378 (2013) 194–199

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Developmental Biology

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Duplications of clusters and the emergence of

Natalia Soshnikova a,b, Romain Dewaele a, Philippe Janvier c, Robb Krumlauf d,e, Denis Duboule a,f,n a Department of Genetics and Evolution, University of Geneva, Sciences III, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland b Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany c Muséum National d’Histoire Naturelle, UMR 7207, CP38, 8, rue Buffon 75231, Paris Cedex 05, France d Stowers Institute for Medical Research, Kansas City, MO 64110, USA e Department of Anatomy and , Kansas University Medical Center, Kansas City, Kansas, USA f School of Life Sciences, Ecole Polytechnique Fédérale, 1015 Lausanne, Switzerland article info abstract

Article history: The body plan is characterized by an increased complexity relative to that of all other Received 6 February 2013 and large-scale gene amplifications have been associated with key morphological innovations Accepted 5 March 2013 leading to their remarkable evolutionary success. Here, we use compound full Hox clusters deletions to Available online 15 March 2013 investigate how Hox genes duplications may have contributed to the emergence of vertebrate-specific Keywords: innovations. We show that the combined deletion of HoxA and HoxB leads to an atavistic heart Neo-functionalization , suggesting that the ancestral HoxA/B cluster was co-opted to help in diversifying the Genome duplication complex organ in vertebrates. Other phenotypic effects observed seem to illustrate the resurgence of 2R ancestral (plesiomorphic) features. This indicates that the duplications of Hox clusters were associated Gene regulation with the recruitment or formation of novel cis-regulatory controls, which were key to the evolution of Enhancer evolution many vertebrate features and hence to the evolutionary radiation of this group. & 2013 Elsevier Inc. All rights reserved.

Introduction associated with the evolution of morphological complexity (though see (Donoghue and Purnell, 2005) for a different view). During vertebrate development, genes belonging to the Hox Genetic studies targeted either to individual Hox genes, or to family of transcription factors organize structures along the major paralogy groups, have revealed a strong functional complemen- body axis (Krumlauf, 1994). As in many other bilateria, these genes tarity, not only amongst paralogous genes (Wellik, 2009; Xu and are organized in genomic clusters. However, while most verte- Wellik, 2011) but also between neighboring genes or even gene brates have four such genomic loci (HoxA to HoxD), clusters (Rancourt et al., 1995; Davis and Capecchi, 1996; Favier only have one cognate cluster (Duboule and Dollé, 1989; Graham et al., 1996; de la Cruz et al., 1999). Duplications also facilitated the et al., 1989). Because cephalochordates also have a single Hox gene opportunity for the gene clusters to acquire specific global reg- cluster (Garcia-Fernandez and Holland, 1994) and urochordates ulatory inputs, via the emergence of strong regulatory sequences only one complement of these factors (Ikuta et al., 2004; Seo et al., able to control the expression of several genes in cis, in the same 2004), it appears that an ancestral gene cluster was duplicated tissue or structure. Such meta-genic regulations are correlated twice, during the emergence of vertebrates, likely as a conse- with vertebrate-specific traits (Di-Poi et al., 2007; Duboule, 2007; quence of full (2R) genome duplications (Ohno, 1970; Hart et al., Montavon et al., 2011), leading to the suggestion that Hox cluster 1987; Holland et al., 2008). As a result, paralogous Hox genes, duplications accompanied the emergence of key vertebrate fea- those located at the same relative positions within each of the four tures (Holland et al., 1994). clusters, show high sequence similarities and, consequently, some functional equivalence (Krumlauf, 1994). It is believed that genome duplications provided the necessary Materials and methods material to evolve novel gene functions (neo-functionalisation), The production of the various mutant strains used in this study was previously described (Medina-Martinez et al., 2000; Suemori and Noguchi, 2000; Spitz et al., 2001; Kmita et al., 2005). Whole- n Corresponding author at: Department of Genetics and Evolution, University of mount in situ hybridization was carried out using standard Geneva, Sciences III, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland. procedures. Apoptotic cells were detected using a DeadEnd™ Fax: þ41 22 379 6795. ' E-mail addresses: [email protected], Colorimetric TUNEL System (Promega) according to manufactures Denis.Duboule@epfl.ch (D. Duboule). instructions. Skeletons of E15.5 embryos were prepared and

0012-1606/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ydbio.2013.03.004 N. Soshnikova et al. / Developmental Biology 378 (2013) 194–199 195 stained with Alcian blue 8GX and Alizarin Red. Whole-mount regarded as a phenotypic reversion, reminiscent of the ancestral in situ hybridization was performed using digoxygenin-labeled condition. In this view, both HoxA and HoxB clusters were (DIG) RNA probes (Roche) as described (Montavon et al., 2011). necessary for complete vertebrate heart evolution. In support of this, anterior Hox genes such as Hoxa1, Hoxb1 and Hoxa3 are expressed in the secondary heart field (SHF) prior to heart looping Results and discussion (Makki and Capecchi, 2010; Bertrand et al., 2011). Interestingly, these anterior genes were lost from the HoxC cluster (Kuraku and We experimentally challenged this view by using combined Meyer, 2009), such that HoxA/B mutants embryos are left with a cluster deletions in mice, thus trying to produce animals carrying a single gene complement from HoxD, as in early chordata. HOX more ‘ancestral’ complement of Hox genes. We initially focused on proteins may have been present in an ancestral heart field and the HoxA and HoxB ‘sister’ clusters, which were proposed to derive increasing their amounts through cluster duplication may have from a late duplication event (Kappen and Ruddle, 1993; Amores triggered this important modification. Alternatively, cluster dupli- et al., 1998; Ravi et al., 2009). The deletion of the complete HoxA cation allowed for variations in specificity of expression, changing cluster is embryonic lethal after 11–12.5 days of gestation (E11.0– the fate of the heart field. To test these possibilities, we generated E12.5) due to placental insufficiency and premature stenosis of the compound HoxB/D mutants, which in this case also have a single umbilical artery (Fig. S1a and b; see (Warot et al., 1997; Shaut et al., complement of Hox1-3 genes, from the HoxA cluster. These double 2008; Scotti and Kmita, 2012)). Mice lacking the HoxB cluster mutant embryos (n420) had normal hearts and mostly displayed develop extreme edemas and hemorrhages in the internal jugular the addition of those observed in single cluster veins by E13.5–15.5, suggesting circulation defects (Fig. S1c and d deletions (Fig. S2a–d). Hence, functions from gene complements (Medina-Martinez et al., 2000)). The combined loss of both HoxA of HoxD are not able to compensate for the loss of HoxA. and HoxB sister clusters, however, resulted in growth retardation We conclude that correct heart looping requires the combined and embryonic lethality by E9.5, likely due to severe defects in specific functions of both HoxA and HoxB sister gene clusters. heart development (Fig. 1a–d). In all HoxA/B compound mutants In embryos with the combined loss of both HoxA and HoxB analyzed (n420), cardiac morphogenesis was blocked before clusters, the formation and the early patterning of the sclerotome, looping, at the linear tube stage (Fig. 1e–h). Mutant embryos which gives rise to vertebrae, as well as the organization of the displayed enlarged, contracting hearts, which remained positioned lateral plate mesoderm into limb-forming and limb-free areas was along the ventral midline, without clear demarcation between as in wild type embryos (Fig. S1e–j). The combined deletion of the atrial and ventricular chambers. As a consequence, compound other two sister clusters, HoxC and HoxD, was hampered by mutant embryos had compromised formation of blood vessels, the sterility of trans-heterozygous animals. We thus turned to leading to focal hemorrhages in the cranial region (Fig. 1a and d). the combined removal of pairs of cousin-clusters. In HoxB/D Looping of the heart tube occurs in all extant vertebrates double mutant specimens, the ventral elements (the centrum) of (Moorman and Christoffels, 2003). In contrast, other chordates both the first (atlas) and second (axis) cervical vertebrae were like tunicates and Amphioxus, develop a straight, contractile fused and transformed into a more anterior, basioccipital-looking tubular heart (Holland et al., 2003; Davidson, 2007). The pheno- bone (Fig. 2a and d). While the dorsal elements (the neural arches) type observed in HoxA/B compound mutant embryos may thus be of both atlas and axis were lost, the lateral elements resembled the

Fig. 1. Cardiac defects in HoxA/B mutant embryos. Left lateral views of wild type (a), HoxA (b), HoxB (c) and HoxA/B (d) mutants at E9.0. HoxA/B mutant embryos show abnormalities in heart looping (d). Whole mount in situ hybridization for Mlc2a expression was used to visualize heart in wild type (e), HoxA (f), HoxB (g) and HoxA/B (h) mutants at E9.0 (ventral views). Note aberrant linear heart tube in the HoxA/B embryo. Scale bar, 200 mm(a–d); 100 mm(e–h). 196 N. Soshnikova et al. / Developmental Biology 378 (2013) 194–199

Fig. 2. Homeotic transformation of the axial skeleton in mouse embryos missing both the HoxB and HoxD clusters. Ventral views of skeletal preparations of a wild type (a), HoxB (b), HoxD (c) and HoxB/D (d) homozygous embryos at E15.5. In double homozygous embryos, ventral elements of the atlas (at) and axis (ax) are transformed into the basioccipital bone (bo). (e–h) Lateral views of the same skeletons. Lateral elements of the atlas and axis resemble the exoccipital (eo) bone in HoxB/D double homozygous embryos (h). Arrow indicates posteriorly expanded basioccipital bone in double mutants. Scale bar, 300 mm(a–d); 450 mm(e–h). exoccipital bones, with an ossification process between them a more efficient articulation between the head and the trunk via (Fig. 2e and h) leading to a caudally expanded braincase. the individualization of the most rostral vertebrae and a comple- In comparison, deletion of HoxB alone leads to the reduction of mentarity between occipital condyles and the atlas. the atlas neural arch, whereas it was increased in the axis (Fig. 2b The posterior half of the HoxB cluster, from Hoxb10 to Hoxb12,is and f and (Medina-Martinez et al., 2000)). HoxD mutants, on the lacking in many vertebrate species (Zeltser et al., 1996; Kuraku and other hand, showed a ‘classical’ anterior transformation of the Meyer, 2009). Therefore, removing any other two gene clusters atlas into a basioccipital-like ossification, as well as a truncation of generates animals with only one set of posterior Hox genes, neural arches of both the atlas and axis (Fig. 2c and g). reflective of the ancestral status before genome duplication. Here again, such an extended fusion of vertebrae with the To model this case, we generated HoxA/C compound mutants. basioccipital region is somewhat reminiscent of the extremely Double homozygous embryos resembled HoxA mutants and devel- elongated post-vagal region of the braincase of certain fossil oped extensive hemorrhages, dying by E12.5 due to circulatory armored agnathans (370–430 Myr-old) like galeaspids and osteos- malfunction. Histological analyses revealed that such embryos tracans (the latter being currently regarded as the sister group of completely lacked metanephric kidneys, unlike embryos mutant jawed vertebrates), which completely lacked a cranio-cervical either for HoxA or for HoxC (Fig. 3a, c, e, g and (Suemori and joint. This condition also recalls even more closely the occipital- Noguchi, 2000)). We observed numerous apoptotic cells in pre- synarcual complex (a fusion of vertebrae behind the occipital sumptive metanephric mesenchyme of compound mutants at condyles), of some living cartilaginous fishes like rays, skates and E12.5 (Fig. 3b, d, f and h), which may thus underlie renal aplasia. holocephalans (chimaeras) and the extinct placoderms (Davis Hox11 group genes play an essential role in the formation of et al., 2012). Also, the oldest osteichthyans show a rostro-caudal metanephric kidneys, which are absent in Hoxa11/d11 mutant extension of the endoskeletal pectoral girdle suggestive of that embryos (Davis et al., 1995; Wellik et al., 2002). However, seen in placoderms (Zhu et al., 2009). Early gnathostome anatomy Hoxa11/c11 mutants embryos display much milder defects than may have thus conserved certain features that are also known in those reported here (Wellik et al., 2002), indicating once again that the jawless osteostracans, although the latter shows no cervico- neighbor genes also contribute to building this important organ cranial articulation and no separation between the braincase and (Di-Poi et al., 2007; Yallowitz et al., 2011). In this context, the the shoulder girdle. These results illustrate that the occipital part critical dosage of HOX products can thus be reached either in trans, of the neurocranium can extend far caudally, by fusing with more via paralogous genes, or in cis amongst neighbors. posterior vertebral modules, thus reducing the articulation Paired appendages are another major trait associated with the between the trunk and the head. Consequently, the gain of success of the vertebrate radiation and functional studies have expression of such genes in early gnathostomes may have led to revealed the critical roles of both HoxA and HoxD clusters during N. Soshnikova et al. / Developmental Biology 378 (2013) 194–199 197

Fig. 3. Morphological abnormalities in HoxA/C mutant embryos. Hematoxylin and eosin stained transverse sections of developing kidneys of E12.5 wild type (a), HoxA (c), HoxC (e) and HoxA/C (g) mutant embryos. TUNEL analysis of wild type (b), HoxA (d), HoxC (f) and HoxA/C (h) developing kidneys at E12.5. The apoptotic cells are labeled in red. DAPI stained nuclei are in blue. Note a massive cell death leading to a loss of the definitive kidney formation in mouse embryos missing both the HoxA and HoxC clusters (h). (i–t) Limb defects in Hox clusters mutants. Whole mount in situ hybridization for Col2a1 expression was used to visualize cartilaginous condensations in wild type (i), HoxA (j), HoxC (K)and HoxA/C (l) forelimbs at E12.5. HoxA mutants miss digit 1 (j). In mildly affected HoxA/C mutants a severely reduced handplate display mirror-image symmetrical digit pattern (l). The forelimb bud is smaller along the a–p and p–daxesinHoxA/C (s and t) embryos compare to wt (m-n) or the single cluster mutants (o–r) at E9.5. Whole mount in situ hybridization for Tbx5 and UncX4.1 expression was used to visualize forelimb buds and somites, respectively. Scale bar, 100 mm(a–h); 200 mm(i–l); 225 mm(m–t).

limb development and evolution (Kmita et al., 2005; Woltering and Duboule, 2010). In contrast, neither HoxC nor HoxB cluster mutant limbs displayed any severely abnormal phenotypes (Fig.3k, Figs. S1d and S3c). Unexpectedly, HoxA/C compound mutants limbs (n¼9) were malformed (Fig. 3l), ranging from a hypoplastic (Fig. S3a–c) to a completely absent (Fig. 3d) handplate. These limbs had reduced numbers of presumptive digits and showed apparent mirror-imaged symmetry along the anterior-posterior axis (Fig. 3l and Fig. S3c). Yet, the expression of Shh was not dramatically affected (Fig. S3i–l), consistent with the presence of Fig. 4. Evolution of vertebrate Hox clusters and their functional specificities. In posterior Hoxd genes, which can on their own activate Shh chordates, Hox genes are essential for the patterning of the neural tube and somites. In vertebrates, duplication of the ancestral Hox cluster led to the (Tarchini et al., 2006). emergence of proximal appendages and nephrogenic structures. Furthermore, fi fi The speci cation of the forelimb eld was correctly established recruitment of HoxA/B cluster is a key step in the development of complex in HoxA/C double mutant, as compared to wild type embryos. vertebrate heart. However, at E10, HoxA/C mutants displayed smaller forelimb buds along both the PD and the AP axes, spanning four instead of six Our results indicate that, within the 2R genome duplication somites (Fig. 3m–t). We conclude that the products of the HoxA events, the multiplication of Hox clusters was an important and HoxC clusters have overlapping activities in promoting the phenomenon to allow the emergence of vertebrate innovations, early extension of the forelimb field. As for metanephric kidneys, as particularly well illustrated by the apparent atavistic nature of genetic cooperativity can be associated either to members of the the heart phenotype. In this particular case, it appears that the same cluster (HoxA/D) double mutants lack limbs (Kmita et al., critical morphological event(s) for these effects on heart formation 2005) or to members of groups of paralogy, since Hox9 genes act in co-evolved with a novel cis-regulatory specialization of the HoxA/B a redundant manner in the lateral plate mesoderm for initiation of ancestor cluster, which was subsequently maintained in both limb bud outgrowth via induction of Shh (Xu and Wellik, 2011). sister copies (HoxA and HoxB; see Fig. 4). It is however more 198 N. Soshnikova et al. / Developmental Biology 378 (2013) 194–199 difficult to trace back the acquisition, by the various Hox clusters, Davis, A.P., Capecchi, M.R., 1996. A mutational analysis of the 5′ HoxD genes: of those global regulations underlying the other phenotypic dissection of genetic interactions during limb development in the mouse. Development 122, 1175–1185. anomalies reported in our compound deletions. For example, Davis, A.P., Witte, D.P., Hsieh-Li, H.M., Potter, S.S., Capecchi, M.R., 1995. Absence of while the cooperation between HoxA and HoxD genes in the radius and ulna in mice lacking hoxa-11 and hoxd-11. Nature 375, 791–795. construction of digits is based on distinct regulatory modalities, Davis, S.P., Finarelli, J.A., Coates, M.I., 2012. Acanthodes and shark-like conditions in fl the last common ancestor of modern gnathostomes. 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