The lateral line system of fishes and aquatic amphibians is a good model for studying the diversification of different organs and cell types, both in development and evolution. In jawed vertebrates, this sensory system ancestrally includes mechanosensory neuromasts and electrosensory ampullary organs (‘ampullae of Lorenzini’), both of which develop - together with their afferent neurons - from individual embryonic lateral line placodes (Northcutt et al., 1995; Modrell et al., 2011a; Gillis et al., 2012). (The jawless lampreys have neuromasts and electroreceptors, but the latter are collected in ‘end buds’ at the surface, rather than recessed in ampullary organs, and their embryonic origin is unknown; the lateral line system of hagfishes, which lack electroreceptors altogether, is secondarily reduced; Braun, 1996; Braun and Northcutt, 1997.) The electrosensory division of the lateral line system was lost independently in the lineages leading to extant neopterygian fishes (gars, bowfin and teleosts) and to anuran amphibians (neither of the major anamniote lab models, i.e., the teleost zebrafish and the frog Xenopus, has electroreceptors). However, electrosensory lateral line organs evolved independently at least twice within the teleosts, most likely from neuromast hair cells (Bullock et al., 1983; Northcutt, 1986; Bodznick, 1989; Alves-Gomes, 2001; Bodznick and Montgomery, 2005; Kawasaki, 2009; Baker et al., 2013). The entire lateral line system was lost in amniotes, with the transition to life on land.

The loss of the electrosensory division of the lateral line system in different vertebrate lineages shows that ampullary organ development must be genetically separable from neuromast development. Indeed, even within the same lateral line placode-derived sensory ridge, neuromasts form first, along the center of the ridge, while ampullary organs form later, on the flanks (Schlosser, 2002; Northcutt, 2005a; Piotrowski and Baker, 2014). Neuromasts and ampullary organs are morphologically distinct: neuromasts contain mechanosensory hair cells, plus supporting cells that secrete a gelatinous cupula; ampullary organs comprise a sensory epithelium of electroreceptor and supporting cells located at the base of a duct filled with conductive jelly, leading to a surface pore (Northcutt, 1986; Jørgensen, 2005; Baker et al., 2013). Ampullary electroreceptor cells generally have an apical primary cilium but either no or few apical microvilli, which are not organized into the ‘hair bundle’ (stair-case array) that characterizes hair cells (Northcutt, 1986; Jørgensen, 2005; Baker et al., 2013). The molecular mechanisms underlying neuromast formation from the migrating posterior lateral line primordium in zebrafish have been intensively studied (Chitnis et al., 2012; Piotrowski and Baker, 2014; Thomas et al., 2015), but our molecular understanding of ampullary organ development is very limited.

Like hair cells (and also retinal and pineal photoreceptors, and retinal bipolar neurons), all vertebrate electroreceptors have electron-dense pre-synaptic bodies surrounded by a large pool of synaptic vesicles (Northcutt, 1986; Jørgensen, 2005). Such ‘ribbon synapses’ respond to graded signals and are capable of sustained neurotransmitter release (Matthews and Fuchs, 2010; Pangršič et al., 2012; Safieddine et al., 2012; Nicolson, 2015; Wichmann and Moser, 2015; Moser and Starr, 2016). While the specific proteins involved in ribbon synapse function are increasingly understood in retinal photoreceptors and hair cells (Matthews and Fuchs, 2010; Pangršič et al., 2012; Safieddine et al., 2012; Nicolson, 2015; Wichmann and Moser, 2015; Moser and Starr, 2016), all that is known about neurotransmission at non-teleost electroreceptor ribbon synapses is from work in dissected skate (cartilaginous fish) ampullary organs, which showed that activation of L-type voltage-gated calcium channels results in the release of a ‘glutamate-like’ neurotransmitter (Bennett and Obara, 1986).

Similarly, our only detailed understanding of non-teleost ampullary organ physiology until very recently had come from current- and voltage-clamp approaches to study epithelial currents in dissected single ampullary organ preparations from skates (Bennett and Obara, 1986; Lu and Fishman, 1995; Bodznick and Montgomery, 2005). These revealed that L-type voltage-gated calcium channels are required both for voltage-sensing in the apical (lumenal, i.e., exterior-facing) electroreceptor membrane, and for neurotransmitter release basally. The basal membrane is repolarized by voltage-gated potassium channels and calcium-dependent chloride channels, while the apical membrane is repolarized by the calcium-gated potassium channel BK (Bennett and Obara, 1986; Lu and Fishman, 1995; Bodznick and Montgomery, 2005; King et al., 2016). BK was recently cloned directly from skate ampullary organs (King et al., 2016), a little over 40 years after its properties were initially discovered using the same preparation (Clusin et al., 1975; Clusin and Bennett, 1977a; Clusin and Bennett, 1977b). While the current manuscript was under review, whole-cell patch-clamp experiments on dissociated electroreceptors from adult skates, together with transcriptome profiling, revealed that the L-type voltage-gated calcium channel Ca_v1.3 mediates the low-threshold voltage-activated inward current, and works together with BK to mediate electroreceptor membrane oscillations (Bellono et al., 2017). Other than this recent exciting advance (Bellono et al., 2017), the specific ion channels and subunits involved in electroreceptor function have not been identified in any vertebrate.

In short, our understanding of the specific molecular basis of both vertebrate electroreceptor development and physiology is still rudimentary. The few candidate gene approaches reported thus far have identified some transcription factors, and a few other genes, expressed in both ampullary organs and neuromasts in larval axolotl (Metscher et al., 1997; Modrell and Baker, 2012), paddlefish (Modrell et al., 2011a, 2011b; Butts et al., 2014), shark and skate (Freitas et al., 2006; Gillis et al., 2012). However, a candidate gene approach will not identify genes important specifically for ampullary organ development or function. Here, we report an unbiased transcriptomic approach in paddlefish (a non-teleost chondrostean fish) to identify such genes, which has yielded novel and wide-ranging insights into the development, physiology and evolution of non-teleost ampullary organs.

We generated transcriptomes at stage 46 (the onset of independent feeding; Bemis and Grande, 1992) from pooled paddlefish opercula (gill-flaps), which are covered in ampullary organs plus some neuromasts, versus fins, which have a generally similar tissue composition but no lateral line organs at all (Modrell et al., 2011a, 2011b). Differential expression analysis yielded 490 genes, excluding duplicates, enriched at least 1.85-fold (log₂fold 0.89) in operculum versus fin tissue, hereafter designated ‘lateral line-enriched’ (Supplementary file 1). Of these genes, 112 were uncharacterized loci or could only be assigned to a protein family, while a further 44 were described as being ‘like’ a specific gene, leaving 334 assigned genes (Supplementary file 1). Figure 1 shows a molecular function analysis using Gene Ontology (GO) terms, where available.

Figure 1

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Conservation in ampullary organs of transcription factors critical for mechanosensory hair cell development

The lateral line-enriched dataset (Supplementary file 1) includes several transcription factor genes whose expression we had previously reported in both ampullary organs and neuromasts in larval paddlefish, suggesting that the differential expression analysis had been successful. These were: the homeodomain transcription factor genes Six1 and Six2 (Modrell et al., 2011a); the HMG-domain SoxB1 class transcription factor gene Sox3 (Modrell et al., 2011b); and the basic helix-loop-helix (bHLH) transcription factor gene Atoh1, whose lateral line organ expression at stages 40–45 was noted, in passing, in a study on cerebellum development (Butts et al., 2014).

Atoh1 is essential for hair cell formation (Bermingham et al., 1999). Paddlefish Atoh1 is 18.6-fold lateral line-enriched (Supplementary file 1), and expressed by stage 33 within the otic and preopercular neuromast canal lines (Figure 2A). It is expressed within most canal lines and the migrating posterior lateral line primordium by stage 37 (Figure 2B), and in the developing ampullary organ fields by stage 39 (Figure 2C), where it persists at stage 46 (Figure 2D).

Figure 2

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In mouse cochlear explants, Six1 and its co-factor Eya1, acting cooperatively with SoxB1 subfamily member Sox2, are sufficient to induce Atoh1 (Ahmed et al., 2012). Like Six1, Eya1 is also expressed in paddlefish ampullary organs (Modrell et al., 2011a), while Sox2 is 3.3-fold lateral line-enriched (Supplementary file 1). Immunostaining with a cross-reactive anti-Sox2 antibody revealed Sox2 expression in developing ampullary organs as well as neuromasts (Figure 2E–H), consistent with Sox2 mRNA expression by in situ hybridization (not shown). Sox2 represses Atoh1 in supporting cells in the mouse cochlea while, conversely, Atoh1 represses Sox2 in developing hair cells (Dabdoub et al., 2008). By stage 46, paddlefish Atoh1 expression is hard to detect in canal neuromasts - perhaps partly due to strong expression in scattered overlying epidermal cells, presumably Merkel cells (Maricich et al., 2009; Whitear, 1989) - but is still strong in ampullary organs (Figure 2I). Sox2 immunoreactivity remains strong in both neuromasts and ampullary organs at stage 46 (Figure 2J), where it is most likely restricted to supporting cells, given its punctate staining pattern.

Of the 66 genes in the lateral line-enriched dataset that encode known transcription factors (three other transcription factor genes in the dataset were assigned only to families) (Supplementary file 1), our top candidate was Pou4f3 (Brn3c, DFNA15), which is 27.5-fold lateral line-enriched (Supplementary file 1). Pou4f3 is a confirmed Atoh1 target in hair cells (Masuda et al., 2011; Ikeda et al., 2015) and required for hearing (Costaridis et al., 1996). In mouse cochlear explants, Six1 and Eya1 - acting independently of Atoh1 - are sufficient to induce Pou4f3, which promotes hair cell differentiation (Ahmed et al., 2012). Like Atoh1, paddlefish Pou4f3 is expressed in ampullary organs, as well as neuromasts (Figure 2K,L).

All three SoxB1 subfamily genes are putative Atoh1 targets in the postnatal mouse cerebellum (Klisch et al., 2011) and present in the lateral line-enriched dataset. Sox3 is 5.2-fold lateral line-enriched (Supplementary file 1); as noted above, we previously reported its expression in paddlefish ampullary organs and neuromasts (Modrell et al., 2011b). Sox1 is 8.6-fold lateral line-enriched (Supplementary file 1) and expressed in ampullary organs from their eruption (Figure 2M–P). It is transiently expressed in the migrating posterior lateral line primordium (Figure 2M,N) and, at later stages, expression is also seen in neuromast canal lines (Figure 2O,P).

Taken together, these data support a high degree of conservation in the transcriptional network regulating the development of both lateral line organ types, including the likely requirement of Atoh1 for electroreceptor as well as hair cell formation.

The proneural transcription factor gene Neurod4 is expressed in ampullary organs but not neuromasts

Another highly lateral line-enriched transcription factor gene is Neurod4 (Ath3, NeuroM) (18.9-fold enriched; Supplementary file 1), a putative cerebellar Atoh1 target (Klisch et al., 2011). In mice, this atonal-related proneural bHLH transcription factor gene is expressed in the brain, spinal cord, retina, trigeminal ganglia and dorsal root ganglia (Takebayashi et al., 1997). It is required for the survival of cerebellar granule cell precursors (Tomita et al., 2000), and cooperates with other bHLH and/or homeodomain transcription factors to regulate the development of retinal bipolar and amacrine cells (see Hatakeyama and Kageyama, 2004), and trigeminal and facial branchiomotor neurons (Ohsawa et al., 2005). In zebrafish, Neurod4 is important for olfactory neuron development (Madelaine et al., 2011); it is transiently expressed during chicken otic neurogenesis, but not hair cell formation (Bell et al., 2008). Although the related gene Neurod1 seems to be important for zebrafish neuromast hair cell differentiation (Sarrazin et al., 2006), we previously showed that paddlefish Neurod1 is not expressed in lateral line organs, only in cranial sensory ganglia (Modrell et al., 2011b). Neurod4 is the only Neurod family member in the lateral line-enriched dataset. At early stages, it is expressed in the brain, olfactory epithelium, eyes and trigeminal ganglion (Figure 3A). The earliest lateral line expression of Neurod4 is in developing ampullary organ fields around stage 39 (Figure 3B), where it persists (Figure 3C–F). Expression is never observed in developing neuromast lines. No other gene has previously been reported to show differential expression in ampullary organs and neuromasts. Hence, Neurod4 may be important for specifying ampullary organ and/or electroreceptor fate.

Figure 3

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We went on to examine the expression of transcription factor genes with known links to Neurod4 in other cell types. In trigeminal neurons, Pou4f1 (Brn3a) directly represses Neurod4 (Lanier et al., 2007). Pou4f1 is a putative Atoh1 target in the postnatal mouse cerebellum (Klisch et al., 2011), and is 20.6-fold lateral line-enriched (Supplementary file 1). Expression was seen in both ampullary organs and neuromasts (Figure 3G), suggesting that additional factors besides Pou4f1 are likely involved in controlling the differential expression of Neurod4 in ampullary organs versus neuromasts.

In the spinal cord, Neurod4 cooperatively interacts with the LIM homeodomain transcription factor Lhx3 to specify motor neurons (Lee and Pfaff, 2003). Lhx3 is expressed in all inner ear hair cells, although its expression is differentially regulated by Pou4f3 in cochlear versus vestibular hair cells (Hertzano et al., 2007); it is also a putative cerebellar Atoh1 target gene (Klisch et al., 2011). Lhx3 is 16.3-fold lateral line-enriched (Supplementary file 1), and proved to be expressed in both ampullary organs and neuromasts (Figure 3H). Hence, it is possible that Lhx3 could interact with Neurod4 in paddlefish ampullary organs to specify electroreceptors, and with another partner in neuromasts to specify hair cells.

Finally, we examined the zinc finger transcription factor gene Myt1, another putative cerebellar Atoh1 target gene (Klisch et al., 2011) that is 6.1-fold lateral line-enriched (Supplementary file 1). We selected Myt1 because its expression is upregulated in Xenopus embryos by Neurod4 (Perron et al., 1999; Hardwick and Philpott, 2015), and it synergizes with Neurod4 to induce neuronal differentiation in this system (Perron et al., 1999). Furthermore, Myt1 transcripts are reportedly enriched in both cochlear and vestibular hair cells in postnatal mice (Elkon et al., 2015). We found expression of paddlefish Myt1 in both ampullary organs and neuromasts (Figure 3I).

Overall, our results suggest that the transcription factor networks underlying hair cell development, which center around Atoh1, are likely to be active in paddlefish ampullary organs as well as neuromasts. This suggests significant conservation between the molecular mechanisms underlying hair cell and electroreceptor development. Furthermore, our unbiased RNA-seq approach has also identified the first transcription factor gene expressed in ampullary organs but not neuromasts, Neurod4. Given the importance of members of the proneural bHLH Neurod family for specifying cell fate, we suggest that Neurod4 may be involved in specifying ampullary organ and/or electroreceptor fate in paddlefish.

Hair cell ribbon synapse genes are also expressed in ampullary organs

Some of the most highly lateral line-enriched genes in our paddlefish dataset are required for synaptic transmission in hair cells, which occurs at specialized ‘ribbon synapses’ characterized by electron-dense, pre-synaptic structures called ‘synaptic ribbons’ (Matthews and Fuchs, 2010; Pangršič et al., 2012; Safieddine et al., 2012; Nicolson, 2015; Wichmann and Moser, 2015; Moser and Starr, 2016). These tether glutamate-filled synaptic vesicles and stabilize L-type voltage-gated calcium channels at the plasma membrane (Ca_v1.3 in hair cells; Ca_v1.4, in retinal photoreceptors; Joiner and Lee, 2015), enabling rapid and sustained glutamate release in response to activation of these calcium channels by membrane depolarization (Matthews and Fuchs, 2010; Pangršič et al., 2012; Safieddine et al., 2012; Nicolson, 2015; Wichmann and Moser, 2015; Moser and Starr, 2016). Electroreceptors also have synaptic ribbons of varying morphology (Northcutt, 1986; Bodznick and Montgomery, 2005): in paddlefish, they were described as synaptic ‘sheets’ (Jorgensen et al., 1972). In dissected skate ampullary organ preparations, activation of L-type voltage-gated calcium channels in the basal electroreceptor membrane results in release of a ‘glutamate-like’ neurotransmitter (Bennett and Obara, 1986).

In hair cells, glutamate is loaded into synaptic vesicles by the vesicular glutamate transporter Vglut3, which is encoded by Slc17a8 (DFNA25) and essential for hair cell synaptic transmission in mouse (Ruel et al., 2008; Seal et al., 2008) and zebrafish (Obholzer et al., 2008). This is unusual: Vglut1 or Vglut2 are used at glutamatergic synapses in the central nervous system, and at photoreceptor and bipolar cell ribbon synapses (see e.g. Zanazzi and Matthews, 2009; Pangršič et al., 2012). Slc17a8 is one of the most highly enriched genes in our paddlefish dataset (30.9-fold enriched; Supplementary file 1), and proved to be expressed in both ampullary organs and neuromasts (Figure 4A). Hence, synaptic vesicles in electroreceptors are likely to be loaded by the same vesicular glutamate transporter as hair cells. Furthermore, this also provides independent evidence that the afferent neurotransmitter released by non-teleost electroreceptors is indeed glutamate, as in hair cells (and photoreceptors), as suggested by electrophysiology experiments on dissected skate ampullary organs (Bennett and Obara, 1986).

Figure 4

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Hair cells are thought to be unique in depending on the multi-C₂ domain transmembrane protein otoferlin for synaptic vesicle exocytosis (Yasunaga et al., 1999; Roux et al., 2006; Pangršič et al., 2010; Chatterjee et al., 2015; Strenzke et al., 2016; Vogl et al., 2016), rather than neuronal SNAREs (Nouvian et al., 2011). This contrasts not only with conventional synapses but also with all other ribbon synapses (see e.g. Zanazzi and Matthews, 2009; Mercer and Thoreson, 2011). Otoferlin is a type II ferlin (Lek et al., 2012) encoded by Otof (DFNAB6, DFNAB9), which is 20.9-fold lateral line-enriched (Supplementary file 1). Otof is expressed in both ampullary organs and neuromasts in paddlefish (Figure 4B). This suggests that synaptic vesicle exocytosis at the electroreceptor ribbon synapse, just as at the hair cell ribbon synapse, is otoferlin-dependent.

The L-type voltage-gated calcium channel whose opening triggers synaptic vesicle exocytosis in hair cells is Ca_v1.3 (Kollmar et al., 1997; Platzer et al., 2000; Brandt et al., 2003; Michna et al., 2003; Dou et al., 2004; Brandt et al., 2005; Baig et al., 2011). This contrasts with retinal photoreceptors, which do express Ca_v1.3, but rely on Ca_v1.4 for calcium influx (Matthews and Fuchs, 2010; Joiner and Lee, 2015). The pore-forming (alpha) subunit of Ca_v1.3 is encoded by Cacna1d, which is required for hearing (Platzer et al., 2000; Dou et al., 2004; Baig et al., 2011), and also for hair cell function in zebrafish (Nicolson et al., 1998; Sidi et al., 2004), where it is expressed in both neuromasts and the inner ear (Sidi et al., 2004). (The two zebrafish cacna1d genes show differential expression: cacna1da is expressed in hair cells plus retinal photoreceptors, while cacna1db is expressed in retinal and pineal photoreceptors, but not hair cells; Sidi et al., 2004.) Paddlefish Cacna1d is 19.3-fold lateral line-enriched (Supplementary file 1) and expressed in both ampullary organs and neuromasts (Figure 4C). Hence, Ca_v1.3 channels in the basal membrane are likely to mediate glutamate release from both paddlefish electroreceptors and hair cells. Given the homology of non-teleost ampullary organs (Bullock et al., 1983; Northcutt, 1986, Northcutt, 1992; Braun, 1996; New, 1997; Baker et al., 2013), this also suggests that the L-type voltage-gated calcium channels involved in neurotransmitter release from skate ampullary organs are likely to be Ca_v1.3 channels.

The abundance and function of Ca_v1.3 channels in inner ear hair cells is regulated by the auxiliary beta subunit Ca_vβ₂, which is required for hearing (Neef et al., 2009). Ca_vβ₂ is encoded by Cacnb2, which is the only other voltage-gated Ca²⁺ channel subunit gene in the lateral line-enriched dataset (2.7-fold enriched; Supplementary file 1). Although neuromast expression of cacnb2a and cacnb2b was not reported in zebrafish (Zhou et al., 2008), paddlefish Cacnb2 is expressed in both ampullary organs and neuromasts, with seemingly stronger expression in ampullary organs (Figure 4D). This suggests that Ca_vβ₂ may be the auxiliary beta-subunit for Ca_v1.3 channels in electroreceptors, as well as hair cells.

Furthermore, given that the voltage sensors in skate electroreceptors are L-type voltage-gated calcium channels in the apical membrane (Bennett and Obara, 1986; Lu and Fishman, 1995; Bodznick and Montgomery, 2005), recently demonstrated to be Ca_v1.3 channels (Bellono et al., 2017), and the fact that an apical calcium conductance is required for the firing of sturgeon ampullary organ afferents (Teeter et al., 1980), it seems likely that apically-located Ca_v1.3 channels, with Ca_vβ₂ auxiliary beta subunits, act as the voltage-sensing channels, as well as mediating neurotransmitter release basally.

Finally, we report the expression in both ampullary organs and neuromasts of Rims2 (Rim2) and Ctbp2 (Ribeye), which encode proteins associated with synaptic ribbons in photoreceptors as well as hair cells (Matthews and Fuchs, 2010; Pangršič et al., 2012; Safieddine et al., 2012; Nicolson, 2015; Wichmann and Moser, 2015) (Figure 4E,F). Rims2 encodes Rab3-interacting molecules 2α and β, which are required in cochlear inner hair cells for Ca_v1.3 channel recruitment to the active zone membrane beneath the synaptic ribbon (Jung et al., 2015). Rims2 is 15.7-fold lateral line-enriched (Supplementary file 1) and expressed in all lateral line organs (Figure 4E). Hence, Rims2 may also be involved in Ca_v1.3 channel recruitment in electroreceptors. Ribeye, the only known synaptic ribbon-specific protein, is the main structural component of synaptic ribbons in both photoreceptors and hair cells, encoded by usage of an alternative start site for the transcription factor gene Ctbp2 that generates an N-terminal A-domain unique to Ribeye (Matthews and Fuchs, 2010; Nicolson, 2015; Wichmann and Moser, 2015). Ribeye is important in zebrafish neuromast hair cells for Ca_v1.3 channel recruitment to synaptic ribbons and stabilizing synaptic contacts with afferent neurons (Sheets et al., 2011; Lv et al., 2016). Recently, deletion in mice of the exon encoding the A-domain showed that Ribeye is essential in the retina (the ear was not examined) both for ribbon formation per se, and for rapid and sustained neurotransmitter release (Maxeiner et al., 2016). Ctbp2 is not in the lateral line-enriched dataset, but was present in the combined transcriptome, so was easily cloned. As expected, a riboprobe that exclusively recognizes the Ribeye-specific A-domain sequence of paddlefish Ctbp2 reveals expression in both ampullary organs and neuromasts (Figure 4F), suggesting that Ribeye is likely to be a key component of synaptic ribbons in electroreceptors (and hair cells), where it may also be important for Ca_v1.3 channel recruitment.

Taken together, these data suggest that the mechanisms of neurotransmission at the ribbon synapse in paddlefish electroreceptors are essentially identical to those at the hair cell ribbon synapse, involving otoferlin-dependent exocytosis of synaptic vesicles, loaded with glutamate by Vglut3, in response to the activation of basal Ca_v1.3 channels. Since non-teleost ampullary organs are homologous (Bullock et al., 1983; Northcutt, 1986, Northcutt, 1992; Braun, 1996; New, 1997; Baker et al., 2013), we predict that these mechanisms will be conserved across all other non-teleost ampullary organs.

Differential expression of beta-parvalbumin genes in ampullary organs and neuromasts

The paddlefish lateral line-enriched dataset contains two parvalbumin (Pvalb) genes: one, annotated as being related to zebrafish ‘pvalb8’, is enriched 22.8-fold; the other, annotated as being related to zebrafish ‘pvalb3’, is enriched 2.1-fold (Supplementary file 1). Parvalbumins are cytosolic EF-hand Ca²⁺-buffering proteins (Schwaller, 2010). Ca²⁺ is essential for multiple aspects of hair cell function (Lenzi and Roberts, 1994; Mammano et al., 2007; Ceriani and Mammano, 2012) and different hair cell subtypes are distinguished by different complements of EF-hand Ca²⁺ buffers, including different parvalbumin family members. Mammals have a single alpha-parvalbumin, encoded by Pvalb, and a single beta-parvalbumin, oncomodulin, encoded by Ocm (Schwaller, 2010). Alpha-parvalbumin is restricted to cochlear inner hair cells, while oncomodulin is restricted to cochlear outer hair cells, and is also expressed in vestibular hair cells (Sakaguchi et al., 1998; Yang et al., 2004; Simmons et al., 2010; Pangršič et al., 2015; Tong et al., 2016). Non-mammalian species have variable numbers of parvalbumin genes (nine in zebrafish; Friedberg, 2005), and the nomenclature for both genes and proteins is inconsistent and confusing. This led us to undertake a phylogenetic analysis of selected vertebrate parvalbumin proteins, including the two predicted paddlefish proteins (Figure 5A). This revealed three distinct clades: one comprising the alpha-parvalbumins (including zebrafish ‘Pvalb6’ and ‘Pvalb7’; Friedberg, 2005), and two containing beta-parvalbumins (Figure 5A). One of the beta-parvalbumin clades includes the oncomodulins (i.e., mammalian beta-parvalbumins), chicken Pvalb3/thymic CPV3 (encoded by Ocm) (Hapak et al., 1994), bullfrog Pvalb3 (Heller et al., 2002) and zebrafish ‘Pvalb8’ and ‘Pvalb9’, which were originally named Pvalb3a and Pvalb3b owing to their similarity to chicken Pvalb3/CPV3 and oncomodulin (Hsiao et al., 2002; Friedberg, 2005) (Figure 5A). The second beta-parvalbumin clade includes chicken thymic Pvalb (avian thymic hormone, encoded by Ocm2) (Brewer et al., 1989), zebrafish ‘Pvalb1-5’ (Friedberg, 2005) and various other beta-parvalbumins (Figure 5A).

Figure 5

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Following our phylogenetic analysis, it was clear that both lateral line-enriched Pvalb genes encode beta-parvalbumins. We named the most highly lateral line-enriched gene Pvalbβ1/Ocm (22.8-fold enriched; ‘pvalb8’ in Supplementary file 1), because the predicted protein groups in the beta-parvalbumin clade containing mammalian oncomodulins (Figure 5A). Pvalbβ1/Ocm is expressed in both ampullary organs and neuromasts (Figure 5B). Interestingly, ‘Pvalb8’ was reported to be the most highly expressed transcript in skate ampullary organs: the authors suggest that, following the voltage-dependent Ca²⁺ influx via Ca_v1.3 that depolarizes the electroreceptor and activates BK, this parvalbumin could bind Ca²⁺, thus blocking BK-mediated hyperpolarization and enabling further oscillations (Bellono et al., 2017).

We named the less highly lateral line-enriched gene Pvalbβ2 (2.1-fold enriched; ‘pvalb3’ in Supplementary file 1), as it falls into the second beta-parvalbumin clade (Figure 5A). At all stages examined, Pvalbβ2 proved to be expressed in ampullary organs but not neuromasts (Figure 5C). Therefore, in addition to Neurod4, we have identified another transcript expressed by ampullary organs but not neuromasts. We suggest that this beta-parvalbumin is likely to be involved in ampullary organ-specific aspects of Ca²⁺ regulation.

Identification of ampullary organ-specific voltage-gated potassium channel subunits

The oscillatory character of the basal membrane voltage of skate electroreceptors depends on voltage-gated potassium channels, which contribute to repolarization of the basal membrane (Bennett and Obara, 1986; Lu and Fishman, 1995; Bodznick and Montgomery, 2005). Noisy voltage oscillations have been recorded from ampullary organ canals in adult paddlefish in vivo (Neiman and Russell, 2004). Hence, we were very interested to find two highly lateral line-enriched shaker-related voltage-gated potassium channel subunit genes in our paddlefish dataset: Kcna5, encoding the pore-forming alpha subunit of K_v1.5 (12.4-fold enriched) and Kcnab3, encoding the beta subunit K_vβ3 (21.4-fold enriched). In contrast to the L-type voltage-gated calcium channel subunit genes Cacna1d and Cacnb2, which are expressed in both ampullary organs and neuromasts (Figure 4C,D), Kcna5 (Figure 6A–D) and Kcnab3 (Figure 6E–H) are only expressed in ampullary organs. Expression of both genes is first seen around stages 38–39, slightly after the eruption of the first ampullary organs at the surface at stage 37 (Modrell et al., 2011a).

Figure 6

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Voltage-gated potassium channels are tetramers of four alpha (pore-forming) subunits, each containing six transmembrane segments, of which S4 contains a high density of positively charged residues and is the main transmembrane voltage-sensing component (Barros et al., 2012). Intriguingly, paddlefish K_v1.5 has a number of amino acid substitutions at otherwise highly conserved positions in S4, as well as in the S3-S4 linker, at the top of the S5 helix, and in the channel pore (Figure 6I,J). How the sum of these substitutions affects paddlefish K_v1.5 channel behavior must await studies of channel expression.

Here, we took advantage of the abundance of ampullary organs (plus some neuromasts) on the operculum (gill-flap) of late-larval Mississippi paddlefish, and the absence of lateral line organs on the fins (Modrell et al., 2011a, 2011b), to generate a dataset of over 400 identified genes whose transcripts are enriched at least 1.8-fold in operculum versus fin tissue (i.e., lateral line-enriched). The dataset is not exhaustive: it does not include some genes whose expression we have validated in developing ampullary organs and neuromasts in paddlefish, whether via previous candidate gene approaches (e.g. Eya family members and Six family members other than Six1 and Six2; Modrell et al., 2011a), or some genes cloned in this study from transcripts present in the combined operculum plus fin transcriptome (e.g. Ctpb2, encoding the synaptic ribbon protein Ribeye). Nevertheless, this unbiased dataset provides an important foundation for investigating the molecular basis of ampullary organ development. Validation of a selection of genes from the dataset revealed significant molecular conservation between developing paddlefish ampullary organs and neuromasts, both in their expression of transcription factor genes critical for hair cell development, and also of genes essential for transmission specifically at the hair cell ribbon synapse. For the first time in any vertebrate, we also identify genes expressed in ampullary organs but not neuromasts, including a transcription factor, a beta-parvalbumin and voltage-gated potassium channel subunits consistent with predictions from skate ampullary organ electrophysiology.

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Author details

1. Melinda S Modrell

   Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

   Contribution

   MSM, Conceived the project with CVHB and designed the experiments, Performed all cloning, almost all in situ hybridization and all immunostaining experiments and analysis, and generated all related figures, Wrote the manuscript, with significant contributions from CVHB and HHZ

   Competing interests

   The authors declare that no competing interests exist.

2. Mike Lyne

   1. Cambridge Systems Biology Centre, University of Cambridge, Cambridge, United Kingdom
   2. Department of Genetics, University of Cambridge, Cambridge, United Kingdom

   Contribution

   ML, Assembled the transcriptome with ARC and performed differential expression analyses, Read and commented on the manuscript

   Competing interests

   The authors declare that no competing interests exist.

3. Adrian R Carr

   1. Cambridge Systems Biology Centre, University of Cambridge, Cambridge, United Kingdom
   2. Department of Genetics, University of Cambridge, Cambridge, United Kingdom

   Contribution

   ARC, Assembled the transcriptome with ML and performed differential expression analyses, Read and commented on the manuscript

   Competing interests

   The authors declare that no competing interests exist.

4. Harold H Zakon

   1. Department of Neuroscience, The University of Texas at Austin, Austin, United States
   2. Department of Integrative Biology, The University of Texas at Austin, Austin, United States

   Contribution

   HHZ, Performed the phylogenetic and sequence analyses for Kcna5, and generated the schematic in Figure 6, Made a significant contribution to writing the manuscript

   Competing interests

   The authors declare that no competing interests exist.

5. David Buckley

   1. Departmento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales-MNCN-CSIC, Madrid, Spain
   2. Department of Natural Sciences, Saint Louis University - Madrid Campus, Madrid, Spain

   Contribution

   DB, Performed the phylogenetic analysis for the parvalbumin genes, Read and commented on the manuscript

   Competing interests

   The authors declare that no competing interests exist.

6. Alexander S Campbell

   Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

   Contribution

   ASC, Contributed in situ hybridization data for the parvalbumin genes, Read and commented on the manuscript

   Competing interests

   The authors declare that no competing interests exist.

7. Marcus C Davis

   Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, United States

   Contribution

   MCD, Contributed to the collection and maintenance of paddlefish embryos and the sequencing of the fin transcriptome, Read and commented on the manuscript

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-2462-0138

8. Gos Micklem

   1. Cambridge Systems Biology Centre, University of Cambridge, Cambridge, United Kingdom
   2. Department of Genetics, University of Cambridge, Cambridge, United Kingdom

   Contribution

   GM, Provided overall guidance for ML and ARC, Read and commented on the manuscript

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-6883-6168

9. Clare VH Baker

   Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

   Contribution

   CVHB, Conceived the project with MSM and helped design the experiments, Made a significant contribution to writing the manuscript

   For correspondence

   cvhb1@cam.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-4434-3107

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