Clustered brachiopod Hox genes are not expressed collinearly and are associated with lophotrochozoan novelties

Significance

Hox genes pattern the anteroposterior axis of all animals that have left and right body sides. In many animals, Hox genes are clustered along the chromosomes and expressed in spatial and temporal order. This coordinated regulation is thought to have preserved the cluster through a developmental constraint. Our study of the genomic organization and the embryonic spatial and temporal expression of Hox genes in sessile marine animals called lampshells (brachiopods) shows that along with having a broken Hox cluster, they lack both temporal and spatial collinearity. Furthermore, we present molecular evidence that the hard tissues (chaetae and shells) of segmented worms, mollusks, and brachiopods share a common origin that dates back to the Early Cambrian.

Abstract

Temporal collinearity is often considered the main force preserving Hox gene clusters in animal genomes. Studies that combine genomic and gene expression data are scarce, however, particularly in invertebrates like the Lophotrochozoa. As a result, the temporal collinearity hypothesis is currently built on poorly supported foundations. Here we characterize the complement, cluster, and expression of Hox genes in two brachiopod species, Terebratalia transversa and Novocrania anomala. T. transversa has a split cluster with 10 genes (lab, pb, Hox3, Dfd, Scr, Lox5, Antp, Lox4, Post2, and Post1), whereas N. anomala has 9 genes (apparently missing Post1). Our in situ hybridization, real-time quantitative PCR, and stage-specific transcriptomic analyses show that brachiopod Hox genes are neither strictly temporally nor spatially collinear; only pb (in T. transversa), Hox3 (in both brachiopods), and Dfd (in both brachiopods) show staggered mesodermal expression. Thus, our findings support the idea that temporal collinearity might contribute to keeping Hox genes clustered. Remarkably, expression of the Hox genes in both brachiopod species demonstrates cooption of Hox genes in the chaetae and shell fields, two major lophotrochozoan morphological novelties. The shared and specific expression of Hox genes, together with Arx, Zic, and Notch pathway components in chaetae and shell fields in brachiopods, mollusks, and annelids provide molecular evidence supporting the conservation of the molecular basis for these lophotrochozoan hallmarks.

Hox genes are transcription factors that bind to regulatory regions via a helix-turn-helix domain to enhance or suppress gene transcription (1, 2). Hox genes were initially described in the fruit fly Drosophila melanogaster (3, 4) and later reported in vertebrates (5–7) and the nematode Caenorhabditis elegans (8). In all of these organisms, Hox genes were shown to provide a spatial coordinate system for cells along the anteroposterior axis (9). Remarkably, the Hox genes of Drosophila and vertebrates are clustered in their genomes and exhibit a staggered spatial (3) and even temporal—as in mammals (10, 11)—expression during embryogenesis that corresponds to their genomic arrangement (3, 12, 13). These features were used to classify Hox genes into four major orthologous groups—anterior, Hox3, and central and posterior Hox genes—and have been proposed as ancestral attributes of all bilaterally symmetrical animals (1, 13, 14).

Further study of the genomic arrangements and expression patterns of Hox genes in a broader phylogenetic context has revealed multiple deviations from that hypothesized ancestral state. Hox genes are prone to gains (15–17) and losses (18–21), and their arrangement in a cluster can be interrupted, or even completely disintegrated (22–25). Furthermore, the collinear character of the Hox gene expression can fade temporally (24, 26, 27) and/or spatially (28). In addition, Hox genes have diversified their roles during development, extending beyond providing spatial information (29). They also are involved in patterning different tissues (30), and often have been recruited for the evolution and development of novel morphological traits, such as vertebrate limbs (31, 32), cephalopod funnels and arms (28), and beetle horns (33).

Thus, it is not surprising that Hox genes show diverse arrangements regarding their genomic organization and expression profiles in the Spiralia (34), a major animal clade that includes highly disparate developmental strategies and body organizations (35–39). A striking example of this is the bdelloid rotifer Adineta vaga, which belongs to the Gnathifera, the possible sister group to all remaining Spiralia (38, 39). Owing to reduced tetraploidy, the A. vaga Hox complement includes 24 genes, although it lacks posterior Hox genes and a Hox cluster (40). The freshwater flatworms Macrostomum lignano and Schmidtea mediterranea also lack a Hox cluster (41, 42), and parasitic flatworms have undergone extensive Hox gene losses, likely associated with their particular lifestyle (21). Interestingly, the limpet mollusk Lottia gigantea (16) shows a well-organized Hox cluster. Other mollusks (e.g., the Pacific oyster Crassostrea gigas) and the segmented annelid Capitella teleta exhibit split Hox clusters (43, 44). On the other hand, the cephalopod mollusk Octopus bimaculoides has lost several Hox genes and lacks a Hox cluster (22), and the clitellate annelids Helobdella robusta and Eisenia fetida do not have a Hox cluster but have greatly expanded certain Hox classes (16, 17).

Although Hox gene expression is known for a handful of spiralian species (26, 41, 43, 45–54), the relationship between genomic organization and expression domains is known for only three of these species, namely the annelids C. teleta and H. robusta and the planarian S. mediterranea. Consistent with their disintegrated Hox clusters, H. robusta and S. mediterranea show no temporal collinearity and only remnants of spatial collinearity (41, 51, 52). Conversely, C. teleta, which apparently has a split cluster, does exhibit these features (43). In general, these observations suggest that the presence of collinearity—in particular, temporal collinearity—may be associated with the retention of a more or less intact spiralian Hox cluster, as seems to be the case for the vertebrate cluster (14, 23, 55, 56). Nonetheless, more studies combining genomic and expression information, and including the vast spiralian morphological diversity, are essential to draw robust conclusions about Hox gene evolution and regulation in Spiralia and Metazoa (57). These studies also would allow investigators to test whether hypotheses about the correlation between collinearity and cluster organization observed in deuterostomes (23) hold true for protostomes as well.

Here we report a comprehensive study of the genomic arrangement and expression of Hox genes in Brachiopoda, a lineage of Spiralia with origins dating back to the Lower Cambrian (58). We use two brachiopod species—the “articulate” Terebratalia transversa and the “inarticulate” Novocrania anomala—that belong to the two major brachiopod lineages, thereby allowing the reconstruction of putative ancestral characters for Brachiopoda as a whole (Fig. 1A). Our findings demonstrate that the split Hox cluster in the Brachiopoda is not associated with a temporally collinear expression of Hox genes. Furthermore, the spatial expression of Hox genes, together with other transcription factors, such as Zic, Aristaless-related (Arx), and members of the Notch pathway, provide molecular evidence supporting the homology of annelid, brachiopod, and mollusk chaetae and shell fields.

Fig. 1.

Genomic organization of Hox genes in Brachiopoda. (A) Images of adult T. transversa and N. anomala, and phylogenetic position of these species within Brachiopoda and Lophotrochozoa. (B) The 10 Hox genes of T. transversa are ordered along three genomic scaffolds and are flanked by external genes (vertical lines); gene orthology is based on best blast hit. Thus, T. transversa has a split Hox cluster composed of three subclusters. No predicted ORFs were identified between the Hox genes in scaffolds A and B. A colored box represents each Hox gene, and below each box are the direction of transcription and the exon-intron composition. The genomic regions containing Hox genes are represented in scale. (C) The genomic organization of brachiopod Hox genes in a phylogenetic context. Adapted with permission from ref. 22. The genomic order of Hox genes in T. transversa is similar to that observed in other spiralians (e.g., C. teleta, L. gigantea), suggesting that the translocation of the Antp gene upstream to lab is a lineage-specific feature of L. anatina. (In T. transversa and L. anatina, the arrows below the genes show the direction of transcription.) A degenerate-primer screening for Hox genes reported the presence of Lox2 and Lox4 in L. anatina (15). Blastn searches against the sequenced L. anatina genome only confirmed the presence of Lox4, in the same scaffold as Post1 and Post2, although genome annotation pipelines failed to predict this gene (69). The low contiguity of the draft genome assembly of N. anomala hampered the recovery of genomic linkages between the identified Hox genes. Each ortholog group is represented by a specific color.

Results

The Hox Gene Complement of T. transversa and N. anomala.

Transcriptomic and genomic searches resulted in the identification of 10 Hox genes in T. transversa and 7 Hox genes in the transcriptome and two additional fragments corresponding to a Hox homeodomain in the draft genome assembly in N. anomala. Attempts to amplify and extend these two genomic sequences in the embryonic and larval transcriptome of N. anomala failed, suggesting that these 2 Hox genes might be expressed only during metamorphosis and/or in the adult brachiopod. Maximum likelihood orthology analyses resolved the identity of the retrieved Hox genes (Fig. S1 and Table S1). The 10 Hox genes of T. transversa were orthologous to labial (lab), proboscipedia (pb), Hox3, deformed (Dfd), sex combs reduced (Scr), Lox5, antennapedia (Antp), Lox4, Post2, and Post1. The 9 Hox genes identified in N. anomala corresponded to lab, pb, Hox3, Dfd, Scr, Lox5, Antp, Lox4, and Post2.

Fig. S1.

Orthology analysis of T. transversa and N. anomala Hox genes. Maximum likelihood phylogenetic analysis of bilaterian Hox and ParaHox genes, using as outgroup the even-skipped (EVX) subfamily. Colored boxes indicate Hox ortholog groups present in spiralian representatives. Hox gene sequences of a representative selection of bilaterian lineages (Table S1) were aligned with MAFFT v.7. The multiple sequence alignment (available on request) was trimmed to include the 60 amino acids of the homeodomain. ProtTest v.3 was used to determine the best-fitting evolutionary model (LG+G+I). Orthology analyses were conducted with RAxML v.8.2.6 using the autoMRE option. T. transversa sequences are highlighted by green boxes; N. anomala sequences, by red boxes. Only bootstrap values of at least 70 at nodes that supposedly represent distinct orthology groups are shown. High support values within orthogroups (e.g., between paralogs) were omitted for the sake of clarity.

Table S1.

Sequences and accession numbers used for Hox orthology assignment

Organism	Gene	Database	Accession no.
Homo sapiens	HoxA1	GenBank	AAB35423.2
	HoxB1		AAH99633.1
	HoxD1		AAG44444.1
	HoxA2		NP_006726.1
	HoxB2		NP_002136.1
	HoxA3		NP_705895.1
	HoxB3		AAD10852.1
	HoxD3		CAA71102.1
	HoxA4		NP_002132.3
	HoxB4		AAG45052.1
	HoxC4		AAG42145.1
	HoxD4		NP_055436.2
	HoxA5		CAG47052.1
	HoxB5		NP_002138.1
	HoxC5		EAW96748.1
	HoxA6		NP_076919.1
	HoxB6		NP_061825.2
	HoxC6		CAG33235.1
	HoxA7		CAA06713.1
	HoxB7		NP_004493.3
	HoxB8		AAG42143.1
	HoxC8		AAG42146.1
	HoxD8		AAG42152.1
	HoxA9		NP_689952.1
	HoxB9		AAG42144.1
	HoxC9		AAG42151.1
	HoxD9		NP_055028.3
	HoxA10		AAH07600.1
	HoxC10		NP_059105.2
	HoxD10		NP_002139.2
	HoxA11		NP_005514.1
	HoxC11		NP_055027.1
	HoxD11		AAF79045.1
	HoxC12		AAK16717.1
	HoxD12		AAF79044.1
	HoxA13		AAC50993.1
	HoxB13		AAH70233.1
	HoxC13		AAF73439.1
	HoxD13		AAC51635.1
	Gsx1		NP_663632.1
	Gsx2		NP_573574.1
	Pdx1		NP_000200.1
	Cdx-1		NP_001795.2
	Cdx-2		NP_001256.3
	Cdx-4		NP_005184.1
	Evx-1		NP_001291448.1
	Evx-2		NP_001073927.1
Branchiostoma floridae	Hox1	GenBank	BAA78620
	Hox2		BAA78621
	Hox3		X68045
	Hox4		BAA78622
	Hox5		CAA84517
	Hox6		CAA84518
	Hox7		CAA84519
	Hox8		CAA84520
	Hox9		CAA84521
	Hox10		CAA84522
	Hox11		AAF81909
	Hox12		AAF81903
	Hox13		AAF81904
	Hox14		AAF81905
	Hox15		ACJ74394.1
	Gsx		AAC39015.1
	Xlox		AAC39016.1
	Cdx		AAC39017
	Evx-a		AAK58953.1
	Evx-b		AAK58954.1
Ptychodera flava	Hox1	GenBank	AAR07634.1
	Hox4		AAR07635.1
	Hox5		AAR07636.1
	Hox6		AAR07637.1
	Hox9/10		AAR07638.1
	Hox11/13a		AAR07639.1
	Hox11/13b		AAR07640.1
	Hox11/13c		AAR07641.1
	Xlox1		AAR07643.1
	Xlox2		AAR07644.1
Saccoglossus kowalevskii	Gsx	Uniprot	A0A0U2UDE9
	Cdx	GenBank	NP_001158415.1
	Evx		NP_001164694.1
Flaccisaggita enflata	Hox1	GenBank	ABS18809.1
	Hox3		ABS18810.1
	Hox4		ABS18811.1
	Hox5		ABS18812.1
	Hox6		ABS18813.1
	Hox8		ABS18814.1
	MedPost		ABS18817.1
	Posta		ABS18815.1
	Postb		ABS18816.1
Priapulus caudatus	lab	GenBank	AAD40640.1
	pb		AAD40641.1
	Hox3		AAD40642.1
	Dfd		AAD40643.1
	Ubx		AAD40647.1
	Abd-B		AAD40649.1
Drosophila melanogaster	lab	GenBank	CAB57787
	pb		CAA45271
	Zen		AAF54087.1
	Zen2		P09090.2
	Dfd		P07548
	Scr		NP_524248
	ftz		NP_477498
	Antp		CAA27417
	Ubx		CAA29194
	Abd-A		P29555
	Abd-B		CAB57859
	Ind		NP_996087.2
	Cad		AAA28409.1
	Eve		NP_523670.2
Tribolium castaneum	lab	GenBank	EEZ99257.1
	Mxp		NP_001107807.1
	Zen1		NP_001036813
	Zen2		AAK16425.1
	Dfd		AAK16423.1
	Cx		NP_001034523.1
	ftz		AAK16421.1
	Ptl		NP_001034505.1
	Utx		EEZ99249.1
	Abd-A		EEZ99248.1
	Abd-B		EEZ99247.1
	Ind		AAW21974.1
	Cad-1		NP_001034498.1
	Cad-2		XP_008191732.1
	Eve		NP_001034538.1
Capitella teleta	lab	GenBank	ABY67952
	pb		ABY67953
	Hox3		ABY67954
	Dfd		ABY67955
	Scr		ABY67956
	Lox5		ABY67957
	Antp		ABY67962
	Lox4		ABY67958
	Lox2		ABY67959
	Post1		ABY67961
	Post2		ABY67960
	Gsx		AAZ23124.1
	Cdx		AAZ95508
	Xlox		AAZ95509.1
	Evx		ABG82164
Hellobdella robusta	lab-a	Simakov et al. (16)
	lab-b	Simakov et al. (16)
	Scr-a	Simakov et al. (16)
	Scr-b	Simakov et al. (16)
	Scr-c	Simakov et al. (16)
	Dfd-a	Simakov et al. (16)
	Dfd-b	Simakov et al. (16)
	Lox5	Simakov et al. (16)
	Antp	Simakov et al. (16)
	Lox4-a	Simakov et al. (16)
	Lox4-b	Simakov et al. (16)
	Lox2	Simakov et al. (16)
	Post2	Simakov et al. (16)
Lingula anatina	lab	ENSEMBL	g10891
	pb		g10890
	Hox3		g10889
	Dfd		g10888
	Scr		g10887
	Lox5		g10886
	Antp		g10892
	Post1		g12396
	Post2		g12399
Crassostrea gigas	Hox1	ENSEMBL	CGI_10024083
	Hox2		CGI_10024086
	Hox3		CGI_10024087
	Hox4		CGI_10024091
	Lox5		CGI_10026565
	Lox2		CGI_10018592
	Lox4		CGI_10026562
Lottia gigantea	lab	Simakov et al. (16)
	pb	Simakov et al. (16)
	Hox3	Simakov et al. (16)
	Dfd	Simakov et al. (16)
	Scr	Simakov et al. (16)
	Lox5	Simakov et al. (16)
	Antp	Simakov et al. (16)
	Lox2	Simakov et al. (16)
	Lox4	Simakov et al. (16)
	Post1	Simakov et al. (16)
	Post2	Simakov et al. (16)
Octopus bimaculoides	Hox1	ENSEMBL	Ocbimv22030263
	Scr		Ocbimv22018468
	Lox5		Ocbimv22010205
	Antp		Ocbimv22036189
	Lox2		Ocbimv22033340
	Lox4		Ocbimv22009726
	Post1		Ocbimv22015181
	Post2		Ocbimv22031197
Gibbula varia	HoxA	GenBank	ACX84671.1
	Hox2		ADJ18233.1
	Hox3		ADJ18232.1
	Hox4		ACX84672.1
	Hox5		ADJ18234.1
	Lox5		ADJ18235.1
	Hox7		ADJ18236.1
	Lox2		ADJ18238.1
	Lox4		ADJ18237.1
	Post1		ACX84673.1
Euprymna scolopes	lab	GenBank	AY330184
	Hox3		AY330185
	Scr		AY330186
	Lox5		AY330187
	Antp		AY330188
	Lox4		AY330189
	Post1		AY330190
	Post2		AY330191
Micrura alaskensis	lab	GenBank	KP762174
	pb		KP762176
	Hox3		KP762173
	Dfd		KP762180
	Scr		KP762177
	Lox5		KP762179
	Antp		KP762171
	Lox4		KP762175
	Post2		KP762178
Bugula turrita	pb	GenBank	AAS77225
	Hox3		AAS77226
	Dfd-a		AAS77227
	Dfd-b		AAS77228
	Lox5		AAS77229
	Post2		AAS77230
Lepidodermella squamata	Lox4*	This study

^*> Lepidodermella_squamata_Lox4.

IITNAVTGANNGSSGKLMGAAHRTAPMYAWMAVVGPNSSQKRRGRQTYTRHQTIELEKEFAFCHYLARKRRIELAAALSLSERQVKIWFQNRRMKLKKEKQQIADMNHISTSTTSTSNSSHSKSNRHDDYNDVNDASSSDEDHLD

Genomic Organization of Hox Genes in T. transversa and N. anomala.

We used the draft assemblies of T. transversa and N. anomala genomes to investigate the genomic arrangement of their Hox genes. In T. transversa, we identified three scaffolds containing Hox genes (Fig. 1B). Scaffold A spanned 81.7 kb and contained lab and pb in a genomic region of 15.4 kb, flanked by other genes with no known linkage to the Hox cluster in other animals. Scaffold B, the longest (284.8 kb) scaffold, included Hox3, Dfd, Scr, Lox5, Antp, Lox4, and Post2, in that order (Fig. 1B), along with the microRNA mir-10 between Dfd and Scr. As in scaffold A, other genes flanked the Hox genes, which occupied a genomic region of 76.2 kb. Finally, Post1 aligned to various short scaffolds. We could not recover any genomic linkage between the identified Hox genes in N. anomala owing to the low contiguity (N50 of 3.5 kb) of the draft genome assembly. Taken together, these data demonstrate that T. transversa has a split Hox cluster broken down into three subclusters, each of them with an organized arrangement. Importantly, the potential genomic disposition of these three subclusters is similar to that observed in other spiralians, such as C. teleta and L. gigantea (Fig. 1C), which suggests that the lineage leading to the brachiopod Lingula anatina experienced genomic rearrangements that modified the order and linkage of the Hox genes.

Hox Gene Expression in T. transversa.

To investigate the presence of temporal and/or spatial collinearity in the expression of the clustered Hox genes in T. transversa, we first performed whole-mount in situ hybridization in embryos from blastula to late, competent larval stages (Fig. 2).

Fig. 2.

Whole-mount in situ hybridization of each Hox gene during embryonic and larval stages in T. transversa. The Hox genes lab and Post1 are expressed during chaetae formation. The genes pb, Hox3, and Dfd are expressed collinearly along the mantle and pedicle mesoderm. The Hox genes Scr and Antp are expressed in the periostracum, the shell-forming epithelium. Lox5, Lox4, and Post2 are expressed in the posterior ectoderm of the pedicle lobe. These expression patterns are described in detail in the text. Black arrowheads indicate expression in the chaetae sacs. Orange arrowheads highlight mesodermal expression. Green arrowheads indicate expression in the periostracum. The genomic organization of the Hox genes is shown on the left. On top are schematic representations of each analyzed developmental stage on its respective perspective. In these schemes, the blue area represents the mesoderm. Drawings are not to scale. The red line indicates the onset of expression of each Hox gene based on in situ hybridization data. The blastula stage is a lateral view (Inset in Ca is a vegetal view). For each other stage, the left column is a lateral view and the right column is a dorsoventral view. The asterisk demarcates the animal/anterior pole. al, apical lobe; bp, blastopore; ch, chaetae; em, endomesoderm; gp, gastral plate; gu, gut; me, mesoderm; ml, mantle lobe; mo, mouth; pl, pedicle lobe.

Anterior Hox genes.

The anterior Hox gene lab was first detected in the mid gastrula stage in two faint, bilaterally symmetrical dorsal ectodermal domains (Fig. 2A, d and e). In late gastrula stages, lab expression consisted of four dorsal ectodermal clusters corresponding to the position at which the chaetae sacs form (Fig. 2A, f and g). In early larva, lab expression was strong and broad in the mantle lobe (Fig. 2A, h and i), and in late larvae it became restricted to a few mantle cells adjacent to the chaetae sacs (Fig. 2I, j and k). These cells do not colocalize with tropomyosin, which labels the muscular mesoderm of the larva (Fig. 3A). This finding suggests that lab-expressing cells are likely ectodermal, although we cannot exclude the possibility of localization in nonmuscular mesodermal derivates.

Fig. 3.

Hox expression in mesoderm and periostracum of T. transversa. Double fluorescence in situ hybridization of lab, pb, Hox3, Dfd, and Scr with tropomyosin (Tropo, in green) in late larval stages of T. transversa. (A) The gene lab is expressed in relation to the chaetae sacs, but does not overlap with the tropomyosin-expressing mesoderm. (B–D) The Hox genes pb, Hox3, and Dfd show spatial collinearity along the mantle and pedicle mesoderm. (E) The gene Scr is expressed in the periostracum, the epithelium that forms the shell.

The Hox gene pb was first detected asymmetrically on one side of the ectoderm of the early gastrula (Fig. 2B, b and c). In the mid gastrula, the ectodermal domain was located dorsally and extended as a transversal stripe (Fig. 2B, d and e). Remarkably, this domain disappeared in late gastrula embryos, where pb was detected in the anterior mantle mesoderm (Fig. 2B, f and g). This expression was maintained in early and late larva (Figs. 2 B, h–k and 3B).

Hox3.

The gene Hox3 was detected already in blastula embryos in a circle of asymmetric intensity around the gastral plate (Fig. 2C, a). In early gastrulae, Hox3 was restricted to one half of the vegetal hemisphere, the prospective posterior side (Fig. 2C, b and c). With axial elongation, Hox3 was expressed in the anterior mantle mesoderm and in the ventral ectoderm, limiting the apical and mantle lobe (Fig. 2C, d and e). This expression was maintained in late gastrula stages and in the early larva (Fig. 2C, f–i). In the late larva, Hox3 was detected in part of the ventral internal mantle ectoderm and in the anteriormost part of the pedicle mesoderm (Figs. 2C, j and k and 3C).

Central Hox genes.

The Hox gene Dfd was asymmetrically expressed on one side of the vegetal pole of the early gastrula of T. transversa (Fig. 2D, b and c). This expression was maintained in the mid gastrula and corresponded to the posteriormost region of the embryo (Fig. 2D, d and e). In the late gastrula, Dfd was strongly expressed in the posterior mesoderm (Fig. 2D, f and g). In the early larva, expression remained in the pedicle mesoderm, but new domains in the posterior ectoderm and in the anterior ventral pedicle ectoderm appeared (Fig. 2D, h and i). These expression domains were observed in the late larva as well (Figs. 2D, j and k and 3D).

The central Hox gene Scr was first expressed in the medial dorsal ectoderm of the mid gastrula (Fig. 2E, d and e). In late gastrula stages, Scr expression expanded toward the ventral side, forming a ring (Fig. 2E, f and g). In the early larva, Scr was detected in a ring encircling the anteriormost ectoderm of the pedicle lobe and extending anteriorly on its dorsal side (Fig. 2E, h and i). With the outgrowth of the mantle lobe in the late larva, Scr expression became restricted to the periostracum, the internal ectoderm of the mantle lobe that forms the shell (Fig. 2E, j and k and 3E).

The Hox gene Lox5 was expressed on one side of the early gastrula (Fig. 2F, b and c). During axial elongation, the expression became restricted to the posteriormost ectoderm of the embryo (Fig. 2F, d–g). This domain remained constant in larval stages, where it was expressed in the entire posterior ectoderm of the pedicle lobe (Fig. 2F, h–k).

The Antp gene was weakly detected at the mid gastrula stage, in one posterior ectodermal domain and one dorsal ectodermal patch (Fig. 2G, d and e). In the late gastrula, the posterior expression was maintained and the dorsal domain extended ventrally, encircling the embryo (Fig. 2G, f and g). These two domains remained in the larvae; the ectodermal anteriormost ring-like domain localized to the periostracum, and the posterior domain was limited to the posteriormost tip of the larva (Fig. 2G, h–k).

The Hox gene Lox4 was first detected in the dorsal posterior end of the late gastrula and early larva (Fig. 2H, f–i). In the late larva, Lox4 was expressed dorsally and posteriorly, although it was absent from the posteriormost end (Fig. 2H, j and k).

Posterior Hox genes.

The posterior Hox gene Post2 was first detected in mid gastrula stages at the posterior tip of the embryo (Fig. 2I, d and e). This expression was maintained in late gastrulae (Fig. 2I, f and g). In early larva, Post2 expression extended anteriorly and occupied the dorsoposterior midline of the pedicle lobe (Fig. 2I, h and i). In late, competent larvae, Post2 was detected in a T domain on the dorsal side of the pedicle ectoderm (Fig. 2I, j and k). The Hox gene Post1 was transiently detected in late gastrula stages in the four ectodermal chaetae sacs (Fig. 2J, f and g).

We verified the absence of temporal collinearity in Hox gene expression in T. transversa by real-time quantitative PCR (qPCR) and comparative stage-specific RNA-seq data (Fig. S2).

Fig. S2.

Quantitative expression of Hox genes in T. transversa developmental stages. For comparative stage-specific transcriptomic analyses, total RNA was used for constructing Illumina single end libraries and sequenced in four lanes of a HiSeq 2000 platform. Samples were randomized among the lanes. To estimate the abundance of transcripts per stage, we mapped the single end reads to the transcriptome of T. transversa with Bowtie, calculated expression levels with RSEM, and generated a matrix with TMM normalization across samples by running Trinity’s utility scripts. For real-time qPCR, total RNA was DNase-treated and preserved at −80 °C. Gene-specific primers bordering an intron splice site and defining an amplicon of 80–150 bp were designed for each gene (Table S2). Expression levels of two technical replicates performed in two biological replicates were calculated based on absolute quantification units. (A) RNAseq expression levels calculated by fragments per kilobase of exon per million reads mapped (FPKM). As shown by whole-mount in situ hybridization, Hox3 is the first gene up-regulated in the two biological replicates (female 1, F1; female 2, F2). The Hox genes pb and Post2 are detected at low levels compared with other Hox genes, which obscures their temporal variation during development in the heat map. (B) Real-time qPCR expression levels based on absolute quantification units (AU). PCR was not performed for stages 10–14 (white cells). qPCR confirmed the absence of temporal collinearity, although higher levels of Hox3 were not detected at late blastula (S04), as observed by RNAseq and in situ hybridization, likely owing to technical issues with the qPCR. Stages: S01, oocytes; S02, 8 h mid blastula; S03, 19 h late blastula; S04, 24 h moving late blastula; S05, 26 h early gastrula; S06, 37 h mid gastrula; S07, 51 h late gastrula; S08, 59 h bilobed late gastrula; S09, 68 h trilobed late gastrula; S10, 82 h early larva; S11, 98 h late larva; S12, competent larva; S13, 1 d juvenile; S14, 2 d juvenile. Expression levels obtained by real-time qPCR and comparative stage-specific transcriptomics were plotted with R.

Hox Gene Expression in N. anomala.

To infer potential ancestral Hox expression domains for the Brachiopoda, we investigated the expression of the 9 Hox genes of N. anomala during embryogenesis and larval stages (Figs. 4 and 5).

Fig. 4.

Whole-mount in situ hybridization of the Hox genes during embryonic and larval stages in N. anomala. The gene lab is expressed in the chaetae. The Hox genes Hox3 and Dfd are expressed collinearly in the mantle mesoderm. The genes Scr and Antp are expressed in the prospective shell-forming epithelium. The genes pb and Lox5 are detected in the ectoderm of the mantle lobe. The genes Lox4 and Post2 were not detected in transcriptomes and cDNA during embryonic stages. These expression patterns are described in detail in the text. Black arrowheads indicate expression in the chaetae sacs. Orange arrowheads highlight mesodermal expression. Green arrowheads indicate expression in the periostracum. On top are schematic representations of each analyzed developmental stage on its respective perspective. In these schemes, the blue area represents the mesoderm. Drawings are not to scale. The red line indicates the onset of expression of each Hox gene based on in situ hybridization data. The blastula stage is a lateral view (Inset in Ba is a vegetal view). For each other stage, the left column is a lateral view and the right column is a dorsoventral view. The asterisk demarcates the animal/anterior pole. al, apical lobe; bp, blastopore; ch, chaetae; em, endomesoderm; gp, gastral plate; gu, gut; me, mesoderm; ml, mantle lobe; mo, mouth.

Fig. 5.

Summary of Hox gene expression in T. transversa and N. anomala. Schematic drawings of late larvae of T. transversa (A) and N. anomala (B) depicting the expression of each Hox gene. The Hox genes pb (not in N. anomala), Hox3, and Dfd show staggered expression, at least in one of their domains, associated with the mesoderm (light-blue box). In both brachiopods, the genes Scr and Antp are expressed in the periostracum, or the shell-forming epithelium (red boxes), and lab and Post1 are associated to the developing chaetae (green boxes; asterisk in Post1). Post1 is expressed in the chaetae only during late embryonic stages, not in the mature larva, and only in T. transversa. The expression of Lox4 and Post2 in N. anomala could not be determined in this study. The gene Post1 is missing in N. anomala. Drawings are not to scale.

Anterior Hox genes.

The Hox gene lab was first detected at the mid gastrula stage in three bilaterally symmetrical ectodermal cell clusters that appear to correlate with the presumptive site of chaetae sac formation (Fig. 4A, d and e). The expression in the posteriormost pair was stronger than that in the two anteriormost pairs. This expression level was maintained in the late gastrula stages (Fig. 4A, f and g). In larval stages, lab was detected in the two anteriormost chaetae sacs of the mantle lobe (Fig. 4A, h and i), with expression diminishing in late larvae (Fig. 4A, j and k).

The Hox gene pb was expressed asymmetrically already at blastula stages, in the region that putatively gives rise to the posteriormost body regions (Fig. 4B, a). With the onset of gastrulation, the expression of pb extended around the vegetal pole, almost encircling the entire blastoporal rim (Fig. 4B, b and c). During axial elongation, pb was first broadly expressed in the region that forms the mantle lobe (Fig. 4B, d and e) and later in the ventral mantle ectoderm of the late gastrula (Fig. 4B, f and g). In early larvae, pb was detected in the anterior ventral mantle ectoderm (Fig. 4B, h and i). This domain was not detected in late, competent larvae (Fig. 4B, j and k).

Hox3.

The Hox gene Hox3 was asymmetrically detected around one-half of the vegetal pole of the early gastrulae (Fig. 4C, b and c). In mid gastrulae, the expression almost encircled the entire posterior area and the blastoporal rim (Fig. 4C, d). In addition, a domain in the midposterior mesoderm became evident (Fig. 4C, e). By the end of the axial elongation, Hox3 was strongly expressed in the posterior mesoderm and weakly expressed in the ventral posterior mantle ectoderm (Fig. 4C, f and g). Notably, the posteriormost ectoderm showed no Hox3 expression. This expression pattern was maintained in early and late larval stages (Fig. 4C, h–k).

Central Hox genes.

The central Hox gene Dfd was first detected in the posterior ectodermal tip of mid gastrulae (Fig. 4D, d and e). In late gastrula stages, Dfd was expressed in the posterior ectodermal end (Fig. 4D, f) and in the posterior mesoderm (Fig. 4D, g). Early larvae showed expression of Dfd in the posterior mesoderm and posterior mantle ectoderm (Fig. 4D, h and i). This expression remained in late larvae, although the posteriormost ectodermal end was devoid of expression (Fig. 4D, j and k).

The Hox gene Scr was detected only in late larval stages, in a strong dorsal ectodermal domain (Fig. 4E, j and k). The gene Lox5 was detected asymmetrically around one-half of the blastoporal rim in early gastrula stages (Fig. 4F, b and c). During axial elongation, the expression progressively expanded around the blastoporal rim (Fig. 4F, d and e) and became limited to the ventral midline (Fig. 4F, f and g). In the larvae, Lox5 was expressed in the ventral posteriormost midline (Fig. 4F, h–k).

The Hox gene Antp was first expressed asymmetrically in one lateral side of the early gastrula (Fig. 4G, b and c). In the mid gastrula, Antp was detected in the dorsal ectodermal mantle in a cross-configuration, in the dorsal midline and the mantle cells closer to the apical-mantle lobe boundary (Fig. 4G, d and e). In late gastrulae, Antp was expressed only in a middorsal ectodermal region (Fig. 4G, f and g). This expression pattern also was observed in early larval stages, although the size of the domain was reduced (Fig. 4G, h and i). In late larvae, Antp was detected in a small middorsal patch and a weak ventroposterior ectodermal domain (Fig. 4G, j and k).

We could neither identify nor amplify Lox4 in a transcriptome and cDNA obtained from mixed embryonic and larval stages, suggesting that it is either only very transiently and weakly expressed during embryogenesis or expressed only in later stages (metamorphosis and adulthood).

Posterior Hox genes.

The only posterior Hox gene present in N. anomala, Post2, could not be amplified in cDNA obtained from mixed embryonic and larval stages, suggesting that it is not expressed—or at least expressed only at very low levels—during these stages of the life cycle. The absence of larval expression of Lox4 and Post2 could be related to the lack of the pedicle lobe of craniiform brachiopod larva, which is a characteristic of the lineage (59, 60).

Expression of Arx, Zic, and Notch Components in Brachiopod Chaetal Sacs.

Brachiopods and annelids share the expression of lab and Post1 in the chaetal sacs (26, 43, 61). To further analyze these molecular similarities, we identified and studied the expression of the homeodomain-containing transcription factor Arx, the zinc finger Zic, and components of the Notch signaling pathway, all of which are associated with the development of chaetae in annelids (62–65). Arx was specifically expressed in the developing and mature chaetal sacs (Fig. 6 A and B), whereas Zic was expressed in anterior mesodermal and ectodermal domains as well as in the developing chaetae (Fig. 6 C and D). Similarly, the core components of the Notch pathway, Notch and Delta, were expressed in the developing chaetae, although salt-and-pepper expression in the ectoderm and mesoderm was detected as well (Fig. 6 E–H). The downstream component of the Notch pathway Hes1 was expressed in the mantle lobe ectoderm of the gastrula, where the chaetae sacs form, but was not detected in the larva (Fig. 6 I and J). Finally, the Hes2 ortholog was expressed in the developed chaetae sacs of the larva of the brachiopod T. transversa (Fig. 6 K and L).

Fig. 6.

Expression of chaetae-related genes during T. transversa embryogenesis. (A–L) Whole- mount in situ hybridization of Arx, Zic, Notch, Delta, and two Hes genes in gastrula embryos and larvae of T. transversa. (A) In mid gastrulae, Arx is expressed in the ectoderm of the prospective chaetae sac territories (black arrows) and in a ventral domain. (B) In early larvae, Arx is expressed in the chaetae sacs (black arrows). (C) In late gastrulae, Zic is expressed in the mesoderm of the chaetae sacs (black arrows), apical lobe mesoderm, and anterior ectoderm. (D) In early larvae, Zic is detected in the chaetae sacs (black arrows), in a domain in the pedicle lobe, and in the anterior mesoderm and anterior ectoderm. (E and F) Notch is broadly expressed in the ectoderm and mesoderm of the late gastrula and early larva, particularly in a cluster of a few cells of the developing chaetae (black arrows). (G and H) Delta is strongly expressed in the apical lobe and in a salt and pepper manner in the mantle and pedicle lobe, including the chaetae (black arrows). (I and J) Hes1 is observed in the lateral ectoderm of the gastrula (black arrows), in the area that will subsequently form the chaetae and mantle lobe ectoderm. It is not detected in the larva. (Black arrowheads indicate background expression in J.) (K and L) Hes2 is detected in a salt and pepper manner in the ectoderm and mesoderm of the T. transversa embryo, and in the chaetae of the larva (black arrows). The images are dorsal views (except I and K), with the anterior pole at the top. (M) Morphological similarities between brachiopod and annelid chaetae. Drawing adapted with permission from ref. 84. (N) The shared morphological and molecular characters of chaetae in Brachiopoda and Annelida, together with the presence of chaetae-like structures (chaetae sign with a question mark) in the Mollusca and Bryozoa, support the homology of this lophotrochozoan novelty.

Discussion

The Brachiopod Hox Complement and Evolution of Hox Genes in Spiralia.

Our findings regarding T. transversa and N. anomala reveal an ancestral brachiopod Hox gene complement consistent with what has been hypothesized to be ancestral for Spiralia and Lophotrochozoa based on degenerate PCR surveys (15, 66–68). This ancient complement comprises ten Hox genes—lab, pb, Hox3, Dfd, Scr, Lox5, Antp, Lox4, Post2, and Post1—and has been confirmed by genomic sequencing of representative annelids and mollusks (16, 22, 44), rotifers and platyhelminthes (21, 40–42), and the linguliform brachiopod L. anatina (69). Whereas T. transversa and L. anatina have retained this ancestral Hox complement, N. anomala has apparently lost Post1 (Fig. 1). Although a previous analysis based on degenerate PCR primers reported the presence of Lox2 in the brachiopod L. anatina (15), current high-throughput sequencing approaches seem to restrict Lox2 to Annelida and Mollusca (Fig. S3). Whether multiple independent losses in diverse spiralian lineages shaped the evolutionary history of this gene, or whether this represents a molecular synapomorphy of annelids and mollusks, requires further study, specifically involving a thorough genomic sequencing of a larger number of lophotrochozoan taxa.

Fig. S3.

Evolution of Hox organization and expression across Metazoa. The table presents the features of the Hox gene complement of each animal lineage in a phylogenetic framework. The Hox complement is summarized by the presence of at least one representative each of the anterior, Hox3, central, and posterior ortholog groups. The Hox number indicates the possible ancestral number, but can vary between species. In Craniata, the number corresponds to the human Hox complement, which consists of four clusters (asterisk). The cluster organization can be of one of three types: organized (O), disorganized (D), or split (S). For species with an atomized cluster, the cluster is described as absent (no presence). Question marks indicate unknown data, and dashes indicate absences. In Cephalochordata, the asterisks indicate that Hox6 and Hox14 do not follow the general temporal and collinear expression in Branchiostoma lanceolatum. See the main text for references.

Our genomic information shows that the Hox cluster of T. transversa is split into three parts, with lab and pb separate from the major cluster and Post1 also on a separate scaffold (Fig. 1B). Overall, this Hox cluster extends for >100 kb, which is significantly shorter than clusters of other lophotrochozoans, such as C. teleta (∼345 kb) (43) and L. gigantea (∼455 kb) (16). Its compact size is related to short intergenic regions and introns, comparable to the situation observed in vertebrate Hox clusters (23). The order and orientation of the Hox genes in T. transversa are preserved and more organized compared with the Hox cluster reported for the brachiopod L. anatina, which exhibits a genomic rearrangement that places a portion of the cluster upstream of lab and in reverse orientation (69). Indeed, the split Hox clusters reported so far in lophotrochozoan taxa exhibit multiple different conformations, indicating that lineage-specific genomic events have shaped Hox gene clusters in Spiralia.

Noncollinearity of Hox Expression in the Split Cluster of T. transversa.

Our analysis of Hox clustering in different animal species together with the temporal and spatial expression patterns of their Hox genes supports the hypotheses that the regulatory elements required for their collinearity—mostly temporal—maintain the clustered organization of the Hox genes in vertebrates and possibly other animals as well (13, 23, 55–57, 70, 71) (Fig. S3). Although there are cases in which spatial collinearity is exhibited in the absence of a cluster, as in the appendicularian chordate Oikopleura dioica (24), all investigated clustered Hox genes show at least one type of collinearity that could account for their genomic organization (23, 57). Given that there are exceptions to the spatial collinearity in vertebrates—e.g., Hoxa2 and Hoxb2 are expressed more anteriorly than Hox1 genes in the vertebrate hindbrain (72)—temporal collinearity is seen as a manifestation of Hox clustering; however, whether temporal collinearity is the agent keeping the cluster together [e.g., through enhancer sharing (73)] remains a matter of debate.

Within Spiralia, this evolutionary scenario appears to be supported by the staggered temporal and spatial expression of the Hox genes in the split cluster of the annelid C. teleta (43). In the other investigated spiralians, there is only either genomic information (e.g., the mollusks L. gigantea and C. gigas) or expression analysis (e.g., the mollusks Gibbula varia and Haliotis asinina) (16, 44, 49, 54). Most of these previous gene expression studies have demonstrated coordinated spatial or temporal expression of Hox genes along the animal’s anteroposterior axis (45, 46, 61) or in organ systems, such the nervous system (49, 54). However, the absence of studies revealing a correlation between Hox gene expression and their genomic organization in these animals hinders reconstruction of the putative mechanisms that preserve Hox clusters in Lophotrochozoa, and thus preclude generalizations about possible scenarios of Hox cluster evolution across all animals.

Our findings robustly demonstrate that overall, the Hox genes of the split Hox cluster of T. transversa exhibit neither strictly spatial nor temporal collinearity (Figs. 2 and 3), and lack quantitative collinearity (57), as has been shown in mice, for example (74). These observations are also supported by the absence of coordinated spatial and temporal expression of the Hox genes in N. anomala (Fig. 4). Although a general trend toward spatial collinearity is present (e.g., the posterior Hox genes are expressed in posterior tissues), the early expression of Hox3 breaks temporal collinearity in T. transversa, whereas it is pb that is first expressed in N. anomala. In both species, Lox5 is also expressed before Scr, as it is also the case in the annelid Nereis virens (61). Ectodermal spatial collinearity is absent in the two brachiopods even when the future location of the larval tissues after metamorphosis is considered (75, 76). The anteriormost class gene lab is expressed exclusively in the chaetae of T. transversa and N. anomala, and thus is not affiliated with anterior neural or foregut tissues as in other lophotrochozoans, such as annelids (43, 77). Similarly, the posteriormost Hox gene, Post1, is very transiently expressed in the chaetae sacs, which occupy a midposition in the larval body. We detected strict spatial collinearity only in the staggered expression of the Hox genes pb, Hox3, and Dfd along the anteroposterior axis of the developing larval mesoderm in both T. transversa and N. anomala (Fig. 5). Thus, the absence of strict temporal and spatial collinearity exhibited by the split Hox cluster in the brachiopod T. transversa supports the idea that temporal collinearity might help keep spiralian Hox genes clustered, as seems to be the case in vertebrates and at least some arthropods (14, 23, 25, 55–57, 78). However, other factors, such as unequal rates of genome rearrangements in different lineages and shared enhancers between genes, also might contribute to the genomic evolution of Hox genes.

Recruitment of Hox Genes for Patterning Lophotrochozoan Chaetae and Shell Fields.

The bristle-like chaetae (or setae) of annelids and brachiopods and shell valves in mollusks and brachiopods are the most prominent hard tissues found in lophotrochozoan spiralians (79) and provide fossilized hallmarks of the Cambrian explosion (80). Chaetae-like structures are also present in the sensory organs of polyplacophoran mollusks (81), Kölliker’s organ of juvenile octopods (82), and the gizzard teeth of some bryozoans (83). The ultrastructural morphology of the brachiopod and annelid chaetae are known to be nearly identical (84–86) (Fig. 6M), and with the placement of brachiopods as close relatives of annelids and mollusks (87), the homology of these structures appears more likely (88). In this context, the anterior Hox gene lab is expressed in the chaetae of Chaetopterus sp. (26) and in the ectoderm around chaetal sacs in N. virens (61), and Post1 is expressed in the chaetae of C. teleta, Platynereis dumerilii, and N. virens (43, 61). Our results show that, similarly, lab and Post1 are expressed specifically in the chaetal sacs of the brachiopods T. transversa and N. anomala (Figs. 2 and 4) and follow the different arrangements of the chaetae in both species. Further evidence of a common, and likely homologous, molecular profile comes from expression of the homeodomain gene Arx, the zinc finger Zic, and components of the Notch signaling pathway. These genes are expressed at each chaetae sac territory in the Platynereis larva (62, 64), in C. teleta (63, 65), and also in the region of the forming chaetae sac territories in T. transversa (Fig. 6 A–L and Fig. S4). Therefore, the expression of the Hox genes lab and Post1 and the homeodomain gene Arx indicate that a similar molecular signature underlays the development of chaetae in annelids and brachiopods. This property, together with the evident and striking morphological similarities shared by brachiopod and annelid chaetae, lend support to considering these two structures homologous and thus common lophotrochozoan innovations (Fig. 6N). This would be consistent with placing the iconic Cambrian fossil Wiwaxia, which contains chaetae, as a stem group lophotrochozoan (89).

Fig. S4.

Orthology assignment of the T. transversa Arx gene. (A) Amino acid alignment of the homeodomain region of representative sequences of the PRD gene class. Aristaless-related genes (ARX) are highlighted with a rectangle, and all of them share a unique tyrosine (Y) at position six of the alignment (indicated by a red arrow on top). (B) Maximum likelihood tree of Aristaless-related (ARX), Aristaless-like (ALX), and Homeobrain (HBN; also termed ARX-like) genes. Although the monophyly of ARX genes is dubious, the T. transversa ARX gene is not an ALX or HBN member. Orthology analyses were conducted with RAxML v.8.2.6 using the autoMRE option and LG+G substitution model. Bootstrap values are indicated for each node.

The protective shell is a mineralized tissue present in brachiopods and mollusks. In the gastropod mollusk G. varia, the Hox genes lab, Post1, and Post2 are first expressed in the shell field, and later Dfd is expressed in the shell field as well (53). In H. asinina, lab and Post2 also are related to shell formation (49). In brachiopods, Dfd is associated with the adult shell in L. anatina (69); however, during embryogenesis of T. transversa and N. anomala, only Scr and Antp are expressed in the shell fields, and lab and Post1 are not; here lab and Post1 are expressed in the chaetae sacs. These properties could support the homology of the chitin network formed at the onset of brachiopod and mollusk shell fields. However, the differing deployment of Hox genes in the shell fields of brachiopods and mollusks might indicate that these genes do not have an ancient role in the specification of the shell-forming epithelium. Instead, their consistent deployment during shell development might reflect a more general, conserved role in shaping the shell fields according to their position along the anteroposterior axis.

Conclusion

In this study, we have characterized the Hox gene complement of the brachiopods T. transversa and N. anomala, demonstrating that the last common ancestor to all brachiopods likely had 10 Hox genes (lab, pb, Hox3, Dfd, Scr, Lox5, Antp, Lox4, Post2, and Post1). Noticeably, brachiopod Hox genes do not exhibit global temporal and spatial collinearity, although T. transversa exhibits a split Hox cluster. Only the genes pb (in T. transversa), Hox3 (in both brachiopods), and Dfd (in both brachiopods) show spatial collinearity in the “trunk” mesoderm. The dramatic divergence of the expression of Hox genes from the supposed ancestral state for Hox expression while still retaining a relatively intact Hox cluster might indicate that the loss of constraint on the organization of the Hox cluster in T. transversa is relatively recent. In addition, the Hox genes lab and Post1, as well as the homeobox Arx, are expressed in the developing chaetae, as also has been reported for some annelid species (43, 50, 61). These molecular similarities, together with evident morphological resemblances (85), lend support to considering brachiopod and annelid chaetae homologous structures and reinforce the classification of the fossil Wiwaxia as a stem group lophotrochozoan (89).

Materials and Methods

Animal Cultures.

Gravid adults of T. transversa (Sowerby, 1846) were collected around San Juan Island, WA, and those of N. anomala (Müller, 1776) were collected around Bergen, Norway. Animal husbandry, fertilization, and larval culture were conducted following previously published protocols (90–92).

Hox Cluster Reconstruction in T. transversa and N. anomala.

Male gonads of T. transvesa and N. anomala were preserved in RNAlater (Life Technologies) for further genomic DNA (gDNA) isolation. Paired-end and mate pair libraries of 2 kb and 5 kb insert sizes of T. transversa gDNA were sequenced using the Illumina HiSeq 2000 platform. We first trimmed Illumina adapters with Cutadapt 1.4.2 (93), then assembled the paired-end reads into contigs, scaffolded the assembly with the mate pair reads, and closed the gaps using Platanus 1.21 (94). The genomic scaffolds of T. transversa including Hox genes are available in the GenBank database (accession nos. KX372775 and KX372776). We sequenced the paired-end libraries of N. anomala gDNA using the Illumina HiSeq 2000 platform. We then removed Illumina adapters as above and assembled the paired-end reads with MaSuRCA 2.2.1 (95).

Gene Isolation.

Genes were identified by BLAST searches on public transcriptomes of T. transversa and N. anomala developmental stages (National Center for Biotechnology Information Sequence Read Archive; T. transversa, GenBank accession no. SRX1307070; N. anomala, GenBank accession no. SRX1343816) and their respective draft genomes (see above). All gene sequences have been uploaded to GenBank (accession nos. KX372756–KX372774 and KY124237–KY124242).

Gene Expression Analyses.

Single colorimetric whole-mount in situ hybridization was carried out following an established protocol (detailed protocol available in Protocol Exchange, 2008; www.nature.com/protocolexchange/protocols/480) (96, 97). Double fluorescence in situ hybridization was conducted as described elsewhere (98, 99).

Quantitative Hox Gene Expression in T. transversa.

Thousands of synchronous T. transversa embryos collected at 14 specific stages [oocytes, 8 h mid blastula, 19 h late blastula, 24 h moving late blastula, 26 h early gastrula, 37 h asymmetric gastrula, 51 h bilateral gastrula, 59 h bilobed, 68 h trilobed, 82 h early larva (first chaetae visible), 98 h late larva (long chaetae, eye spots), 131 h competent larva, 1 d juvenile, and 2 d juvenile] were pooled together and preserved in RNAlater (Life Technologies). The same material was used for real-time qPCR and stage-specific transcriptomes (Fig. S2 and Table S2).

Table S2.

Primers used for real-time qPCR experiments

Gene	Forward	Reverse
lab	CAAAGCTCCGTAGCCACTTA	TCGAGCTCTGTCAATTGCTT
pb	AACAAATCGGATGGCTCTG	TTCATGGTCTGCTTCCTCTG
Hox3	ACTTCGCGTTAGCCAATCA	TGCAGGAACCCTTCAGAAA
Dfd	ATGCCGAGTATAAGCCGTTC	TATACCCGTGGATGAAACGA
Scr	ACGTCTGATGCCTGGTGTAG	ATAGCCATGAACAAATGCCA
Lox5	GTGTACGTTTGCCTGGTACG	GCATGTCGCAAGCGTATAGT
Antp	TCTCAAGCTCGAGTGTTTGG	GGAGACGCAGATAACGACAG
Lox4	GTTTGTCGACCGCGTCTT	AAATGGATACGGGTCTGCTC
Post2	GCTCCTGTGGCATTGTGTAG	AGCAAGCAAGCCCTGTAGAT
Post1	AACGTTGTCCCATTCTCTCC	CGATATACTATGCGGACCCA