Analysis of C. elegans NR2E nuclear receptors defines three conserved clades and ligand-independent functions
© Weber et al.; licensee BioMed Central Ltd. 2012
Received: 6 December 2011
Accepted: 31 May 2012
Published: 12 June 2012
The nuclear receptors (NRs) are an important class of transcription factors that are conserved across animal phyla. Canonical NRs consist of a DNA-binding domain (DBD) and ligand-binding domain (LBD). While most animals have 20–40 NRs, nematodes of the genus Caenorhabditis have experienced a spectacular proliferation and divergence of NR genes. The LBDs of evolutionarily-conserved Caenorhabditis NRs have diverged sharply from their Drosophila and vertebrate orthologs, while the DBDs have been strongly conserved. The NR2E family of NRs play critical roles in development, especially in the nervous system. In this study, we explore the phylogenetics and function of the NR2E family of Caenorhabditis elegans, using an in vivo assay to test LBD function.
Phylogenetic analysis reveals that the NR2E family of NRs consists of three broadly-conserved clades of orthologous NRs. In C. elegans, these clades are defined by nhr-67, fax-1 and nhr-239. The vertebrate orthologs of nhr-67 and fax-1 are Tlx and PNR, respectively. While the nhr-239 clade includes orthologs in insects (Hr83), an echinoderm, and a hemichordate, the gene appears to have been lost from vertebrate lineages. The C. elegans and C. briggsae nhr-239 genes have an apparently-truncated and highly-diverged LBD region. An additional C. elegans NR2E gene, nhr-111, appears to be a recently-evolved paralog of fax-1; it is present in C. elegans, but not C. briggsae or other animals with completely-sequenced genomes. Analysis of the relatively unstudied nhr-111 and nhr-239 genes demonstrates that they are both expressed—nhr-111 very broadly and nhr-239 in a small subset of neurons. Analysis of the FAX-1 LBD in an in vivo assay revealed that it is not required for at least some developmental functions.
Our analysis supports three conserved clades of NR2E receptors, only two of which are represented in vertebrates, indicating three ancestral NR2E genes in the urbilateria. The lack of a requirement for a FAX-1 LBD suggests that the relatively high level of sequence divergence for Caenorhabditis LBDs reflects relaxed selection on the primary sequence as opposed to divergent positive selection. This observation is consistent with a model in which divergence of some Caenorhabditis LBDs is allowed, at least in part, by the absence of a ligand requirement.
The nuclear receptors (NRs) constitute a class of transcriptional regulators that are conserved throughout the animal kingdom, where they function in a wide variety of physiological and developmental roles, including metabolic regulation, xenobiotic defense, and development [1–4]. Archetypal NRs have a DNA-binding domain (DBD), which contains two C4 zinc fingers and mediates binding to specific DNA sequences and receptor dimerization, and a more C-terminal ligand-binding domain (LBD), which may bind a lipophilic ligand and functions in dimerization, nuclear localization, and transcriptional trans-activation. Across the animal kingdom, amino acid sequence similarity is strongly conserved in the DBD, and more weakly conserved in the LBD. The majority of NRs that have been identified on the basis of phylogenetic sequence relationship have no known ligand, despite having a recognizable LBD and are sometimes referred to as “orphan receptors” .
The NR superfamily has proliferated and diverged to a striking degree in the nematode Caenorhabditis elegans: while the C. elegans genome boasts 284 predicted NRs, the human genome has only 48 and the Drosophila melanogaster genome only 21 [5–8]. Only 15–20 C. elegans NRs are clearly orthologous to NRs that are broadly conserved among animal phyla . The remaining (approximately 265) C. elegans predicted NRs appear to have evolved from an HNF4 ancestor and do not have clear orthologs in non-nematodes or distantly-related nematodes, suggesting that these genes have proliferated within the nematode phylum [5, 9, 10]. Nematode species that are closely related to C. elegans display a similar expansion of the NR gene family. Caenorhabditis briggsae and Caenorhabditis remanei, two species that are separated from C. elegans by about 100 million years and are morphologically indistinguishable from C. elegans, contain 232 and 256 NR genes, respectively . Furthermore, only about half of C. elegans NRs are conserved in both C. briggsae and C. remanei as three-way orthologs, while an additional 10% are present as two-way orthologs . Thus, the rapid expansion of the NR family appears to have begun before the separation of C. elegans C. briggsae and C. remanei, and continued separately in all three species. The more distantly related nematode Pristionchus pacificus also has an expanded complement of 167 nuclear receptors [13, 14], (R. Sommer, personal communication). The driving evolutionary explanation for this proliferation is unclear, but it could reflect adaptation to the complex chemical and xenobiotic environment of soil life [5–8]. In contrast, this expansion is not present in the genome of the nematode Brugia malayi, a human parasite: it contains only 27 NR genes, most of which are conserved across animal phyla . Therefore, evolution of NRs is unexpectedly dynamic among free-living nematodes, suggesting that proliferation and divergence in the NR family may play a major role in nematode speciation. This possibility elevates the importance of understanding NR evolution and function in nematodes.
One explanation for the wide variety of Caenorhabditis NRs and the radiating divergence of LBD sequences is a lack of hormone-responsiveness. NRs that are known to bind ligands in other invertebrates, such as the ecdysone receptor, are absent in Caenorhabditis. However, a bona fide ligand has been identified for the C. elegans NR DAF-12 , and most C. elegans NRs have a recognizable LBD, raising the possibility that other C. elegans NRs may also bind as yet unknown ligands. Even if some Caenorhabditis NRs are not hormone-responsive, the LBD may be retained for its other functions, such as transcriptional modulation, receptor dimerization, and/or nuclear localization. In any of these cases, the wide variety of Caenorhabditis LBD sequences could reflect a wider diversity of inputs or outputs, or a relaxation of selection on LBD primary sequences.
The nuclear receptors have been grouped into phylogenetically conserved families on the basis of amino acid sequence relationship [7, 17]. Members of the NR2E group include tailless (tll) and unfulfilled (unf) in Drosophila Tlx and PNR in vertebrates, and nhr-67 and fax-1 in C. elegans. These NRs have been systematically defined as NR2E1 through NR2E5. Another family member, designated NR2E6, has been identified in insects . The NR2E nuclear receptors that have been functionally characterized have a common theme of function in nervous system development. While mutations in the tll gene of Drosophila were first identified based on their disruption of anterior-posterior patterning, subsequent analysis demonstrated functions in embryonic CNS and larval eye development [19, 20]. The mouse Tlx gene functions in limbic system and eye development, but is not required for overall patterning of the embryo [21–23]. C. elegans fax-1, its vertebrate homolog PNR, and Drosophila homolog unf all play key roles in regulating neuron development [24–29]. The NR2E class is of particular importance from an evolutionary perspective as well; it includes members from the cnidaria to vertebrates, indicating that it is of ancient evolutionary ancestry . DNA-binding studies suggest that DBD properties of FAX-1 and NHR-67 are at least partially evolutionarily-conserved . However, like other C. elegans LBDs, the NR2E class LBDs are more highly-diverged from each other and from orthologous LBDs.
We have studied the conserved NR2E class of NRs in C. elegans to examine how the low level of LBD sequence conservation affects function using an in vivo functional assay. Our analysis of C. elegans NR2E-related sequences identified one unstudied “satellite” nuclear receptor, nhr-111, which is related to fax-1, but is not present in any other genome sequenced to date, and another NR, nhr-239, which is a member of new, relatively uncharacterized clade of conserved NRs found in non-vertebrate animals that includes insect Hr83. Using an in vivo functional assay, we tested whether LBD function was required for fax-1 and if LBD functions of other NR2E genes could be substituted for the fax-1 LBD function.
NR2E family receptors can be grouped into three major conserved clades
Systematic classifications of NRs have grouped vertebrate Tlx-like genes into an NR2E1 subgroup, insect TLL-like genes into an NR2E2 subgroup, vertebrate PNR-like genes into an NR2E3 subgroup, and C. elegans FAX-1 as an NR2E5 subgroup [17, 31, 32]. Additional genes were designated NR2E4 and NR2E6, but these appear to be absent from both nematodes and vertebrates and are not considered further in this analysis. NR2E1 and NR2E2 subfamily members have very similar DBDs and DNA-binding activities, as do NR2E3 and NR2E5 subfamily members [30, 33, 34]. Ecdysozoans have an NR2E2, but not an NR2E1, and vice versa for vertebrates. The simplest explanation for this relationship is the existence of a single common ancestral gene for NR2E1/NR2E2. A similar scenario is likely for the NR2E3/NR2E5 group.
Percent identities between NRs by domain
CeFAX-1 compared to:
DmUNF compared to:
CeNHR-67 compared to:
DmTLL compared to:
CeNHR-239 compared to:
Annotation of NR2E orthologs by clade
Nematostella NvR5 (2)
Apis AmTll (3)
Bombyx Tll (4)
Tribolium Tll (5)
Brugia BmNHR-15 (6)
Schmidtea Tlx (7)
Strongylocentrotus Tll (8)
Saccoglossus NP 001158362;Tll (9)
Nematostella NvR6-NvR9 (2)
Apis AmHr51 (3)
Bombyx BmHr51 (4)
Tribolium Hr51 (5)
Brugia BmNHR-16 (6)
Strongylocentrotus Pnr (8)
Saccoglossus NP 001158447; PNR (9)
Nematostella NvR6-NvR9 (2)
Apis AmHr83 (3)
Bombyx ortholog not identified (4)
Tribolium Hr83 (5)
Brugia BmNHR-C* (6)
Strongylocentrotus “Fax1” (8)
Saccoglossus XM 002740611; “NR6A1-like” (9)
As expected, the Caenorhabditis NR2E LBDs aligned more poorly than the DBDs with insect and vertebrate orthologs (Table 1; Additional file 2: Figure S2). Ka/Ks ratios for LBD regions (Figure 1) at branches that lead to nematode NRs were high (0.72 to 0.84), in comparison to branches that lead to insect or vertebrate NRs (0.17 to 0.31). In contrast, Ka/Ks ratios for DBD regions (Additional file 1: Figure S1) were relatively low at nematode and non-nematode branches (0.04 to 0.21 for the more strongly-conserved PNR/FAX-1 and TLX/TLL clades). These data suggest strong purifying selection on the DBD across phyletic groups and very weak purifying selection on nematode LBDs. Furthermore, some key positions in the LBD signature domain and other regions that are conserved in insects and vertebrates have diverged in Caenorhabditis (Additional file 2: Figure S2). The Brugia malayi orthologs of FAX-1 and NHR-67 seem to have followed different evolutionary histories. While the LBD of Brugia FAX-1 looks similar to Drosophila UNF (45.9% identical) and human PNR (50.8%), the LBD of B. malayi NHR-67 looks more similar to C. elegans NHR-67 (27.3% identical) than to D. melanogaster (21.4%; Table 1). Therefore, the B. malayi FAX-1 LBD appears to have followed an insect-like path, while the B. malayi NHR-67 LBD appears to have followed a free-living nematode-like path. The NHR-239/Hr83 clade LBDs are considerably more diverged from each other and the LBDs of other clades (Table 1; Additional file 2: Figure S2). This was particularly true for C. elegans and C. briggsae NHR-239, which align very poorly in the LBD signature region with other NR2E LBDs and are apparently truncated (Additional file 2: Figure S2). This observation suggests that Caenorhabditis NHR-239 may lack a bona fide LBD, like C. elegans ODR-7 and Drosophila KNIRPS [48, 49].
Analysis of NR2E nuclear receptors of the nematode Pristionchus pacificus suggested extensive sequence divergence of LBDs in this species. While the predicted P. pacificus FAX-1 ortholog aligned well with PNR/FAX-1 clade DBDs (Figure. 2; Table 1), the predicted LBD aligned very poorly (Table 1; Additional file 2: Figure S2). In particular, key features of the LBD signature domain that were present in other nematode LBDs were not found in the P. pacificus FAX-1 LBD. Analysis of P. pacificus genomic sequence also identified candidate NHR-67 and NHR-239 orthologs by conservation of DBD sequences (Figure 2; Table 1), but failed to identify coding sequences or gene assembly models with similarity to TLX/TLL and NHR-239/Hr83 clade LBDs (data not shown). The P. pacificus genomic sequence is mostly complete , however gene assembly and confirmation by cDNA analysis is not yet comprehensive. Nonetheless, the absence of LBD sequences with a clear relationship to Caenorhabditis or Brugia NR2E LBDs suggests that LBD divergence of P. pacificus NR2E LBDs may be much greater than that observed in Caenorhabditis. This conclusion contrasts with the relatively strong conservation of LBD sequences reported for P. pacificus RXR (NR2B) and ecdysone (NR1H) receptors .
The C. elegans genome project also identified another potential NR2E gene family member, nhr-111. No clear ortholog of nhr-111 has been identified in any other species, including the fully-sequenced and closely-related C. briggsae and C. brenneri nematodes. The NHR-111 sequence is included in the PNR/FAX-1 clade when full-length proteins are considered (Figure. 1), but is not included when the DBD alone is considered (Additional file 1: Figure S1). Alignment of the NHR-111 DBD revealed divergence of the NHR-111 DBD from other NR2E family members at multiple positions (Table 1; Figure 2). However, the NHR-111 DBD was still more similar to NR2E DBDs than to the DBDs of other nematode NRs (data not shown). Inclusion of NHR-111 in the full-length NR2E clade occurred due to extensive similarity between the NHR-111 LBD and FAX-1 LBD (25% identical in Table 1; Additional file 2: Figure S2). From these observations, we propose that the nhr-111 gene arose from a relatively recent duplication of the fax-1 gene in the C. elegans evolutionary lineage, followed by extensive divergence of the DBD.
The novel nhr-111 and nhr-239 NRs are expressed genes
In order to examine cell-specific expression of both genes in living nematodes, we constructed nhr-111::gfp and nhr-239::gfp transgenes (Figure 3A). The nhr-111::gfp reporter was consistently expressed in at least eight pairs of neurons in the head, the sensory PVD neurons of the posterior lateral body wall, the pharynx, intestine (most often in the posterior- and anterior- most cells), the dorsal peri-vulva region of adults (which may be either uterine or vulval cells), and the somatic gonad precursor cells (Figure 3C). Among the head neurons was one prominent pair of sensory neurons just posterior to the nerve ring and at least one pair of neurons or support cells that appear to be inner or outer labial sensory cells. We also observed weak and variable expression in a subset of ventral nerve cord motorneurons. The temporal dynamics of nhr-111::gfp were consistent with nhr-111 qRT-PCR results: expression was very bright in the Z1 and Z4 somatic gonad precursor cells in embryos and early L1, but decreased in the developing gonad at later stages and was relatively faint in other cells. Therefore, despite its relatively recent evolutionary origin, the nhr-111 gene is fairly broadly expressed.
The nhr-239::gfp reporter was weakly expressed in three to four pairs of neurons in the head and a pharyngeal neuron in late stage embryos and all larval and adult stages (Figure 3D). One pair of dorsal neurons express nhr-239::gfp very consistently and appear to be sensory, as do one pair of pharyngeal cells that appear to be the MC, NSM or M3 neurons. We observed faint fluorescence in the pharynx (which may be an artefact), but did not observe nhr-239 expression in other cells at any stage. As expected from the modest expression levels observed in qRT-PCR experiments, fluorescence from the nhr-239::gfp transgene was very faint. While it is possible that our translational fusion did not recapitulate the entire nhr-239 expression pattern, this result suggests that nhr-239 is expressed only in a very limited subset of neurons. Taken together, these data demonstrate that both genes are expressed, and that both may play roles in neuron development like other NR2E family members.
FAX-1, NHR-67, and NHR-111 have predicted LBD structures that are similar to defined vertebrate LBDs
Structural predictions for Caenorhabditis LBDs by sequence threading
Best structural match
H. sapiens NR2C2 TR4 LBD
1 x 10-6
H. sapiens NR2C2 TR4 LBD
9 x 10-7
H. sapiens NR2F2 COUP LBD
H. sapiens NR2F2 COUP LBD
H. sapiens NR2F2 COUP LBD
1 x 10-5
No significant matches.
No significant matches.
The C. elegans FAX-1 LBD is not required for some functions in vivo
The higher substitution rate of Caenorhabditis LBDs raised the possibility that nematode LBDs have broadly diverse functions—that diversifying selection has led to the evolution of highly-specific LBDs. In order to test this possibility, we used well-characterized fax-1 mutants and rescuing plasmids to serve as a basis for testing the functional requirements of the FAX-1 LBD [24, 26]. A null mutation in the fax-1 gene causes a distinctive movement defect. C. elegans normally moves with a smooth sine wave, but fax-1 mutants are unable to generate coordinated muscle contractions in their posterior half, leading to severely compromised forward movement. Backing movement is more rapid, but inevitably leads to “coiling”—animals back into a circle on themselves instead of progressing straight backward. These defects may be due to underlying defects in the differentiation of the command interneurons that coordinate forward and backward motility [26, 54]. fax-1 mutations affect multiple interneuron types. In the AVK interneuron pair, fax-1 mutations cause a high-penetrance defect in axon pathfinding: instead of the AVKR and AVKL axons extending on the respective left and right sides of the ventral nerve cord, the axons are misrouted to the dorsal nerve cord, a lateral nerve bundle, or extend as a pair along the right ventral nerve cord . The anatomy of the AVK neurons can be easily evaluated using anti-FMRFamide antisera [55, 56]. fax-1 function in AVK development is presumably a separate function from the fax-1 function in movement, since the AVK neurons are not known to be required for movement coordination.
In order to test the ability of the fusion and deletion constructs to provide in vivo fax-1 function, we injected each plasmid into fax-1(gm83) lin-15(n765ts) C. elegans hermaphrodites, along with a co-transforming plasmid that provides wild-type lin-15 function. For each transgenic line, we evaluated fax-1 function by measuring forward movement rates (Figure 4B). While wild-type worms had a forward movement rate of 19.4 (±5.6, N = 10) μm/sec, fax-1(gm83) mutants, fax-1(gm83) mutants transformed with the lin-15- rescuing plasmid alone, and fax-1(gm83) mutants transformed with the FAX-1::INV FAX-1 negative control had forward movement rates of 2.40 (s.d. 1.6, N = 10), 2.34 (s.d. 1.5, N = 10), and 1.23 (s.d. 1.3, N = 10) μm/sec, respectively (differences among speeds for negative controls were not statistically significant). The FAX-1::FAX-1 positive control provided a significantly rescued (p < 0.00001) forward movement of 9.6 (s.d. 4.2, N = 14) μm/sec. When the fax-1 DBD region was fused to another Caenorhabditis LBD, we obtained significant rescue (p < 0.00001) of the fax-1 movement phenotype: forward movement rates ranged from 9.33 (s.d. 3.4, N = 10) μm/sec (for FAX-1::NHR-111) to 13.7 (s.d. 4.0, N = 10) μm/sec (for FAX-1::NHR-67). Likewise, the FAX-1::Δ LBD construct, which contains no LBD at all, was able to provide a robust forward movement rate of 14.7 (s.d. 6.2, N = 17) μm/sec. Differences between C. elegans FAX-1, C. briggsae FAX-1, and NHR-111 fusion pairs were not statistically significant (p > 0.5), but the increased rescue with the NHR-67 LBD or Δ LBD constructs as compared to other LBDs was significant (p = 0.008 to 0.05). The reason for the improved rescue by the NHR-67 LBD and Δ LBD constructs is not clear. It may reflect a larger number of copies in the transgene, array-specific variation in transcription levels, or structural differences at the level of protein fusion. Therefore, despite a conserved LBD structure and ligand-binding signature region, the FAX-1 LBD is not required for fax-1 function in the development of neurons that control movement.
Rescue of AVKR pathfinding defects
% AVKR DEFECT
fax-1 9 kb genomic DNA
fax-1 (gm83) lin-15 (n765)
fax-1 (gm83) lin-15 (n765)
fax-1 (gm83) lin-15 (n765)
fax-1 (gm83) lin-15 (n765)
fax-1 (gm83) lin-15 (n765)
fax-1 (gm83) lin-15 (n765)
fax-1 (gm83) lin-15 (n765)
We confirmed expression and subcellular localization of our fusion and deletion products by immunofluorescence staining. A mouse anti-FAX-1 antiserum raised to full-length FAX-1 protein detected a strong signal in fax-1(gm83) lin-15(n765) embryos that carried a FAX-1 DBD-contained transgene (Figure 4D). Because fax-1(gm83) mutants produce no detectable FAX-1 protein , the protein detected by the antiserum must reflect expression of the FAX-1 DBD that is common to all fusion and deletion construct transgenes. We detected strong expression by all four fusion transgenes (FAX-1::FAX-1, FAX-1::CbFAX-1, FAX-1::NHR-111, and FAX-1::NHR-67) and the FAX-1::Δ LBD transgene in a spatial and temporal pattern that was nearly identical to the FAX-1 expression pattern in wild-type embryos , indicating that our constructs produced stable protein at the correct time and place (Figure 4D; data not shown). The FAX-1::Δ LBD, FAX-1::FAX-1, FAX-1::CbFAX-1 and FAX-1::NHR-67 constructs also showed excellent nuclear localization, suggesting that the LBD is not required for import of FAX-1 protein to the nucleus. The FAX-1::NHR-111 construct was expressed at very high levels, but many embryos showed significant accumulation of protein in the cytoplasm (Figure 4D). It is unclear whether this reflects an artefact of high expression levels or a “dominant” effect of the NHR-111 LBD that inhibits nuclear localization of the FAX-1::NHR-111 fusion protein. Despite the nuclear localization issue, the FAX-1::NHR-111 construct still provided functional rescue, although it is possible that the somewhat weaker rescue by this construct may be accounted for by the compromised nuclear localization.
Analysis of LBD function in the C. elegans NR2E subfamily demonstrates the functional independence of some fax-1 functions on the presence or sequence of the LBD. The simplest interpretation of this result is that the key FAX-1 functions that we have assayed require only binding of the DBD moiety to its cognate recognition site. This is consistent with our previous finding that the FAX-1 DBD alone possesses both sequence specificity and homodimerization function . Furthermore, the DBD also appears to possess sufficient transcription regulation activity to confer normal function. In addition, the DBD expressed in vivo without an LBD appears to be efficiently localized to the nucleus, indicating that the LBD is not required for efficient localization. Finally, the absence of a requirement for the FAX-1 LBD demonstrates that it is not ligand-dependent—at least for the functions we have tested. The LBD of the Drosophila ortholog of fax-1, UNF, has been shown to bind heme , although there is no known developmental or physiological requirement for heme-binding. If the FAX-1 LBD can also bind heme-related ligands, our results suggest that this binding is not essential for key FAX-1 functions in C. elegans.
The independence of fax-1 functions from the presence or identity of an LBD suggests that the relatively high level of primary sequence divergence of Caenorhabditis LBDs may not reflect diversifying selective pressure toward highly specific functional roles. Instead, the relatively high sequence divergence may result from a release of positive selection on the primary sequence, with sequence differences reflecting drift. Nonetheless, protein sequence threading suggests that some general aspects of secondary and tertiary LBD structure may be maintained in Caenorhabditis. Changes in selective pressure on nematode LBDs may have allowed a greater degree of tolerance for substitution within the context of conserved structure.
Analysis of C. elegans LBDs and the Caenorhabditis genomes has cast doubt on the hypothesis that C. elegans NRs mediate transcription using the same components as vertebrate systems. For example, the LBD of several vertebrate nuclear receptors have been shown to bind a common set of coactivators and corepressors, which in turn mediate the effect of the nuclear receptor on transcription . However, an AF2 domain, which is contained within the many vertebrate LBDs and responsible for interaction with p160 coactivators, is absent from most Caenorhabditis NRs, including the fax-1 and nhr-239 orthologs in this study, and most of the known vertebrate NR coactivator and corepressor genes were absent from a survey of predicted genes in C. elegans. Therefore, significant differences between vertebrates and Caenorhabditis in the mechanisms of transcriptional control may account for the apparent differences in LBD function in C. elegans.
The lack of a requirement for LBD function for fax-1rescue in our assay does not necessarily indicate that the FAX-1 LBD provides no function or that C. elegans NR2E proteins in general are ligand-independent. On the contrary, the structural conservation of the C. elegans LBDs (Table 3), weak purifying selection on LBD sequences (Figure 1), and the general absence of truncated LBDs among conserved NRs in the C. elegans genome (save perhaps nhr-239), combine to argue that LBDs have been retained because they provide function. It is possible that the FAX-1 LBD is required for subtle aspects of developmental control that are not revealed by our rescue assay. Alternatively, the developmental functions that we have tested could be entirely LBD-independent, but other functions are LBD-dependent. This might be the case if FAX-1 provides an unknown physiological, ecological, or behavioural function during larval and/or adult stages. A precedent for both ligand-dependent and ligand-independent functions exists for vertebrate steroid NRs . It is worth noting that all the C. elegans NR2E genes in this study are expressed in larval and adult cells (Figure 3, [26, 61], which certainly allows for later functions in addition to the major embryonic developmental functions.
Our preliminary analysis of expression of nhr-111 and nhr-239 reveals some commonalities among members of the NR2E family in Caenorhabditis. First, all NR2E family members are expressed in subsets of neurons. In the two best studied cases, fax-1 and nhr-67, they play important roles in neuron differentiation and specificity [26, 61], functions that are also maintained in flies and vertebrates [23, 27, 62]. While we have not yet determined functions for nhr-111 and nhr-239, the expression of both genes in subsets of neurons at a time in embryogenesis when neuronal specification is occurring suggests that they may also function in neuronal development. Furthermore, a genomic study focusing on neuron-specific transcriptional complexes identified 15 candidate gene targets for NHR-111, including the well-studied neuronal developmental control genes unc-30 and unc-86. In addition, both NHR-111 and NHR-67 were found to bind a common target promoter, suggesting that they may be co-ordinately involved in regulating overlapping neuron-specific genes. A second feature common to all Caenorhabditis NR2E genes, except for nhr-239, is that they are expressed in the somatic gonad: fax-1 in the distal tip cells during larval gonadogenesis , nhr-67 in the larval ventral uterine cells, anchor cell, and linker cell of the male [64, 65] and nhr-111 in the Z1 and Z4 gonad precursors and their descendants. Of these, only nhr-67 has a clearly defined function in uterine development. Nonetheless, the nexus of NR2E expression in somatic gonadal tissues raises the possibility for combinatoric functions within the NR family. At the very least nhr-111 expression overlaps with nhr-67 expression in the ventral uterine lineages and anchor cell, and with fax-1 expression in the distal tip cells. The identification of common targets for both NHR-67 and NHR-111 supports the notion that these transcription factors may have overlapping functions. Arda et al.  have identified metabolic gene regulatory networks that are highly-enriched with NRs, both as components of transcription factor networks that regulate genes involved in metabolism, homeostasis, and environmental response, and as targets of regulation by NRs and other transcription factors. Both NHR-67 and NHR-111 were components of modules implicated in coordinated regulation of metabolic genes, similar to what was found with neuron-specific target genes. NHR-111 was also implicated in six additional smaller regulatory complexes, consistent with the relatively broad expression pattern we describe here.
The C. elegans NR2E genes that have been studied most thoroughly play various roles in development. Deletion of nhr-67 results in early developmental arrest . Loss of fax-1 causes significant movement and nervous system defects, but does not cause lethality . Like nhr-67, nhr-111 is expressed fairly broadly, but its deletion does not cause lethality or obvious morphological phenotypes (KW, GMB, SC, and BW, unpublished observations). This observation raises the possibility that NHR-111 may partner with other transcription factors in a highly-redundant and overlapping manner to fine-tune gene regulation in many cells. The observation that NHR-111 is a major node in the C. elegans interactome map of predicted protein interactions, partnering with itself (suggesting possible homodimerization) and 53 other proteins , many of which are potential transcription factors, provides some support for this hypothesis.
Finally, our phylogenetic analysis using a larger set of NR2E sequences from many species argues for three significant evolutionarily-conserved clades: TLL/TLX, FAX-1/PNR, and NHR-239/Hr83. Our data call into question the hypothesis that FAX-1 and PNR represent different genes in the urbilateralian ancestor followed by subsequent loss of PNR in nematodes and loss of FAX-1 in vertebrates . Not only do our phylogenetic data support an orthologous evolutionary origin for FAX-1 and PNR, direct examination of DBD alignments identify consistent conserved loci within each clade, a situation that is less likely to exist due to convergent evolution. A more parsimonious interpretation is a single urbilateralian ancestor for both PNR and FAX-1. To date, we know of no genome that includes a PNR ortholog, a FAX-1 ortholog and an NHR-239/Hr83 ortholog. Instead most animal genomes boast a single PNR-like gene or a single FAX-1-like gene, with a single NHR-239/Hr83 clade member and a single TLL/TLX clade member. Our interpretations of the relationship of NR2E receptors call into question the utility of the systematic nomenclature system for NRs  when applied to divergent C. elegans receptors. In this case, a version of long-branch attraction or similar artefact has resulted in a confusing situation in which C. elegans FAX-1 and insect Hr83 both have been assigned NR2E5 even though FAX-1 is clearly more similar to insect NR2E3 and C. elegans NHR-239 is more similar to Hr83. This change in the evolutionary assumptions of the NR2E subfamily does not change predictions of the original number of NRs as described by Bertrand et al., , since the “new” NHR-239/Hr83 clade effectively replaces the “lost” FAX-1 clade that results from fusing FAX-1 and PNR into a single clade. In this case, a larger data set was important for drawing the clearest evolutionary inferences. Therefore, complex evolutionary patterns in large gene families may benefit substantially from large-scale sequencing projects that examine closely-related species, such as the current 959 Nematode Genomes project (http://www.nematodes.org).
We define three conserved clades of NR2E receptors, only two of which are represented in vertebrates. This observation suggests that there were three ancestral NR2E genes in the urbilateria. Additional genes have spawned from descendants of these three ancestral genes, including nhr-111, which is a broadly-expressed paralog that appears to have arisen within the Caenorhabditis evolutionary lineage. LBD function is not required for at least some important developmental functions of one NR2E family member. This result suggests that the relatively high level of sequence divergence for Caenorhabditis LBDs reflects relaxed selection on the primary sequence, rather than highly diversifying positive selection.
Phylogenetics and computational analysis
We aligned protein sequences using the alignment utility of MEGA 5.0 . Alignments were performed with the Clustal W algorithm  using a BLOSUM matrix, a pairwise gap penalty of 10, with extension penalty 0.1, and a multiple alignment gap penalty of 10, with extension penalty 0.2. We developed phylogenetic trees using the Maximum Likelihood method based on the Dayhoff matrix based model  and by Neighbor-Joining  using MEGA 5.0 software. All positions containing gaps were eliminated. The bootstrap consensus trees were inferred from 500 replicates. Trees were rooted manually to the Homo sapiens RAR gamma outgroup sequence. The trees were drawn to scale, with branch lengths measured in the number of substitutions per site. We evaluated LBD structures of NR2E proteins using PSIPRED and the threading utility pGenTHREADER [52, 53] at the Bloomsbury Centre for Bioinformatics at University College London . We calculated Ka/Ks ratios for DBD and LBD regions separately by generating matched Maximum Likelihood trees based on amino acid alignments and nucleotide alignments using Clustal W and MEGA 5.0 as described above. Not all sequences shown in Figure 1 and Additional file 1: Figure S1 were included in the Ka/Ks analysis. Newick-formated trees were generated with MEGA 5.0 and edited by hand to create binary files. Ka/Ks ratios were calculated using the Ka/Ks utility at the Bergen Center for Computational Science, University of Bergen (http://www.bccs.uni.no/units/cbu/).
Nematode strains and GFP reporter analysis
C. elegans were cultured as described by Brenner  and Stiernagle . Strains were obtained from the Caenorhabditis Genetics Center, the National Bioresource Project of Japan, the C. elegans Gene Knockout Consortium. nhr-111::gfp and nhr-239::gfp constructs were created as illustrated in Figure 4. The nhr-111::gfp plasmid, pG3.9GFP1, was constructed by amplifying a 2.7 kb region from genomic cosmid clone F44G3 using oligonucleotides [Additional file 3: Table S1] and ligating the product to the GFP expression vector pPD95.79 using BamHI and SphI. The resulting plasmid fused the entire 5’ flanking region of nhr-111, 1.2 kb from the predicted start codon to the predicted 3’ end of the immediately adjacent upstream gene, and the genomic nhr-111 coding region into exon 5 to the coding sequence for GFP. The nhr-239::gfp plasmid, pNHR239GFP1, was constructed by amplifying a 2.8 kb region from wild-type C. elegans genomic DNA using oligonucleotides [Additional file 3: Table S1] and ligating the product to pPD95.79 using XbaI and XmaI. The resulting plasmid fused 2.1 kb of 5’ flanking DNA, which includes the last intron and exon of the adjacent upstream feh-1 gene, and the first three predicted exons of the nhr-239 gene to the coding sequence for GFP. We introduced plasmid constructs into nematodes following standard microinjection techniques  using a Nikon UD Optiphot 2 microscope. Transgene-positive progeny were identified by the Roller phenotype conferred by the pRF4 co-transforming marker that bears a dominant mutant version of the rol-6 gene. We employed a Nikon Eclipse TE 2000 U inverted microscope to examine nematodes using DIC Nomarski microscopy and captured images using a Nikon DMX 1200 camera. To examine and record GFP fluorescence patterns, we used a Nikon UD Optiphot 2 microscope and captured images using a Nikon DS camera. We cropped images and adjusted for optimum contrast and brightness using Adobe Photoshop software.
Quantitative real-time qPCR
We used quantitative real-time PCR (qRT-PCR)  to estimate relative levels of nhr-111 and nhr-239 expression. Taqman probe/primer mixtures were purchased from Applied Biosystems/Life Technologies (Carlsbad, CA). For both nhr-111 and nhr-239, manufacturer-designed probes were used. In addition, we designed a confirmatory probe/primer mixture for nhr-239 using Applied Biosystems Primer Express 3.0 software. We prepared staged worm preparations for wild-type C. elegans nematodes as follows: embryos, treatment with 20% chlorine bleach and 0.1 M NaOH; L1, bleach treatment followed by overnight incubation in M9 buffer; L2/L3, as for L1 followed by 24 hour feeding on OP50 bacteria at 20°C; L4, as for L1 followed by 48 hour feeding on OP50 bacteria at 20°C. Samples were washed in M9 exhaustively and frozen in RNALater solution (Ambion, Austin, TX). Total RNA was prepared using a RiboPure Yeast RNA kit (Ambion) as directed by the manufacturer. We synthesized cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) as directed by the manufacturer. Real-time qRT-PCR was conducted on a StepOnePlus system (Applied Biosystems) using Fast Taqman Master Mix (Applied Biosystems). All samples were tested in triplicate or quadruplicate. We measured expression levels in arbitrary units calculated as 2-ΔCtx1010 relative to 18 S rRNA controls (18 S Eukaryotic Taqman Probe/Primer mix, Applied Biosystems).
LBD swap and deletion experiments
Plasmid pFXCDSp.8 was constructed by cloning an 809 bp SphI fragment from a pFXCD5 fax-1 cDNA clone into vector pUC18, creating a basic fax-1 cDNA backbone clone for the purpose of replacing the fax-1 LBD coding region with cDNA for LBD coding regions of other NRs. To produce FAX-1::FAX-1 and FAX-1::INV FAX-1 constructs, we ligated the 809 bp SphI fragment from pFXCDSp.8 into genomic clone pF56SH9  digested with SphI, thereby replacing the fax-1 genomic region with a fax-1 cDNA in the correct (FAX-1::FAX-1) or inverted (FAX-1::INV FAX-1) orientation. To produce FAX-1::ΔLBD, we digested the parent rescuing plasmid pF56SH9 with EcoNI and EagI (which deletes the entire LBD and a portion of the DBD), following by amplification of the 3’ coding portion of the fax-1 DBD from a fax-1 cDNA clone using EcoNI and EagI- tailed oligonucleotides (Additional file 3: Table S1) and reconstitution of the intact DBD by ligation of the amplification product into the deleted pF56SH9 plasmid. In addition to a deletion of the LBD, this construct also deleted 468 bp of 3’ UTR and flanking DNA. The resulting FAX-1::ΔLBD plasmid construct was sequenced across the amplified region to confirm wild-type sequence and deletion of the LBD coding region. The LBD swap constructs were created by amplifying the corresponding LBD coding regions from C. briggsae fax-1, nhr-67, and nhr-111, using corresponding cDNA clones as template, and ligating each to vector pGEM-T Easy using oligonucleotides tailed with BclI, BamHI and/or EcoRI recognition sites (Additional file 3: Table S1). Each LBD cDNA construct was sequenced on both strands to confirm wild-type sequence. We created cDNA fusions for each swap construct by excising the target LBD cDNA with either BclI and EcoRI or BamHI and EcoRI, and ligating the fragment to BamHI/EcoRI-digested pFXCDSp.8. The resulting family of clones created FAX-1::swap LBD cDNA fusion clones with flanking SphI sites. Each cDNA fusion clone was digested with SphI and ligated to the SphI-digested fax-1 genomic pF56SH9 clone in the proper orientation to create a family of genomic clones that contained replaced LBD cDNA regions. We used a lin-15 marker for transformation experiments since the more commonly-used Rol-6 marker would interfere with subsequent movement assays. Each plasmid construct (at 50 μg/ml) and the lin-15-rescuing marker plasmid pSK1 (at 50 μg/ml), was microinjected into fax-1(gm83) lin-15(n765ts) hermaphrodites that had been grown at 15°C. The lin-15 mutation results in a multi-vulva (Muv) phenotype at the non-permissive temperature of 25°C, but is near wild-type when grown at 15°C. Injected hermaphrodites were grown at 25°C to allow identification of transformed non-Muv animals in the next generation. Each transgene was maintained as one or more independent extrachromosomal arrays by picking wild-type animals at 25°C. For each construct we obtained at least two dozen transient F1 non-Muv transformants and established between two and twenty independent stable lines. Transgenic lines and control strains were evaluated for movement using a Nikon SMZ800 stereo dissection microscope outfitted with a Hitachi CCD camera KP-D20BU. Video was captured for 30 seconds per trial using Mitotic Images Plus software. Worm movement was measured by digital calibration of distance moved per second during bursts of forward movement. Some fax-1(gm83) and non-rescued transgenic strains made no forward progress during the trial period, which was scored as 0 μm of movement over the period of apparent attempted movement. We calculated forward movement speeds as μm/sec. The AVKR axon pathfinding phenotype of transgenic and control strains was evaluated using immunofluorescence and an anti-FMRFamide antibody as described previously [24, 56]. We evaluated expression of deleted and fusion proteins from each transgene using immunofluorescence and a mouse anti-FAX-1 polyclonal antibody, and a Cy3-labeled Goat anti-mouse IgG secondary antibody as described previously . The anti-FAX-1 antibody was raised to full-length FAX-1 protein and was able to detect transgene-expressed protein from each fusion construct and the deletion, indicating that the antiserum contains antibodies that recognize the DBD. Different transgenic lines containing the same construct differed somewhat in the rate at which the array transgene was transmitted, but did not differ substantially from each other in rescue or expression experiments. For each construct, the data reported here are from one representative transgenic strain.
Jennifer Baldwin, Dan Shpilksy, Jessica Tanis, and Matt Stein made early contributions to some of the data or constructs in this study. Marten Edwards, Amy Hark, Elizabeth McCain, and Jeremy Teissere of the Muhlenberg College Biology Department generously shared equipment, reagents and resources. Scott Clark provided pSK1 plasmid. We appreciate helpful discussions about this project with Ann Sluder, Chris Gissendanner, Piali Sengupta, and Marc Van Gilst. The anonymous reviewers provided critical and much-appreciated feedback. Wormbase, Wormatlas, and the Caenorhabditis Genetics Center provided critical resources for this work. Robert Barstead, Shohei Mitani, the National Bioresource Project, and the C. elegans Gene Knockout Consortium produced the knockout strain referred to in this study. KPW, KR, and GMB received funding from National Science Foundation Research Experiences for Undergraduates (REU) awards. This project was supported by Research at Undergraduate Institution (RUI) grants 0234716, 0640483, and 0948367 from the National Science Foundation to BW. The qRT-PCR experiments were performed using equipment purchased under Course, Curriculum and Laboratory Improvement grant 0836869 from the National Science Foundation to BW.
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