A set of meis2 CNEs drive expression in the hindbrain and cranial ganglia in zebrafish and lamprey embryos

We previously identified a genomic region, downstream of the developmental gene meis2, containing a number of CNEs that are conserved between jawed vertebrates and lamprey [28] (Additional File 1). We grouped these CNEs into five separate elements (Additional File 2) for functional testing in a zebrafish tol2 reporter assay [29]. Four of these elements drive discreet and complementary patterns of reporter expression in the hindbrain of zebrafish embryos, with homologous zebrafish and lamprey elements driving highly similar expression patterns (Figure 1). These patterns of reporter expression are consistent with the endogenous expression of meis2 in the hindbrain [30, 31], where Meis proteins play a crucial patterning role by interacting with Hox and Pbx transcription factors [32, 33]. Lamprey and zebrafish CNE 3285 elements both drive GFP expression in the cranial ganglia and primary neurons of the hindbrain and spinal cord. CNE 3288 elements of zebrafish and lamprey drive GFP in neurons of the hindbrain posterior to rhombomere 4 (r4), as shown by comparison to RFP expression in r3 and r5 in a transgenic line containing RFP under the control of a krox20 regulatory element [34]. CNE 3299 elements up-regulate GFP in the anterior hindbrain - r2-4 for the lamprey homolog and r3-4 plus neural crest migrating into the hyoid pharyngeal arch for the zebrafish homolog. CNE 329X of lamprey and zebrafish both drive GFP expression in the anterior hindbrain and neurons of the midbrain.

We have developed a parallel reporter assay in lamprey embryos (in submission) to assess the functional conservation of CNEs across vertebrates. Using this assay, we have tested lamprey CNEs 3285 and 3299 for enhancer activity during lamprey embryogenesis. In lamprey embryos, CNE 3285 drives GFP expression in the cranial ganglia and CNE 3299 in the anterior hindbrain (Figure 1). Thus, for both of these elements, the pattern of reporter expression driven in lamprey embryos is almost identical to the pattern driven in zebrafish embryos (Figure 1). This provides compelling evidence that these CNEs are part of a gene-regulatory network for hindbrain patterning that is conserved across all vertebrates.

Some meis2 CNEs contain deeply conserved Pbx-Hox TFBS motifs

Because of its clear and specific expression pattern in the hindbrain of zebrafish and lamprey embryos, we chose element 3299 as a starting point for the identification of putative transcription-factor binding-sites by phylogenetic footprinting. A number of studies have documented a role for the anterior Hox proteins in regulating rhombomere-specific gene expression by binding as hetero-dimers and -trimers with the TALE-class homeodomain proteins Pbx and Meis [17, 18, 35, 36]. These complexes bind to characteristic binding-sites composed of partially overlapping Pbx-Hox half-sites, frequently in conjunction with a distal Meis/Prep site [17, 18, 35, 36]. In some cases it has been shown that the pbx-hox motif is both necessary and sufficient for highly specific patterns of reporter expression, for instance for activity of a mouse hoxb1 enhancer in r4 in the mouse hindbrain [17] and for r4 and pharyngeal arch activity of a mouse hoxb2 enhancer [36].

We identified two Pbx-Hox motifs within CNE 3299, conforming to the TGATNNAT consensus [37, 38], that are conserved across all sequenced vertebrate genomes, each closely associated with conserved Meis motifs (TGACAG/A) [39] (Figure 2). In the zebrafish sequence, the first pair of Pbx-Hox and Meis motifs is also preceded by a Pbx-Meis motif (TGATTGACAG/A) [39]. We verified the essential nature of these motifs for rhombomere-specific activity of the enhancer through mutagenesis of the zebrafish element followed by reporter assay (Methods). Mutating the first cluster of motifs (sub1) resulted in a loss of the neural crest expression of the wild type enhancer and less anteriorly restricted expression in the hindbrain compared to the wild type element (Figure 2b, c). Mutation of the second Pbx-Hox and Meis motif cluster abrogated reporter expression by this enhancer altogether, whilst a construct in which both motif clusters were mutated (sub12) also drove no GFP expression. Interestingly, CNEs 3285, 3288 and 329X were also found to harbour conserved Pbx-Hox and Meis motifs. Together, the expression patterns of these elements in the hindbrain (3285, 3288, 3299, 329X), as well as in the pharyngeal arch neural crest (3299), suggests that these motifs may represent a common feature of CNEs that drive segment-specific expression patterns in the vertebrate hindbrain and pharyngeal arches.

Pbx-Hox motifs are enriched in CNEs and in other sets of conserved vertebrate enhancers

In order to address how widespread Pbx-Hox motifs are across conserved vertebrate enhancers, we performed a systematic scan for these motifs in vertebrate CNEs. We searched for instances of the canonical Pbx-Hox motif, TGATNNAT, that are completely conserved across CNE multiple sequence alignments. In a set of 246 alignments of CNEs between human, zebrafish, fugu and lamprey (Methods), we identified 61 conserved motifs, representing 22 fold enrichment over shuffled alignments (Methods and Additional File 3). Furthermore, in a set of 4259 gnathostome CNE alignments (of human, fugu and zebrafish sequences), 712 conserved motifs were identified; a 9 fold enrichment compared to shuffled alignment controls.

Further analysis of Pbx-Hox motifs in the gnathostome set reveals a paucity of cytosines at variable positions 5 and 6 (Figure 3). This is a feature of characterised Pbx-Hox binding-sites, where T, A or G at these positions contribute to determining the Hox specificity of the binding site [38, 40, 41]. Furthermore, positions 9 and 10, immediately 3' to the canonical Pbx-Hox motif, show strong bias towards G/T and A/G respectively, thereby defining a more stringent TGATNNATKR (KR) consensus motif that is also consistent with previously characterised Pbx-Hox binding-sites [17, 18, 37, 38] (Figure 3). Further analysis of the lamprey and gnathostome CNE alignment sets results in even stronger enrichment for this 'KR' motif (Additional File 3).

Pbx-Hox motifs are associated with hindbrain and pharyngeal arch CNE enhancer function

Next, we tested whether Pbx-Hox motifs within CNEs associate with segment-specific reporter expression in the hindbrain and pharyngeal arches. To do this, we assayed 21 zebrafish CNEs containing conserved Pbx-Hox motifs for reporter expression in zebrafish. All of these CNEs are conserved across gnathostomes, with 11 also identifiable in lamprey (Additional File 2). Elements were chosen to represent a range of different genes from the lamprey and gnathostome CNE sets. 12 of these 21 elements consistently up-regulate patterns of reporter expression, comprised of 8 from the lamprey set and 4 from the gnathostome set. It should be noted that some of the elements from which no consistent expression patterns were obtained may act as enhancers in-vivo, but not in our transient transgenic reporter assay, possibly due to being taken out of their genomic context. Remarkably, 11 of the 12 GFP-expressing elements (91.6%) drive expression either in the hindbrain, pharyngeal arches or both, with one element expressing in the trunk musculature (Figure 4). In support of the hypothesis that these elements are directly regulated by specific Hox proteins, which have segmentally-restricted expression patterns, the majority of the elements expressing in the hindbrain do so in particular rhombomeres, as shown by comparison with r3r5 RFP expression (Figure 4). Hindbrain reporter expression driven by these elements is often further restricted dorso-ventrally (e.g. Nkx6-1_4281), medio-laterally (e.g. Pax2_217) and temporally (e.g. Tshz3_43509).

We next examined functional data from the VISTA Enhancer Browser (EB). Compared to shuffled motifs, the KR motif was found to be significantly enriched in those elements annotated as hindbrain positive as well as those positive for either hindbrain, branchial arch or cranial nerve expression (Table 1). Investigating those EB elements that overlap CNEs from the CONDOR set, we found significant enrichment for the KR motif in those with hindbrain expression (HB+, 64 motifs in 112 kb) compared with those with no hindbrain annotation (HB-, 85 in 238 kb) (chi-square p = 0.0042). We then focused upon those sub-regions within EB enhancers that align directly with CNEs. Within these deeply conserved regions, there was more than two-fold enrichment for the stringent Pbx-Hox motif (30 occurrences in 24990 bp of HB+ elements compared with 32 occurrences in 60341 bp of HB- elements; p = 0.001). Importantly, this enrichment demonstrates that Pbx-Hox motifs in ancient CNEs show a correlation with hindbrain reporter expression. We also analysed a smaller dataset from the cneBrowser [21] that contains evolutionarily conserved enhancers associated with genes expressed in forebrain and hindbrain during zebrafish development. Although only 18 of 146 enhancers are annotated as hindbrain positive, 7 out of a total of 17 identified KR motifs reside in hindbrain positive enhancers (p = 3 × 10^-4) (Table 1).

CNEs containing Pbx-Hox motifs are associated with genes that have roles in A-P patterning of the hindbrain and head

There is good agreement between the genes highlighted by our in-silico binding-site search and by microarray screens for downstream targets of hoxb1 in rhombomere 4 of zebrafish [52] and mouse [53]. Specifically, the expression levels of znf503, tshz2, evi1, zic4, shox, and meis2.1 are decreased upon knock-down of HoxB1 in zebrafish, with Znf503, Nkx6-1, Atbf1 and Mab21l2 down-regulated in HoxB1-/- mouse embryos. Accordingly, the CNEs around each of these genes are enriched in Pbx-Hox motifs (Table 2 and Additional File 6). Thus, both microarray datasets are consistent with our prediction that Pbx-Hox motifs in CNEs represent direct regulatory links between Hox genes and their targets during development.

Discovery of Pbx-Hox motif enrichment is a further step toward de-coding CNEs

Despite a pervasive assumption that CNEs bind transcription factors in order to elicit gene activation, there is, perhaps surprisingly, very little direct evidence to confirm this. We sought to identify TFBS motifs in CNEs through phylogenetic footprinting, reasoning that the relatively high divergence of lamprey CNEs would highlight important motifs. The utility of this approach is confirmed by our identification of conserved Pbx-Hox and Meis TFBS motifs in CNEs. The enrichment of the Pbx-Hox TFBS motif in the jawed vertebrate CNE set reveals this motif to be a regulatory signature that is utilised by a large proportion of highly conserved cis-regulatory elements (the 6, 693 CONDOR CNEs contain 562 KR motifs and 1416 TGATNNAT motifs). Whilst enriched motifs identified in mammalian conserved elements include a few that show partial overlap with variants of the Pbx-Hox consensus motif [23, 24], the link between those enriched motifs and Hox factors had not been made, and their strong enrichment in more ancient CNEs had not been characterised. This enrichment agrees with the crucial, conserved roles of Hox factors in development of the vertebrate body plan. Indeed, the association of these motifs with hindbrain and pharyngeal arch enhancer function is in keeping with the characterised roles of Pbx, Hox and Meis factors in patterning these domains.

Despite the crucial roles of Hox factors in patterning the vertebrate embryo, relatively few downstream target genes, other than the hox genes themselves, have been identified. Our data suggests that Pbx-Hox motifs in CNEs can identify such targets. The striking manner in which Pbx-Hox and Meis TFBS motifs are highlighted as conserved sequence blocks in multiple alignments, especially when lamprey sequences are included, leads us to predict that this footprinting approach will be useful for further deciphering the regulatory code within vertebrate enhancers. In combination, our in-silico and functional analyses form an important link between well characterised cis-regulatory motifs and a large proportion of relatively uncharacterised ancient CNEs, helping to better place these elements within a developmental context. This represents a significant further step in systematically de-coding the enhancers responsible for development of the vertebrate body plan and highlights the utility of the lamprey as a model system for investigating vertebrate gene regulation.

The diversity of expression patterns driven by our tested elements suggests that Pbx-Hox TFBSs are just one component of a complex cis-regulatory logic encoded within these enhancers. Whilst responding to A-P patterning cues by interacting with particular Hox factors through Pbx-Hox TFBSs, these elements concomitantly determine the tissues in which they are active (e.g. hindbrain vs pharyngeal arch) and limit the expression patterns dorso-ventrally, medio-laterally and temporally. An example of this is the CNE Pax2_403, which drives reporter expression that is restricted to a ventro-lateral population of neurons in r2-3 of the hindbrain (Figure 4). Furthermore, whilst some of our functionally characterised CNEs drive reporter expression in domains with sharp boundaries that are co-incident with rhombomere boundaries -similar to that of previously characterised Pbx-Hox regulated elements - this is not the case for all of them. This could be due to the Pbx-Hox input establishing a competence for the enhancer to drive expression within particular rhombomeres, which is further restricted to specific sub-domains within the rhombomeres by the influence of other regulatory inputs to the enhancer. This would result in expression domains that do not encompass the whole area of expression of the regulating Hox factor. It is likely that the reason why many previously characterised Pbx-Hox regulated elements show expression domains across whole rhombomeres and with tight boundaries co-incident with rhombomere boundaries is that the majority of these elements are regulating Hox factors, and thus setting up or maintaining the rhombomere-specific Hox expression patterns. Many of the elements described in this study may be acting downstream of this Hox network, utilising these AP patterning cues along with other cues to further pattern the hindbrain. The tissue specificity of these enhancers, as well as the restriction of expression to specific domains and time points, is presumably due to other factors acting as specifiers by binding to nearby TFBSs. Identifying these specifiers and characterising their TFBSs, as well as the nature of their interactions with Hox factors, are key tasks toward understanding the cis-regulatory logic underlying vertebrate development. The set of putative Hox-responsive cis-regulatory elements identified in this study provides a powerful resource that will facilitate efforts toward this end.

Our expression data from the mutated versions of zebrafish CNE 3299 suggests that the multiple Pbx-Hox and Meis sites predicted in this enhancer may interact with each other, to co-operatively modulate and restrict reporter expression. The two clusters of Pbx-Hox and Meis motifs do not contribute equally to the expression driven by this enhancer in the hindbrain and pharyngeal arch neural crest. The second Pbx-Hox and Meis motif cluster appears to be necessary for the general function of this enhancer, as its mutation results in the loss of reporter expression in both hindbrain and neural crest. In contrast, the first Pbx-Hox and Meis motif cluster appears to be necessary (but not sufficient) for neural crest expression, but not for hindbrain expression. Conversely, it appears to restrict the hindbrain expression, as reporter expression is seen more anteriorly when this cluster is mutated. This is reminiscent of interactions between Pbx-Hox and Meis/Prep binding sites within a Hoxb1 enhancer, which direct expression of this gene to r4 the hindbrain in mouse and chick [54]. In that case, it was found that the formation of a Pbx-Hox-Meis/Prep ternary complex on Pbx-Hox and Meis sites within this enhancer could be restricted by the binding of a Pbx1-Prep1 heterodimer to a nearby site, thus limiting the expression driven by this enhancer to r4. This highlights the complexity of the regulatory interactions between transcription factors that are likely to bind to CNEs, a complexity that could well underlie their high sequence constraint.

A potential role for CNEs in the evolution of vertebrate head patterning

A strength of identifying conserved cis-regulatory elements is that they can provide compelling evidence for conserved GRNs. Our reporter assay data from zebrafish and lamprey embryos clearly demonstrate functional conservation of enhancers shared between the most distantly related extant vertebrate lineages. We deduce that all vertebrates share aspects of a GRN for hindbrain patterning, downstream of nested Hox expression. As the sea lamprey is from a vertebrate lineage that diverged prior to the evolution of many jawed vertebrate innovations, such as paired appendages and jaws [55], we predict that the lamprey reporter assay will be a crucial tool for investigating the gene regulatory changes involved in vertebrate evolution.

Without detailed knowledge of the function or mechanism of action of CNEs it has been difficult to derive scenarios of how they evolved and became fixed in vertebrate genomes. The findings from our in silico and functional analyses, coupled with previous characterisation of Pbx-Hox and Meis transcription-factor complexes, enable us to propose a hypothesis regarding the role of a large number of CNEs in vertebrate evolution. Recognising the same TFBS motifs in worms, flies and vertebrates, the Pbx, Hox and Meis factors are part of an ancient regulatory language shared across bilaterians [17, 36, 37, 56, 57]. Nevertheless, none of the CNEs containing these motifs are identifiable in invertebrate genomes, leading us to speculate that many of these elements may have arisen in the vertebrate lineage. Accordingly, our functional data suggest that many of these CNEs have roles in patterning an elaborate head and brain - key vertebrate innovations [54]. We hypothesise that the fundamental role of head patterning in vertebrates led to the functional conservation of these elements and that their reliance upon the precise organisation of TFBSs necessitated their strict sequence conservation. The mechanisms through which new cis-regulatory elements arise in the genome are still largely unresolved. In this case, the finding that simple Pbx-Hox sites are sufficient to drive robust and specific, but modifiable, expression [17] hints that these particular TFBSs may pioneer new cis-regulatory elements, functioning as one of the fundamental seeds from which many CNEs were able to grow.

Identification of CNEs

6, 693 non-redundant human CNEs (average length 116 bp) were retrieved from the CONDOR database [43] at http://condor.nimr.mrc.ac.uk (Additional File 7). We used these to search lamprey sequence reads available from the NCBI trace server at http://www.ncbi.nlm.nih.gov/Traces/trace.cgi with sensitive parameters (-W 7 -q -1 -e 5e-4) as described previously [28]. The lamprey trace sequences were searched because they represent a greater coverage of the lamprey genome than the publicly available draft genome assembly, which consists of many short contigs and thus provides little advantage with regard to identification of conserved syntenic regions. Lamprey sequences satisfying the initial parametric threshold were further analysed for contamination, and those with > 90% homology to human or chicken across the whole read (i.e. extending outside the evolutionarily conserved region in other vertebrates) were removed.

Alignments

The sequences of human, fugu and most zebrafish CNEs were retrieved from the CONDOR database [57]. Additional zebrafish CNEs that were not previously included in the CONDOR database due to absence from earlier assemblies were identified using BLAST against a more recent zebrafish genome assembly (Zv8 release 58). Sequences in each alignment were clipped to the same size to prevent unaligned edges. To align the sequences we used ClustalW version 1.83. These alignments formed the lamprey CNE set (comprised of alignments of CNEs from human, fugu, zebrafish and lamprey) and the gnathostome CNE set (containing alignments of CNEs from human, fugu and zebrafish). As a control, for each CNE we also generated 1000 multiple alignments by randomly shuffling the columns of each alignment using the seqboot implementation in Phylip version 3.67. The sequences of lamprey and zebrafish CNEs in these datasets are given in Additional File 8 and Additional File 9 respectively. The sequences of CNEs from the EB and cneBrowser datasets are given in Additional File 10 and Additional File 11 respectively.

Scanning CNEs and control sets for Pbx-Hox motifs

We searched for Pbx-Hox motifs in two different types of datasets. Firstly, in multiple sequence alignments of CNEs from the CONDOR CNE database (the lamprey and gnathostome CNE sets). Secondly, in datasets consisting of sequences from just one species (the EB CNEs, shark CNEs, human elements from the CONDOR CNE set, cneBrowser CNEs). The two different types of datasets required different types of control to test for Pbx-Hox motif enrichment. For the alignment sets, we generated control sets of shuffled alignments. To find evolutionarily conserved Pbx-hox motifs (TGATNNAT and TGATNNATKR) we employed the software Cis-Finder [42] on our two alignment sets and their respective shuffled alignment controls. A motif match was only considered if it matched all aligned species and occurred at the exact same aligned position. For the single species sequence sets we generated shuffled motifs, based upon the KR motif, as a control to search across the same sets. In parallel we also employed a de-novo motif finding strategy implemented in Cis-Finder on the CONDOR CNE set. It scans a set of DNA sequences for over-represented position frequency matrices (PFMs), clusters these and then estimates significance using the false discovery rate [42]. The TGATNNAT and TGATNNATKR motif occurances in the CONDOR CNE set are detailed in Additional File 12 and Additional File 13.

To characterise the frequency of Pbx-Hox KR motifs in different gene loci, we used the CONDOR CNE set, in which CNEs are grouped according to gene locus as specified in the CONDOR database [57]. For each gene locus, we counted the frequency of Pbx-Hox KR motifs in the associated CNEs and compared this to the average frequency in 1000 sets of randomised versions of CNEs from that locus. A markov chain model of order zero was used to generate shuffled sequences. To model DNA sequences, 4 states (A, C, G, T) and 4 transitions were used. Transition probabilities were retrieved from the CNE set by calculating the relative frequencies of the bases.

Measuring relative enrichment of Pbx-Hox motifs

To measure the enrichment, we compared the occurrence of Pbx-Hox motifs in a test set against shuffled versions (Table 1), calculated mean and standard deviation and generated z-scores. The z-scores were then transformed into p-values under a normal distribution model. We also counted Pbx-Hox occurrence and shuffled versions in a number of control regions and across the whole human genome (Additional File 4).

Overlap with other evolutionarily conserved 'enhancer' sets

There is inevitably some overlap between the different sets of evolutionarily conserved sequences. 482/1307 EB human sequences overlap 994 CONDOR CNEs, covering a total of 146226 bases (7.4% of the EB sequence; 18.8% of CNE sequence). 1632 human sequences identified through comparison with Callorhinchus milii [13] overlap 2172 CONDOR CNEs, covering a total of 271260 bases (26.5% of the Callorhinchus dataset; 34.9% of CNEs). Finally, 69/146 zebrafish cneBrowser sequences overlap 83 CONDOR CNEs, covering a total of 11496 bases (20.5% of the cneBrowser sequence; 1.5% of CNE sequence).

Zebrafish transgenesis

CNEs were amplified from zebrafish and lamprey genomic DNA by PCR, sub-cloned into the Pcr8/GW/TOPO vector (Invitrogen) and then into a Tol2 construct (pGW_cfosEGFP) [29, 58, 59], using the Gateway LR Clonase II enzyme (Invitrogen). The Tol2 reporter assay was performed as described previously [29]. Transient transgenic zebrafish embryos were screened for GFP expression at 24-30hpf, 48-54hpf and 72-78hpf using a Leica M165FC microscope and photographs taken with a Leica DFC310FX camera. Expression patterns were deemed consistent when found in > 20% of founders, consistent with previous studies [25, 60].

CNE Mutagenesis

Mutations in zebrafish CNE 3299 were introduced by PCR from genomic DNA with primers containing the desired mutations either through conventional PCR (for sub1) or megaprimer PCR (for sub2) [61]. Mutated CNE PCR products were then cloned for zebrafish transgenesis as described above.

Lamprey transgenesis

The transgenesis protocol was based upon that developed in Xenopus [62]. Lamprey CNEs were amplified from genomic DNA and cloned into the cFos-I-sceI-EGFP plasmid, which contains the mouse cFos minimal promoter and EGFP coding sequence flanked by I-sceI restriction sites. Plasmids were extracted using the EndoFree Plasmid Maxi Kit (Qiagen) and eluted with water through QIAQuick columns (Qiagen). Fresh restriction digests (20 μl containing 400 ng plasmid, 15 units I-SceI enzyme (NEB), 1 × I-SceI buffer + BSA, digested for 40 minutes at 37°C) were micro-injected into 5-6hpf lamprey embryos using a Pico-Spritzer with drop volume of 2-3 nl. Lamprey husbandry was performed as described previously [63]. Embryos were screened for GFP expression between embryonic days 7-16. Typical survival rates ranged from 20-50% of injected embryos. The promoter alone drives highly mosaic background expression in the ectoderm in roughly 50% of surviving embryos. Enhancer-specific expression was seen in approximately 10% of surviving embryos.