Ancestral role of Pax2/5/8 in molluscan brain and multimodal sensory system development

Background Mollusks represent the largest lophotrochozoan phylum and exhibit highly diverse body plans. Previous studies have demonstrated that transcription factors such as Pax genes play important roles during their development. Accordingly, in ecdysozoan and vertebrate model organisms, orthologs of Pax2/5/8 are among others involved in the formation of the midbrain/hindbrain boundary, the auditory/geosensory organ systems, and the excretory system. Methods Pax2/5/8 expression was investigated by in situ hybridization during the development of representatives of the two major molluscan subclades, Aculifera and Conchifera. Results Compared to the investigated polyplacophoran and bivalve species that lack larval statocysts as geosensory organs and elaborate central nervous systems (CNS), cephalopods possess highly centralized brains and statocysts. Pax2/5/8 is expressed in regions where sensory cells develop subsequently during ontogenesis. Expression domains include esthetes and the ampullary system in polyplacophorans as well as the eyes of cephalopods. No Pax2/5/8 expression was observed in the less centralized CNS of bivalve, polyplacophoran, and gastropod embryos, thus arguing for a loss of Pax2/5/8 involvement in CNS development in these lineages. In contrast, Pax2/5/8 is expressed among others in brain lobes along the trajectory of the esophagus that divides the cephalopod brain. Conclusions Our results, along with those on Otx- and Hox-gene expression, demonstrate that the cephalopod condition is similar to that in mouse and fruit fly, with Otx being expressed in the anterior-most brain region (except for the vertical lobe) and a Pax2/5/8 expression domain separating the Otx-domain from a Hox-gene expressing posterior brain region. Thus, Pax2/5/8 appears to have been recruited independently into regionalization of non-homologous complex brains of organisms as different as squid, fruit fly, and mouse. In addition, Pax2/5/8 is expressed in multimodal sensory systems in mollusks such as the esthetes and the ampullary system of polyplacophorans as well as the eyes of cephalopods. Pax2/5/8-expressing cells are present in regions where the future sensory cells such as the polyplacophoran esthetes are situated and hence Pax2/5/8 expression probably predates sensory cell development during ontogeny. In mollusks, Pax2/5/8 is only expressed in derivatives of the ectoderm and hence an ancestral role in molluscan ectoderm differentiation is inferred.


Background
A coordinated tempo-spatial expression of transcription factors is required for cell type specification and the differentiation of the three germ layers into distinct organ systems during bilaterian ontogeny [1]. Studies on ecdysozoan and deuterostome model organisms have elucidated the multifold roles of transcription factors during development; however, a major gap in knowledge does exist for the Lophotrochozoa, one of the three bilaterian superphyla [2]. For inferences concerning the evolutionary conservation versus plasticity of transcription factor expression patterns and their putative roles, a broad comparative approach is imperative. Among the lophotrochozoans, Mollusca is the most speciose phylum and exhibits extremely diverse body plans and adaptative capacities ranging from worm-like spicule-bearing aplacophorans and sessile bivalved clams to highly motile squids (Fig. 1). No less diverse are the ontogenies, with direct and indirect developers and larval forms ranging from trochophore and pericalymma larvae to veligers [3]. For the detection of settlement cues, predator avoidance, location of prey, as well as navigation in a complex environment, the larvae are equipped with sensory organs. The most prominent larval sensory organ is the apical organ that is present in representatives of all molluscan classes investigated so far, except for cephalopods [4][5][6]. It is equipped with flask-shaped (ampullary) cells and in some mollusks with additional peripheral cells. A so-called ampullary system has been described in polyplacophoran larvae and appears to constitute an apomorphy of the class [5]. It comprises four pairs of sensory cells that are located dorsolaterally and ventrolaterally in the episphere of the trochophore. Cells are of the ampullary sensory cell type, exhibit serotonin-like and FMRFamide-like immunoreactivity and are innervated by the cerebral commissure [5]. Another larval sensory organs, the so-called post-anal organ, only appears to exist in protobranch bivalves [4]. It is composed of sensory cells with two types of cilia and a cavity in proximity to the anus. Coleoid cephalopods do not possess larvae as direct developers but a plethora of sensory organs such as complex camera eyes. These have been subject of a recent study suggesting that a vast number of genes expressed in the squid camera-type eye is not found in the transcriptomes of the nautiloid pinhole eye [7]. Sensory organs as well as other epidermal epithelia or the nervous system are ectodermal derivatives [8]. In mollusks, sensory organs have been described by histological and immunochemical techniques, however, the molecular underpinnings of their development and evolution remain largely obscure (but see [9]).
Members of the paired box (Pax) gene family encode transcription factors that are involved in a variety of processes such as the development of excretory organs, the differentiation of the musculature, neuroectoderm specification, and the development of auditory/geosensory systems [10,11]. In bilaterians, nine orthologous groups of Pax proteins are known that can be distinguished on the basis of the presence or absence of highly conserved structural domains [12]. Members of the Pax2/5/8 group exhibit a conserved N-terminal paired domain, an octapeptide, a partial homeodomain, and a C-terminal transactivation domain [10]. The N-terminal domain and the homeodomain serve as DNA binding sites, the octapeptide is the site for activation or repression of the gene, and the C-terminal domain is the protein/protein interaction domain [10]. In vertebrates and ecdysozoans, Pax2/5/8 is involved among others in the genesis of the auditory/geosensory system as well as in the formation of the midbrain/hindbrain boundary [13,14]. For lophotrochozoans, so far only one study has been published on the expression domains and the putative function of Pax2/5/8 during development. In this study, Pax2/5/8 expression was found in the statocysts, i.e. the geosensory organ, of the gastropod veliger larva [9].
In order to elucidate putative roles of Pax2/5/8 during molluscan development, we investigated the expression of its orthologs in representatives of both major molluscan lineages, the Aculifera and the Conchifera [15,16] (Fig. 1). As a polyplacophoran, Acanthochitona crinita has been shown to exhibit putative ancestral features of the Aculifera [17], while the conchiferan Nucula tumidula is a protobranch that holds a basal position among the bivalves and possesses a unique pericalymma-type larva [4,18]. A. crinita and N. tumidula do not possess statocysts in their larvae and exhibit a less centralized CNS. In contrast, the conchiferan cephalopod Idiosepius notoides has a highly centralized CNS and statocysts. This study will test whether Pax2/5/8 is expressed in ectodermal, endodermal, or mesodermal derivatives during the ontogeny of all three molluscan species and it will shed light on the shared Pax2/5/8 expression among bilaterian representatives.

Pax2/5/8 gene orthologs and phylogenetic analysis
The multiple sequence alignment of Pax2/5/8 orthologs shows the highly conserved N-terminal paired domain, the octapeptide domain, and the lysine-arginine rich region (Fig. 2b). In particular the octapeptide is less conserved among the bilaterian Pax2/5/8 orthologs. The C-terminal partial homeodomain was not included in the alignment since it is even less well conserved. The phylogenetic analysis included proteins of all Pax families and demonstrates that all three molluscan Pax2/5/8 amino acid sequences cluster with their bilaterian orthologs (Fig. 3).

Acanthochitona crinita
The gastrula stage is reached between 3-5 h post fertilization (hpf) at 20°C. During this stage Acr-Pax2/5/8 is expressed in ectodermal cells of the apical region ( Fig. 4a-c). Early trochophore larvae hatch at approximately 12 hpf. They are lecithotrophic and are slightly ovoid in shape, with an apical organ and apical tuft in the episphere (Fig. 4d-h). The apical organ comprises three serotonin-like immunoreactive flask-shaped cells and faint FMRFamide-like immunoreactivity (Fig. 4d, e). Two other groups comprising four FMRFamide-like immunoreactive cells each are located in the episphere close to the prototroch (Fig. 4e). The episphere is separated from the hyposphere by two rows of trochoblasts that give rise to the prototroch. Acr-Pax2/5/8 is expressed in different domains, here referred to as "groups", located mainly in the larval episphere ( Fig. 4e-l). Several Acr-Pax2/ 5/8-expressing cells are located in the ectoderm of the dorsal episphere adjacent to both rows of trochoblasts (group 1 in Figs. 4e, j, k, l and 5). In addition, two other groups are located bilaterally and anterior to group 1 (group 2 in Figs. 4f, j, l and 5). Each group 2 comprises approximately four ectodermal cells that are located subepidermally and project dendrites through the epidermis (group 2 in Figs. 4f, j and 5). Two single Acr-Pax2/5/8-expressing cells are located bilaterally, immediately adjacent to the two rows of trochoblasts and more ventrally to group 2 in the episphere (group 3 in Figs. 4g, i and 5). In the center of the ventral episphere, a group of 5-10 ectodermal Acr-Pax2/5/8-expressing cells is present (group 4 in Figs. 4h, k, l and 5). The only Acr-Pax2/5/8 expression domain that is located in the hyposphere of the early trochophore is situated in the region of the nascent shell fields in the dorsal hyposphere (arrowheads in Figs. 4f, g, j, k and 5). The apical organ does not express Acr-Pax2/5/8 (red dashed circle in Fig. 4f, j, l). In further developed trochophore larvae (35 hpf), Acr-Pax2/5/8-expression is less pronounced in the episphere, while Acr-Pax2/5/8 expression in the hyposphere gains in intensity ( Fig. 5; arrowheads in Fig. 6a, c). This and other Acr-Pax2/5/8-expressing cell groups are located in similar expression domains as described for earlier developmental stages ( Fig. 5; cf. arrowheads in Fig. 4j, k with arrowheads in Fig. 6a, c). Acr-Pax2/5/8expressing cells are present in the expression domains of groups 1, 2, and 4 of previous stages (c.f. Figs. 5 and 6a, c, d). Furthermore, serotonin-like immunoreactive cell somata are located in corresponding regions, such as the lateroventral ampullary cells corresponding to Acr-Pax2/5/8-expressing group 2, or the additional sensory cells that are located in the dorsal episphere that correspond to group 1 (Fig. 6b). In the expression domains of Acr-Pax2/5/8-expressing groups 3 and 4, FMRFamide-like immunoreactive cell somata are located (not shown). In further developed trochophore larvae (35 hpf ), the apical organ does not exhibit Acr-Pax2/5/8 expression, however, very close to it, Acr-Pax2/5/8-expressing cells are located (Fig. 6c). In metamorphic competent trochophore larvae (65 hpf ), the Acr-Pax2/5/8 expression pattern is similar to the one of earlier trochophores (35 hpf ) (Figs. 5 and 6e-k). Strong Acr-Pax2/5/8 expression is present in cells of the nascent shell fields (Figs. 5 and 6e). These Acr-Pax2/5/8-expressing cells are located in the epidermis and project dendrites to the periphery (inset in Fig. 6j). In addition, Acr-Pax2/5/8-expressing cells are numerous in groups 1-4 during earlier development (Figs. 5 and 6f-j). Interestingly, all these Acr-Pax2/5/8 expression ; arguing for the Aculifera-Conchifera concept (but see [41] for a different view) Fig. 2 Alignments of the molluscan Pax2/5/8 domains and their bilaterian orthologs. a Paired domain alignment of the predicted amino acids of various metazoan Pax genes including Acr-Pax2/5/8, Ntu-Pax2/5/8, and Ino-Pax2/5/8 (highlighted in red). This alignment was used for the maximum-likelihood consensus tree shown in Fig. 3. b Alignment of Pax proteins of various metazoans including Acr-Pax2/5/8, Ntu-Pax2/5/8, and Ino-Pax2/5/8 (highlighted in red). The conserved N-terminal paired domain (red), the octapeptide (blue), and the lysine-arginine-rich region (green) are shown, while the partial homeodomain and the C-terminal transactivation domain are omitted. GenBank accession numbers of all encoding genes used are listed in Table 1 domains house FMRFamide-like-immunoreactive (usually flask-shaped) cells (Fig. 6j).
An additional Acr-Pax2/5/8 expression domain is present close to the lateral borders of the foot (Fig. 5, group 5 in Fig. 6h, i). Both groups 5 are interconnected via a thin band of Acr-Pax2/5/8-expressing cells that runs along the abapical side of the mouth (Figs. 5 and 6h, i). Few Acr-Pax2/5/8-expressing cell are present in the posterior mantle fold (Figs. 5 and 6k), while no Acr-Pax2/5/8-expressing cells are located in the region of the apical organ (dashed circle in Fig. 6k).
Settled animals (130 hpf ) commence to reduce their episphere and the foot becomes more pronounced. Metamorphosis is largely completed with the shedding of the prototroch, although the eighth shell plate is not formed until probably several weeks after settlement. Early settled animals solely possess few faintly stained Acr-Pax2/5/8-expressing cells in the region of the shell fields ( Fig. 6l) and only non-specific staining was observed in the perinotum that surrounds the plates (Fig. 6l).
Pax2/5/8 expression in the bivalve Nucula tumidula Embryos of N. tumidula reach the 2-4 cell stage by 12 hpf at 6.5°C and the zygotes gastrulate by approximately 24 hpf. During gastrulation, Ntu-Pax2/5/8 is expressed in approximately five ectodermal cells close to the blastopore (Fig. 7a). Early pericalymma larvae swim by approximately 48 hpf, are roundish, and possess 5-7 Ntu-Pax2/5/ 8-expressing cells on the future dorsal side below the test-cells (Fig. 7b-d). Slightly further developed  Fig. 2a. Note that Acr-Pax2/5/8, Ntu-Pax2/5/8, and Ino-Pax2/5/8 (all highlighted in red) cluster with the predicted proteins of their bilaterian Pax2/5/8 orthologs (red branches/tips). Pax protein families are labeled in different colors pericalymmae are characterized by an apical organ with a ciliary tuft, while mouth and anus are located on the abapical side (Fig. 5). They are covered with large, multiciliary calymma (test) cells. Subsequently, three rows of test cells bear three bands of longer cilia, while shorter cilia still cover the area around the apical tuft as well as the abapical side around mouth and anus. These larvae express Ntu-Pax2/5/8 in the area of the dorsal mantle (Fig. 7e) and the expression domain also includes the shell gland that develops as an invagination after gastrulation on the future dorsal side (Fig. 7f, g). During subsequent development the apical-abapical axis elongates and the foot develops on the ventral side. Meanwhile, the shell field on the dorsal side has secreted two shell valves. Further developed larvae express Ntu-Pax2/5/8 in more ventral and apical mantle regions (Fig. 7h). Late pericalymma larvae express Ntu-Pax2/5/8 along the dorsal mantle region as well as in ventral domains of the mantle (Figs. 5 and 7i-k). ). c Confocal reflection scan highlighting same Acr-Pax2/5/8-expressing cells (arrowheads) in apical ectoderm as shown in a-b d Three serotonin-like immunoreactive neurons of the apical organ in a 12 hpf early trochophore. e-h Optical sections from dorsal to ventral through an early trochophore (12 hpf) showing Acr-Pax2/5/8-expressing domains 1-4 (termed "group 1-4" in the following) in the episphere and single Acr-Pax2/5/8-expressing cells in the hyposphere (arrowheads in (f-g)). e Upper inset: Several Acr-Pax2/5/8-expressing cells (1) are located in the ectoderm of the dorsal episphere adjacent to the prototroch (pt). Lower inset: This very early trochophore (10 hpf) exhibits faint FMRFamide-like immunoreactive signal (red staining) in the region of the apical organ and in two groups of four FMRFamide-like immunoreactive ectodermal cells each in the episphere close to the prototroch. f Two other bilateral groups of Acr-Pax2/5/8-expressing cells (2) are located anterior to group 1 and additional Acr-Pax2/5/8-expressing cells lie in the region of the nascent shell fields in the hyposphere (arrowhead). g Two Acr-Pax2/5/8-expressing ectodermal cells (3) are located bilaterally in the episphere adjacent to the trochoblasts. h A group of 5-10 ectodermal Acr-Pax2/5/8-expressing cells (4) lies in the central ventral episphere. i-k Same specimen as shown in e-h from the left side (i) to the mid-sagittal plane (k). l Apical view of same specimen as shown in e-k with Acr-Pax2/5/8 expression domains 1, 2, and 4. Abbreviations: bc, blastocoel; f, foot; pt, prototroch. Scale bars : 20 μm Larvae that are metamorphic competent are more ovoid in shape than earlier larvae and express Ntu-Pax2/5/8 mostly in dorsal domains but also in ventral ones (summarized in Figs. 5 and 7l). Lecithotrophic larvae swim until 15-20 dpf in the water column and finally settle. During the settlement process that may last for several hours, larvae appear to probe the substrate and metamorphosis commences with shedding and ingestion of the

Pax2/5/8 expression in the cephalopod Idiosepius notoides
As a typical cephalopod, I. notoides is a direct developer with discoidal cleavage. Hatching of the juvenile occurs within 9-10 days after oviposition at 25°C. In the present study, we adhere to the common designation of  (1), the shell fields (arrowheads in (a & c)), the dorso-lateral (2) and ventral episphere (4), and close to the apical organ (inset in c) in 35 hpf old trochophores (optical sections from dorsal (a) to ventral (d)). b A further developed, 35 hpf old, trochophore (left micrograph) and a slightly younger specimen (right micrograph) with serotonin-like immunoreactive latero-ventral ampullary cells (las), additional sensory cells (yellow dashed circles), apical organ cells (red dashed circles), and cells in the ventral hyposphere (arrowheads). e-h In competent trochophores (65 hpf) (optical sections from dorsal (e) to ventral (h)), Acr-Pax2/5/8 is expressed in cells of the shell fields (arrowheads), in cells of group 1, 2, and 4 (c.f. Fig. 4), and in two groups of cells (5) along the developing foot (f), which are connected via a slender bridge of cells. Inset in g Acr-Pax2/5/8-expressing cells close to the apical organ. i-k Lateral view of trochophore (65 hpf) shown in (e-h). j Dashed box marks location of the small inset that depicts a confocal reflection scan of Acr-Pax2/5/8-expressing subepidermal flask-shaped cells (red) in the shell fields. Right large inset: FMRFamide-like immunoreactive cell somata (red) in the shell fields (arrowheads) and close to the trochoblasts. k Acr-Pax2/5/8 expression in the posterior mantle fold (arrowhead). l No Acr-Pax2/5/8 expression was observed in postmetamorphic (settled) specimens (130 hpf). The staining of the perinotum is unspecific (arrowhead). Abbreviations: at, apical tuft; pt, prototroch. Scale bars: 20 μm (except inset in (j) 10 μm) embryological orientation in cephalopods, i.e., the mantle apex is considered dorsal and the cephalic region including the brachial crown that surrounds the mouth is considered ventral. The funnel is located posteriorly, while the opposite side including the supraesophageal mass is considered anterior.

Evolution of Pax2/5/8 proteins
Our phylogenetic analysis confirms the identities of all three molluscan Pax2/5/8 proteins presented herein. The sequences of Acanthochitona crinita, Idiosepius notoides, and Nucula tumidula exhibit a paired domain, an octapeptide, the lysine-arginine rich region, as well as the partial homeodomain (not shown) diagnostic for Pax2/5/ 8.
Pax2/5/8 is expressed in the development of multimodal sensory systems of mollusks Among bilaterians, Pax2/5/8 expression is particularly well-investigated in the Deuterostomia and Ecdysozoa [13,20]. Studies on various deuterostomes revealed that orthologs of Pax2/5/8 play a role during development of auditory/ geosensory organs [21][22][23]. Interestingly, the phylogenetically distantly related gastropod mollusk Haliotis asinina also expresses Pax2/5/8 in its geosensory organ, the statocysts [9]. This present study demonstrated that homeobox genes may be recruited into the development of organs with similar function but of different evolutionary origin (i.e., in non-homologous systems). It also poses the question as to how Pax2/5/8 is recruited during the ontogeny of organisms without auditory and equilibrial sense.
In our study, we included the chiton Acanthochitona crinita and the bivalve Nucula tumidula, both lophotrochozoan representatives without larval statocysts, in order to assess the role of Pax2/5/8 during their development. Pax2/5/8 is expressed in several groups of ectodermal cells in the episphere and the hyposphere of the polyplacophoran trochophore. Although A. crinita and N. tumidula possess larval apical (sensory) organs, no Pax2/5/8 expression was detected in components of this organ. In A. crinita cells of groups 2-3 are located in a similar location as cells of the larval ampullary sensory system that is an autapomorphy of Polyplacophora (Figs. 5 and 6 in present study; [5]). Cells of the ampullary system are flask-shaped and probably chemosensory rather than mechanosensory [5]. Pax2/5/8 expression and FMRFamide-like immunoreactivity may be colocalized in ectodermal cells in the dorsal episphere adjacent to the prototroch (group 2, c.f. Figs. 5 and 6j). Further Pax2/5/8-expressing ectodermal cells (group 4) are located in the ventral episphere and might correspond to the four serotonin-like immunoreactive flaskshaped cells (c.f. group 4 in Figs. 4h, 5, 6b). The immunochemical and molecular phenotype, their flask-shaped somata, and their subepithelial location argue for a sensory role of the above-mentioned cells [5]. This might also be true for the Pax2/5/8-expressing groups 5 that are located on the ventral side along the borders of the foot of advanced trochophore larvae (Fig. 5). Both groups are connected to each other via a small band along the abapical side of the mouth/ apical side of the foot (Fig. 5). This expression domain also includes a region where the "Schwabe organ", a putative chemosensory organ of a polyplacophoran ingroup, is located [24]. Although the chitonid A. crinita has not been studied with respect to the presence of this organ, it is likely that it is not present in A. crinita since other congeneric species of the Chitonida lack this sensory structure. Pax2/5/8 expressing cells are also located in the posterior mantle fold that exhibits the chemosensory osphradia of adult chitons (arrowheads in Fig. 6k; see also [24]. In both described expression domains, however, no Pax2/5/8 expression is found in postmetamorphic chitons. A prominent group of Pax2/5/8-expressing cells is present in the shell fields of A. crinita (arrowheads in Figs. 4j, k, 5 and 6a). The majority of these cells is located in central regions of each shell field and exhibits processes that penetrate the epidermis and the nascent calcareous layers of the shell plates (inset in Fig. 6j). These processes exhibit FMRFamide-like immunoreactivity in earlier developmental stages than the associated cell somata (c.f. Figs. 4j, 5 and 6a, j). Judging by their  & c)). e A further developed specimen (8 dpf) with Ntu-Pax2/5/8 expression in the mantle (m). f Ntu-Pax2/5/8 is expressed in the mantle including the shell gland (sg) (same specimen as shown in (e). g Confocal reflection (Ntu-Pax2/5/8 expression in red) and autofluorescence (grey) scan of a single optical section of the same specimen as shown in (f). h This further developed larva (8 dpf) expresses Ntu-Pax2/5/8 bilaterally along the dorsal and apical mantle (arrowhead). i Late larvae (12 dpf) express Ntu-Pax2/5/8 along the dorsal and ventral mantle (arrowhead). j 12 dpf old larva of the same stage as the one shown in I with stronger Ntu-Pax2/5/8 expression in the dorsal and ventral (arrowhead) mantle. k Same specimen as shown in J with Ntu-Pax2/5/8 expression in the dorsal and ventral mantle (arrowheads) but not the foot (f). l Sketch drawing summarizing the Ntu-Pax2/5/8 expression domains (blue) in a metamorphic competent pericalymma larva before settlement. Abbreviations: an, anus; at, apical tuft; e, esophagus; f, foot; pc, perivisceral cavity; tc, test cell; s, shell; stm, stomach. Scale bars: 30 μm location and morphology, these Pax2/5/8-expressing and FMRFamide-like immunoreactive cells are probably esthetes. Esthetes are primarily photoreceptive organs that constitute a polyplacophoran autoapomorphy and penetrate the surface of the shell plates of adult specimens [25][26][27]. During ontogeny, esthetes may first be identified by the narrow holes of their canals in shell plates of metamorphosed specimens [28].
In the pygmy squid, Pax2/5/8 is expressed in various organs that are known to be equipped with sensory cells. During a short developmental time frame (stages 24-25), Ino-Pax2/5/8 is expressed in the eyes (Figs. 5 and 10b, c), resembling the situation of the adult gastropod Haliotis asinina [29]. The brachial crown, i.e. all five arm pairs, expresses Pax2/5/8 in epidermal cells until stage 24 (Fig. 10a, b). In stage 25 individuals, Pax2/5/8 expression is restricted to the proximal part of each arm/tentacle. Expression fades in subsequent developmental stages.
Cephalopod arms and tentacles house a plethora of sensory cells. Although cell proliferation experiments during cephalopod arm growth are still lacking, it is tempting to speculate that Pax2/5/8 is expressed in ectodermal sensory cells in regions of high proliferation activity, such as the proximal portion of the brachial crown in stage 25 specimens. The funnel shows a similar pattern as the brachial crown. During early development the entire funnel expresses Pax2/5/8, while only the proximal portion expresses Pax2/5/8 in subsequent stages until it fades entirely in late prehatching stages. Here, Pax2/5/8 appears to be expressed in the epidermis as it has also been reported for vertebrates [30]. Along the lateral sides of the foot the future gills develop and Pax2/5/8 expression predates their development. Pax2/5/8 is expressed in different regions of the developing mantle of the aculiferan polyplacophoran Acanthochitona crinita, the basal bivalve Nucula tumidula, and the cephalopod Idiosepius notoides. This corroborates data on the veliger larva of Haliotis asinina, where Pax2/5/8 is expressed in the anterior region of the mantle that is known to be richly equipped with sensory cells [9]. In contrast to the esthetes as distinct expression domain of A. crinita, Pax2/5/8 is rather broadly expressed in the mantle of N. tumidula and I. notoides. During earlier development of N. tumidula, Pax2/5/8 expression is, however, first restricted to the dorsal mantle epithelium, including the shell gland. In contrast, the shell gland of H. asinina does not exhibit Pax2/5/8 expression (present study and [9]). Shortly before metamorphosis, Pax2/5/8 expression shifts to the ventral side of the mantle and disappears in the dorsal region after metamorphosis. This may be explained by the onset of sensory cell development on the ventral side of the mantle that is exposed to water currents in the postmetamorphic, settled animal. Other ectodermal expression domains comprise the cephalopedal epidermis of stage 24 individuals of I. notoides (Fig. 10a).

Pax2/5/8 plays a role in cephalopod brain regionalization
Studies on mouse and Drosophila embryos revealed that several gene orthologs play a role in brain regionalization.
The anterior-most brain regions of both organisms, i.e. the murine forebrain/midbrain as well as the fruit fly's protocerebrum/deuterocerebrum, express Otd/Otx ( Fig. 11; [14]). The adjacent more posterior brain regions, that are patterned in a spatially collinear fashion by anterior Hox gene orthologs, are separated from the anterior-most brain regions by a Pax2/5/8 expression domain ("midbrain/ hindbrain boundary"; Fig. 11; [14]). Interestingly, a similar molecular fingerprint is manifested in the developing brain of the cephalopod Idiosepius notoides (present study) and other cephalopods investigated. Here, Pax2/5/8 expression is restricted to the cerebral ganglia during early development. In subsequent developmental stages transcripts are restricted to the anterior basal and the interbasal lobes that are located slightly anterior to the esophagus, thus dividing the cephalopod brain into a supraesophageal and a subesophageal mass (Fig. 11). Anterior portions of the posterior subesophageal mass that are located close to the esophagus also exhibit faint Pax2/5/8 expression. Notably, brain lobes that are located more anteriorly, such as the median basal lobe or the vertical lobe complex, do not express Pax2/5/8 in I. notoides. In late-stage prehatching individuals, Pax2/5/8 is only expressed in the interbasal lobes as well as in the posterior perikaryal layer of the middle subesophageal mass.
Otx is expressed in the supraesophageal mass of the cuttlefish Sepia officinalis and the pygmy squid I. notoides  [31]; Wollesen, unpublished data). Compared to Pax2/5/8 it is, however, expressed in more anterior regions of the supraesophageal mass; i.e., in components of the vertical lobe complex (i.e. the superior frontal and subpedunculate lobe) and the median basal lobe of the supraesophageal mass. In contrast to Pax2/5/8 expression, no Otx expression was found in the subesophageal mass (present study, [31], Wollesen, unpublished data). In cephalopods, Hox genes are expressed in the CNS with exception of the cerebral ganglia/supraesophageal mass [32]. Although no collinear spatial expression is reported from cephalopods, the Otxand Pax2/5/8 expression patterns adhere to the overall pattern reported for Drosophila and mouse [14]. Data on other bilaterians suggest a Six3 expression domain in the anterior-most region of the cephalopod brain, i.e. the vertical lobe complex (white box in Fig. 11; [33]). Surprisingly, Pax2/5/8 is not expressed in the developing nervous system of the polyplacophorans, bivalves, or gastropods investigated so far (present study; [9]). In contrast to cephalopods, the fruit fly, or mouse none of the latter mollusks possess a highly centralized brain.

Pax2/5/8 expression is restricted to the ectoderm in mollusks
Among lophotrochozoans, Pax2/5/8 expression during development has only been studied in mollusks (present study; [9]; but see [34] on the role of Pax2/5/8 in regenerating tissue of the adult polychaete Platynereis dumerilii). In all mollusks investigated so far, Pax2/5/8 is exclusively expressed in ectodermal domains such as the epidermis, the shell gland, the mantle, sensory cells, as well as the nervous system (present study; [9]). Considering that Pax2/5/8 is consistently expressed in ectodermal epithelia in all metazoans investigated so far, with solely the Onychophora and the Chordata expressing Pax2/5/8 in mesodermal domains such as the nephridia, the most parsimonious explanation is an independent recruitment of Pax2/5/8 in mesodermal epithelia development in these clades (Fig. 12). In nematodes and cnidarians the Pax2/5/8 orthologs are involved in the formation of endodermal structures such as the hindgut or the endoderm at the aboral end of the pharynx (Fig. 12). Since the anterior-most and posterior-most portions of the digestive tract are located at the endodermal-ectodermal boundary, the Pax2/5/8 expression domains in the nematode hindgut and the aboral end of the pharynx of the cnidarian Nematostella vectensis might be an evolutionary reminiscent of ancestral ectodermal expression domains in the ancestors of these taxa (see Fig. 12).

Conclusions
The present study shows that Pax2/5/8 is expressed in multimodal sensory systems in mollusks such as the esthetes and the ampullary system of polyplacophorans, the eyes of squids, but not in the larval apical organ of A. crinita and N. tumidula. Pax2/5/8 expression probably predates sensory cell development during ontogenesis, since Pax2/5/8-expressing cells are present in regions where the future sensory cells are situated. Compared to other bilaterians, Pax2/5/8 is not expressed in the development of the less centralized nervous system of bivalves, gastropods, and polyplacophorans, and hence it most likely lost its role in brain development in these mollusks. Interestingly, Pax2/5/8 is expressed along the trajectory of the esophagus that divides the cephalopod brain into a supraesophageal and a subesophageal mass. Together with Otx and Hox genes Pax2/ 5/8 might have been recruited into brain regionalization, thus representing an extreme case of convergent evolution of gene function to the situation found in vertebrates (mouse) and insects (fruit fly). Since Pax2/5/8 is largely expressed in ectodermal domains throughout the Bilateria, its ancestral role was most likely in the differentiation of this outer most germ layer of bilaterians.

Collection and culture of animals
Adults of the polyplacophoran Acanthochitona crinita were collected in the intertidal zone close to the Station Biologique Roscoff in Roscoff, France, during the summers of 2013 and 2014. Adults were kept at 20°C in running seawater. Prior to spawning, adults were isolated and maintained separately in glass dishes. Spawning was induced by exposing adults to sunlight for 3-4 h or thrice each for 15 min to alternating water temperatures, i.e. 25-30°C and 10-15°C. Released sperm and oocytes were separated and the latter rinsed several times in Millipore-filtered seawater (MFSW) and fertilized immediately. Early cleavage stages were kept at 20°C in running seawater, while post-gastrulation stages were kept in MFSW with the addition of antibiotics against Fig. 11 Pax2/5/8 has been independently recruited in brain regionalization in various bilaterian subgroups. Expression of Otx, Pax2/5/8, and anterior Hox genes during brain formation in a lophotrochozoan (cephalopod), an ecdysozoan (Drosophila melanogaster), and a deuterostome (Mus musculus). Anterior-most brain regions face up and posterior-most brain regions face down. The cephalopod vertical lobe does not express Otx, Pax2/5/8, or Hox genes. The superior frontal, subpedunculate, and median basal lobes express Otx in Sepia officinalis [31] and the anterior basal and interbasal lobes express Pax2/5/8 in Idiosepius notoides (present study). The posterior subesophageal mass and the posterior perikaryal layer of the middle subesophageal mass express Pax2/5/8 in I. notoides (present study). Anterior Hox genes are expressed in the palliovisceral and pedal ganglia/ subesophageal mass of other cephalopods [32]. Sketch modified after [14] bacterial and fungal growth (50 mg streptomycin sulfate (Sigma-Aldrich) and 60 mg penicillin (Sigma-Aldrich) per liter MFSW). Water was changed every other day and settlement of metamorphic competent larvae was induced by the addition of substrate on which adult animals had been found.
Adults of the pygmy squid Idiosepius notoides were dip-netted in the sea grass beds of Moreton Bay, Queensland, Australia. Adults were kept in closed aquaria facilities at the School of Biological Sciences of the University of Queensland in Brisbane and fed with various crustaceans. Fertilization is internal and females attached egg clutches to sea grass or to the glass surface of the aquaria. Embryos were cultured and staged as described previously [35].

RNA extraction and fixation of animals
Several hundred individuals of early cleavage stages, larvae, metamorphic competent individuals to early juveniles of Acanthochitona crinita and Nucula tumidula were collected and stored in RNAlater (Lifetechnologies, Vienna, Austria) at −20 to −80°C. RNA was extracted with a RNA extraction kit (Qiagen, Roermond, Netherlands) and stored at −80°C. For I. notoides, egg jelly and chorion of embryos were removed and RNA from approximately 300 specimens including freshly laid zygotes (stage 1) to hatchlings (stage 30) was extracted using TriReagent according to the manufacturer's instructions (Astral Scientific Pty. Ltd., Caringbah, Australia; see also [36]). Several hundred individuals of representative developmental stages of all three species were fixed for in situ hybridization experiments as previously described [36,37]. Specimens were either stored in 75 % EtOH or in 100 % Methanol at −20°C.

RNAseq and transcriptome assembly
Pooled total RNA of all above-mentioned developmental stages was used for the preparation of amplified short insert cDNA libraries (150-250 bp insert size) for Nucula tumidula and Acanthochitona crinita (Eurofins, Ebersberg, Germany). Both libraries (Kit version TruSeq SBS Kit v3) were sequenced together with another two bar-coded libraries in 1 channel of HiSeq 2000 with Illumina chemistry v3.0. Sequences were demultiplexed according to the 6 bp index code with 0 mismatch allowed. In both cases a PhiX library was added before sequencing to estimate the error rate of the sequences. Sequencing resulted in a total amount of 8.160 Mbp for N. tumidula and 7.147 Mbp for A. crinita. Paired-end reads with an average read length of 100 bp were obtained. These reads were subsequently filtered (rRNA removal) and adapter and low quality sequences were trimmed, normalized, and assembled de novo into contigs with the assembler Trinity [38]. The transcriptomes of A. crinita and N. tumidula comprised 166,556 contigs and 224,633 contigs, respectively. The sequencing strategy for developmental stages of I. notoides was described previously [36]. Briefly, RNA of developmental stages was sequenced by 454 technology by Eurofins (Ebersberg, Germany). After filtering, adapter and low quality read trimming, the reads were normalized and assembled de novo by Eurofins.

Alignment and phylogenetic analysis
Amino acid sequences of bilaterian Pax2/5/8 orthologs retrieved from NCBI were used in blastp searches against the assembled transcriptomes of A. crinita, N. tumidula, and I. notoides. The amino acid sequences with the highest sequence similarities of these blastp hits were aligned in ClustalX 2.1 (for accession numbers see Table 1) and edited manually in Geneious Pro 5.5.6 (Biomatters, Auckland, New Zealand, www.geneious.com). One alignment including only metazoan Pax2/5/8 orthologs was performed in order to highlight the Pax2/5/ 8-specific domains (Fig. 2b), while another alignment comprising amino acid sequences of various Pax genes was done to show that Acr-Pax2/5/8, Ntu-Pax2/5/8, and Ino-Pax2/5/8 cluster with their metazoan orthologs (Fig. 2a). The conserved motifs of the paired domains of the latter alignment were used to construct a maximumlikelihood consensus tree (100 bootstrap replicates) with the program Phylip v.3.695 [39].
Molecular isolation of Pax2/5/8 sequence orthologs RNA pooled from different developmental stages of A. crinita, N. tumidula, and I. notoides, respectively, was used for first-strand cDNA synthesis by reverse transcription using the First strand cDNA Synthesis Kit for rt-PCR (Roche Diagnostics GmbH, Mannheim, Germany). Gene-specific primers were designed from identified Pax2/5/8 orthologs of A. crinita, N. tumidula, and I. notoides and transcripts were amplified via standard PCR. PCR products were size-fractioned by gel electrophoresis and gel bands of the expected length were excised and cleaned up using a QIAquick Gel Extraction Kit (Qiagen). Subsequently, cleaned-up products were cloned by insertion into pGEM-T Easy Vectors (Promega, Mannheim, Germany). Plasmid minipreps were grown overnight, cleaned-up, and sent for sequencing. All Pax2/5/8-gene orthologs were identified using the BLASTx algorithm screening the database of the National Center for Biotechnology Information (NCBI). All sequences and phylogenetic data that have been obtained in this study have been deposited in appropriate data bases.

Probe syntheses and whole-mount in situ hybridization
From the miniprepped plasmids the probe template was amplified via standard PCR using M13 forward and reverse primers, and in vitro transcription reactions were performed with these templates, digoxigenin-UTP (DIG RNA Labeling Kit, Roche Diagnostics GmbH), and SP6/ T7 polymerase (Roche Diagnostics GmbH) for the syntheses of antisense riboprobes, according to the manufacturer's instructions. Whole-mount in situ hybridization experiments were carried out as described previously [36,37]. Briefly, specimens were rehydrated into PBT (PBS + 0.1 % Tween-20). They were treated with Proteinase-K (50-60 μg/ml for Acanthochitona crinita, 10 μg/ml for Nucula tumidula, and 25 μg/ml for Idiosepius notoides) in PBT at 37°C for 15 min and prehybridized in hybridization buffer for 4 h or overnight at 65°C for A. crinita and I. notoides, and at 56°C for N. tumidula. Hybridization with a probe concentration of 0.5 to 1 μg/ml was carried out overnight at the same temperatures as those for prehybridization. For I. notoides, a minimum of 20 individuals per stage was investigated and for A. crinita and N. tumidula approximately 50 individuals per stage. In addition, negative controls were carried out with sense probes for all genes and developmental stages. The majority of whole-mount preparations were cleared in a 3:1  solution of benzyl-benzoate and benzyl alcohol, mounted on objective slides, and analyzed. The results were documented with an Olympus BX53 microscope (Olympus, Hamburg, Germany) and polyplacophoran and bivalve developmental stages were additionally scanned with a Leica confocal SP5 II microscope (Leica Microsystems,Wetzlar, Germany) using bright-field and autofluorescence scans as well as the reflection mode [40]. If necessary, images were processed with Adobe Photoshop 9.0.2 software (Adobe Systems, San Jose, USA) to adjust contrast and brightness. Sketch drawings were done with Adobe Illustrator CS5 software (Adobe Systems).

Immunochemistry
Specimens stored in 75 % EtOH or 100 % MetOH at −20°C were rehydrated and washed several times in PBS. Samples were rinsed thrice for 20 min each in PBS with 2 % Triton X-100 (PBT) to increase tissue permeability and were then blocked for 4 h in PBT with 3 % normal swine serum (NSS; Jackson ImmunoResearch, West Grove, USA) at RT. Specimens were incubated for 12 to 24 h at RT in a cocktail of primary antibodies directed against serotonin (raised in rabbit, polyclonal, Immunostar, Hudson, USA) or FMRFamide-related peptides (raised in rabbit, polyclonal; Biotrend, Cologne, Germany), and a primary antibody against acetylated α-tubulin (raised in mouse, monoclonal; Sigma Aldrich), all diluted 1:800 in PBT with 2 % NGS. After rinsing specimens five times for 20 min each in PBS at RT, secondary fluorochrome-coupled antibodies were applied in a 1:1.000 dilution over night at RT. These cocktails contained PBT with 1 % NGS and Alexa Fluor 488 (anti-rabbit; Invitrogen) and Alexa Fluor 633 (anti-mouse, Invitrogen), as well as 1 % Hoechst 33342 ((2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate); Thermo Scientific) for cell nuclei staining. Samples were rinsed in PBS five times for 20 min each at RT and subsequently mounted on glass slides in Fluoromount G (Southern Biotech, Birmingham, Alabama, USA). Glass slides were stored in the dark at 4°C until the mounting medium had hardened. Specimens were investigated and results analyzed as described above. Isosurfaces of FMRFamide-like reactive and serotonin-like immunoreactive elements were created by surface rendering algorithms using the 3D reconstruction imaging software IMARIS (Bitplane, Zurich, Switzerland). Specificity of the FMRFamidelike and serotonin-like antibodies was tested by omission of the primary antibodies and rendered no signal.

Statement of ethical approval
Animals were collected, anesthetized, and fixed according to internationally recognized standards (University of Queensland Animal Welfare Permit No. 158/09 "The cultivation of Idiosepius (pygmy squid) for studies in developmental biology" to BMD). Field work permission was granted by the ethics committee for every collection site of I. notoides used in this study.