR-91 is dependent on NHR-23 expression as identified by whole genome expressional microarrays [143] (and was confirmed by qPCR, unpublished). From the C. elegans conserved NRs, NHR-23 is involved in a regulatory pathway that is strongly reminiscent of the regulatory pathways in both vertebrates as well as insects (Fig. 3). NHR-23 is strongly conserved at the sequence level and functionally. It binds the RORα/HR3 response element. NHR-23/ROR/HR3 response element is present in promoters of C. elegans hedgehog related genes with expression dependent on NHR-23. Interestingly RORα also regulates expression of one of three mammalian hedgehog related genes, the Sonic Hedgehog [144]. In RORα-deficient mice, the reduced production of sonic hedgehog by these cells appears to be the major cause of the decreased proliferation of granule cell precursors and the observed cerebellar atrophy [144]. Comparison of the regulatory cascades of NHR-23, RORα and DHR3 suggests that these pathways developed in parallel (Fig. 3). NHR-23, RORα and DHR3 are in the centers of these cascades. Interestingly, the NR6 subfamily member GCNF homologues in nematodes and insects are part of the cascade. NHR-91, the nematode orthologue of GCNF is downstream of NHR-23 [143], and in insects HR4 is a negative regulator transmitting the negative regulation upstream of HR3 [145,146]. GCNF is widely expressed in vertebrate tissues, similarly as RORs and it is therefore likely that such interplay is employed in various tissues. GCNF is required for the repression of pluripotency genes during retinoic acid-induced embryonic stem cell differentiation [147]. Considering the wide expression of GCNF especially in tissues involved in immunity [148] and the roles of RORα and RORγ in the regulation of T cell differentiation [149], it is likely that this interplay between ROR homologues (NR1F) and NR6 (GCNF homologues) is a more general theme employed during the evolution of metazoans. 2.2.3. NR1H Nematode NRs related to EcR Homologues of EcR and USP, the major regulators of arthropode development are not present in known genomes of Caenorhabditis species. EcR and RXR/USP were identified in the parasitic nematodes Dirofilaria immitis [35], Brugia malayi [21,90,91] and Pristionchus pacificus [92]. The presence of a functional EcR was shown in Haemonchus contortus which responds to high concentrations of 20E (20-hydroxyecdysone). The cloned receptor, however, did not heterodimerize with the ligand binding domain of phthirapteran USP [150]. 2.2.4. NR1I–NR1J nematode NRs related to VDR and LXR: DAF-12, NHR-48 and NHR-8 Vitamin D receptor (VDR) is a critical vertebrate NR involved in a wide range of metabolic processes, from bone metabolism to cell differentiation and cancer [151]. It is activated by vitamin D3. LXRs (α and β) are oxysterol-activated NRs involved in the regulation of cholesterol homeostasis [152,153]. VDR and LXRs are expressed in most physiological systems and their functions project to a wide range of metabolic, reproductive and developmental processes. 2.2.4.1. DAF-12. DAF-12, shows a close relationship to two vertebrate NRs, VDR and LXR. DAF-12 is expressed from embryo to adulthood and its expression is localized in many tissues; in epidermis, vulva, somatic gonad, intestine, pharynx, sex myoblasts, neurons, body wall muscles. daf-12 expression

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

M. Kostrouchova, Z. Kostrouch / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

Nematodes

Vertebrates

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Insects

TRH

miRNAs

NR1H (ECR) TSH NR1D (E75A) NR1D (E75B)

TR

NR6A (DHR4) NR1F (NHR-23)

Collagens

NR6A (NHR-91)

Metabolic processes

NR2A (NHR-60)

NR1F1 (RORα)

NR1F (DHR3)

Late genes

NR5A (βFTZ-F1)

Collagens Sonic Hedgehog

Hedgehog related genes

Fig. 3. Comparison of regulatory cascades of NHR-23, ROR α and DHR3. The regulatory core is represented by NHR-23, ROR α and DHR3 in nematodes, vertebrates and insects, respectively. DHR3 pathway is adopted from [208].

is up-regulated during mid-larval stages. In starving laboratory cultures or at high density worm populations, DAF-12 promotes formation of a stress-resistant long lived dauer larva, an alternate L3 larval stage. At favorable conditions, dauer larvae develop into L4 stage and adult hermaphrodites. DAF-12 is a critical regulator in this regulatory switch [154,155]. DAF-12 also influences lipid metabolism, fertility, and adult life span [156–160]. DAF-12 directly regulates expression of let-7 microRNAs mir-84 and mir-241 [161,162]. Favorable conditions promote the biosynthetic pathway for DAF-12 ligands Δ4- and Δ7-dafachronic acid (DA) [163]. Genetic screens indicate that DAF-9, a p450 cytochrome acts upstream of DAF-12 during production of DAF-12 ligands whose presence is necessary for DAF-12 dependent bypassing the dauer larva development [159,160,163]. A recently published metabolomic screen for endogenous ligands revealed that Δ1,7-DA and 3α-OH-Δ7-DA and Δ7-DA are naturally occurring, while Δ4-DA, although found as a possible ligand is not detected in laboratory cultures [164]. A role of DAF-12 function in the regulation of adult life span has been proposed. Animals with ablated germline have a longer life span. When germline stem cells are ablated, the expression of DAF-36 is upregulated and increases the production of DAF-12 ligands. This activates DAF-12 and upregulates miR-84 and miR-241 and subsequently down-regulates their targets akt-1 and lin-14 and leads to activation of DAF-16/Foxo [165]. Unliganded DAF-12 was found to bind co-regulator DIN-1, a homologue of human co-repressor SHARP. DIN-1 is expressed as two isoforms. They are both localized in the nucleus. The long isoform (DIN-1 L) functions during embryonic and larval development, while the short isoform (DIN-1S) together with DAF-12 is important for longevity. The short isoform is also important for the regulation of lipid metabolism, larval stage-specific programs and diapause. It was proposed that the DIN-1S/DAF-12 complex serves as a molecular switch that allows the dauer alternatives in response to diminished hormonal signals [166]. Orthologues of C. elegans DAF-12 have been identified in the human parasitic nematodes Strongyloides stercoralis, Ancylostoma duodenale, Ancylostoma Ceylanicum, Ancylostoma caninum, Necator americanus and other parasitic nematodes [167,168]. The ligands of C. elegans DAF-12, Δ4 dafachronic acid (Δ4-DA) and Δ7 dafachronic acid (Δ7-DA) [163] activate AcDAF-12 and SsDAF-12 (Ancylostoma caninum and Strongyloides stercoralis, respectively) and reduce infectivity of L3 larvae suggesting a very promising lead towards new therapy of helminthiases [168].

2.2.4.2. NHR-48. NHR-48, together with DAF-12 and NHR-8, comprise the C. elegans homologues of Drosophila HR96 and the vertebrate NRs VDR, PXR, and CAR. The LBD of NHR-48 is highly diverged from other members of the NR1 subfamily. The expression of an nhr-48::GFP reporter is in the pharyngeal gland in larvae and adults and in the spermatheca in L4 larvae. NHR-48 is also expressed in the epidermis [61]. NHR-48 represses expression of Y8A9A.2 in the g1A pharyngeal gland cells indicating that very specific regulatory circuits are likely to exist for diversified nematode NRs [169]. 2.2.4.3. NHR-8. NHR-8 is the third closest homologue of NR1I-J group members. The nhr-8::GFP is expressed in intestinal cells. The expression starts in embryos and continues until adulthood. nhr-8(ok186) mutants have a DNA binding domain of nhr-8, but the LBD is completely missing. Homozygous mutants are viable and develop normally during standard conditions. The mutant animals are more sensitive to colchicine and chloroquine. Inhibition of nhr-8 function by RNAi also results in increased toxin sensitivity [170]. Loss of function of nhr-8 results in the deficiency of dafachronic acid, a bile acid-like steroids and ligand for DAF-12. The loss of nhr-8 function also results in deficiency of unsaturated fatty acids, reduced fertility and developmental arrest. These phenotypes are rescued with cholesterol supplementation. NHR-8 is involved in the regulation of cholesterol levels by interaction with daf-16/FOXO and is important for the regulation of cholesterol balance and metabolism [171]. NHR-8 thus forms a metabolic network with DAF-12 in the regulation of cholesterol metabolism, development, reproduction, and aging. 2.3. NR3 — lost in the evolution of nematodes The NR3 subfamily includes major endocrine receptors in vertebrates. Their function projects to many critical developmental and metabolic processes including sexual dimorphism, growth and metabolism. The NR3 subfamily includes ER, ERR, GR, MR, PR and AR (Fig. 2). This group is likely to have its origin set before the separation of Cnidaria and deuterostomes/protostomes [12,25,172,173]. The ancestral member of this subfamily is Estrogen Related Receptor, ERR, which is found in the genomes of Trichoplax adhaerens [11] as well as in several species of mollusks. In octopus, ERR (named ER) is more similar to vertebrate ERRs than to ER and was shown to bind estrogen [174,175]. Octopus ER is a constitutive activator of transcription [176]. The NR3 subfamily

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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members are not detected in nematodes and are assumed to have been lost during the beginning of nematode evolution [12,25,172,173]. 2.4. NR4 — gonadal functions of the NR4 subfamily member in nematodes 2.4.1. NHR-6 NHR-6 shows close relation to NR4 subfamily members NUR77/ NGFIB (NR4A1), Nurr1 (NR4A2) and NOR-1 (NR4A3) in vertebrates and HR38 in insects (DHR38 in Drosophila). Vertebrate members are expressed in a wide range of tissues and have pleiotropic functions ranging form regulation of development and homeostasis of dopaminergic neurons, immediate stress response, cellular proliferation, apoptosis to regulation of glucose metabolism, inflammation and cancer (reviewed in [75,76,177,178]). NR4 proteins can bind response elements as monomers, homodimers and NUR77 and Nurr1 also as heterodimers with RXR [179]. NUR77 promotes glucose utilization [180] and NOR-1 increases oxidative metabolism in skeletal muscle in response to β-adrenergic stimulation [181,182]. Vertebrate NR4 subfamily members have distinct tissue-specific functions in modulation of glucose homeostasis. NUR77, Nurr1 and NOR-1 up regulate gluconeogenesis [183]. NUR77 and NOR-1 also increase sensitivity to insulin in adipose tissue [184]. In insects, HR38 functions in cuticle formation during metamorphosis [185] and has also prominent metabolic functions including regulation of expression of phosphoglucomutase, an enzyme that critically regulates glycogenesis [145]. In C. elegans, NR4A proteins are represented by only one member of the NR4 subfamily, NHR-6. NHR-6 was originally cloned as CNR8 as a developmentally regulated conserved NR with the highest expression in L3 hermaphrodites [3]. The integrated transgene of nhr-6 fused with GFP is expressed in a subset of somatic gonad cells in the late L3 and L4 stage. In later L4 stage, the transgene is expressed in developing spermatheca cells of the anterior and posterior spermathecae and in a pair of head chemosensory neurons (from 3 fold embryos to adulthood). It is also weakly expressed in the intestine in late embryos and L1 larvae. The function of NHR-6 in the somatic gonad is specific to hermaphrodites. nhr-6 transgene is not expressed in male somatic gonad and males of nhr-6 null mutants have normal gonads. The function of NHR-6 has been studied on nhr-6 homozygous hermaphrodites lacking functional NHR-6. They are viable, but exhibit severe reproduction defects. NHR-6 is necessary for development of the spermatheca. In NHR-6 mutants the size of the spermatheca is reduced, because the normal number of cells is reduced by half. The decreased number of cells is very likely due to failure of proliferation. The NHR-6 mutant hermaphrodites have ovulation defects; they have decreased brood size and abnormal cell morphology oocytes and embryos. These defects vary from retained embryos and internal hatching of larvae to abnormal round embryos that arrest during development and fragmentation of oocytes. A large proportion of embryos have abnormal morphology but later develop into larvae that appear normal. In the wild type nematodes, the most proximal oocyte in the gonad arm moves to the spermatheca, where it is fertilized, and then to the uterus. The process of consecutive events in oocyte fertilization is guided by the anatomical arrangement of the gonad and the spermatheca– uterine valve formed by spermaheca–uterine junction core (sujc) cells. The spermatheca–uterine valve is not properly formed in nhr-6 mutants. Mutant animals have fever sujc cells that are spatially disorganized resulting in the absence of a functional spermatheca–uterine valve. NHR-6 thus promotes proliferation of sujc cells and the normal differentiation of spermatheca lineages. The ovulation defect is a likely secondary consequence of abnormal spermatheca development. Cell proliferation and differentiation is the prominent function of NR4 members in mammals [186–188]. This function is conserved between vertebrate and nematode NR4 members. A search for modifiers of NHR-6 loss of function phenotype identified c-jun homologue jun-1 as a strong enhancer of partial nhr-6 loss of function phenotype and a direct interactor of NHR-6 in a yeast two-hybrid assay [189]. This is likely to constitute a novel function

for NR4 subfamily members. c-Jun is a constituent of a composite transcription factor AP-1 together with c-Fos. AP-1 and individually also both c-Jun and c-Fos are critical regulators of cell proliferation, differentiation and apoptosis and were shown to interact with NRs in a number of ways including competition and interactions on DNA binding through overlapping DNA binding sites. The interaction between AP-1 and NRs has often opposing effects on cellular proliferation and differentiation as shown on the interaction with GR [190] and RARs [191,192]. Since the critical role of NR4A members in the promotion of cell proliferation is likely to be conserved between nematodes and vertebrates, this direct involvement of a complex c-jun and NR4A proteins may also be conserved between nematodes and vertebrates. A direct physical and functional interaction of c-jun was found in the case of AR [193–195]. Nematode NR4A proteins thus are likely to predominantly support cell proliferation and differentiation roles, while insect NR4A proteins are likely to keep supporting the metabolic functions of their common ancestor. It is tempting to speculate that the metabolic functions of the NR4A ancestor were adopted in nematodes by some of the multiplied descendants of the ancestral HNF4 (NR2A). One member of the NR4A the subfamily was also identified in Brugia malayi (BmNHR9) [91]. Its functions has yet to be determined. 2.5. NR5 — conservation and evolvability of SF1-like NHR-25 2.5.1. NHR-25 NHR-25 is the single member of the NR5 subfamily in C. elegans and is highly similar to vertebrate SF-1 and LRH-1 and arthropod Ftz-F1. It is expressed as two mRNA isoforms coding for two proteins, of which only one has a DNA binding domain. An nhr-25::GFP fusion gene is expressed in the epidermis, the developing somatic gonad, and a subset of other epithelial cells. Its inhibition by RNA-mediated interference indicates its involvement in broad developmental processes including embryonic development, molting and development of the somatic gonad [196, 197]. A mutant carrying a deletion in the DNA binding domain shows that NHR-25 is essential for embryogenesis, molting and differentiation of the gonad and vulva [197]. NHR-25 regulates, together with NHR-23 the expression of acn-1, which codes for a multi-domain angiotensin-converting enzyme (ACE)-like protein in Caenorhabditis elegans, an enzyme that is essential for larval development, molting and adult morphogenesis [142]. Impairment of nhr-25 activity results in defective cell–cell fusions of hypodermal syncytial cells, seam cells, and vulva precursor Pn.p cells. NHR-25 appears to cooperate with lin-39 Hox gene in the regulation of vulval cell differentiation [198]. NHR-25 is expressed in seam cells and is required for proper regulation of their asymmetric divisions. Inhibition of nhr-25 function leads to defects of anterior daughter seam-cell that normally fuse and form lateral epidermis [199]. The role of NHR-25 in the regulation of gonad development has been shown to be dependent on the interplay with beta-catenin signaling. Beta-catenin signaling promotes distal fate in asymmetrically dividing somatic gonad precursor cells. Inhibition of nhr-25 leads to the formation of extra DTCs (distal tip cells) from cells that would otherwise develop into proximal cells. In mutants that have defective fertility and DTC-1 formation, pop-1, sys-1 and nhr-25 inhibition reverses DTC defects and restores fertility. This suggests that NHR-25 and betacatenin activities are required to establish both proximal and distal fates. It has been proposed that this nuclear receptor–beta-catenin interaction may be an ancient mechanism of cell-fate decision [200,201]. In this regulatory cascade, both NHR-25 and NHR-23 are under the control of let-7 and mir-84 [141]. NHR-25 dependent regulatory events in differentiation programs include both promoting as well as inhibiting heterochronic gene pathways. NHR-25 regulates positively the larva-to-adult transition as visualized in seam cell fusion, regulation of cell division and formation of alae, an acellular epidermal structure formed by adult seam cells. On

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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the other hand, NHR-25 regulates negatively collagen 19 (col-19) in seam cells. This illustrates the complexity of the regulatory network of NHR-25 even in one cell type [202]. NHR-25 may be regulated by a ligand or ligands. Impaired function of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid-rich intestinal depots. Aside from the intestine, ACS-3 is expressed in seam cells, that are in epidermal cells anatomically distinct from sites of fat uptake and storage. Restoration of ACS-3 function in seam cells is sufficient to reverse the mutant acs-3 phenotype on the level of whole animal suggesting involvement of mediators acting cell non-autonomously. The acs-3 mutant phenotype can be rescued by additional mutations in enzymes consuming acyl-CoAs or by inactivation of NHR-25 suggesting that the products of ACS-3 modulate the function of NHR-25 in regulation of fat uptake and synthesis [203]. The complex regulatory network that includes NHR-25 was illustrated on the regulatory cascade that defines the male-tip morphogenesis. This network is proposed as a so called bow-tie regulatory architecture in which regulatory inputs and outputs are connected through a conserved core. For male tail formation, this network includes NHR-25 together with transcription factors DMD-3 (homologue of vertebrate DMRT1 and transcriptional repressor MAB-3). This regulatory mechanism is likely to be conserved between different phyla [204]. The regulatory potential of NHR-25 was recently shown to be modulated by a highly spatially and temporary coordinated sumoylation visualized on its critical role in maintaining specific cell fate during vulval development [205]. NHR-25 regulates development of the epidermis together with ELT-3, a GATA transcription factor. NHR-25 and ELT-3 bind different response elements which were identified in close proximity of responsive genes suggesting their involvement in a regulatory complex that is likely to execute their activation as well as inhibitory roles on target genes [206]. Thus, NHR-25 is not only a highly conserved member of the NR family but it is likely to illustrate the potential of NR5 orthologues for evolvability of new species specific functions. 2.6. NR6 — an important emerging player GCNF-like NHR-91 The NR6 subfamily is an evolutionarily old and highly conserved branch of orphan NRs with members found in Cnidaria [8], and in all deuterostome and protostome phyla that had been studied to date. All species studied contain only one member of this subfamily in their genome. Mammalian GCNF (germ cell nuclear receptor) (NR6A1) is essential for embryonic development. GCNF is an orphan nuclear receptor, has no known ligand and is likely to function ligand-independently based on its lack of the AF2 activation domain. Its prominent function is transcriptional repression through binding to the DR0 response element. It is critically important for restriction of Oct4 to primordial germ cells after gastrulation. GCNF is expressed in ES/EC (embryonic stem cells and embryocarcinoma cells respectively) and during their differentiation and has been reported to be required for pluripotency gene repression during retinoic acid (RA)-induced mES (mouse embryonic stem) cell differentiation. GCNF can interact with DNA methylation proteins, and has been suggested to recruit DNA methylation complexes to repress and silence Oct4 expression (reviewed in [148]). The insect homologue of NR6A is HR4 (DHR4 in Drosophila) [103, 207,208]. DHR4, together with E75A, E78B and betaFTZ-F1 are expressed in a mutually dependent way forming a regulatory cascade which is triggered by pulses of the steroid hormone ecdysone at the onset of metamorphosis [207]. DHR4 plays a central role in this cascade acting as both a repressor of the early ecdysone-induced regulatory genes and an inducer of the betaFTZ-F1 midprepupal competence factor. DHR4 coordinates growth and maturation by mediating endocrine responses to the critical size of the developing larva [208]. DHR4 establishes temporal restrictions by terminating ecdysone pulses. These

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regulatory functions have morphological basis in physical oscillation of DHR4 molecules between the nucleus and cytoplasm in prothoracic gland (PG) cells. At low ecdysone level DHR4 is absent from PG cell nuclei. This oscillatory behavior is blocked when prothoracicotropic hormone (PTTH) or Torso (a receptor tyrosine kinase that regulates embryonic terminal cell fate) are abolished, resulting in nuclear accumulation of DHR4. Hyper-activation of the PTTH pathway results in cytoplasmic retention of DHR4. Increasing DHR4 levels in the PG can delay or arrest development. In contrast, reducing DHR4 function in the PG triggers accelerated development caused by precocious ecdysone signaling, due to failure to repress ecdysone pulses. DHR4 negatively regulates the expression of a cytochrome P450 gene, Cyp6t3 which is necessary for the ecdysone biosynthesis pathway [209]. 2.6.1. NHR-91 The nematode NR6 is represented by NHR-91 in Caenorhabditis. The nhr-91::GFP transgene is expressed in embryos, larvae and adults. In adults the expression is observed in head and tail neurons, hypodermis, vulva, spermatheca, seam cells and in the excretory duct cell and larval intestine [61]. NHR-91 is likely to be important for numerous developmental events. NHR-91 loss of function was visualized by RNAi on mutant larvae eri-1(mg366) that are susceptible to RNAi in the nervous system. The affected worms exhibited a slow-growing phenotype with defects in vulvae development and molting [210]. NHR-91 was found in the NHR-23 regulatory cascade under NHR-23 [143], where it is likely to restrict regulatory pulses of NHR-23. This is similar to the insect Ecdysone cascade, where DHR4 represses E75 and EcR in a negative feedback regulation. NHR-91 has been found to dimerize with NHR-25 in yeast twohybrid screens indicating its possible additional regulatory potential in the NHR-25 regulatory cascade [205]. This suggests that NHR-91 may be a negative regulator of gene expression at critical regulatory points similarly as in insects and in vertebrates. An interesting and important fact resulted from genome wide screens for targets of regulatory RNAs. nhr-91 was identified as a target of miR-235. NHR-91 transcript was increased in starved L1 mutated for miR-235 compared to wild type larvae. This coincides with a decrease of miR-235 expression. The expression of a reporter transgene composed of Pnhr-91::gfp-pest::nhr-91 3′UTR is increased in the hypodermis of fed L1 mutants and its expression is further elevated in larvae that are deficient in miR-235 expression. Loss of nhr-91 significantly suppressed the defects caused by miR-235 mutants in L1 diapause [211]. 2.7. Non-conserved NR ODR-7 in the regulation of sensory identity of olfactory neurons (NR0A4) The family of NRs in C. elegans includes a member that is strikingly divergent in sequence, ODR-7. It lacks LBD and its DBD is displaced to the C-terminus. The expression of odr-7::GFP was detected in two AWA olfactory neurons and was detected in all larval stages and in adults. Its expression was localized to the nuclei. By RT-PCR the expression was detected not only in larval stages and also in embryos [212]. The odr-7 expression is activated by lin-11 (LIM homeobox gene), which regulates AWA olfactory neuron differentiation [213]. ODR-7 has orthologues in C. briggsae, C. remanei and C. japonica but not in C. brenneri and other nematodes. odr-7 null mutants do not express AWA-specific signaling genes: odr-10 diacetyl receptor and osm-9 TRPV (transient receptor potential vanilloid)-like channel genes and fail to respond to odorants diacetyl and pyrazin, which are sensed by the AWA neurons [214]. The mutants ectopically express the str-2 olfactory receptor in AWA neurons instead of the AWG olfactory neurons [215,216]. The detail study of specific residues and domains of odr-7 demonstrates that this receptor utilizes multiple mechanisms for the regulation of gene expression and that these domains are conserved in the C. briggsae ODR-7 orthologue

Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016

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[217]. ODR-7 illustrates species specific regulatory circuits important for neuronal development. It has also been shown that mutation of odr-7 significantly increases the lifespan of nematodes in a temperature-independent way. The prorogated lifespan in odr-7 mutant was suppressed by daf-16 mutation and enhanced by daf-1 or age-1 mutations. These findings document that ODR-7 regulates longevity in an insulin/IGF signaling dependent way [218].

EcR/USP in most nematode species is coincidental and is accompanied with the burst of successive duplication of NR2A members in Rhabditidae. Some of the multiplied and diversified nematode NRs are likely to acquire the heterodimerization potential (as documented on NHR-49). It seems likely that many multiplied nematode NRs will have specific functions evolved by nematodes as part of their evolutionary strategy. This ongoing process of natural experimentation with the potential of NRs in the regulation of development of nematode species is an impressive example of evolution in action.

3. Concluding remarks Acknowledgements Nematode NRs, although excessively multiplied in comparison to other phyla, represent prime targets of biological research. Conserved NRs as well as members of the multiplied NR2 subfamily are involved in regulatory processes that reflect fundamental mechanisms of regulation by NRs. The multiplied network of nematode NRs is likely to provide a very complex view on the interplay between metabolic pathways and regulation of animal development. It is likely, that there is a particular mechanistic pressure that results in the multiplication of one of the most ancient members of the NR family, HNF4. Multiplication of members within other NRs families is observed in several phyla but in a substantially smaller extent. In the majority of documented cases, such multiplications are linked to well understandable metabolic or developmental purposes. 3.1. Nematode nuclear receptors support the existence of the Ecdysozoa clade Molecular data support the division of protostomes into two major clades, Ecdysozoa and Lophotrochozoa [219]. The most prominent feature of the ecdysis is the orchestrated chain of behavioral, structural and mechanistic events that is in insects and other arthropods regulated by steroidal hormones ecdysteroids through their binding to the dimeric receptor EcR/USP. Molting in nematodes resembles molting in arthropods but the initiation players of arthropod molting, the ecdysteroid and its receptor, EcR, were not identified in most nematode species. The presence of EcR and RXR/USP homologues was however confirmed in several evolutionarily basal nematode species (discussed above). Sterols were shown to be indispensable for normal molting, suggesting that a steroid hormone may be an important regulator of molting in nematodes [220]. However, sterols are also structural constituents of the hedgehog related pathway which is implicated in molting regulation [143,221,222]. The function of hedgehog related proteins in sterol metabolism and sterol based developmental signaling was not yet proven in nematodes. The molting cascade in nematodes includes homologues of other NRs that participate in the regulation of molting in arthropodes, especially NHR-23 (homologue of HR3) and NHR-25 (homologue of FTZ-F1) (Fig. 3). NHR-23 was found to be indispensable for all four molts in C. elegans [140]. It has been proposed that a regulatory loop consisting of NHR-23 on one side of the loop and NHR-25 and LIN-42A on the other side of the loop may constitute the molting timer in rhythmic molting cycles together with endocrine and behavioral regulators [196,197,223]. In Drosophila, the timing boundaries of ecdysone pulses are regulated by NR6A group member DHR4 [208,209]. This pathway interacts with the mechanisms which sense the size and metabolic state of the organism (reviewed in [224,225]). A similar pathway is likely to function in nematodes. NRs that are involved in the regulation of molting in both nematodes and insects seem to be positioned differently in regulatory cascades or may be absent in some species as is exemplified by EcR/USP in nematodes. Nevertheless, the regulatory pathway as a whole is determining the function and the evolution of species. Nematode NRs involved in the regulation of molting thus strongly support the classification of nematodes in the Ecdysosoa clade in agreement with other phylogenomic data [226]. Interestingly, EcR and USP show signs of strong co-evolution and favorization of heterodimerization in insect species [226]. The loss of

MK and ZK are supported by grants PRVOUK-P27/LF1/1 from Charles University in Prague and by the project “BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec” (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund. The funders had no role in study design, decision to publish or preparation of the manuscript. Authors are grateful to Marketa Kostrouchova, Veronika Kostrouchova, Ahmed Chughtai and Jan Philipp Novotny for valuable help during manuscript preparation, critical reading of the manuscript and advice and Veronika Kostrouchova for language editing. The support from WormBase is gratefully acknowledged. Authors thank two anonymous reviewers and Dr. Vincent Laudet for very helpful comments, suggestions and advice. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagrm.2014.06.016. References [1] F. Rastinejad, T. Perlmann, R.M. Evans, P.B. Sigler, Nature 375 (1995) 203–211. [2] K. Nakata, Comput. Appl. Biosci. 11 (1995) 125–131. [3] Z. Kostrouch, M. Kostrouchova, J.E. Rall, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 156–159. [4] M. Robinson-Rechavi, H. Escriva Garcia, V. Laudet, J. Cell Sci. 116 (2003) 585–586. [5] W. Wu, E.G. Niles, N. El-Sayed, M. Berriman, P.T. LoVerde, Gene 366 (2006) 303–315. [6] W. Wu, E.G. Niles, H. Hirai, P.T. LoVerde, BMC Evol. Biol. 7 (2007) 27. [7] A. Jackson, P. Panayiotidis, L. Foroni, Genomics 50 (1998) 34–43. [8] A.M. Reitzel, A.M. Tarrant, BMC Evol. Biol. 9 (2009) 230. [9] J.M. Wurtz, W. Bourguet, J.P. Renaud, V. Vivat, P. Chambon, D. Moras, H. Gronemeyer, Nat. Struct. Biol. 3 (1996) 87–94. [10] P. Germain, B. Staels, C. Dacquet, M. Spedding, V. Laudet, Pharmacol. Rev. 58 (2006) 685–704. [11] M. Srivastava, E. Begovic, J. Chapman, N.H. Putnam, U. Hellsten, T. Kawashima, A. Kuo, T. Mitros, A. Salamov, M.L. Carpenter, A.Y. Signorovitch, M.A. Moreno, K. Kamm, J. Grimwood, J. Schmutz, H. Shapiro, I.V. Grigoriev, L.W. Buss, B. Schierwater, S.L. Dellaporta, D.S. Rokhsar, Nature 454 (2008) 955–960. [12] J.T. Bridgham, G.N. Eick, C. Larroux, K. Deshpande, M.J. Harms, M.E. Gauthier, E.A. Ortlund, B.M. Degnan, J.W. Thornton, PLoS Biol. 8 (2010). [13] K. King-Jones, C.S. Thummel, Nat. Rev. Genet. 6 (2005) 311–323. [14] V. Laudet, J. Mol. Endocrinol. 19 (1997) 207–226. [15] S. Bertrand, F.G. Brunet, H. Escriva, G. Parmentier, V. Laudet, M. Robinson-Rechavi, Mol. Biol. Evol. 21 (2004) 1923–1937. [16] H. Escriva, L. Manzon, J. Youson, V. Laudet, Mol. Biol. Evol. 19 (2002) 1440–1450. [17] M. Van Gilst, C.R. Gissendanner, A.E. Sluder, Crit. Rev. Eukaryot. Gene Expr. 12 (2002) 65–88. [18] M. Robinson-Rechavi, C.V. Maina, C.R. Gissendanner, V. Laudet, A. Sluder, J. Mol. Evol. 60 (2005) 577–586. [19] W. Haerty, C. Artieri, N. Khezri, R.S. Singh, B.P. Gupta, BMC Genomics 9 (2008) 399. [20] K. Yook, T.W. Harris, T. Bieri, A. Cabunoc, J. Chan, W.J. Chen, P. Davis, N. de la Cruz, A. Duong, R. Fang, U. Ganesan, C. Grove, K. Howe, S. Kadam, R. Kishore, R. Lee, Y. Li, H.M. Muller, C. Nakamura, B. Nash, P. Ozersky, M. Paulini, D. Raciti, A. Rangarajan, G. Schindelman, X. Shi, E.M. Schwarz, M. Ann Tuli, K. Van Auken, D. Wang, X. Wang, G. Williams, J. Hodgkin, M. Berriman, R. Durbin, P. Kersey, J. Spieth, L. Stein, P.W. Sternberg, Nucleic Acids Res. 40 (2012) D735–D741. [21] A.E. Sluder, S.W. Mathews, D. Hough, V.P. Yin, C.V. Maina, Genome Res. 9 (1999) 103–120. [22] A. Antebi, WormBook (2006) 1–13. [23] D.D. Shaye, I. Greenwald, PLoS One 6 (2011) e20085. [24] J.M. Maglich, A. Sluder, X. Guan, Y. Shi, D.D. McKee, K. Carrick, K. Kamdar, T.M. Willson, J.T. Moore, Genome Biol. 2 (2001) (RESEARCH0029). [25] G.V. Markov, M. Paris, S. Bertrand, V. Laudet, Mol. Cell. Endocrinol. 293 (2008) 5–16.

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Please cite this article as: M. Kostrouchova, Z. Kostrouch, Nuclear receptors in nematode development: Natural experiments made by a phylum, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.06.016