Electroreception is an ancient subdivision of the lateral line sensory system, found in all major vertebrate groups (though lost in frogs, amniotes and most ray-finned fishes). Electroreception is mediated by 'hair cells' in ampullary organs, distributed in fields flanking lines of mechanosensory hair cell-containing neuromasts that detect local water movement. Neuromasts, and afferent neurons for both neuromasts and ampullary organs, develop from lateral line placodes. Although ampullary organs in the axolotl (a representative of the lobe-finned clade of bony fishes) are lateral line placode-derived, non-placodal origins have been proposed for electroreceptors in other taxa. Here we show morphological and molecular data describing lateral line system development in the basal ray-finned fish Polyodon spathula, and present fate-mapping data that conclusively demonstrate a lateral line placode origin for ampullary organs and neuromasts. Together with the axolotl data, this confirms that ampullary organs are ancestrally lateral line placode-derived in bony fishes.
The mechanosensory and electrosensory divisions of the lateral line system enable fishes and amphibians to detect changes in local water flow and weak electric fields, respectively1,2. Lateral line mechanosensory hair cells, which are essentially identical to inner ear vestibular hair cells, are collected in neuromasts, small sense organs arranged in characteristic lines over the head and trunk (both superficially and in canals linked to the surface by pores). Neuromasts function as displacement detectors of local water flow, used for prey/predator detection, obstacle avoidance and behaviours such as schooling. Electrosensory hair cells are collected in ampullary organs3,4, either at the surface or recessed in pores filled with mucous jelly of low electrical resistance, distributed in fields flanking the lines of mechanosensory neuromasts5,6. Transduction is direct: cathodal stimuli open voltage-gated calcium channels in the apical membrane3; the information is used for prey/predator detection, orientation and migration. Afferent innervation for mechanosensory hair cells in a given neuromast line, and for electrosensory hair cells in the ampullary organ fields flanking that line, is provided by neurons within a specific lateral line ganglion7. The mechanisms underlying the development of the mechanosensory lateral line system are increasingly well understood6,8,9,10,11,12. However, the development of electrosensory hair cells, and the extent to which it is similar to the development of mechanosensory hair cells, remain less explored.
The evolutionary history of the lateral line system shows multiple independent losses of the electrosensory subdivision5,13,14,15, suggesting that its development must be independent of the mechanosensory subdivision, at least to some extent. Both mechanosensory and electrosensory systems are found in all major vertebrate groups, including jawless fishes (lampreys, although not hagfishes), cartilaginous fishes (chondrichthyans) and bony fishes (osteichthyans) (Fig. 1a). Within the lobe-finned bony fishes (sarcopterygians), frogs lack electroreceptors, whereas amniotes seem to have lost the entire lateral line system with the transition to terrestrial life5,13,14,15. Within the ray-finned bony fishes (actinopterygians), electroreception was lost in the radiation leading to the neopterygian clade, that is, holostean fishes16 (gars, bowfins) and teleosts, but subsequently evolved independently at least twice in several teleost groups, for example, catfishes (siluriforms), knifefishes (gymnotiforms) and elephantnose fishes (mormyriforms)5,13,14,17.
Neuromasts, and the afferent neurons for both neuromasts and ampullary organs, develop from cranial lateral line placodes, that is, paired patches of thickened head ectoderm (primitively, three preotic and three postotic placodes5,8,15). Neurons delaminate from one pole of each placode18,19,20 and collect (with neural crest-derived glia) to form a cranial lateral line ganglion; the other pole forms a primordium that elongates or migrates over the head or trunk, tracked by axons of lateral line neurons, and deposits a line of neuromasts5,6,9,11. Ampullary organ development is much less well understood, at least partly because the main model systems for studying lateral line development (namely, the teleost zebrafish and the frog Xenopus) are from lineages that have lost the electrosensory subdivision (Fig. 1a). Grafting and ablation studies in the urodele amphibian Ambystoma mexicanum (axolotl), provided clear experimental evidence that lateral line placodes form both ampullary organs and neuromasts in sarcopterygians21,22. In contrast, a neural crest origin for ampullary organs was proposed (based on gene expression data alone) for a cartilaginous fish, the shark Scyliorhinus canicula23, whereas lateral line axons have been suggested to induce electroreceptors from local ectoderm in an electroreceptive teleost, the catfish Silurus glanis24 (also see ref. 25). Before we can hope to understand the extent to which the mechanisms underlying electrosensory and mechanosensory hair cell development are conserved, and the extent to which the mechanisms underlying electrosensory hair cell development are conserved across different taxa, it is essential to define their embryonic origins experimentally in phylogenetically informative taxa.
The North American paddlefish, Polyodon spathula, is a basal actinopterygian fish with easily the largest number of ampullary organs of any living vertebrate species: 50,000–70,000 ampullae per adult26,27. The characteristic rostrum, or paddle (an extension of the cranium), is covered in ampullary organs and is used as an electrosensory 'antenna' for feeding28,29. Paddlefishes and sturgeons together comprise the chondrosteans30,31,32,33,34, the sister group to the neopterygian clade (gars, bowfins, teleosts) whose ancestors lost the electrosensory subdivision of the lateral line system (but within which specific teleost groups independently evolved electroreceptors) (Fig. 1a). Here, we provide an embryological, molecular and experimental analysis of lateral line development in Polyodon. We show that multiple members of the Eya (eyes absent) and Six (sine oculis) gene families (characteristic placode markers in all vertebrates35) are expressed throughout lateral line placode development and in both ampullary organs and neuromasts. Using in vivo fate mapping (the first such data reported for any electroreceptive actinopterygian fish), we experimentally demonstrate a lateral line placode origin for ampullary organs as well as neuromasts. Together with previously published data showing a lateral line placodal origin for sarcopterygian ampullary organs22, our findings strongly support an ancestral lateral line placode origin for ampullary organs in all bony fish. Because all available evidence suggests that the ampullary organs found in sarcopterygians and basal actinopterygians are homologous to the ampullae of Lorenzini of chondrichthyan elasmobranchs (sharks and rays)5,13,14, our data also support the hypothesis that ampullary organs are primitively derived from lateral line placodes in all jawed vertebrates.
The lateral line system in juvenile paddlefish
The organization of the mechanosensory and electrosensory divisions of the lateral line was clear in scanning electron micrographs (SEMs) of an early juvenile paddlefish (∼14 days poststaging) (Fig. 1b,c). In general, the neuromast canals followed a similar pattern to that observed in other gnathostomes. We adopted the following terminology to refer to the lines: supraorbital, infraorbital, otic, preopercular, middle, supratemporal and posterior (Fig. 1c). A derived condition of the preopercular line in Polyodon (as compared with other chondrosteans7) is that it joins the infraorbital line ventral to the spiracle, instead of joining the otic line posterior to the spiracle. Ampullary organs are confined to the head, and most numerous on the rostrum and opercular flap. Fields of ampullary organs flank the supraorbital, infraorbital, otic and preopercular lines: these fields were named primarily according to their innervation pattern (see below) and position adjacent to specific canals, for example, the dorsal infraorbital and ventral infraorbital fields flank the infraorbital canal, whereas the anterior preopercular and posterior preopercular fields flank the preopercular canal (Fig. 1c).
The pores of ampullary organ clusters were clearly seen on the surface, whereas the neuromasts were recessed in canals (Fig. 1d). Individual neuromasts were identifiable within the canals (Fig. 1e) by their morphology and characteristic protrusions of kinocilia from the apical surfaces of the sensory hair cells (Fig. 1f). Individual neuromasts of the posterior trunk line were also easily distinguishable (Fig. 1g). Transverse sections revealed the organization of the sensory receptor cells within the different organs (Fig. 1h,i). In neuromasts, the sensory hair cells (three in this section) were located in the centre of the organ, where kinocilia could be observed on the apical surface (Fig. 1h). In ampullary organ clusters, a larger number of sensory cells (large, round nuclei) were organized as an epithelium with support cells wedged between them (Fig. 1i), consistent with previous anatomical descriptions27. Neuromasts appeared smaller than the ampullary organs by this point in development.
Sudan Black staining of whole specimens (ranging from late larval to juvenile stages, plus analysis of sectioned material and an osmicated dissection of juvenile cranial nerves) enabled reconstruction of the lateral line ganglia and nerves of the head and anterior trunk and the innervation patterns of the neuromast canals and ampullary organ fields (Fig. 1k,j). Three of the lateral line ganglia are preotic, and three are postotic, a generally conserved feature found across the osteichthyans5,36. Anteriorly, the anterodorsal lateral line ganglion and nerve innervate the neuromasts of the supraorbital and infraorbital lines and their flanking fields of ampullary organs (Fig. 1j). This is the largest lateral line ganglion because of the very large number of ampullary organs in these fields. Closely adjoining the anterodorsal ganglion is the much smaller otic lateral line ganglion and nerve, which innervates neuromasts of the otic lateral line and its flanking fields of ampullary organs, as well as the spiracular organ, a specialized mechanosensory lateral line organ located in a recess dorsal to the spiracle (Fig. 1j). The anteroventral lateral line ganglion and nerve innervate the neuromasts of the preopercular canal and its flanking fields of ampullary organs. There are so many ampullary organs on the operculum that the anteroventral lateral line ganglion and nerve are the second-largest component of the lateral line system. The anterodorsal and anteroventral ganglia are also closely associated (Fig. 1k). The preotic lateral line nerves send a dorsal root to enter the brain at the level of the dorsal octavolateral nucleus (associated with electroreception) and a ventral root to enter at the level of the medial octavolateral nucleus (associated with mechanoreception) (both roots circled in green in Fig. 1k). In contrast, the three postotic lateral line nerves (middle, supratemporal and posterior), which, respectively, innervate the neuromasts of the middle, supratemporal and posterior canals (which lack flanking fields of ampullary organs), only send a common root (circled in blue in Fig. 1k) into the brain at the level of the medial octavolateral nucleus, suggesting these are only associated with mechanoreception.
Spatiotemporal development of functional neuromasts
Primordial neuromasts developing from the inner layer of ectoderm were observed on sections at stage 32 (Fig. 2a), but SEM revealed the emergence of the first neuromasts through the epidermal layer at stage 33 in the otic lateral line canal (Fig. 2b,c). We also followed neuromast development in live embryos using FM1-43, a voltage-sensitive fluorescent styryl dye that is internalized (putatively via mechanosensitive ion channels) by mechanosensory hair cells of the inner ear and lateral line37. Consistent with the SEM data, the first lateral line hair cell clusters visible with FM1-43 were within neuromasts of the otic lateral line canal (Fig. 2d): at stage 36, three to four FM1-43-positive hair cell clusters were observed. By stage 40, supraorbital and preopercular canal line neuromasts were also evident (Fig. 2e). FM1-43-positive neuromasts progressively emerged in both directions along the anterior–posterior axis. By stage 42, supratemporal and middle canal line neuromasts were FM1-43-positive (Fig. 2f). By stage 44, all neuromast canal lines contained FM1-43-positive hair cell clusters, including the posterior line, in which the clusters appeared in an anterior to posterior progression along the main trunk line (Fig. 2g,h). At this stage there were ∼10–12 hair cells per cluster (Fig. 2g, inset). FM1-43 uptake was never seen in ampullary organs, consistent with the proposed uptake of FM1-43 via mechanosensory ion channels37, which may therefore be absent in electrosensory hair cells.
Development of ampullary organ fields
Primordial ampullary organs beneath the epidermis were seen on sections at stage 35 (Fig. 3a; for comparison with a neuromast, see Fig. 2a), but SEM analysis showed that the first ampullary organs to open to the surface only emerged at approximately stage 39 (well after hatching, at stage 36), mainly on the operculum (Fig. 3b,c), after which the number of open ampullary organs rapidly increased. By stage 42, ampullary organs had begun to erupt from all fields (Fig. 3d). By stage 44, nearly all ampullary organs were open to the surface and fully differentiated (Fig. 3e). Ampullary organ fields and neuromast canals were beautifully revealed at stage 46 (the start of feeding) by staining skin mounts with the nuclear markers Sytox Green (Fig. 3f) and DAPI (4′,6-diamidino-2-phenylindole) (Fig. 3g). Confocal images of DAPI-stained ampullary organs from the surface (Fig. 3g) and in transverse section (Fig. 3h) showed the shallow depth of paddlefish ampullary organs and the large number of sensory cells lining the epithelium (in comparison with a neuromast, which has fewer sensory cells; Fig. 3i). Staining for the F-actin marker phalloidin also highlighted the microvilli (stereocilia) on the apical surface of the ampullary organ sensory epithelium (Fig. 3h). Immunostaining in both whole mount (Fig. 3j,k) and on sections (Fig. 3l,m) revealed that parvalbumin-3 (Pv3), a Ca2+-binding protein thought to be the major calcium buffer in inner ear and lateral line mechanosensory hair cells38, is also expressed in electrosensory hair cells.
Development of lateral line placodes
Using SEM and reconstructions of histological sections, lateral line placodes were first discernable around stage 25, and well developed by stage 32 (Fig. 4a; summarized schematically in Fig. 4b). Although the boundaries of individual placodes were difficult to distinguish precisely, we recognized three preotic lateral line placodes (anterodorsal, anteroventral and otic) and three postotic lateral line placodes (middle, supratemporal and posterior; Fig. 4a,b). This is consistent with sturgeons7, and thought to be the primitive condition for jawed vertebrates5. The first lateral line placode to be morphologically discernable was the otic, consistent with the appearance of the first neuromasts in the otic lateral line canal (Fig. 2). The largest lateral line placode was the anterodorsal, which formed a visible bulge in SEM, ventral and slightly posterior to the eye (Fig. 4a). The anteroventral placode, located ventral and posterior to the anterodorsal placode, was the second largest lateral line placode; the three postotic placodes (middle, supratemporal and posterior) were the smallest. Sample sections through the anterodorsal placode showed the extent of the thickened epithelium, extending from the level of the developing eye (Fig. 4c) to the otic vesicle (Fig. 4d). At stage 32, the placodes were already beginning to elongate or spread over the head to form sensory ridges, and neuromast primordia were beginning to differentiate from the central zones of these ridges (Fig. 4c).
Lateral line expression of Eya and Six family members
Cranial placodes originate from a common 'preplacodal region' or 'pan-placodal primordium', characterized by expression of members of the Eya, Six1/2 and Six4/5 families of transcription factors or cofactors, whose expression is maintained in all cranial placodes and their derivatives12,39. Zebrafish Eya1 is expressed in lateral line placodes and neuromasts20, whereas shark Eya4 is expressed in lateral line placodes, neuromasts and ampullary organs40, making these excellent candidate markers for the developing lateral line system in paddlefish. We used degenerate PCR to clone paddlefish Eya1-4, Six1, Six2 and Six4, and examined their expression by in situ hybridization. All seven genes were expressed in lateral line placodes, neuromasts and ampullary organs. We present data for Eya1, Eya4, Six1 and Six4 in Figure 4e–j; Eya2, Eya3 and Six2 were similarly expressed.
At stage 32, Eya1, Six1 and Six4 (Fig. 4e) were all expressed in multiple placodes, including the otic vesicle, olfactory placode, epibranchial placodes and lateral line placodes, in which their expression patterns were consistent with the elongation and spreading of lateral line primordia to form sensory ridges. In contrast, Eya4 expression was specific to the otic vesicle and the elongating lateral line placodes (Fig. 4e). At stage 36, placodal expression of all these genes was maintained, although at weaker levels, and individual neuromast canal lines could be distinguished (Fig. 4f). By stage 39, expression was seen in all neuromast lines and revealed the formation of ampullary organ fields (Fig. 4g); by stage 42, individual ampullary organs could be seen (Fig. 4h). At stage 46, expression was maintained in both neuromasts and ampullary organs (Fig. 4i). The overall expression pattern of these genes was consistent with our morphological reconstructions of lateral line placodes. Analysis of sections at different stages showed that patches of placodal tissue progressively lose expression, whereas the regions that give rise to the organs retain expression (Fig. 4j). Expression of all seven genes in ampullary organs, as well as in neuromasts and lateral line placodes, supports the hypothesis that ampullary organs in this basal ray-finned fish are lateral line placode-derived. However, gene expression data alone cannot be taken as proof of cell lineage.
Paddlefish lateral line placodes generate ampullary organs
Our analysis of paddlefish lateral line development by SEM, reconstruction of histological sections, as well as Eya and Six gene expression and innervation patterns, together suggest that preotic lateral line placodes form both neuromast canal lines and their flanking fields of ampullary organs. (In contrast, the postotic placodes seem only to give rise to neuromasts and their associated nerves; this is also consistent with the observed size difference of the individual placodes.) To test the hypothesis that ampullary organs, as well as neuromasts, are lateral line placode-derived in paddlefish, we focally labelled the two largest lateral line placodes (anterodorsal and anteroventral) with the fluorescent lipophilic dye DiI at stages 32–34 and followed their development to stage 46 (see Fig. 5a,c). Examples are shown in Figures 5 and 6.
In the embryo shown in Figure 5b, DiI-labelled cells in the anterodorsal placode ultimately contributed to the infraorbital neuromast canal line as well as to ampullary organs in the ventral infraorbital field adjacent to the eye (Fig. 5g). Imaging the same embryo at successive stages revealed the elongation and spreading of DiI-labelled cells (Fig. 5d–g). Figure 6 shows four additional examples of placode labelling experiments (two anterodorsal, two anteroventral). Transverse sections through an embryo after labelling the anterodorsal placode (Fig. 6a,b) showed DiI in individual infraorbital canal neuromasts (Fig. 6c) and ampullary organs in the flanking fields (Fig. 6d). In the embryo shown in Figure 6e, DiI-labelled anteroventral placode cells contributed to preopercular canal neuromasts and flanking ampullary organs (Fig. 6f). After isolating the operculum, counterstaining with Sytox Green and flat mounting the sample (Fig. 6g), DiI was clearly observed in both neuromasts (Fig. 6h) and ampullary organs (Fig. 6i). In another labelled embryo (Fig. 6j,k), confocal imaging of transverse sections revealed DiI-positive cells within the ampullary organ sensory epithelium (Fig. 6l,m). Sectioning in some cases also revealed DiI in the lateral line ganglia as expected, because neurons in these ganglia are lateral line placode-derived18,19,20. In the example shown in Figure 6n, DiI-labelled anterodorsal placode cells contributed not only to infraorbital and supraorbital line neuromasts and flanking fields of ampullary organs (Fig. 6o) but also (as observed on sections) to cells within the anterodorsal lateral line ganglion (Fig. 6p,q).
In total, we successfully labelled ampullary organs in 50 embryos after focally injecting DiI into the anterodorsal or anteroventral placodes between stages 32 and 34. DiI was seen in neuromasts as well as in ampullary organs in 24 of these embryos, confirming successful targeting of lateral line placodes. In the remaining 26 embryos, DiI was only visible in ampullary organs. We suggest that in these cases, the label only targeted the lateral region of the placode, as ampullary organs develop from the lateral zones of lateral line primordia in the axolotl, whereas neuromasts develop from the central ridges21,22.
Overall, our fate-mapping data confirm that ampullary organs are derived from lateral line placodes in Polyodon, an actinopterygian. Taken together with the previous experimental demonstration of a lateral line placodal origin for ampullary organs in the axolotl22, a sarcopterygian, this strongly supports an ancestral lateral line placodal origin for ampullary organs in all bony fishes.
Our morphological, molecular and in vivo fate-mapping data demonstrate a lateral placode origin for both electrosensory and mechanosensory divisions of the lateral line system in a basal actinopterygian, the North American paddlefish P. spathula. The developmental origin of electrosensory ampullary organs had previously only been experimentally tested in a sarcopterygian, the axolotl A. mexicanum, in which grafting and ablation experiments confirmed that individual lateral line placodes form both ampullary organs and neuromasts22. A neural crest origin was recently proposed for ampullary organs in a chondrichthyan, the shark S. canicula23, but this proposition was based on gene expression data alone, which is not sufficient to confirm cell lineages. Lateral line axons were proposed to induce electroreceptor formation from non-placodal ectoderm in a teleost, the catfish S. glanis24. However, teleost electroreceptors evolved independently (ampullary organs were lost in the lineage leading to neopterygians5,13,14) and no fate maps have yet been undertaken in electroreceptive teleosts. Our demonstration that lateral line placodes form ampullary organs and neuromasts in a basal actinopterygian provides the strongest possible support for an ancestral lateral line placode origin for ampullary organs in bony fishes (see phylogeny in Fig. 1a). Furthermore, the homology of ampullary organs between bony fishes and chondrichthyans (sharks, rays and chimaeras) is supported by all the available evidence: they are excited by cathodal stimuli and innervated by lateral line nerves projecting to a dorsal octavolateral nucleus in the medulla. To this we can now add embryonic expression of Eya4 (paddlefish, this paper; shark40). If this homology is accepted, then our data also support an ancestral lateral line placode origin for ampullary organs in all jawed vertebrates (and perhaps even all vertebrates: although electroreceptor organs in lampreys have a significantly different morphology, they are innervated by a branch of the anterior lateral line nerve and adult lampreys have a dorsal octavolateral nucleus in the medulla4,41,42).
Although a neural crest contribution to neuromasts was reported for both Xenopus (a sarcopterygian) and zebrafish (an actinopterygian) after DiI-labelling presumptive neural crest precursors43, the nature and extent of this contribution has been debated7,19,22,44. For example, these cells may represent neural crest-derived glial cells, which are intimately associated with the lateral line nerves that innervate neuromasts45. It would be useful to re-visit these neural crest labelling experiments using known hair cell markers such as Pv3.
Almost all the information we have on the molecular mechanisms underlying vertebrate hair cell development, whether in the inner ear or lateral line, has been obtained from a handful of model systems: two amniotes (mouse and chick), the frog Xenopus and the teleost zebrafish. None of these species has electroreceptors, which are modified hair cells with a limited range of morphologies4. Hence, our understanding of the potential range of mechanisms underlying vertebrate hair cell development is very incomplete. The expression during paddlefish lateral line placode development of all Eya, Six1/2 and Six4/5 family members examined, and their maintenance in both neuromasts and ampullary organs, suggests at least some conservation of the molecular mechanisms underlying the formation of these distinct sense organs. Further studies of lateral line development in the paddlefish will enable us to discover the extent to which the mechanisms underlying the development of mechanosensory and electrosensory hair cells are conserved.
Despite having the most ampullary organs of any vertebrate species, Polyodon may also be an excellent model for investigating the possible developmental basis for the evolutionary loss of ampullary organs, as occurred in various groups, including frogs and the lineage leading to the neopterygian clade (gars, bowfins, teleosts)5,13,14,15. We have shown here that paddlefish ampullary organ formation is restricted to the three preotic placodes (anterodorsal, anteroventral and otic): the three postotic placodes (middle, supratemporal and posterior) only generate neuromasts. In the sturgeon Scaphirhynchus platorynchus (a member of the sister group to paddlefishes within the chondrosteans; Fig. 1a), ampullary organ fields are associated with the three preotic lines and also with the supratemporal line7. In the axolotl (a sarcopterygian tetrapod), the preotic lines plus the supratemporal and middle lines form ampullary organs21. The strict relationship between each of the six lateral line placodes and their developmental derivatives (placode-nerve-receptor organ line/field) offers support for the theory that these six placodes and their derivatives should be regarded as phylogenetically independent components of the lateral line system, subject to individual modification through evolution. Both the extreme number of ampullary organs derived from the anterodorsal and anteroventral placodes in paddlefish, and the failure of all postotic lateral line placodes to form ampullary organs in this species, would seem to offer an ideal opportunity to discover direct evidence about the ways in which such evolutionary modification of placodes and their derivatives might occur.
Embryos were obtained from Osage Catfisheries, Inc. over multiple spawning seasons. Embryos were raised at approximately 22 °C in tanks with filtered and recirculating water (pH 7.2±0.7, salinity of 1.0±0.2 p.p.t.) to desired stages. Staging was performed according to ref. 46.
cDNA synthesis and cloning
RNA from embryos stages 26–46 was extracted using the Trizol reagent (Invitrogen) and used to generate single-strand cDNA using the Superscript III First Strand Synthesis kit (Invitrogen), as per the manufacturer's instructions. The following degenerate primers were used (F, forward; R, reverse): Eya1F: 5′-ACGGTTCAAGCTTTACAACC-3′, Eya1R: 5′-TGCTACAAGTAATCAAGTTCC-3′; Eya2F: 5′-AACCTCCTACCTACAGCAGG-3′, Eya2R: 5′-TGGAGTTGATGAGAGTCAGC-3′, Eya3F: 5′-AGACCGTGATGGATGAAGC-3′; Eya3R; 5′-ATGCATCCGATCGCTTCC-3′, Eya4F: 5′-GCATGACAATTCTTAATACTGC-3′, Eya4R: 5′-TCACACCAGTGGCCAGGCAGAGG-3′; Six1F: 5′-TTCACKCARGARCARGTSGCGTGYGTSTG-3′, Six1R: 5′-TGYCKYCKRTTYTTRAACCARTT-3′, Six2F: 5′-AGAGGCGATCATGTCAATGC-3′, Six2R: 5′-TTGTGGGTGTACCACTCC-3′, Six4F: 5′-TCTGTGAGGCTTTGCAGCAAGG-3′, Six4R: 5′-GAGTTCAAAGGGTCTGAATTGCC-3′. PCR fragments were cloned into the pDrive cloning vector (Qiagen). Plasmids were purified and sequenced by the Department of Biochemistry DNA Sequencing Facility, University of Cambridge. Sequence results were analysed using MacVector and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). We confirmed the orthology of the different Eya and Six family members through a phylogenetic analysis using Bayesian inference (MrBayes3 (ref. 47)). GenBank accession numbers are as follows: PsEya1 JF949727; PsEya2 JF949728; PsEya3 JF949729; PsEya4 JF949730; PsSix1 JF949731; PsSix2 JF949732; PsSix4 JF949733.
For plastic sections, embryos were fixed in paraformaldehyde, rinsed in distilled water, dehydrated stepwise in an ethanol series and embedded using commercially available methylacrylate kits (Polysciences, Inc.) before microtome sectioning and processing in 1% toluidine blue in sodium borate buffer and 2% basic fuchsin in water. Transverse serial sections were cut at 1.5 μm, unless otherwise noted. The nerves of cleared larval and juvenile whole specimens were stained using Sudan Black48. These samples were then dissected and photographed for camera lucida tracings for schematic reconstructions.
Scanning electron microscopy
Embryos were fixed in 2% glutaraldehyde followed by dehydration through ethanol, and subsequent processing with dimethoxypropane and CO2 or Freon 113 (1,1,2 trichlorotrifluoroethane) and Freon 13 (chlorotrifluoromethane). Specimens were then mounted and sputter coated with gold palladium alloy (300–400 Å thick) and imaged.
Live embryos were immersed in a solution of 3 μM of FM1-43 (Invitrogen) in water for 45 s, then immediately rinsed in three changes of water. Embryos were anaesthetized with tricaine before imaging.
Embryos were dechorionated, immobilized and oriented in troughs made in agarose dishes to the approximate width of stage 32 embryos. Embryos between stages 32 and 34 were injected with 0.5–1 μg μl−1 Cell Tracker-CM-DiI (Invitrogen) diluted in 0.3 M sucrose (from a 5 μg μl−1 stock diluted in ethanol), using a pressure injector. The ectoderm in paddlefish is a stratified epithelium. Therefore, to penetrate the superficial layer of cells, DiI-filled glass capillary needles were gently pushed through the surface ectoderm before release of pressure. Embryos were allowed to recover and develop to stage 46. Embryos were then fixed in 4% paraformaldehyde and embedded in 20% gelatin in PBS. Transverse serial sections were cut at 50–75 μm using a vibratome. Sections were counterstained with the nuclear markers Sytox Green or DAPI (Invitrogen) before microscopy.
In situ hybridization and immunohistochemistry
Embryos were fixed in 4% paraformaldehyde in PBS or modified Carnoy's fixative (6 volumes ethanol: 3 volumes 37% formaldehyde: 1 volume glacial acetic acid) for 12–24 h at 4 °C. Embryos were then dehydrated through a methanol or ethanol series and stored at −20 °C. Antisense RNA probes were generated using T7 or SP6 polymerases (Roche) and digoxigenin dUTPs (Roche). For in situ hybridization, embryos were rehydrated through a stepwise series into PBS with 0.1% Tween-20 (PTw), bleached under strong white light in 0.5% SSC, 10% H2O2, 5% formamide for 30–60 min (depending on stage), then rinsed several times in PTw. After treating for 10–15 min at room temperature with proteinase K (14–22 μg ml−1 of Roche proteinase K), followed by two rinses in PTw, embryos were fixed again for 1 h in 4% paraformaldehyde with 0.02% glutaraldehyde, followed by four rinses in PTw. Embryos were then graded into hybridization solution (1× salt solution (0.2 M NaCl, 10 mM Tris, pH 7.5, 5 mM NaH2PO4.H2O, 5 mM Na2HPO4, 5 mM EDTA), 50% formamide, 10% dextran sulphate, 1 mg ml−1 yeast tRNA, 1× Denhardt's solution). After prehybridizing for at least 3 h at 70 °C, embryos were incubated for at least 18 h at 70 °C with probe diluted in hybridization solution. After four 30–60-min rinses in wash solution (50% formamide, 1× SSC, 0.1% Tween-20) at 70 °C, embryos were graded into MABT (1× maleic acid buffer with 0.1% Tween-20) (10× MAB: 1 M maleic acid, 1.5 M NaCl, pH 7.5). After four 30-min washes in MABT, embryos were blocked in MABT with 20% filtered sheep serum and 2% Boehringer blocking reagent (Roche) for 3 to 4 h at room temperature before incubation in 1:2,000 dilution of anti-DIG-AP antibody (Roche) overnight at 4 °C. Embryos were washed with MABT six times for 1 h each at room temperature, and then overnight at 4 °C. For the colouration reaction, embryos were rinsed three times in NTMT buffer (0.1 M NaCl, 0.1 M Tris, pH 9.5, 50 mM MgCl2, 0.1% Tween-20), before incubation in 0.02% of nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, toluidine salt stock solution (Roche) in NTMT, until desired signal strength was obtained. Embryos were then rinsed in PBS to stop the reaction, postfixed, if necessary, and then processed for sectioning or graded into 70% glycerol for whole-mount imaging. Embryos were then cryosectioned as described in the following section or sectioned using a vibratome, as described above. For immunohistochemistry, the polyclonal Pv3 antibody (gift from A. Hudspeth) was used at 1:30,000, followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson Labs) used at 1:600. For the histochemical reaction, embryos were incubated in 600 μl of DAB+Ni solution (0.3 mg ml−1 3,3′-diaminobenzidine (Sigma) in 1× PBS/0.05% Tween-20 plus 5 μl of an 8% NiCl2 solution) for 5 min. The reaction was started by adding H2O2 to a final concentration of 0.03%, and monitored for up to 15 min before stopping the reaction with 1× PBS/0.1% Tween-20. Embryos were then cleared through a glycerol series into 70% glycerol/PBS.
Sectioning embryos after whole-mount in situ hybridization
After 5 h incubation at room temperature in 5% sucrose in PBS, embryos were placed in 15% sucrose in PBS and incubated overnight at 4 °C. They were transferred into prewarmed 7.5% gelatin in 15% sucrose in PBS, then incubated overnight at 37 °C before being embedded in moulds in the appropriate orientation for transverse sectioning. After freezing the gelatin by immersing the moulds in a dry ice and isopentane solution for 1 min, the embryos were cryosectioned at 20 μm. After 24 h at room temperature, the slides were incubated for 5 min in PBS prewarmed to 37 °C to remove the gelatin, followed by counterstaining with the nuclear marker DAPI (1 ng ml−1). Slides were mounted in Fluoromount G (Southern Biotech).
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This work was supported by the BBSRC (BB/F00818X/1 to C.V.H.B.), NIH (NS24869 and NS24669 to R.G.N.), NSF (BRS 8806539 to R.G.N; 8806539, 8901132 and 9119561 to W.E.B.), the Whitehall Foundation (to W.E.B.) and the Tontogany Creek Fund (to W.E.B.). We also thank the Kahrs family (Osage Catfisheries, Inc.), Jerry Hamilton, Kim Graham, Charlie Suppes (Missouri Department of Conservation), Eric Findeis and Sue Commerford.
The authors declare no competing financial interests.
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Modrell, M., Bemis, W., Northcutt, R. et al. Electrosensory ampullary organs are derived from lateral line placodes in bony fishes. Nat Commun 2, 496 (2011). https://doi.org/10.1038/ncomms1502
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