409 (2016) 530–542

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Developmental Biology

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Evolution of Developmental Control Mechanisms Analyses of fugu hoxa2 genes provide evidence for subfunctionalization of neural crest cell and rhombomere cis-regulatory modules during evolution

Jennifer A. McEllin a, Tara B. Alexander a, Stefan Tümpel a,1, Leanne M. Wiedemann a,b, Robb Krumlauf a,c,n a Stowers Institute for Medical Research, Kansas City, MO 64110, USA b Department of Pathology and Laboratory Medicine, Kansas University Medical Center, Kansas City, KS 66160, USA c Department of Anatomy and , Kansas University Medical Center, Kansas City, KS 66160, USA article info abstract

Article history: Hoxa2 gene is a primary player in regulation of craniofacial programs of head development in verte- Received 3 October 2015 brates. Here we investigate the evolution of a Hoxa2 neural crest enhancer identified originally in mouse Received in revised form by comparing and contrasting the fugu hoxa2a and hoxa2b genes with their orthologous and 8 November 2015 mammalian sequences. Using sequence analyses in combination with transgenic regulatory assays in Accepted 8 November 2015 zebrafish and mouse embryos we demonstrate subfunctionalization of regulatory activity for expression Available online 26 November 2015 in hindbrain segments and neural crest cells between these two fugu co-orthologs. hoxa2a regulatory Keywords: sequences have retained the ability to mediate expression in neural crest cells while those of hoxa2b Hox genes include cis-elements that direct expression in rhombomeres. Functional dissection of the neural crest Neural crest regulatory potential of the fugu hoxa2a and hoxa2b genes identify the previously unknown cis-element Hindbrain NC5, which is implicated in generating the differential activity of the enhancers from these genes. The Gene regulation Enhancers NC5 region plays a similar role in the ability of this enhancer to mediate reporter expression in mice, Evolution of cis-elements suggesting it is a conserved component involved in control of neural crest expression of Hoxa2 in ver- tebrate craniofacial development. & 2015 Elsevier Inc. All rights reserved.

1. Introduction jaw elements, such as Meckel’s cartilage (Gendron-Maguire et al., 1993; Rijli et al., 1993; Santagati et al., 2005). Conversely, Hoxa2 In , Hoxa2 is an important regulator of both hind- gain-of-expression experiments in a number of vertebrates, in- brain development and cranial neural crest patterning. Mouse cluding mice, Xenopus and chickens have shown that ectopic loss-of-function studies have revealed multiple roles for Hoxa2 Hoxa2 represses jaw formation (Grammatopoulos et al., 2000; during central nervous system (CNS) development, including Kitazawa et al., 2015; Pasqualetti et al., 2000), in part through its regulation of properties of specific hindbrain segments or rhom- ability to prevent chondrogenesis and inhibit bone formation bomeres (r) (Davenne et al., 1999; Gavalas et al., 1997; Oury et al., (Kanzler et al., 1998). In compound mouse mutants where Hoxa2 2006; Ren et al., 2002). In head development, Hoxa2 expression is and Hoxb2 are both lost, there appears to be very little difference also spatially-restricted in cranial neural crest cells where con- in , suggesting the Hoxb2 paralog has a relatively minor vincing evidence implicate it as a master regulator of craniofacial input into cranial neural crest patterning (Santagati and Rijli, 2003). However, deletions of entire Hox clusters indicate that programs and jaw formation (Couly et al., 2002, 1998; Hunt et al., other HoxA genes and HoxB genes do contribute to craniofacial 1991a). Mouse Hoxa2 / mutants display duplications of lower regulatory programs (Minoux et al., 2009; Vieux-Rochas et al., 2013). There is evidence in zebrafish (Danio rerio), based on mor- n Corresponding author at: Stowers Institute for Medical Research, 1000 50th, pholino experiments, that the functional roles for hoxa2 are par- Kansas City, MO 64110, USA. tially compensated for by its paralog hoxb2, as both genes must be fl E-mail addresses: tuempel@ i-leibniz.de (S. Tümpel), knocked down to generate phenotypes analogous to those ob- [email protected] (R. Krumlauf). 1 Current address: Leibniz Institute for Age Research, Fritz Lipmann Institute, served in mouse Hoxa2 mutants alone (Hunter and Prince, 2002). Beutenbergstrasse 11, 07745 Jena, Germany. In most vertebrates Hoxa2 is proposed to be a primary node of http://dx.doi.org/10.1016/j.ydbio.2015.11.006 0012-1606/& 2015 Elsevier Inc. All rights reserved. J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 531 transcriptional regulation in controlling craniofacial programs to temporally dynamic patterns of expression in cranial neural crest facilitate proper formation of the vertebrate jaw and pharyngeal cells important for craniofacial patterning, has been identified arches (Minoux and Rijli, 2010; Santagati and Rijli, 2003). upstream of the mouse gene (Maconochie et al., 1999). This neural Following their divergence from the , the or crest enhancer contains four separate regions (NC1-NC4) which ray-finned fish underwent genome duplication (Meyer and Mala- are all required for regulatory activity. The NC4 cis-element con- ga-Trillo, 1999; Meyer and Schartl, 1999; Meyer and Van de Peer, tains binding sites for the AP2 family of transcription factors, 2005; Ravi and Venkatesh, 2008; Smith and Keinath, 2015; Volff, known to be important for neural crest development (Mitchell 2005). This led to the duplication of a number of genes relative to et al., 1991; Morriss-Kay, 1996; Schorle et al., 1996). mammals, including Hox genes. The mouse genome has a single The Hoxa2 neural crest enhancer is embedded within a highly Hoxa2 gene that is expressed in the second branchial arch and in conserved enhancer that mediates segmental expression of the hindbrain rhombomeres, while there are two hoxa2 co-ortholo- gene in r3 and r5 (Maconochie et al., 1999, 2001; Nonchev et al., gous genes present in striped bass (Morone saxatilis), fugu (Taki- 1996a, 1996b; Parker et al., 2014). Hence, it has been challenging fugu rubripes), Nile tilapia (Oreochromis niloticus), cichlid (Astato- to characterize the evolution of this neural crest enhancer in- tilapia burtoni), and medaka (Oryzias latipes). These duplicated dependent of constraints on the rhombomeric enhancer. In reg- hoxa2 genes can display divergent patterns of expression in their ulatory analyses of the two fugu Hoxa2 co-orthologous genes, respective teleost species. For example, in striped bass hoxa2a is hoxa2a and hoxa2b, using reporter genes in transgenic chicken expressed in r2-r7 and in the second pharyngeal arch while hox- embryos, evolutionary divergence in key cis-elements of the en- a2b is expressed in r2-r5; whereas in fugu hoxa2a is expressed in hancer regions that mediate segmental expression in hindbrain thin stripes in r1-r2 while hoxa2b is expressed in r2-r5 (Scemama rhombomeres has previously been shown to account for their et al., 2002, 2006). This implies that there has been significant differential (Tümpel et al., 2006). However, no alteration in the cis-elements that govern hindbrain and phar- reproducible neural crest regulatory activity with either enhancer yngeal arch expression leading to divergent patterns for hoxa2 co- was detected in these assays in chicken embryos. It is surprising orthologs in several teleosts. Through mutation some teleosts have that the regulatory assays of the fugu hoxa2a and hoxa2b enhancer lost one of the duplicated hoxa2 genes or one co-ortholog has in chicken embryos failed to uncover any robust cranial neural become a non-functional pseudogene. Examples of this are me- crest regulatory potential in either enhancer. In light of the fact daka, with a single functional hoxa2a gene and a hoxa2b pseudo- that in zebrafish Hoxb2 partially compensates for the function of gene, and zebrafish with a functional hoxa2b and a hoxa2a pseu- Hoxa2 in neural crest cells (Hunter and Prince, 2002), it is possible dogene (Davis et al., 2008; Hunter and Prince, 2002). In both these that the fugu Hoxa2 genes are not expressed in neural crest and instances the single functional hoxa2 ortholog has an expression their functional roles have been taken over by Hoxb2. Alter- pattern which matches that of mouse Hoxa2, in contrast to the natively, cis-elements directing neural crest expression in fugu variant expression patterns seen with the two functional co-or- may not function effectively in the chicken embryo or there may thologs in striped bass and fugu. be species-specific differences in their requirements. This gene duplication, divergence, and differential expression In this study, since Hoxa2 has such a key role in craniofacial in teleosts can be informative from a regulatory standpoint in patterning in many vertebrates, we addressed this important helping to identify cis-elements required for tissue-specific gene problem by investigating the evolution of the neural crest reg- expression. Functional domains within and around a gene have a ulatory potential of the fugu hoxa2a and hoxa2b enhancers. Our slower rate of change compared to non-functional domains, pre- findings reveal that there has been subfunctionalization of neural sumably due to selective pressures. However, following gene du- crest activity between the two co-paralogs, such that fugu hoxa2a plication, functional regions of one gene can be free to mutate if its appears to retain the ability to mediate expression in neural crest paralog preserves or fulfills the relevant functions. Through this cells while hoxa2b possesses the cis-elements that direct expres- process, paralogous genes can adopt new functions (neofunctio- sion in rhombomeres. Furthermore, our sequence comparisons nalization) or partition ancestral functions between the duplicate and regulatory analyses have uncovered an additional cis-element genes (subfunctionalization) (Jimenez-Delgado et al., 2009). An (NC5) which plays a conserved role in potentiating neural crest example of regulatory subfunctionalization can be seen when regulatory activity of this enhancer. comparing mouse Hoxb1 to the zebrafish genes, hoxb1a and hoxb1b. Zebrafish hoxb1b is expressed in early gastrula stage em- bryos, like its mouse counter-part, through a conserved pair of 2. Materials and methods response elements (RAREs) located downstream of both the mouse and zebrafish genes (Marshall et al., 1994; 2.1. Transgenic zebrafish and mouse reporter assays McClintock et al., 2001, 2002). In contrast, the zebrafish hoxb1a gene is the co-ortholog expressed in r4, like the mouse gene, Slusarski AB (wild type) and egr2b:KalTA4BI-1UASkCherry (r3r5- through an equivalent highly conserved Hox auto- and cross-reg- mCherry) (Distel, Wulliman, Koster 2009) lines were maintained ulatory cis-element (McClintock et al., 2002; Pöpperl et al., 1995; at 28 °C. Embryos were raised in Embryo Medium (Nüsslein-Vol- Studer et al., 1998, 1994). Therefore, each individual zebrafish hard and Dahm) and staged according to hours post-fertilization hoxb1 co-ortholog has maintained separate functions of the an- (hpf). Enhancer regions to be tested were inserted, using Gateway cestral Hoxb1 gene, in part through conservation and divergence of cloning, into a Tol2 transposon based vector containing a cFos cis-regulatory elements essential for modulating distinct aspects of minimal promoter-EGFP reporter cassette (pGW-cfos-EGFP) the expression pattern. Therefore, non-coding sequences con- (Fisher et al., 2006). Transient transgenic fish embryos (F0)were served only in teleost paralogs, whose gene expression matches injected at the 1-cell stage with an injection mix of phenol red specific aspects of their counter-parts, have the potential (0.05%), Tol2 transposase (Fisher et al., 2006) and the expression to be associated with distinct regulatory activities. vectors. Each embryo was injected with a bolus (visualized by the Compared with hindbrain rhombomeres and , phenol red) one-fifth the size of the cell formed once the cyto- very little is known about the upstream regulatory network, sig- plasm begins to separate from the yolk towards the animal pole. A nals and transcription factors that couple Hox expression to cranial minimum of 350 embryos were injected to account for mosaicism neural crest patterning (Hunt et al., 1991a, 1991b). With respect to and position effects. Embryos expressing GFP were raised to Hoxa2, an enhancer that mediates its spatially-restricted and adulthood and crossed to wild-type fish to create stable transgenic 532 J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 lines. Embryos were screened and imaged with either a Leica 1996b; Tümpel et al., 2006). Fugu hoxa2 hybrid constructs were M205FA microscope coupled to a Leica DFC360FX camera with LAS designed initially based on positioning of elements homologous to AF imaging software or a Zeiss confocal LSM-510 and ZEN soft- known regulatory elements of the mouse enhancer and subse- ware. Images were cropped and altered for contrast and brightness quently in response to data obtained from previous hybrids. For a using Adobe Photoshop CS5.1 and NIH ImageJ. schematic of the hybrids created, complete with the exact bases Transgenic mice were generated as previously described (Ma- used from the fugu hoxa2a and hoxa2b enhancers, see Table 1. conochie et al., 2001). Candidate regulatory regions were inserted Constructs were created by PCR with primers (Table 1) designed to in the pBGZ40 reporter cassette which contains a minimal human create products with overlapping sequence for the formation of β-globin promoter linked to the β-galactosidase gene (Yee and constructs with DNA from multiple sources. Topo cloning (Fisher Rigby, 1993). Inserts were released from the vector by digestion et al., 2006) was used to introduce constructs into a cloning vector with the appropriate restriction enzyme followed by separation followed by Gateway site cloning (Hartley et al., 2000) to move the via gel electrophoresis and recovery of the insert by agarose gel construct into the expression vector pGW-cfos-EGFP. extraction. The recovered DNA was injected into the pronucleus of All original source data are deposited in the Stowers Institute fertilized eggs which were reimplanted into foster animals. Em- Original Data Repository at the time of publication and available bryos were harvested and analysed at 9.5 days post coitum. All online at http://odr.stowers.org/websimr/. animal experiments were performed in accordance with the re- commendations and guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and 3. Results protocols were approved by the Institutional Animal Care and Use Committees of the Stowers Institute (RK protocols #2013-0110 and 3.1. Identification of a fugu hoxa2a neural crest cell enhancer mouse protocol number #2013-0114). We sought to investigate the regulatory potential of putative 2.2. Enhancer elements rhombomeric and neural crest enhancers upstream of fugu hoxa2a and hoxa2b to determine whether activity of this enhancer cap- Fugu hoxa2a and hoxa2b NCC r3/5 enhancers were identified ability has been lost, retained, or altered in the duplicate fugu based on sequence alignment compared to the known murine NCC hoxa2 genes. In previous regulatory studies using chicken em- r3/5 enhancer (Maconochie et al., 2001; Nonchev et al., 1996a, bryos, no robust neural crest regulatory activity with either the

Table 1 Primers used to generate specific regions for hybrid construction. Hybrids were generated by combining specific sequences from fugu hoxa2a and hoxa2b r3/5 NC enhancers using PCR fragments to produce chimeric enhancers. The numbers are base pairs coordinates within the enhancers with 1 as the 5′ most base pair.

Hybrid Composition

Construct Fugu hoxa2a (red) Fugu hoxa2b (gray) Primers used

fr-hoxa2a #1 1-1485 0 FW: tggcttaatgcaaacgctat REV: ccattaagttaacactgacagatat fr-hoxa2b 0 1-1263 FW: tgctgtaatgccaaaacctcagattaaaag REV: cctgcctcgccttcgtgccg Hybrid 1 1-814 682-1263 FW: tggcttaatgcaaacgctat REV: ttcacgccaaaaggtctgacagctcggtgccctcgcaaca FW: tgttgcgagggcaccgagctgtcagaccttttggcgtgaa REV:cctgcctcgccttcgtgccg Hybrid 2 1-441 401-1263 FW: tggcttaatgcaaacgctatatttaatatatgtattttttg REV: gatgaagatcggaattgttttgctcagagatgttcagggaa FW: caatctcttcccctgaacatctctgagcaaaacaattccgatcttcatc REV: cctgcctcgccttcgtgccgcgtgcgtggccgtttcctgcg Hybrid 3 441-1485 1-402 FW: ccgcggtgctgtaatgccaaaacctc REV: gaacccagaccttgtagtgaaggacttagtgctatagaaac FW: gatatgagatcgtcttcaaacgtgtcatttgggctgtc REV: gcggccgcccattaagttaacactgaca Hybrid 4 794-1485 1-669 FW: ccgcggtgctgtaatgccaaaacctc REV: ttactcgccaaaaggtctgacagctccctgccctaagaaca FW: tgttcttagggcagggagctgtcagaccttttggcgagta REV: gcggccgcccattaagttaacactgaca Hybrid 5 1-441; 814-1485 402-682 FW: tggcttaatgcaaacgctatatttaatatatgtattttttg REV: gtgcgcgatcaatcttactcacagctccctgccctaagaacaca FW: tgtgttcttagggcaagggagctgtgagtaagattgatcgcgcac REV: ccattaagttaacactgacagatat Hybrid 6 441-814 1-401; 682-1263 FW: tgctgtaatgccaaaacctcagattaaaagctgaaacag REV: gtgatcaatctttcacgccaaaaggtctgacaggccaaaaggtctgacagctcgg FW: ccgagctgtcaaccttttggcctgtcagaccttttggcgtgaaaga REV:cctgcctcgccttcgtgccgcgtgcgtggccgtttcctgcg Hybrid 7 441-670 1-402; 579-1263 FW: tgctgtaatgccaaaacctcagattaaaagctgaaacag REV: ggtgtgcataggacacattttcttttcgctggagggatcttttaaga FW: cctatttatctcttaaaagatccctccaaaagaaaatgtgtcc REV: cctgcctcgccttcgtgccgcgtgcgtggccgtttcctgcg fr-hoxa2a #2 441-662 0 FW: cgctggagggatcttttaagag REV: gtccttcactacaaggtctggg fr-hoxa2a #3 441-814 0 FW: cgctggagggatcttttaagag REV: cgccaaaaggtctgacagctcgg

For schematics of hybrid constructs see Figs. 4 and 5. All constructs begin with 1 as the most 5’ component J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 533

Fig. 1. A conserved enhancer upstream of the duplicated fugu hoxa2 co-orthologs displays subfunctionalization of regulatory activity between neural crest cells and hindbrain segments. (A) Schematic of an enhancer from the mouse Hoxa2 gene mapping the known regulatory elements. Boxes mark neural crest cell activity (NC) in green and r3/r5 activity (RE) in purple. (B-D) F0 transgenic zebrafish embryos at 48 h post fertilization (hpf) expressing a GFP reporter gene under the control of enhancers from zebrafish hoxa2b (zf-Hoxa2b) (B), fugu hoxa2a (fr-Hoxa2a#1) (C) or fugu hoxa2b (fr-Hoxa2b#1) (D). (E-G) F0 transgenic mouse embyos at 9.5 dpc expressing the LacZ reporter gene under the control of enhancers from mouse Hoxa2 (m-Hoxa2) (E), fugu hoxa2a (fr-Hoxa2a#1) (F) or fugu hoxa2b, fr-Hoxa2b#1 (G). Red¼RFP expression driven by a Krox20 enhancer in r3 and r5; green¼GFP expression; dotted line¼otic vesicle (OV). In B-G neural crest (NC) cells are marked with arrows and r3/r5 with arrowheads. fugu hoxa2a or hoxa2b enhancer was observed (Tümpel et al., zebrafish reporter assay, whereas the chicken enhancer did not 2006). Therefore, we explored the basis of this finding in more (data not shown). These results imply that in the chicken, Hoxa2 detail. Initially, we tested the regulatory activity of both the mouse expression in cranial neural crest may have evolved some distinct and chicken Hoxa2 neural crest enhancer regions. Surprisingly, the regulatory features that differ from its orthologs in other enhancer from the chicken gene did not function in mouse em- vertebrates. bryos and the enhancer from mouse displayed no neural crest Next, we tested the fugu enhancers for regulatory activity in activity in chicken embryos (data not shown). This uncovered zebrafish embryos. Consistent with our previous study (Tümpel species-specific differences in regulatory requirements. In an at- et al., 2006), the enhancer from fugu hoxa2b directed robust re- tempt to minimize such factors in analysis of the fugu Hoxa2 porter expression in hindbrain rhombomeres (r3–r5), but not in genes, we performed reporter assays in zebrafish to evaluate its neural crest (Fig. 1D). In contrast, the enhancer from fugu hoxa2a potential. The putative neural crest enhancer of the zebrafish mediated robust reporter-generated fluorescence in cranial neural hoxa2b gene, based on sequence homology to mouse, was linked crest and little or no rhombomeric expression (Fig. 1C). To explore to a GFP reporter in a Tol II vector and assayed for activity (Fig. 1A the degree to which this neural crest regulatory activity is con- and B). This region mediated robust expression in both cranial served, we also tested these same fugu enhancers on lacZ reporter neural crest cells and hindbrain rhombomeres (Fig. 1B). Intrigu- constructs in transgenic mice and compared them with the mouse ingly, the mouse neural crest enhancer also functioned in this enhancer (Fig. 1E–G). In agreement with the zebrafish assays, fugu 534 J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 hoxa2a mediated robust reporter staining in mouse cranial neural et al., 1999, 2001). Fig. 1A is a schematic showing the nested ele- crest cells, while hoxa2b directed weak expression in r3 and r5 of ments and binding sites associated with re- hindbrain. These results demonstrate that following duplication spective domains of enhancer activity. There is a 5′ bias in the and divergence, only one of the two fugu hoxa2 duplicates, hoxa2a, rhombomeric cis-elements, a 3′ bias in neural crest regulatory has maintained the potential to direct expression in neural crest elements and extensive overlap of both in the core of the cells, which corresponds to only one of the functions of the or- enhancer. thologous Hoxa2 enhancers of mouse and zebrafish. This supports Sequence alignment (AlignX; Lu and Moriyama, 2004) of this a model where both fugu hoxa2 co-orthologs were retained after enhancer amongst placental mammals reveals a high degree of duplication at least in part through subfunctionalization in reg- conservation over the entire regulatory region (Fig. 2A). Sequence ulatory activity, whereby the neural crest activity has been re- identity is particularly high in the specific cis-elements previously tained by the fugu hoxa2a gene, and the rhombomeric activity by shown to be essential for mediating activity of the mouse en- the hoxa2b gene. hancer in rhombomeres (RE1-RE5) and neural crest cells (NC1- NC4). The relatively small breaks in conservation are concentrated 3.2. Sequence alignment of the neural crest enhancer in areas between the known cis-elements, however, overall se- quence similarity even in these areas remains high. Many of these Based on the functional identification of a fugu hoxa2a neural breaks in identity correspond to small insertions or deletions in crest enhancer we generated sequence comparisons between some species which disrupt alignments (Fig. 2A). Expanding the species in an attempt to examine conservation and differences in sequence alignment beyond placental mammals to include more cis-regulatory elements that underlie the neural crest regulatory evolutionarily distant vertebrates, including mouse, chick, zebra- activity. Previous analyses have defined a series of cis-elements fish and both fugu hoxa2 genes, reveals a very different level and necessary for r3, r5 and neural crest expression within an 809 bp pattern of conservation (Fig. 2B). Very little of the enhancer is region in a 5′ flanking enhancer of mouse Hoxa2 (Maconochie conserved, and the sequence identity is largely confined to the

Fig. 2. Sequence alignments of the Hoxa2 enhancer from different groups of vertebrate. (A) Alignment of the Hoxa2 r3/5-NC enhancer from representative placental mammals. This homology is particularly high in regions known to be necessary for neural crest or hindbrain expression. In A and B yellow indicates identical sequence in all aligned species and blue indicates identical sequence in a majority of compared sequences. (C) Drawing illustrating the relative size, in bases, of the representative r3/5 and NC regulatory elements using rhombomere element (RE1) and the ATG start site as key landmarks. Rhombomere elements: RE, purple boxes; neural crest elements: NC, green boxes. Gene and species are noted at the left. J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 535

Krox20/BoxA sites and the core region of the enhancer, char- suggests that these neural crest elements may be specific for acterized by the overlap of rhombomeric elements RE3/RE2 and mammals. NC2/NC3. Only one of the two Krox20 binding sites, conserved in This overall decrease in sequence conservation is due at least in all mammals characterized to date, is present in these broader part to variations in the relative size and positioning of key cis- vertebrate alignments. There is minimal sequence similarity in the elements within the enhancer of these species. This is illustrated most 3′ region of the enhancer corresponding to the mouse NC4 by using the RE1 element as the most 5′ boundary and NC1 as the and NC1 elements. This lack of conservation in NC4 and NC1 most 3′ border to estimate the size of the respective enhancers for

Fig. 3. Sequence alignments of the enhancer from teleost paralogs of hoxa2a and hoxa2b. (A,B) Sequence alignments of the hoxa2a enhancer (A) and the hoxa2b enhancer (B) from teleost species fugu, tilapia, cichlid, zebrafish and medaka. Sequence highlighted in yellow indicate identical sequence for all four species while blue represents identical sequence for three and green two of the species. Thick lines above the sequence indicate rhombomeric elements (RE) in purple and below neural crest (NC) elements in green. 536 J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 each species (Fig. 2C). Using these parameters, the mouse en- enhancer, represented by enhancers driving the single Hoxa2 hancer is 809 bp while fugu hoxa2a enhancer is the largest at genes found in most tetrapods. Comparing sequences from the 1347 bp (Fig. 2C). This size difference results in alignment gaps remaining functional teleost hoxa2a enhancer further highlights arising from insertions and deletions in the mouse, chick and the areas of high conservation of sequences in cis-elements or- zebrafish sequences relative to the fugu sequences, particularly in thologous to the mouse regions: RE1, Krox20, BoxA and the core the 5′ region of the enhancer (Fig. 2B and C). In addition, the re- region with RE2-RE5, NC2 and NC3 (Fig. 3A). In addition, con- lative positions of the enhancer upstream of the ATG varies within servation at a lower level extends both upstream of RE4/NC2 and each species based on the location of the most 3′ element, NC1. downstream of RE5/NC3. A different pattern is observed in align- The zebrafish and fugu enhancers all ended less than 1 kb from the ing the sequences of teleost hoxa2b enhancers. There is a greatly hoxa2 ATG start site and as close as 423 bp for fugu hoxa2b, while reduced overall degree of conservation compared to teleost hoxa2a the mouse and chick enhancers ended significantly farther up- (Fig. 3B). Sequence conservation of the Krox20 and BoxA binding stream, 1795 bp and 1643 bp respectively (Fig. 2C). These differ- sites, RE2, RE3, RE5 and NC3 remains high. However, there is re- ences in spacing and conservation may be a contributing factor in duced identity in the RE1, RE4/NC2, NC1 and NC4 cis-elements and the species specific differences in regulatory activity of the the remainder of the sequence upstream and downstream of the enhancers. core components of the enhancer displays very little conservation, We extended our sequence comparisons to include other tel- especially when compared to the hoxa2a alignment (Fig. 3). eost fishes (tilapia, cichlid and medaka) to search for novel con- This analysis implies that the enhancer for the teleost hoxa2a served regions in the fugu hoxa2a and hoxa2b enhancers because a genes has been relatively fixed since the fish-specific duplication, number of teleost family members also have duplicated Hoxa2 with less evidence for change. In contrast, there is more evidence genes (Fig. 3). However, hoxa2a in zebrafish and hoxa2b in medaka for variation of the hoxa2b enhancer, based on the sequence di- (Davis et al., 2008) have become pseudogenes. In these species, the vergence inside and outside of the core region of the enhancer enhancer upstream of the remaining functional hoxa2 ortholog sequence. Despite the variation there is sufficient conservation to would be predicted to retain all of the functions of the ancestral maintain key cis-elements required for regulating hindbrain

Fig. 4. Transgenic analysis of chimeric fugu hoxa2a/hoxa2b enhancers in zebrafish embryos. (A-D) GFP reporter expression mediated by Hybrids 1-4 in neural crest cells (NC) in pharyngeal arch tissue (arrows) or rhombomeres 3 and 5 (arrowheads) in 48hpf zebrafish embryos. In A-D red¼RFP expression in r3 and r5 driven by a control Krox20 enhancer; green¼GFP expression driven by enhancer construct. (E) Drawing of the constructs mapping the portion of the hoxa2a and hoxa2b enhancers in each hybrid compared to the annotated mouse Hoxa2 enhancer at top. The purple box¼ rhombomere elements; green box¼ neural crest elements; red¼hoxa2a enhancer sequence; gray¼hoxa2b enhancer sequence. The table at the right of each construct describes the F0 transgenic expression data marking presence (þ) or absence () of GFP expression in neural crest or r3/r5; # of embryos with expression in neural crest and/or r3/5 (#relevant expression); # of embryos with any GFP expression; total # embryos injected. The number of stable transgenic lines created (F1s) are noted, far right. J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 537 expression in r3 and r5 (Maconochie et al., 2001; Nonchev et al., 1 and 2, which combine two different 5′ regions of fugu hoxa2a 1996a; Tümpel et al., 2006). with the remaining 3′ portion of fugu hoxa2b (Fig. 4A, B and E). GFP expression is detected in neural crest cells with Hybrid 1 but 3.3. Mapping differences in neural crest elements of the fugu hoxa2a not the hindbrain (Fig. 4A and E), indicating that sequences in this and hoxa2b enhancers 814 bp region are sufficient to confer regulatory activity in neural crest on the remaining region of the Hoxa2b enhancer. In contrast, These sequence comparisons open the possibility that the Hybrid 2 containing the first 441 bp of hoxa2a, displays hindbrain conservation in flanking regions of known cis-elements in hoxa2a expression but lacks reporter expression in neural crest cells but not hoxa2b might be important for regulation of neural crest (Fig. 4B and E). These hybrids indicate the presence of a key cis- activity in the enhancer of fugu and other teleosts. To test this idea element between base pairs 441 and 814 in hoxa2a important for and search for additional cis-regulatory elements underlying the regulation in neural crest cells (Fig. 4). differential ability of these enhancers to mediate neural crest ex- To evaluate the contributions of 3′ regions of the hoxa2a en- pression, we generated a series of chimeric constructs of the fugu hancer to neural crest activity, two additional hybrids were gen- hoxa2a and hoxa2b enhancers and evaluated their regulatory ac- erated (Hybrids 3 and 4). We utilized 3′ portions of fugu hoxa2a, tivity in zebrafish reporter assays (Fig. 4.). which holds the conserved neural crest elements as well as re- The 5′ range of neural crest elements was tested using Hybrids gions homologous to r3/5 elements, and combined it with the 5′

Fig. 5. Transgenic analysis of chimeric fugu hoxa2a/hoxa2b enhancers in zebrafish embryos define NC5 as a cis-element important in neural crest expression. (A-E) GFP reporter expression mediated by Hybrids 5-7 and isolated regions of hoxa2a (fr-Hoxa2a #2 and fr-Hoxa2a #3) in neural crest cells (NC) in pharyngeal arch tissue (arrows) or rhombomeres 3 and 5 (arrowheads) in 48hpf zebrafish embryos. In A-D: red¼RFP expression in r3 and r5 driven by a control Krox20 enhancer; green¼GFP expression. (F) Drawing of the hybrid constructs mapping the portion of the hoxa2a and hoxa2b enhancers in each hybrid or the small isolated regions compared to the annotated mouse Hoxa2 enhancer at top. The purple box¼ rhombomere elements; green box¼neural crest elements; red¼hoxa2a enhancer sequence; gray¼hoxa2b enhancer sequence. The table at the right of each construct describes the F0 transgenic expression data marking: presence (þ) or absence () of GFP expression in neural crest or r3/r5; # of embryos with expression in neural crest and/or r3/5 (#relevant expression); # of embryos with any GFP expression; total # embryos injected. The number of stable transgenic lines created (F1s) are noted, far right. 538 J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542

Fig. 6. Sequence alignment of the NC5 neural crest element from fugu hoxa2a with other teleosts and vertebrates. (A) Alignment of fugu NC5 with six teleosts which have maintained both hoxa2a and hoxa2b duplicates in their genomes. The bottom most sequence is the fugu hoxa2b sequence in the equivalent position of NC5. The yellow boxes highlight identical sequences in the majority of aligned sequences. (B) Alignment of NC5 homologous region of placental mammals. This NC5 region is a light green to differentiate it from the identified NC5 in fugu hoxa2a. (C) Alignment of the fugu hoxa2a NC5 element with that of the mouse to illustrate areas of conservation and divergence. In B,C the yellow boxes highlight those sequences which are identical between all aligned sequences, while the blue boxes highlight base pairs which are identical in a majority of the aligned sequences. (D) A schematic of the enhancer mapping NC5 relative to other cis-elements. The second krox20 site is not conserved in fish, and is hatched as a geographic marker. The NC1 and NC4 are illustrated as light green and dotted due to their lack of significant conservation in fish. J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 539 most part of fugu hoxa2b, containing the Krox20 binding site and conservation in the corresponding region of fugu Hoxa2b (Figs. 3A RE1 and BoxA sites (Fig. 4E). Transgenic zebrafish embryos carry- and 6A), in line with differences in its ability to mediate expression ing Hybrid 3 resulted in GFP expression in r3 and r5 of the hind- in neural crest cells. The homologous NC5 region of the mamma- brain, as well as in neural crest cells and pharyngeal arches (Fig. 4C lian Hoxa2 enhancer lies between the second Krox20 binding site and E). The expression in neural crest further supports the position at the 5′ end and NC2 at the 3′ end (Fig. 6B and C). Aligning this of essential elements for this tissue in the 441–1485 bp 3′ region of sequence from representative placental mammals shows that the the hoxa2a enhancer. Somewhat unexpectedly, these data reveal NC5 region is highly conserved (Figs. 2A and 6A), which could be that the 5′ hoxa2b sequences in the construct are able to confer indicative of a need for this region in neural crest enhancer activity rhombomeric activity to the remaining part of the enhancer from in mammals as well as teleosts. The NC5 region is smaller in hoxa2a. This is surprising because there is reduced sequence mammals (150 bp) compared with teleosts (226 bp) (Fig. 2B and identity of RE1 in hoxa2b compared to hoxa2a but these differ- C). ences appear to be important in potentiating rhombomeric activity Our initial analyses of this region from the whole enhancer (Fig. 3B). Hybrid 4, is similar to Hybrid 3 except that the hoxa2a indicated a relatively low degree of conservation (Fig. 2B), but the region between 441 and 794 bp is replaced with that of hoxa2b. smaller size of NC5 in mammals and the presence of indels makes This eliminates reporter expression in both neural crest cells and it challenging to precisely align the mammalian and teleost NC5 the hindbrain (Fig. 4D) suggesting that the sequence differences in regions (Fig. 6C). Therefore, to address whether NC5 has a con- both the overlapping RE4 and NC2 enhancer elements of Hybrid served functional role in neural crest regulation, we tested in 4 between 441 and 794 bp impact both r3/5 and neural crest en- transgenic mice many of the same hybrid constructs of fugu hox- hancer activities. a2a and hoxa2b assayed in transgenic zebrafish embryos. We in- We have named the region we identified between 441 and serted the candidate regulatory regions from Hybrid 1-4 and 7 of 814 bp as NC5 to denote its contribution to activity in neural crest the fugu enhancers into a vector containing lacZ and scored for cells. To further examine the properties of the neural crest reg- reporter activity in transgenic mice (Fig. 7). With respect to ex- ulatory element(s) of NC5, we replaced the homologous region of pression in neural crest cells these reporter assays gave very si- fugu hoxa2a with that of fugu hoxa2b (Hybrid 5). GFP expression is milar results to the transgenic assays using zebrafish. The NC5 detected in r3/5 but there is a complete absence of any reporter region of fugu hoxa2a is an important determinant in the ability of expression in neural crest cells (Fig. 5A). This clearly shows that this enhancer to mediate expression in mouse neural crest cells elements in this region of hoxa2a are necessary for activity of the (Fig. 7A–D). There were some differences in the expression in enhancer in neural crest cells. In a reciprocal experiment, NC5 of hindbrain rhombomeres from these constructs in mice versus hoxa2a was placed in the context of hoxa2b (Hybrid 6). This re- zebrafish, indicating some level of species-specific variation in sulted in robust GFP expression in neural crest cells and the rhombomeric regulatory potential. pharyngeal arches and a loss of rhombomeric expression (Fig. 5B). Together, these results imply that the upstream regulatory These replacement experiments demonstrate that differences in network involved in mediating neural crest expression of mouse sequences in this 441–814 bp region are sufficient to direct neural Hoxa2 is able to read the cis-elements in NC5 of the fugu hoxa2a crest versus rhombomeric expression in the contexts of the re- enhancer to direct expression in neural crest. These analyses maining elements of these two fugu enhancers. suggest that NC5 contains an additional set of conserved cis-ele- In an attempt to refine the size of NC5 we placed the region ments important for regulation of Hoxa2 and hoxa2a orthologs in from 441 to 667 of fugu hoxa2a into fugu hoxa2b (Hybrid 7). Re- neural crest cells. porter expression from Hybrid 7 is apparent in neural crest cells and the pharyngeal arches, however it is mosaic (Fig. 5C). This pattern of reporter expression indicates that cis-elements in the 4. Discussion 441–667 bp region of hoxa2a are able to confer neural crest ac- tivity to the other elements of the fugu hoxa2b enhancer, but that In this study we have examined the evolution of a Hoxa2 neural additional sequences in the 667–814 bp region may contribute to crest enhancer by comparing and contrasting the fugu hoxa2a and levels or efficiency of enhancer activity. Together, these hybrid hoxa2b genes with their teleost and mammalian counterparts results indicate that the NC5 region of hoxa2a contains important through sequence and interspecies transgenic regulatory analyses. cis-elements required for mediating neural crest expression and This revealed subfunctionalization of regulatory activity for ex- they are altered or missing from hoxa2b. Conversely, sequences in pression in hindbrain segments versus neural crest cells between this region of hoxa2b which differ from hoxa2a are important for these two fugu co-orthologs. A unique feature of this enhancer is mediating rhombomeric expression. that subfunctionalization is observed, in spite of the fact that the To test whether the hoxa2a NC5 sequences are sufficient to neural crest and rhombomeric regulatory elements overlap and mediate expression in neural crest cells, we linked the 441–667 bp are presumably under dual constraints. We uncovered evidence (hoxa2a #2) and 441–814 bp (Hoxa2a #3) regions to the reporter for species-specific variations of this enhancer based on differ- vector and assayed for regulatory activity (Fig. 5). Neither con- ences in regulatory potential of the enhancer from the chicken struct was able to direct GFP expression in zebrafish embryos. This Hoxa2 locus compared with enhancers from other vertebrates indicates that the NC5 region is necessary for neural crest ex- counterparts. Functional dissection of the neural crest regulatory pression in the context of a larger enhancer, but alone is not suf- potential of the fugu hoxa2a and hoxa2b enhancers revealed that ficient to mediate expression in neural crest. sequence differences in a previously unknown cis-element (NC5) are critical in contributing to the differential activity of the en- 3.4. Sequence and functional conservation of NC5 hancers from these genes. Furthermore, the NC5 region plays a similar role in the ability of this enhancer to direct expression in To explore the degree of conservation of NC5 we first compared mice, suggesting it is a conserved core component involved in NC5 from the fugu hoxa2a enhancer with that of six other teleosts, modulating cranial neural crest expression of Hoxa2 in vertebrate which contain both hoxa2a and hoxa2b genes, and also compared craniofacial development. This work highlights interesting aspects it to the homologous region of fugu hoxa2b (Fig. 6A). With the for how Hox genes are coupled to cranial neural crest expression exception of small indels, the sequences are highly conserved, and patterning head development. particularly in the 3’ half of NC5. There is much less sequence Considerable progress has been made in building a gene 540 J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542

Fig. 7. Transgenic analysis of chimeric fugu hoxa2a/hoxa2b enhancers in mouse embryos. (A-E) LacZ reporter expression mediated by Hybrids 1-4 and 7 in neural crest cells and/or in pharyngeal arch tissue. These were the same hybrid combinations used in transgenic zebrafish assays in Figs. 4 and 5. (F) Drawing of the hybrid constructs mapping the portion of the hoxa2a and hoxa2b enhancers in each hybrid compared to the annotated mouse Hoxa2 enhancer at top. The purple box¼ rhombomere elements; green box¼ neural crest elements; red¼hoxa2a enhancer sequence; gray¼hoxa2b enhancer sequence. The table at the right of each construct describes the F0 transgenic ex- pression data marking: presence (þ) or absence ()oflacZ expression in neural crest or r3/r5; # of embryos with expression in neural crest and/or r3/5; and # of transgenic embryos. regulatory network (GRN) for neural crest development through the relative inputs of the respective NC1-NC4 elements. Instead it evolutionary comparisons, genomics approaches and regulatory appears that these modules work in a context dependent manner studies (Betancur et al., 2010; Gammill and Bronner-Fraser, 2002; cooperating to specify the appropriate regulatory activity in neural Green et al., 2015; Meulemans and Bronner-Fraser, 2004; Nikitina crest. This is analogous to holo-enhancers, which somehow in- et al., 2008; Sauka-Spengler et al., 2007; Simoes-Costa and Bron- tegrate a series of individual elements into a regulatory coherent ner, 2013, 2015). This has defined major steps and associated unit, as described for Fgf8 and the HoxB cluster (Marinic et al., transcription factors that direct the process from induction 2013; Nolte et al., 2013). In this study, discovering an important through specification, migration and differentiation. It is some- role for an additional element, NC5, further illustrates the degree what surprising that it has proved challenging to fully integrate of complexity in this neural crest enhancer. the regulation and coupling of Hoxa2 to this GRN, because of the We have examined NC5 for the presence of sites that could important role it plays as a primary node for regulation of cra- integrate known factors from the neural crest GRN by performing niofacial programs. Our previous analysis demonstrated that four sequence/motif analyses, using Transfac, MEME and Consite. Of cis-modules, NC1-NC4, were required for temporally and spatially- interest are binding sites for Snail and Stat3 sites present only restricted expression in cranial neural crest migrating into the within the fugu hoxa2a and mouse Hoxa2 NC5 element and Sox10, second branchial arch (Maconochie et al., 1999). One element re- FoxD3 and Mef2 sites found within both fugu and the mouse NC5 ceived input from AP2, which is a transcription factor broadly elements. These five transcription factors are known to be re- expressed and important in neural crest development (Zhang quired for proper neural crest development. Early factors including et al., 1996). However, mutation of any of the four NC1-NC4 ele- Snail family members, Stat3 and FoxD3, are expressed and re- ments abolished regulatory activity, so it has not been possible to quired in neural crest cells for cell specification, migration and identify other specific transcriptional inputs. This suggests that the proliferation (Kuriyama and Mayor, 2008; Nichane et al., 2010; Hoxa2 enhancer does not work as a simple modular unit summing Pohl and Knochel, 2001). Later in embryonic development J.A. McEllin et al. / Developmental Biology 409 (2016) 530–542 541 transcription factors including the SoxE family, Sox8-10, and Mef2, basic domain-leucine zipper transcription factor. Cell 79, 1025–1034. are required for migration and formation of later neural crest de- Couly, G., Creuzet, S., Bennaceur, S., Vincent, C., Le Douarin, N.M., 2002. Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in rived structures, such as the pharyngeal arches and craniofacial patterning the facial skeleton in the vertebrate head. Development 129, development (Hong and Saint-Jeannet, 2005; Miller et al., 2007). 1061–1073. The presence of the Sox10, FoxD3 and Mef2 binding sites within Couly, G., Grapin-Botton, A., Coltey, P., Ruhin, B., Le Douarin, N.M., 1998. Determi- fi nation of the identity of the derivatives of the cephalic neural crest: in- the Hoxa2b NC5 element indicate they must not be suf cient to compatibility between expression and lower jaw development. De- modulate activity, as there is a lack of neural crest expression seen velopment 128, 3445–3459. with this enhancer. Davenne, M., Maconochie, M.K., Neun, R., Pattyn, A., Chambon, P., Krumlauf, R., Rijli, A second set of transcription factor binding sites were found F.M., 1999. Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal devel- opment in the rostral hindbrain. Neuron 22, 677–691. only within the fugu hoxa2b NC5 element, and corresponded to Davis, A., Scemama, J.L., Stellwag, E.J., 2008. Japanese medaka Hox paralog group 2: consensus Pbx and Kreisler (MafB) sites. These transcription fac- insights into the evolution of Hox PG2 gene composition and expression in the – tors are necessary for segmentation of the hindbrain (Cordes and Osteichthyes. J. Exp. Zool. B Mol. Dev. Evol. 310, 623 641. Fisher, S., Grice, E.A., Vinton, R.M., Bessling, S.L., Urasaki, A., Kawakami, K., McCal- Barsh, 1994; Moens and Selleri, 2006; Waskiewicz et al., 2002). lion, A.S., 2006. Evaluating the biological relevance of putative enhancers using Kreisler, is a gene expressed early in r5 and r6 and has a conserved Tol2 transposon-mediated transgenesis in zebrafish. Nat. Protoc. 1, 1297–1305. role in directing regulation of Hoxa3 and Hoxb3 in r5 during Gammill, L.S., Bronner-Fraser, M., 2002. Genomic analysis of neural crest induction. Development 129, 5731–5741. hindbrain segmentation (Kim et al., 2005; Manzanares et al., 1999, Gavalas, A., Davenne, M., Lumsden, A., Chambon, P., Rijli, F.M., 1997. Role of Hoxa-2 1997; Parker et al., 2014). The Pbx family of genes encode known in axon pathfinding and rostral hindbrain patterning. Development 124, – cofactors of Hox proteins necessary for auto- and cross-regulation 3693 3702. Gendron-Maguire, M., Mallo, M., Zhang, M., Gridley, T., 1993. Hoxa-2 mutant mice (Mann and Chan, 1996). The presence of these transcription factor exhibit homeotic transformation of skeletal elements derived from cranial binding sites in the hoxa2b NC5 element correlate with its ability neural crest. Cell 75, 1317–1331. to confer r3/r5 expression when placed in the hoxa2a enhancer. Grammatopoulos, G.A., Bell, E., Toole, L., Lumsden, A., Tucker, A.S., 2000. Homeotic transformation of branchial arch identity after Hoxa2 overexpression. Devel- This indicates that changes in NC5 are important for the differ- opment 127, 5355–5365. ential expression of these two fugu co-paralogs in both neural Green, S.A., Simoes-Costa, M., Bronner, M.E., 2015. Evolution of vertebrates as crest and rhombomeres. viewed from the crest. Nature 520, 474–482. fi fi Hartley, J.L., Temple, G.F., Brasch, M.A., 2000. DNA cloning using in vitro site-speci c Finally, in zebra sh embryos, hoxb2a is expressed in a similar recombination. 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