Tissue-specific regulatory elements in mammalian promoters

Transcription factor-binding sites and the cis-regulatory modules they compose are central determinants of gene expression. We previously showed that binding site motifs and modules in proximal promoters can be used to predict a significant portion of mammalian tissue-specific transcription. Here, we report on a systematic analysis of promoters controlling tissue-specific expression in heart, kidney, liver, pancreas, skeletal muscle, testis and CD4 T cells, for both human and mouse. We integrated multiple sources of expression data to compile sets of transcripts with strong evidence for tissue-specific regulation. The analysis of the promoters corresponding to these sets produced a catalog of predicted tissue-specific motifs and modules, and cis-regulatory elements. Predicted regulatory interactions are supported by statistical evidence, and provide a foundation for targeted experiments that will improve our understanding of tissue-specific regulatory networks. In a broader context, methods used to construct the catalog provide a model for the analysis of genomic regions that regulate differentially expressed genes.
Introduction
Reverse engineering mammalian transcriptional regulatory circuits can be achieved using systematic methodology that includes both computational and experimental techniques, working in tandem to generate, refine and verify hypotheses. Understanding tissue-specific transcription is a necessary step for extending regulatory circuit reverse-engineering efforts from single-cell eukaryotes to metazoans. We recently demonstrated that the information in proximal promoters can predict a significant portion of tissue-specific elevated or inhibited expression (Smith et al, 2006). Here, focusing on tissue-specific regulatory pattern identification and prediction accuracy instead of proof of existence, we use refined analysis and data curation methods to discover and catalog high-confidence regulatory interactions and sites. This catalog will assist experimental efforts to reverse engineer tissue-specific transcriptional regulatory networks from the bottom up.
Numerous techniques for analysis of regulatory sequences have been proposed, and the problem of module identification is now receiving due attention (Zhou and Wong, 2004; Gupta and Liu, 2005; Zhu et al, 2005). Previously characterized binding site motifs have been used to infer transcription factor function in certain tissues (Nelander et al, 2005). Xie et al (2005) identified conserved motifs across ortholog promoters of four mammalian genomes. Robertson et al (2006) describe cisRed, a database that integrates genome annotation data, homology data and genome alignments to identify motifs with conserved sites across mammals. We analyzed proximal promoters with evidence for tissue-specific regulation in order to identify tissue-specific motifs, modules and their sites in proximal promoters. We developed a new technique for characterizing tissue-specific modules that ensures that each module component significantly improves tissue-specific module enrichment.
We integrated multiple sources of expression data to identify reliable sets of transcripts that are under tissue-specific regulation in human and mouse. Using transcription start site (TSS) annotation in Cold Spring Harbor Mammalian Promoter Database (CSHLmpd) (Xuan et al, 2005), we compiled sets of proximal promoters corresponding to transcripts with evidence for specific regulation in the selected tissues. Our analysis was based on motifs discovered de novo (called novel motifs) using DME (Smith et al, 2005a) and DME-B (Smith et al, 2006), as well as previously characterized vertebrate binding-site motifs (called known motifs) from TRANSFAC (Matys et al, 2003) and JASPAR (Sandelin et al, 2004). We evaluated motifs according to enrichment in tissue-specific promoters relative to other promoters from the same species. We showed that motifs associated with factors with known tissue-specific roles rank high for enrichment, that motif ranks are significantly correlated between human and mouse and that this same set of motifs and their corresponding cis-elements are unlikely to be identified using traditional, order-preserving alignments of ortholog promoter sequences. We constructed modules of interacting motifs (both novel and known motifs), ensuring that each component contributed significantly to the enrichment of the whole module. We annotated tissue-specific promoters with predicted tissue-specific regulatory elements and demonstrated that these sites are in excellent agreement with experimentally annotated liver-specific sites in the human albumin promoter and skeletal-muscle-specific sites in the human α-actin promoter. Both promoters are particularly well annotated with experimentally verified tissue-specific regulatory elements and permit an informative comparison. In other tissues, we gave predicted sites for tissue-specific motifs in representative promoters. The complete data and analysis are available in TCat: The Catalog of Tissue-Specific Regulatory Motifs (http://rulai.cshl.edu/tcat).
Results
We describe first steps toward cataloging high-confidence tissue-specific motifs, modules and their sites. We first collected and integrated expression and function data from various sources, and identified transcripts that are likely to be under tissue specific regulation. We demonstrated that transcripts with evidence for tissue-specific regulation from multiple expression sources in one species (human or mouse) are significantly more likely to have evidence for tissue-specificity in the other species. We analyzed and annotated proximal-promoter sets in seven representative tissues from both human and mouse, demonstrating that motifs and predicted binding sites are in agreement with experimentally verified data and that analyses in human and mouse are significantly correlated. We also showed that the top-scoring sites in orthologous tissue-specific promoters from human and mouse rarely have significant conservation of site order, suggesting that comparative genomics alone may not be sufficient to decode the regulatory signals in these proximal promoters.
Transcripts under tissue-specific regulation
Few transcripts have expression restricted to a single tissue, but many transcripts appear to be regulated in a tissue-specific manner (Su et al, 2004), and the corresponding promoters are likely to contain tissue-specific regulatory elements. To circumvent problems associated with individual sources of information, we used a voting system that combined information about expression, function and tissue specificity from different sources. Table I gives the number of transcripts with single and multiple sources of evidence (votes) for tissue-specific regulation in each tissue. Orthologs of transcripts with multiple votes for tissue-specific regulation were more likely to have evidence for specific regulation in that tissue, suggesting that the false-positive rate for calling a transcript tissue specific is lower when based on multiple votes. The number of ortholog transcript pairs with multiple votes for tissue-specific regulation in both species ranged from 1 in CD4 T cells to 69 in liver. Table II lists genes and orthologous transcripts with votes for skeletal-muscle-specific regulation in both human and mouse. Gene and transcript lists for other tissues are given in Supplementary Section 1.5.
Enrichment of known tissue-specific motifs
Knowledge of factors and corresponding binding sites that regulate tissue-specific transcription can be used to evaluate motif ranking. We measured motif enrichment in tissue-specific promoter sets using balanced error rates, evaluating motifs for their ability to distinguish tissue-specific sets (foreground sets) from background sets that are composed of non-tissue-specific promoter samples from CSHLmpd. Balanced error rates measure proportions of misclassified promoters after normalization of foreground and background sizes. We ranked motifs according to enrichment and determined whether the ranks assigned to binding-site motifs for factors with known tissue-specific roles are significantly elevated in the corresponding tissues. The results presented in Table III demonstrate that binding-site motifs for these factors ranked significantly high (P<0.01) according to a Wilcoxon signed-ranks test in almost all tissues tested. Excluding DBP in human liver and HNF-3 in mouse liver, these factors had evidence for expression in their respective tissues, and their binding-site motifs were highly enriched in our foreground sets. Results are summarized in Table IV, and the motifs with greatest enrichment in each tissue are given in TCat. Table IV also includes information for HNF-6 in liver. HNF-6 is a known liver regulator, but there is no evidence for its expression in liver based on our data, and its binding-site motif was not enriched in our liver foreground sets. In addition, C/G- or A/T-rich motifs are likely to be enriched in foreground sets that are C/G or A/T rich relative to promoter base composition. To eliminate this potential bias, we adjusted the GC content in background sets to match foreground sets. Some known tissue-specific motifs were identified as enriched only after GC content correction.
Nuclear receptor binding-site motifs and E-box motifs are among the top enriched motifs in 11 and 10 of the 14 human and mouse tissues, respectively. A Wilcoxon signed-ranks test showed that nuclear receptor and E-box motifs (represented by 54 and 39 TRANSFAC motifs, respectively) are enriched in the union of our foreground sets with P-values below 6.07E−14 and 2.02E−13 (nuclear-receptor motifs) and 2.22E−10 and 4.57E−03 (E-box) in human and mouse, respectively. These results suggest a diversity of tissue-specific roles for nuclear receptors and E-box binders, likely mediated by tissue-specific cofactors.
Tissue-specific cis-regulatory elements
Highly enriched motifs, and the associated score thresholds identified by our methods, provide a starting point for targeted experimental annotation of tissue-specific promoters. Figure 1 shows known and predicted sites mapped on the −500 to +100 region of the human albumin promoter and the −250 to +50 region of the human skeletal muscle α-actin promoter. Human albumin has known functional binding sites for HNF-1, C/EBP, AFP and NF-Y (Paonessa et al, 1988; Sawadaishi et al, 1988; Frain et al, 1990; Li et al, 1990), all of which were identified among the top predicted motifs or included in top modules for liver. Locations of known sites for C/EBP (at −437) and NF-Y (at −125) do not perfectly align with the corresponding predicted sites (at −462 and −143, respectively), but current knowledge about binding sites for those factors raises the possibility that the predicted locations are more accurate. The only predicted binding site for these factors in the human albumin promoter that is not depicted in the figure is a C/EBP site at −956. Human skeletal muscle α-actin has known sites for SRF, TEF and a known TATA box (Boxer et al, 1989; MacLellan et al, 1994), all among top predicted motifs for human skeletal muscle (TATA box motifs have high similarity with MEF-2 motifs). Figure 2 gives predictions in a representative human promoter in each of the remaining tissues.
Comparison to previous results
Previous analysis of tissue-specific patterns in regulatory regions includes analysis based on cross-species conservation (Xie et al, 2005) and coexpression (Smith et al, 2006).
Xie et al (2005) identified conserved elements in orthologous promoters of four mammalian genomes. They found 59 experimentally validated motifs that are significantly conserved and enriched in at least one human tissue. These include E-box, ETS, MEF2, MEIS1 and NF-1 in skeletal muscle; Chx10 in kidney; NRF-1, ELK-1, GABP and E12 in CD4 T cells; AP-4 and MEF-2 in heart; and NRF-1 in testis. Our results agree with Xie et al (2005) on the enrichment of E-Box and MEF-2 in skeletal muscle, ETS in CD4 T-cells and E-box in pancreas.
In previous work (Smith et al, 2006), we tested the hypothesis that proximal promoters contain information that can be used to predict tissue-specific expression. We were not concerned with identifying the most significant tissue-specific motifs, modules and sites. Considering the difference between the goals of the two projects, it is not surprising that the predictive models described by Smith et al (2006) have little similarity to our top motifs and modules. The most significant similarities between our top tissue-specific patterns and the predictive models of Smith et al (2006) include the enrichment of ETS in CD4 T-cell-specific promoters and the enrichment of Smith et al (2006) motifs Novel3 and Novel6 in mouse testis and Novel1 in human testis. The three novel testis motifs are very similar to motifs that rank in the top 100 in our analysis, but the enrichment of these motifs was not sufficiently high for inclusion in TCat.
Correlation between human and mouse regulatory regions
We compared motif enrichment ranks in each human foreground set to ranks in the corresponding mouse foreground set using Spearman's rank correlation test, and found that enrichment ranks across species are highly correlated (P<0.001) for all but CD4 T cells (Supplementary Table 15). In CD4 T cells, motif enrichment ranks are similar only for few highly enriched motifs. Despite the motif-enrichment ranks correlation, the order of the top predicted binding sites is not usually conserved between orthologous promoters. Fewer than 10% of orthologous pairs showed significant (P<0.01) conservation of site order. Weak site-order conservation suggests that the top tissue-specific sites would be difficult to identify using traditional cross-species alignment alone, and methods that rely on co-linear promoter alignment may have high false-negative detection rates. This evidence is in agreement with Frith et al (2006), who found that homologous transcription start sites can be separated by more than 100 nucleotides. A list of the nine genes (out of 102 candidates) with significant conservation of site order is given in Supplementary Section 2.3.
Materials and methods
The steps used in creating the catalog include (1) identifying tissue-specific transcripts, (2) identifying factors that are expressed in each tissue, (3) obtaining promoter sequences for tissue-specific transcript, and (4) identifying individual motifs and modules (i.e. sets of interacting motifs) that characterize tissue-specific promoter sets.
Identifying tissue-specific transcripts
To identify motifs and modules that regulate tissue-specific transcription, we analyzed promoters of transcripts that appear to be regulated in a tissue-specific manner. If an information source indicated that a transcript has restricted expression, unusually high expression, or a specific function in the tissue, that source voted for tissue specificity of the transcript. For each tissue, we sorted the transcripts according to the number of votes received, retaining the top 100 with distinct TSS as tissue specific. Ties in the ranking were broken according to intensity values from the GNF SymAtlas expression data (discussed below), which we have found to be the most complete and the most reliable source of tissue-based expression information. We used the same number of transcripts for each tissue to facilitate comparison across tissues, and 100 sequences provided sufficient information for our analysis while allowing identification of well-known tissue-specific motifs.
Microarray data
The GNF SymAtlas microarray data were generated using Affymetrix HG-U133A array and the custom GNF1H and GNF1M Affymetrix arrays, and include expression profiles for 79 human and 61 mouse tissues (Su et al, 2004). Among these are the seven tissues we selected to include in the catalog. Tissues were selected with consideration to data availability in GNF and other sources and interest from Zhang lab members and collaborators. A transcript received a vote for tissue specificity from this information source if it was called present and its intensity exceeded its mean across all tissues by 3 standard deviations.
The Hughes Toronto microarray data (Zhang et al, 2004), which was generated using custom-built oligonucleotide arrays, provide mouse expression profiles for 55 tissues, including all of our tissues but CD4 T cells. A transcript received a vote for tissue specificity if it was called present in the tissue, and had intensity at least 10 standard deviations above its mean across all 55 tissues. This large number of standard deviations was required to limit the number of transcripts receiving positive votes.
The GeneNote expression profiles (Shmueli et al, 2003), which were generated using the Affymetrix GeneChip HG-U95, provide human expression data for 12 tissues, including all of our tissues but CD4 T cells and testis. The GeneNote data were used in the same way as the GNF SymAtlas data, with a transcript being called tissue specific if it is present in that tissue and has intensity at least 3 standard deviations above its mean across all 12 tissues.
EST data
dbEST is a database of expressed sequence tags (Boguski et al, 1993), and contains source information, such as the tissue of origin, for each EST. This information is used to annotate UniGene clusters with the source data, and a UniGene is said to have restricted expression in a tissue if more than half of the ESTs contributing to that UniGene have the same source tissue. A transcript received one vote for specificity in a particular tissue if the corresponding UniGene cluster is annotated as having expression restricted to that tissue.
GO terms
We associated a set of GO Terms with each tissue. This was performed by compiling a set of keywords for each tissue (e.g. ‘renal' was associated with kidney; ‘sperm' was associated with testis), and searching GO Term names and definitions for those keywords. This produced, for each tissue, a set of GO Terms that were subsequently reviewed to ensure that the context of the keywords was appropriate. A transcript of a gene annotated with a GO Term that is associated with a tissue received a vote for specificity in that tissue.
Selecting promoter sequences
Although regulatory elements can exist almost anywhere in the genome, they are concentrated near the TSS (Cooper et al, 2006). We used the CSHLmpd to map transcripts to promoters, using experimentally confirmed promoters from EPD (Perier et al, 1998), DBTSS (Suzuki et al, 2002) and GenBank, as well as computationally predicted promoters. For each promoter, we used the proximal sequence region of −1000 to +100 relative to the TSS.
Each part of our analysis is based on comparing the tissue-specific promoter sets to a background of random promoters from the same species. For each tissue, a background set was constructed by selecting 1000 transcripts uniformly at random from the set of RefSeqs for the corresponding species with TSS annotation in CSHLmpd. For each tissue, transcripts with at least one vote for specificity in that tissue were removed from consideration before selecting the background.
Because our analysis focused on proximal promoters, −1000 to +100 relative to the TSS, if the TSS annotation is off by several hundred base pairs, important promoter regions might be excluded. AKR1D1, identified as liver specific by our voting system, has two known TSSs within 500 bp of the first exon for the corresponding RefSeq (NM_005989) (Charbonneau and Luu-The, 1999). We used the TSS located upstream of the first exon, but could have chosen to use the other promoter, which was annotated by a generally more reliable source (DBTSS versus GenBank). Currently, in such situations, there is little information that identifies the promoter responsible for observed tissue-specific regulation, but comparative genomics and rapidly improving arrays promise better 5′ end identification, thus improving proximal promoter annotation and association between transcripts and promoters (Kim et al, 2005; Carninci et al, 2006). Negative promoter sets can be used to cancel out patterns that are not related to tissue-specific transcription regulation. We use random negative promoter sets with and without GC-content correction; this correction cancels the inuence of genomic GC-content isochore variability.
Identifying and evaluating motifs
Given a motif M (represented as a position–frequency matrix) and a sequence S, the max-score of M in S, max-score (M, S) is the score of the top scoring subsequence of S when aligned against the scoring matrix for M. Details on constructing and using scoring matrices can be found in Stormo (2000). For a fixed threshold λ, the max-score classification method classifies S as belonging to the foreground if max-score (M,S)⩾λ, and the background otherwise. Given a set of foreground sequences FG (i.e. the tissue-specific promoters) and a set of background sequences BG, the sensitivity of M and λ under max-score classification is
and the specificity is
The balanced error rate for M and λ under max-score classification is then
The quantity of interest in our analysis corresponds to the optimal value of λ for M in distinguishing FG from BG:
Many known motifs are similar to each other, usually owing to similar binding specificities for distinct factors or distinct origins for mofits associated with a single factor. We used MATCOMPARE (Schones et al, 2005) to eliminate redundancies in the sets of known and novel motifs.
Identifying and evaluating modules
Modules are used to classify sequences based on the max-score values of the motifs they contain. Let ℳ={M1, …, Mk} be a module and Λ={λ1, …, λk} be an associated set of thresholds. The max-score classification for modules assigns sequence S to the foreground if and only if max-score (Mi, S)⩾λi holds for all i (1⩽i⩽k). Given sets of sequences FG and BG, the sensitivity of ℳ and Λ under max-score classification is
and the specificity is
The balanced-error rate for ℳ and Λ under the max-score classification is
As with mofits, we are interested in the optimal value of Λ and define
Because modules are intended to describe synergistic function of a set of motifs, we are interested in modules whose performance is better than expected given the performance of the individual motifs composing the module. For a module ℳ composed of k motifs, let ℳ′⊂ℳ minimize B(ℳ′, FG, BG) over all size k−1 modules built from motifs in ℳ, and let ℳ′=ℳ\ℳ′. To assess whether ℳ, with balanced-error rate u, significantly improves over ℳ′ and ℳ′, we use the probability
We estimated this probability empirically, by sampling from the distribution of balanced-error rates resulting from intersections of sets with balanced-error rates B(ℳ′, FG, BG) and B(ℳ′, FG, BG).
We used MODULATOR, which is available in CREAD (Smith et al, 2005b), to construct modules. Given a set of motifs, a set of foreground sequences and a set of background sequences, MODULATOR identifies those modules composed of the given motifs that have the best balanced-error rates. A branch-and-bound algorithm is used to simultaneously optimize the score thresholds for the motifs in a module. Modules are constructed by adding motifs to existing modules until a user-specified module size is reached or until motif addition does not significantly improve enrichment. Each time a motif is added to a module, the resulting larger module is retained only if the balanced-error rate of the larger module is improved significantly above expectation. The initial modules of size two are obtained by combining pairs of motifs.
For modules that are entirely composed of known motifs, the top 100 motifs (before eliminating redundancies) were used. Modules were allowed to contain up to four motifs for reasons of computational feasibility, but many top modules are smaller. Novel modules, which must contain at least one novel motif, were constructed using the top 100 novel motifs and the top 100 known motifs. Redundancies were removed from the lists of top modules using a procedure described in Supplementary Section 2.
Measuring the significance of motifs and modules
To measure significance of enrichment for top known motifs we used known motifs to classify randomly assembled promoter sets. We constructed 1000 foreground/background pairs for each species by selecting 100 sequences for each foreground and 1000 for each background uniformly at random from CSHLmpd. For each foreground/background pair we calculated the balanced-error rate of each known motif. The best balanced-error rates overall obtained on random samples for human and mouse were 0.364 and 0.368, respectively. We used the distribution of these error rates to identify the q-value (Storey and Tibshirani, 2003) significance of the error rate of each motif. Tissues whose highest ranking motifs fail the q<0.05 test include CD4 T cells and heart in human, and CD4 T cells in mouse. TCat includes q-value annotation for each ranked known motif. The full set of motifs for which q<0.05 is estimated to include five false leads per 100 predictions. We did not obtain statistical significance measures for novel motifs, because this will require running DME and DME-B more times than is computationally feasible.
Modules were identified by combining motifs whose cooccurrence was enriched in the foreground sets. We measured enrichment of modules using the balanced-error rate (analogous to that of motifs), and we required that each motif in a module contributes significantly to the enrichment of the module as a whole. To test significance, we randomly selected 100 of the 1000 foreground/background pairs used to evaluate individual known motifs, and performed the module identification procedure on each of the 100 selected pairs. The best balanced-error rates for modules that are entirely composed of known motifs (called known modules) in random human and mouse sets were 0.3145 and 0.304, respectively. We used these balanced-error rates as an estimate of the critical value for P<0.01. We opted for using P-value cutoffs instead of computing q-values because accurate q-value estimation for modules is computationally prohibitive. Top known modules in human kidney (0.2955), liver (0.3105), pancreas (0.3125) and testis (0.3) scored better than the cutoff, as did top known modules in mouse kidney (0.3005), liver (0.3025) and testis (0.2945).
Supplementary Material
Supplementary information

tissue-specific regulation
cis-regulatory modules


Hedgehog signaling pathway function conserved in Tribolium segmentation

In Drosophila, maintenance of parasegmental boundaries and formation of segmental grooves depend on interactions between segment polarity genes. Wingless and Engrailed appear to have similar roles in both short and long germ segmentation, but relatively little is known about the extent to which Hedgehog signaling is conserved. In a companion study to the Tribolium genome project, we analyzed the expression and function of hedgehog, smoothened, patched, and cubitus interruptus orthologs during segmentation in Tribolium. Their expression was largely conserved between Drosophila and Tribolium. Parental RNAi analysis of positive regulators of the pathway (Tc-hh, Tc-smo, or Tc-ci) resulted in small spherical cuticles with little or no evidence of segmental grooves. Segmental Engrailed expression in these embryos was initiated but not maintained. Wingless-independent Engrailed expression in the CNS was maintained and became highly compacted during germ band retraction, providing evidence that derivatives from every segment were present in these small spherical embryos. On the other hand, RNAi analysis of a negative regulator (Tc-ptc) resulted in embryos with ectopic segmental grooves visible during germband elongation but not discernible in the first instar larval cuticles. These transient grooves formed adjacent to Engrailed expressing cells that encircled wider than normal wg domains in the Tc-ptc RNAi embryos. These results suggest that the en–wg–hh gene circuit is functionally conserved in the maintenance of segmental boundaries during germ band retraction and groove formation in Tribolium and that the segment polarity genes form a robust genetic regulatory module in the segmentation of this short germ insect.
Introduction
The ontogenic stage at which the body plans of animals belonging to the same phylum reach maximum morphological similarity is called the phylotypic stage. In insects and other arthropods, the phylotypic stage is the elongated germband at which the three germ layers have formed and segments are morphologically evident along the entire anterior–posterior axis (Sander 1997). In Drosophila, segment polarity genes, most of which are components of two major signal transduction pathways (the Wingless and Hedgehog signaling pathways), control the formation of grooves between segments and anterior–posterior patterning within each segment. Initially, pair-rule genes activate engrailed (en) expression at the anterior boundary of each parasegment and wingless (wg) at the posterior boundary DiNardo and O'Farrell (1987; Howard et al. 1988; Ingham et al. 1988). Engrailed protein activates expression of hedgehog (hh), which encodes a secreted protein that signals to surrounding cells (Hidalgo and Ingham 1990; Ingham and Hidalgo 1993). Hh signaling leads to the continued activation of wg, whose secreted protein product is necessary for the continued activation of en. This positive feedback loop ensures the interdependence of these three genes for the maintenance of each other’s expression until embryonic stage 9–10 (Forbes et al. 1993). At the end of stage 10, en expression becomes independent of wg, and it is around this stage that segmental boundaries are morphologically visible at the posterior edge of cells expressing en and hh.
Evidence that this segment polarity network might be conserved is primarily based on the expression patterns of en, wg, and more recently, Hh pathway component genes in other insects as well as non-insect arthropods and annelids (Patel et al. 1989; Brown et al. 1994; Kraft and Jackle 1994; Nagy and Carroll 1994; Grbic et al. 1996; Peterson et al. 1998; Damen 2002; Dhawan and Gopinathan 2003; Simonnet et al. 2004). There is also limited data suggesting this network is functionally conserved among insects. Ectopic expression of Drosophila wg in Tribolium induces ectopic en in the anterior half of the parasegment suggesting functional conservation of the wg–en interaction (Oppenheimer et al. 1999). wg and/or en have been implicated by RNAi analyses in the proper formation of segmental boundaries in the milkweed bug Oncopeltus faciatus (Angelini and Kaufman 2005), the honey bee Apis mellifera (Beye et al. 2002), the blowfly Lucilia sericata (Mellenthin et al. 2006) and the beetle Tribolium (Ober and Jockusch 2006). While RNAi analysis of wg and hh in the cricket Gryllus (Miyawaki et al. 2004) failed to reveal significant effects on segmentation, RNAi analysis of armadillo, a wg pathway component, does implicate wg signaling in segmentation in this short germ insect. In the long germ embryo of Drosophila, where all segments initiate virtually simultaneously, loss of any one of these three genes destabilizes the expression of the others and they eventually fade, resulting in shorter embryos that, in addition to the loss of segmental grooves, also show misspecified epidermal cell fates (Ingham et al. 1991; Forbes et al. 1993). We have investigated genes that encode the Hh-signaling pathway components hedgehog (hh), patched (ptc), smoothened (smo), and cubitus interruptus (ci) to determine whether they function with en and wg in the formation of segmental boundaries in this short germ band insect.
The Hh-signaling pathway is well-conserved between insects and vertebrates (Huangfu and Anderson 2006) and is thus likely to be conserved in Tribolium. The main components were first elucidated in Drosophila, where Hh is secreted by cells in the posterior compartment of embryonic segments and larval imaginal discs. It diffuses to the anterior compartment (Lee et al. 1992; Tabata et al. 1992; Tashiro et al. 1993) where the signal is controlled by two membrane proteins: Patched (Ptc), a twelve pass transmembrane protein (Hooper and Scott 1989; Nakano et al. 1989) and Smoothened (Smo), a seven pass transmembrane protein (Alcedo et al. 1996; van den Heuvel and Ingham 1996). In the absence of Hh signal, Ptc represses Smo activity (Chen and Struhl 1996). Signaling is initiated by binding of Hh to its receptor Ptc, which relieves this repression and allows Smo to signal to a multimeric complex inside the cell. This complex is composed of the serine threonine kinase Fused (Alves et al. 1998), the kinesin related protein Costal-2 (Sisson et al. 1997), a novel cytoplasmic protein Suppressor of fused (Monnier et al. 1998) and a zinc finger transcription factor Cubitus Interruptus (Ci; Motzny and Holmgren 1995). In unstimulated cells, this complex sequesters ci, inhibits nuclear import of the full-length 155 kDa protein and promotes its cleavage to generate an N-terminal 75-kDa form containing the Zn finger DNA-binding domain, which can enter the nucleus and repress transcription of Hh target genes (Aza-Blanc et al. 1997). When Hh signal is transduced, activation of Smo inhibits ci cleavage and activates the full-length protein, which then translocates to the nucleus, resulting in the transcription of Hh-responsive target genes including wg, ptc, gooseberry, and decapentaplegic (Alexandre et al. 1996; Dominguez et al. 1996; Hepker et al. 1997; Ingham and McMahon 2001).
Consistent with reports on other arthropods, we found that the expression patterns of hh, ci, smo, and ptc were largely conserved in Tribolium. Using RNAi to study the function of these genes during segmentation in Tribolium, we followed embryonic development in these embryos using En as a marker of segment development and integrity. When the Hh signal was depleted by RNAi, segments were specified normally in the posterior growth zone and the embryos elongated as fully as wild-type. En and wg expression in these embryos, although properly initiated, was not maintained and defects appeared during germ band retraction, resulting in tiny, sphere-shaped embryos lacking segmental grooves. On the other hand, overactivation of the pathway by ptc RNAi produced embryos with transient ectopic segmental grooves and embryonic cuticles with enlarged heads and thoracic appendages. All together, these results indicate that Hedgehog signaling is an essential component of the segment polarity network in Tribolium, which is necessary to maintain segmental integrity during germband retraction after the segments have been enumerated in the growth zone. The conserved function of an en–wg–hh gene circuit during segmentation suggests that the segment polarity genes constitute a robust gene regulatory module in this short germ insect.
Materials and methods
Beetle husbandry
Tribolium castaneum strain GA-1 was reared in whole wheat flour supplemented with 5% dried yeast at 30 C (Beeman et al. 1989).
Identification of hedgehog pathway component genes in the Tribolium genome
Partial cDNAs of tc-hh, tc-ci, tc-smo and tc-ptc, cloned into the pCR4TOPO vector (Invitrogen), were obtained from Y. Tomoyasu. Orthologs of each gene were identified in the annotation of the Tribolium genome (the Tribolium genome consortium, in review). The sequences of the partial cDNAs matched those deduced from the gene models with minor differences. hh, glean gene number tc01364 or NCBI mRNA accession number XM961615, is located on LG 2; smo, TC05545 or XM966834, is on LG 8; ci, TC03000 or XM965017, is on LG 3 and ptc, TC04745 or XM962700 is on LG 1 = X.
In situ hybridization and immunostaining
Whole mount in situ hybridizations were performed according to established protocols (Tautz and Pfeifle 1989). Expression of Engrailed in Tribolium embryos was determined using the α-Invected antibody, 4D9 which cross-reacts with Tc-En (Brown et al. 1994). Double staining for the different mRNAs in addition to En protein was performed simultaneously according to the protocol of Nagaso et al. (2001).
RNA interference (RNAi)
Templates for dsRNA synthesis were amplified as described (Tomoyasu et al. 2007). Double stranded RNA was synthesized using the T7 MEGAscript kit (Ambion) and purified using the MEGAclear kit (Ambion). Different amounts of dsRNA (Table 1) were mixed with injection buffer (5 mM KCl, 0.1 mM KPO4 pH 6.8) prior to injection. Parental RNAi was performed and affected embryos were analyzed as previously described (Bucher et al. 2002).
Microscopy and imaging
Stained embryos and larval cuticles were documented with a Nikon Digital DXM 1200F camera on an Olympus BX50 microscope using Nikon ACT-1 version 2.62 software. Brightness and contrast of all images were adjusted and some were placed on a white background using Adobe Photoshop 7.0.1 software.
Table 1
Summary of RNAi effects
Gene
DsRNA μg/μl
Class I (%)
Class II (%)
Class III (%)
Undeveloped (%)
Wild type (%)
Totals (n)
Tchh
3.0
0
6
83
11
0
464
1.5
18
73
0
9
0
477
Tcsmo
3.0
0
0
89
11
0
579
1.5
0
43
43
14
0
477
0.75
38
44
0
20
0
230
Tcci
3.0
0
33
61
5
0
694
1.5
0
30
63
7
0
405
0.75
73
19
0
8
0
293
Tcptc
4.0
0
76
–
24
0
271
2.0
13
68
–
19
0
189
Embryos were collected every 48 h for the first 2 weeks after injection. Phenotypic classes are unique for each gene. See text for details. Percentages have been rounded up.
Results
Expression patterns of Tc-hh, Tc-smo, and Tc-ci
Tc-hh transcripts are first detected in the presumptive head lobes on either side of the ventral mesoderm (arrowhead in Fig. 1a) and at the posterior end of the embryo. As the embryonic rudiment condenses, faint stripes of Tc-hh expression appear immediately posterior to the intense stripes in the head lobes and in the presumptive mandibular segment (arrowheads in Fig. 1b). Gnathal and trunk stripes appear in an anterior to posterior progression (Fig. 1c). Double staining for En expression revealed that Tc-hh and Tc-En are coexpressed in cells of the posterior compartment in each segment (Fig. 1d). During germband elongation, expression at the posterior end of the embryo resolves into spots on either side of the mesoderm and eventually into a ring surrounding the proctodeum (Fig. 1b–d and arrowhead in f). In the head, twin spots appear on either side of the presumptive stomodeum. As the head lobes mature, expression in the anterior region of the developing stomodeum increases (Fig. 1c,d and black arrowhead in e), while the anterior-most stripes of Tc-hh expression resolve into spots in the brain that overlap with the Tc-En-expressing cells (Fig. 1c,d and blue arrowhead in e). Expression at the posterior end of the embryo continues throughout germ band extension (Fig. 1b,d and f) eventually surrounding the proctodeum as it invaginates (Fig. 1f). As segments mature, Tc-hh expression at the ventral midline clears and Tc-hh is not expressed in the CNS. In total, there are three gnathal, three thoracic, and ten abdominal Tc-hh stripes with additional expression in the stomodeum, proctodeum, antennae, and brain (Fig. 1d).
Fig. 1Expression of Tc-hh, Tc-ci, and Tc-smo in Tribolium during segmentation. In these ventral views, anterior is to the left. a–cTc-hh; d–fTc-hh (purple) and Tc-En (gold); g–hTc-smo; i–kTc-ci; l, mTc-ci (purple) and Tc-En (gold); a Anterior stripes of Tc-hh (arrowhead) in the presumptive head lobes of a blastoderm embryo. Expression at the posterior end of the embryo is not in the plane of focus. b Weak stripes appear in the antennal and mandibular segments (arrowheads) posterior to the dark stripes in the head lobes. Twin spots of expression flank the mesoderm at the posterior end of the embryo and near the stomodeum. c In addition to expression in the head lobes and antennae, three gnathal stripes and one trunk stripes appear in an elongating germband embryo. d Coexpression of Tc-En (gold) and Tc-hh (purple) in the posterior compartment of each segment. Note Tc-En expression at the ventral midline in the absence of Tc-hh. e–fTc-hh expression in the stomodeum and the proctodeum (arrowheads) in the absence of Tc-En. Expression of Tc-hh in the head lobes has resolved into spots that overlap Tc-En expression (blue arrowhead). g Ubiquitous expression of Tc-smo in an early germ band embryo. h In an elongating germ band embryo, Tc-smo expression appears ubiquitous with some segmental modulation (arrowheads). i Expression of Tc-ci at the anterior edge of the head lobes (arrowhead) of a young germband embryo. j Gnathal stripes appear first in a slightly older embryo. Expression throughout the posterior region of the embryo fades anteriorly. k Wide Tc-ci stripes in the head lobes and antennal segments as well as in the gnathal and thoracic segments of an elongating germ band embryo. l Tc-En (gold) expression does not overlap Tc-ci (purple) expression in cells of the anterior compartment of each segment. m Enlarged view of a few thoracic segments from the embryo shown in l reveals a gap of two or three rows of cells anterior to the Tc-En stripes that do not express Tc-ci (arrowhead)
Tc-smo is expressed ubiquitously in the germband (Fig. 1g) in Tribolium. As the germ band elongates, expression appears to modulate slightly within each segment (arrowheads in Fig. 1h). smo transcripts are expressed in a similar manner in Drosophila (Alcedo et al. 1996).
Tc-ci expression is first detected at the anterior edge of each head lobe in the early embryonic rudiment (arrowhead in Fig. 1i). Dynamic expression of Tc-ci in the head lobes eventually resolves to a wedge of cells in the lateral eye field (Fig. 1j). Expression is detected in the labrum (Fig. 1l) and in a broad posterior region, which fades toward the anterior (Fig. 1j). As the germ band elongates, Tc-ci is expressed in a broad stripe in every segment in an anterior to posterior progression (Fig. 1i–k). Double staining for Tc-En indicates that Tc-ci is expressed in the anterior compartment where the anterior-most Tc-ci expressing cells are immediately posterior to the Tc-En-expressing cells of the preceding segment (Fig. 1m). Segmentally reiterated stripes of ci expression are also observed in the mulberry silkworm Bombyx mori (Dhawan and Gopinathan 2002), the spider Cupiennius salei (Damen 2002), and the millipede Glomeris (Janssen et al. 2004), suggesting that the segmental expression of ci in the anterior region of each segment is conserved among arthropods. Closer examination of Tc-ci expression revealed a gap of two or three rows of cells immediately anterior of the Tc-En-expressing cells that do not express Tc-ci (Fig. lm). In contrast, expression of ci throughout the anterior compartment of each segment in Drosophila is thought to be essential to the function of the Hh-signaling pathway in maintaining wg expression (Orenic et al. 1990; Hepker et al. 1997). Expression of ci immediately anterior to En-expressing cells is conserved in the spider (Damen 2002), but has not been reported for Bombyx or Glomeris. Thus, it is not clear whether this unusual expression pattern is unique to Tribolium.
RNAi analysis of Tc-hh, Tc-smo, and Tc-ci
The conserved expression patterns of Tc-hh and Tc-smo are consistent with the hypothesis that the Hh function in segmentation is conserved in Tribolium, but the lack of Tc-ci gene expression in cells immediately anterior to Tc-hh is not. To determine whether the Hh-signaling pathway is required for proper segmentation, we performed parental RNAi analysis of these Hh pathway components. Three different amounts of Tc-hh and Tc-smo dsRNA were injected, which uncovered a range of hypomorphic phenotypes (Table 1). Similar RNAi phenotypes were produced for both genes, and the RNAi cuticles were classified into three different categories (Class I, II, and III) based on severity as listed in Table 1. Regardless of the severity, none of the RNAi embryos hatched and the cuticle had to be dissected out of the vitelline membranes. Class I cuticles display the weakest phenotypes, in which the head is severely reduced and contains only rudimentary limb structures. The legs are present but slightly warped, and segmental grooves in the abdomen are occasionally fused but all eight abdominal segments are present (Fig. 2b (Tc-hh), f (Tc-smo)). Class II cuticles are small and spherical, with a large protuberance at the anterior end and three small warped pairs of limbs. The presence of the spiracle on the second thoracic segment (arrowhead in Fig. 2c and g) allowed us to identify these structures as rudimentary legs. The small amount of cuticle posterior to these small warped legs is smooth and lacked any abdominal features (Fig. 2c (Tc-hh), g (Tc-smo)). The most severely affected embryos, Class III, produced small spherical cuticles with a large protuberance at the anterior end and no obvious head structures or thoracic limbs (Fig. 2d (Tc-hh), h (Tc-smo)). These cuticles are very smooth on all sides, with no sign of grooves and are quite small relative to the size of similarly aged wild-type cuticles (Fig. 2a Tc-hh, e Tc-smo). The head and gnathal appendages appear to be more sensitive to the depletion of Tc-hh or Tc-smo than the legs, as even in the weakly affected individuals the head, including gnathal segments, failed to form properly (Fig. 2b,f).
Fig. 2Tc-hh, Tc-smo, and Tc-ci RNAi phenotypes in first instar larval cuticles. a–dTc-hh RNAi; e–hTc-smo RNAi; i–lTc-ci RNAi. a Comparison of a severely affected small spherical Tc-hh RNAi cuticle and a wild-type cuticle at the same magnification. b Lateral view of a mildly affected Class I cuticle containing reduced gnathal appendages, normal legs and all abdominal segments. c Ventral view of a Class II cuticle that is similar in size and shape to more severely affected Class III cuticles but retains three pairs of thoracic appendages. Note spiracle on second thoracic segment (arrowhead). d Ventral view of a small spherical Class III cuticle with rudimentary head structures, no appendages and no segmental grooves. e Comparison of a severely affected small spherical Tc-smo RNAi cuticle and a wild-type cuticle at the same magnification. f Class I cuticle with highly reduced head structures and mouthparts but a full complement of body segments. g Class II cuticle with three pairs of legs and rudimentary head structures. Note spiracle on second thoracic segment (arrowhead). h A severely affected Class III Tc-smo RNAi cuticle phenotypically similar to the most severely affected Tc-hh RNAi cuticle. i Comparison of a severely affected small spherical Tc-ci RNAi cuticle and a wild-type cuticle at the same magnification. j Mildly affected Class I cuticle with reduced head structures, three pairs of legs and mildly fused abdominal segments. k Class II cuticles are slightly longer than intermediate Tc-hh or Tc-smo RNAi cuticles with highly fused abdominal segments. l A severely affected Class III Tc-ci RNAi spherical cuticle with three pairs of legs
The phenotypes of Tc-ci RNAi embryos are not as severe as those observed for Tc-hh or Tc-smo. The most weakly affected, Class I Tc-ci RNAi cuticles (Fig. 2j), are shorter and fatter than wild-type with severely reduced heads, but contain all thoracic and abdominal segments. The Class II Tc-ci RNAi cuticles are also shorter than wild-type with a protuberance at the anterior end and fairly normal thoracic limbs, but little to no sign of segmental grooves in the abdomen (Fig. 2k). Similar to Class III RNAi embryos of Tc-hh and Tc-smo, Class III Tc-ci RNAi embryos produced smooth unsegmented cuticles lacking heads and gnathal appendages (Fig. 2l) that are considerably smaller than wild-type cuticles (Fig. 2i). Unlike Tc-hh and Tc-smo Class III RNAi embryos, they still produced fairly normal legs (Fig. 2l). Interestingly, loss of function mutants of ci in Drosophila also produce milder limb phenotypes than do hh mutants (Methot and Basler 1999).
To understand how the segmentation process is affected by loss of Hh signaling, we followed Tc-En expression during elongation and retraction in Class III RNAi embryos (Fig. 3). In RNAi embryos for all three genes, segmental stripes of Tc-En initiate normally during germ band elongation (blue arrowheads in Fig. 3a,d and g). Tc-En expression fades as the segments matured (black arrowheads Fig. 3a,d and g), suggesting that Hh signaling is required for the maintenance of Tc-En. However, Tc-En expression in cells along the midline, presumably in the developing CNS (wild-type, Fig. 3i), is maintained in the RNAi embryos, which allowed us to follow segmentation in RNAi embryos. Tc-hh, Tc-smo, and Tc-ci RNAi embryos completed elongation more or less normally (Fig. 3a,d and g), but abnormalities became evident during retraction. The head lobes fail to mature, and there is no evidence of antennal Tc-En stripes. There is also a protuberance at the anterior end of the embryo that is likely to correspond to the anterior protuberance seen in the RNAi cuticles (Fig. 2d,h and l, and arrow in 3 h). In embryos that completed retraction, the unsegmented germbands are highly compacted, and Tc-En expression pattern is very irregular (Fig. 3c,e and h). Compared to wild-type germ bands at a similar stage, these germ bands occupy only a portion of the egg (Fig. 3j,k). In addition, loss of early Hh signaling at the ventral midline affected cell fate, as indicated by the loss of Tc-En expression here (Fig. 3b,e). Loss of Hh signal in Drosophila similarly affects En expression in midline cells (Bossing and Brand 2006). We also found that Tc-wg expression was initiated but not maintained in these embryos (data not shown), suggesting Tc-wg is a target of the Hh pathway.
Fig. 3En staining in wild-type and severely affected Tc-hh, Tc-smo and Tc-ci RNAi embryos during elongation and retraction. a–cTc-hh RNAi; d–f, kTc-smo RNAi; g, hTc-ci RNAi; i, j wild-type. a Tc-En expression initiated normally in the posterior segments of an elongating Tc-hh RNAi germband embryo (blue arrowhead) but did not persist in older, more anterior segments (black arrowhead). b During germband retraction, patches of Tc-En expression are visible near the ventral midline, and laterally in the posterior segments. c In a highly compacted germ band with fused segments, Tc-En expression is disrupted along the ventral midline. d Expression of Tc-En in an elongating Tc-smo RNAi embryo initiated normally (blue arrowhead) but failed to persist in older, more anterior segments (black arrowhead). e During germband retraction, Tc-En is expressed in patches along the ventral midline, but has faded laterally. f In a highly compacted germ band, persists in a disrupted pattern along the ventral midline. g In an elongating Tc-ci RNAi embryo, similar to Tc-hh and Tc-smo RNAi, Tc-En initiated normally (blue arrowhead) but is not maintained (black arrowhead). h Tc-En expression at the ventral midline of a retracted germband embryo. Note the extended stomodeum (arrowhead) near the anterior end. i Tc-En expression in the CNS along the ventral midline and in the lateral ectoderm of each segment of a wild-type embryo during germband retraction. j Lateral view of a wild-type embryo inside the vitelline membrane at the end of germ band retraction. Tc-En expression persists laterally to the edge of the germband. k Ventral view of a severely affected Tc-smo RNAi embryo located near one end of the egg
hedgehog was first isolated in a screen for mutations that disrupt the Drosophila larval cuticle pattern and identified as one of the segment polarity genes with a ‘lawn of denticles’ phenotype (Nusslein-Volhard and Wieschaus 1980). In segment polarity mutants (e.g., wg, hh, and smo), deletion of a portion of the larval epidermis in each segment is accompanied by a mirror image duplication of the remaining structures. As a result, they contain the normal number of segments, but are smaller than wild-type due to partial deletion of each segment. In our study, we found Tc-hh and Tc-smo RNAi embryos to be smaller in size than the wild-type Tribolium larva (Fig. 2a,e), which may be due to cell death, fusion, or failure of cell division in segments during germ band retraction. The few random bristles produced in severely affected RNAi cuticles do not have any definable polarity suggesting that, unlike Drosophila, loss of function of these genes in Tribolium does not appear to produce mirror image duplications or affect polarity within the segments.
Analysis of Tc-ptc and overactivation of the Hh pathway
Transcripts of Tc-ptc are first detected in a broad domain in the posterior regions of the head lobes in the embryonic rudiment encompassing the antennal segments and the stomodeum, and at the posterior end of the embryo (Fig. 4a). Broad segmental stripes of Tc-ptc appear in an anterior to posterior progression during germband elongation (Fig. 4b). Expression fades in the middle of each initial stripe resulting in two narrow Tc-ptc stripes per segment (Fig. 4c). Double staining with Tc-En and Tc-ptc indicates that in each segment, one of the narrow Tc-ptc stripes marks the anterior boundary of a segment while the other is located immediately anterior to Tc-En-expressing cells (Fig. 4c and arrowhead in inset). This pattern persists even after germ band retraction; the anterior stripe appears stronger than the posterior stripe in each segment. ptc expression during segmentation in Drosophila is similarly dynamic (Nakano et al. 1989; Hidalgo and Ingham 1990).
Fig. 4Analysis of Tc-ptc in Tribolium.a–cTc-ptc expression in wild-type embryos; d–fTc-ptc RNAi cuticles; g wild-type; h–mTc-ptc RNAi. a Expression of Tc-ptc in three gnathal stripes, in a broad posterior region of the head lobes and in the posterior region of an early germ band embryo. b Segmental stripes appear sequentially during elongation and resolve in double stripes. c Tc-En (gold) expression in the posterior compartment abuts, but does not overlap the double Tc-ptc stripes (extent denoted by black lines) in the anterior compartment of each segment. Inset: enlarged view of Tc-ptc stripes adjacent to a stripe of Tc-En (arrowhead). d A mildly affected Class I Tc-ptc RNAi cuticle with deformed legs and head appendages. e A more severely affected Class II cuticle with enlarged misshapen legs but a normal complement of abdominal segments. f Comparison of a Class II Tc-ptc RNAi cuticle and a wild-type cuticle at the same magnification. g Expression of Tc-En (gold) and Tc-wg (purple) in a wild-type embryo during elongation. h Segmental expression of Tc-En and Tc-wg initiated fairly normally in a Tc-ptc RNAi germband. Tc-wg expression domains in the head lobes and around the proctodeum are expanded. i Expanded expression of Tc-wg in a slightly older embryo. j Ectopic stripes of Tc-En expression (blue arrowhead) in a germband undergoing retraction. k Ectopic expression of Tc-En at the ventral midline (arrowhead) in a close up of the embryo in h. l Transient grooves (arrowheads) around expanded Tc-wg expression domains in a close up of the embryo in i. m Ectopic Tc-En stripes (blue arrowhead) between each set of normal Tc-En stripes (black arrowhead) in a close up of the embryo in j. T1, first thoracic segment
As discussed above, Ptc is a negative regulator of the Hh-signaling pathway in Drosophila. Depletion of a negative regulator of the pathway would ectopically activate the pathway. To understand what happens when the Hh pathway is overactivated in Tribolium, we performed parental RNAi using two different amounts of Tc-ptc dsRNA (Table 1). The resulting cuticles (Fig. 4d,e,f) are very different from the ones described above for the other three genes. In mildly affected embryos (Class I), the head appendages are misshapen, while the legs are relatively normal (Fig. 4d). In the most severely affected embryos (Class II), all segments are present, but the head and thoracic appendages are enlarged and misshapen (Fig. 4e,f).
To understand the phenotype of the Tc-ptc RNAi embryos, we examined the expression of Tc-wg and Tc-En. In Tc-ptc-depleted embryos, segmental expression of Tc-En and Tc-wg is initiated normally (Fig. 4h), although the domains of Tc-wg expression in the head and at the posterior end of the embryo are expanded. Closer inspection revealed ectopic Tc-En expression at the ventral midline (Fig. 4k). In slightly older embryos, Tc-wg is expressed more intensely than normal in enlarged domains (Fig. 4i,l). Cells surrounding the expanded Tc-wg domains appear to be invaginating, as if beginning to form grooves. Closer inspection revealed a row of non-Tc-wg-expressing cells between the Tc-wg domain and the grooves (Fig. 4l). Tc-En is expressed in ectopic stripes that, in combination with the normal Tc-En stripes, would surround the expanded Tc-wg domains (Fig. 4j,m). In addition to the normal grooves that form posterior to the normal Tc-En stripe, ectopic grooves initiate anterior to the ectopic Tc-En stripes. Thus, overactivation of the Hh-signaling pathway in Tribolium leads to overexpression of Tc-wg, and ectopic expression of Tc-En, which results in ectopic groove formation. In Drosophila, binding of Hh to Ptc receptor relieves repression of wg and allows expression of target genes. In Drosophila ptc mutants, ectopic induction of wg and En result in the formation of extra grooves (Nakano et al. 1989), suggesting that the functional role of Ptc is highly conserved between these two species. However, unlike Drosophila, the ectopic grooves in Tribolium are transient and it appears that a late regulatory action restores the normal number of segments in this insect.
Discussion
Expression patterns suggest that the function of the Hh-signaling pathway is conserved in several developmental pathways in Tribolium.
In Tribolium, the expression patterns of the four Hh-signaling pathway components we examined are highly similar to those of their Drosophila counterparts. In both Drosophila and Tribolium, the domains of Tc-hh are very similar to those of Tc-En. However, there are some significant differences in their expression dynamics. For example, the antennal Tc-En stripes appear after the three gnathal Tc-En stripes, whereas antennal Tc-hh stripes appear before the gnathal Tc-hh stripes. Each metameric stripe of Tc-hh is laterally continuous across the width of the germband after mesoderm invagination. Later, Tc-hh expression has disappeared at the ventral midline in anterior segments, while in posterior segments the stripes are still continuous. In contrast, Tc-En expression continues in the CNS after Tc-hh expression has faded there. Tc-hh expression in the stomodeum and at the posterior end of the blastoderm embryo in the absence of Tc-En suggests that, similar to hh in Drosophila (Lee et al. 1992; Mohler and Vani 1992; Tashiro et al. 1993), Tc-hh is involved in some En-independent processes in these regions.
In both Drosophila and Tribolium, expression of segment polarity genes is initiated by pair-rule genes (recently reviewed by Damen 2007). Soon thereafter, their expression is controlled by interactions between the segment polarity genes themselves. In Drosophila, Ptc is constitutively active unless or until Hh represses Ptc activity. In the absence of Hh, unbound Ptc keeps the pathway switched off. One of the targets of the Hh pathway is ptc itself. In cells where its activity is antagonized upon binding of Hh, ptc continues to be expressed, but ptc expression disappears in cells that do not receive the signal. Thus, an initially broad domain of ptc expression resolves into two narrow stripes flanking the En expression domain. Tc-ptc expression pattern is similar: each broad stripe splits into two, such that each En stripe is bracketed by two Tc-ptc stripes, suggesting that Tc-ptc itself might also be a target of the Hh pathway in Tribolium.
The expression patterns of Hh pathway component genes are highly conserved between Drosophila and Tribolium, except for that of Tc-ci (Fig. 1l,m). In Drosophila, ci transcripts are initially expressed uniformly in the early cellular blastoderm and persist until the end of germ band elongation. At that point, ci expression is directly repressed by En in cells of the posterior compartment in each segment. In Tribolium, expression of Tc-ci is somewhat different in that there is a narrow region of two to three cells between the Tc-ci and En-expressing cells that does not express Tc-ci. In Tribolium, the absence of Tc-ci transcripts in cells just anterior to En-expressing cells might suggest the existence of an En-independent mechanism regulating Tc-ci expression. Alternatively, it is possible that Tc-ci transcripts turn over rapidly in these cells, but the expression pattern is conserved at the protein level. In Drosophila, ci is regulated post-transcriptionally. At stage 11, ci transcripts are localized throughout the anterior compartment of each segment whereas protein levels are lower at the center of each transcriptional stripe and higher in cells that bracket the En-expressing cells (Motzny and Holmgren 1995; Slusarski et al. 1995) much like what we describe here for ptc transcription in Tribolium and previously for Ptc protein levels in Drosophila. It will be interesting to see if Tc-ci is also post-transcriptionally regulated in Tribolium.
Functional analysis of Hh pathway component genes supports conserved roles in segment boundary formation.
In Drosophila, mutations in genes encoding positive regulators of the Hh pathway including hh, smo, and ci produce smaller than wild-type embryos with asegmental phenotypes (Nusslein-Volhard and Wieschaus 1980). In Tribolium, Tc-hh, and Tc-smo RNAi embryonic phenotypes are nearly identical; both produce highly compacted spherical cuticles with no evidence of appendages or segmental grooves. The most severe Tc-ci RNAi phenotypes are not as severe as those of Tc-hh or Tc-smo RNAi. Interestingly, the ci94 allele in Drosophila is a null allele (Slusarski et al. 1995; Methot and Basler 1999) and these mutant embryos differ considerably from hh mutants. hh mutants are much shorter in length and have a continuous ‘lawn of denticles’ phenotype whereas ci94 mutants are almost normal in size and have alternating naked cuticle and denticle belts on the ventral surface. Target genes of hh are partially derepressed in the absence of ci, producing a ci phenotype that is milder than that of hh (Methot and Basler 2001). An analogous situation has been described for the Wingless signaling pathway in Drosophila where derepression of target genes in the absence of pangolin results in a milder segment polarity phenotype compared to that of wg null mutants (Cavallo et al. 1998; Waltzer and Bienz 1998). In hh and smo mutants, only the repressor function of ci remains, producing the catastrophic phenotype. In contrast, loss of both the activating and repressor forms results in the milder phenotype seen in ci null mutants. In Tribolium, the most severe Tc-ci RNAi phenotype is milder than the most severe Tc-hh RNAi phenotype, suggesting similar regulation of the Hh pathway in the beetle.
In Drosophila, loss of Wg or Hh-signaling results in larvae that are smaller than wild-type due, at least in part, to epidermal cell death during and after germband retraction (Martinez-Arias and Lawrence 1985) or a combination of transformation and cell death (Klingensmith et al. 1989). In Tribolium, hh, smo, and ci RNAi individuals are greatly reduced in size compared to the wild-type (Fig. 2). These embryos go through the events of early embryogenesis normally, producing the full complement of segments and initiating Tc-En and Tc-wg expression. Tc-En and Tc-wg expression fade and the segmental remnants become highly compacted along the anterior–posterior axis during retraction. Tribolium embryos lacking Tc-wg also elongate normally but fail to maintain En expression and form shorter than wild-type embryos during retraction (Ober and Jockusch 2006). While it is likely that cell proliferation and programmed cell death both contribute to shaping the embryo in Tribolium, during normal development cells divide randomly throughout the elongating germ band (Brown et al. 1994); organized patterns of cell division or cell death have not been reported. Similarly, it is likely that excessive cell death or the lack of cell proliferation contributes to the severely compacted terminal phenotype of Tc-hh, Tc-smo or Tc-ci RNAi embryos, but closer examination will be required to determine if this is so.
In Drosophila ptc mutants, deletion of the midregion of each segment is accompanied by a mirror image duplication of the remaining denticles (Nusslein-Volhard and Wieschaus 1980). In contrast, Tribolium Tc-ptc RNAi embryos, which also display the correct number of segments, are characterized by distended gnathal and thoracic appendages. Although there are some random bristles, we could not identify any noteworthy difference in the polarity of these bristles that could be attributed to a characteristic loss of function phenotype for genes belonging to this class. This suggests that unlike Drosophila, the loss of segment polarity gene function in Tribolium does not result in any morphologically identifiable polarity defect in the cuticle.
In the absence of ptc in Drosophila (DiNardo et al. 1988) and Tribolium, En expression is established properly; but later, de novo En stripes appear between the normal stripes. Ectopic expression of En in Drosophila is not due to regulation of ptc by pair-rule factors (DiNardo et al. 1988). In the absence of ptc, the wg expression domain broadens anteriorly. Expanded wg expression induces ectopic En stripes, which cause ectopic groove formation, suggesting that the principal function of Ptc is to repress wg. Similar expression of Tc-ptc in Tribolium, considered with the expanded Tc-wg domains that are surrounded by ectopic Tc-En-expressing cells and the ectopic grooves transiently formed in Tc-ptc RNAi embryos suggest that the role of Tc-ptc is likely to be functionally conserved between Drosophila and Tribolium. However, in Tc-ptc RNAi embryos, ectopic Tc-En is also detected along the ventral midline, a phenotype that has not been described for Drosophila ptc mutants. hh RNAi has been attempted in the orthopteran Gryllus bimaculatus (Miyawaki et al. 2004). Unfortunately, this organism seems to be resistant to hh dsRNA. In the RNAi embryos, the level of hh is not reduced. The embryos develop normally and hatched larvae show no cuticular defects. Lack of ptc analysis in other insects makes it difficult to speculate as to whether this novel expression of Tc-En along the ventral midline is specific to Tribolium, or a general feature related to short germ development. Finally, although ectopic grooves appear to form around the expanded Tc-wg domains, they are not detected in the terminal cuticles, implying that events late in embryogenesis restore the normal number of segmental grooves.
Conservation of the hh–wg–en gene circuit in short germ segmentation
Several lines of evidence suggest that the segment polarity gene circuit, in which the expression of the hh, wg, and en genes are dependent upon one another in the long germ mode of segmentation elucidated in Drosophila, is conserved in the short germ mode of segmentation found in Tribolium. As in Drosophila, in the absence of smo, hh, ci (this paper), or wg (Ober and Jockusch 2006) in Tribolium, En expression is not maintained. In addition, Tc-wg mRNA fails to persist in Tc-hh, Tc-smo and Tc-ci RNAi embryos (data not shown). Furthermore, expression patterns and RNAi phenotypes of the Hh pathway components we examined (Tc-hh, Tc-smo, Tc-ci, and Tc-ptc) suggest that the regulation of the Hh pathway is also conserved. Segment polarity genes, which function last in the segmentation gene hierarchy, are expressed in segmental fields that have been predefined by genes at higher levels (gap and pair rule). Conservation of the segment polarity gene circuit in Tribolium suggests that that segment polarity genes form a robust regulatory module in the short germ mode of segmentation in this beetle and their expression patterns in numerous insects and chilicerates suggest that this module is likely to be conserved among the Insecta, and perhaps the Arthropoda.
While the expression patterns of segment polarity orthologs are highly conserved, the expression patterns of pair-rule gene orthologs vary greatly among insects and other arthropods (recently reviewed in Tautz 2004; Peel et al. 2005; Damen 2007). Functional analysis in Tribolium indicates that interactions among pair-rule gene homologs differ from those of their Drosophila counterparts (Choe et al. 2006) and that some secondary pair-rule genes function in opposite parasegmental registers in Drosophila and Tribolium (Choe and Brown 2006). These findings suggest that the inputs from pair-rule genes to the segment polarity gene module are likely to be quite different in each of these insects. Functional analysis of how pair-rule genes regulate the alternating expression of en and wg in Tribolium will provide insight into these differences, which will ultimately help us understand the evolution of genetic regulatory networks. Interestingly, the segment polarity gene module seems to be resilient enough to withstand such evolutionary changes in input from the pair-rule gene module.

hedgehog
segmentation
segmental groove
smoothened
patched
cubitus interruptus