Notch and Fgf signaling during electrosensory versus mechanosensory lateral line organ development in a non-teleost ray-finned fish

Abstract

The lateral line system is a useful model for studying the embryonic and evolutionary diversification of different organs and cell types. In jawed vertebrates, this ancestrally comprises lines of mechanosensory neuromasts over the head and trunk, flanked on the head by fields of electrosensory ampullary organs, all innervated by lateral line neurons in cranial lateral line ganglia. Both types of sense organs, and their afferent neurons, develop from cranial lateral line placodes. Current research primarily focuses on the posterior lateral line primordium in zebrafish, which migrates as a cell collective along the trunk; epithelial rosettes form in the trailing zone and are deposited as a line of neuromasts, within which hair cells and supporting cells differentiate. However, in at least some other teleosts (e.g. catfishes) and all non-teleosts, lines of cranial neuromasts are formed by placodes that elongate to form a sensory ridge, which subsequently fragments, with neuromasts differentiating in a line along the crest of the ridge. Furthermore, in many non-teleost species, electrosensory ampullary organs develop from the flanks of the sensory ridge. It is unknown to what extent the molecular mechanisms underlying neuromast formation from the zebrafish migrating posterior lateral line primordium are conserved with the as-yet unexplored molecular mechanisms underlying neuromast and ampullary organ formation from elongating lateral line placodes. Here, we report experiments in an electroreceptive non-teleost ray-finned fish, the Mississippi paddlefish Polyodon spathula, that suggest a conserved role for Notch signaling in regulating lateral line organ receptor cell number, but potentially divergent roles for the fibroblast growth factor signaling pathway, both between neuromasts and ampullary organs, and between paddlefish and zebrafish.

Keywords: Ampullary organs; Electroreceptors; Fgf signaling; Hair cells; Neuromasts; Notch signaling.

Fig. 1

Timeline for paddlefish development, with an emphasis on lateral line development. Events defining boundaries between stages of development (embryo, yolk-sac larva and feeding larva) are marked in red, with approximate timings (days post fertilization [dpf]) given for development at 18 °C. Adapted from Bemis and Grande (1992) and Modrell et al. (2011a). Abbreviations: AO, ampullary organs; dpf, days post-fertilization; LL, lateral line; NM, neuromasts.

Fig. 2

Notch signaling pathway genes are expressed in the developing lateral line system in paddlefish. Whole-mount in situ hybridization for paddlefish Notch1, Jag1 and Hes-5-like at different stages, with schematic representations of lateral line development, modified from Modrell et al. (2011a), showing lateral line placode/organ development at different stages in shades of blue, elongating placodes in light blue and emerging neuromast canal lines/ampullary organs in darker blue. Notch pathway gene expression is indicated within those tissues or organs, depending on stage, in black. (A-D) At stages 30–32, Notch1, Jag1 and Hes5-like all seem to be expressed in the developing otic neuromast line between the eye and otic vesicle; Notch1 and Hes5-like are strongly expressed in the brain, eye and otic vesicle, and in the region of the pre-otic lateral line placodes. Jag1 expression is restricted to the developing otic neuromast line and prospective opercular line, plus the otic vesicle. (E-H) At stages 36–37, the lateral line expression of these Notch pathway transcripts includes the other pre-otic neuromast lines and post-otic lateral line primordia. Weak, patchy expression of Jag1 ventral to the pre-otic neuromast lines likely represents developing ampullary organ fields (F). Similarly, Hes5-like expression is observed in the developing ventral infraorbital ampullary organ field (arrowhead in G). (I-L) At stages 39–41, although lateral line expression of Notch1 (I) is difficult to observe, expression of Jag1 (J) and Hes5-like (K) is present in all neuromast lines, including the posterior lateral line, while expression of Jag1 and Hes5-like is also seen in the flanking ampullary organ fields (J,K: arrowheads indicate the ventral infraorbital ampullary organ field). (M-P) At stages 44–45, expression of Notch1 (J), Jag1 (K) and Hes5-like (L) is seen in both neuromasts and ampullary organs. Abbreviations: adp, anterodorsal lateral line placode; app, anterior preopercular ampullary field; avp, anteroventral lateral line placode; dot, dorsal otic ampullary field; di, dorsal infraorbital ampullary field; ds, dorsal supraorbital ampullary field; e, eye; epi, epibranchial placode region; io, infraorbital lateral line; LL, lateral line; m, middle lateral line; mlp, middle lateral line placode; ol, otic lateral line; olf, olfactory; otp, otic lateral line placode; ov, otic vesicle; pll, posterior lateral line; plp, posterior lateral line placode; pop, preopercular lateral line; ppp, posterior preopercular ampullary field; S, stage; so, supraorbital lateral line; st, supratemporal lateral line; stp, supratemporal lateral line placode; vi, ventral infraorbital ampullary field; vot, ventral otic ampullary field; vs, ventral supraorbital ampullary field. Scale bars: 200 µm.

Fig. 3

DAPT treatment during lateral line development in paddlefish results in irregularly spaced sensory organs with supernumerary receptor cells. Whole-mount immunostaining using an antibody raised against bullfrog parvalbumin-3 (Heller et al., 2002), which labels paddlefish hair cells and electroreceptors (Modrell et al., 2011a). (A-D) DMSO control embryos at stages 36 (A), stage 39 (B; inset shows higher power view of preopercular neuromast line) and stage 43 (C,D), for stage-matched comparison with drug-treated embryos. Panel D shows a higher-power view of the region caudal to the eye from the embryo in C, showing the infraorbital neuromast line and the flanking ampullary organ fields. Dotted lines indicate approximate boundaries of the neuromast line. (E-H) Embryos treated with 50 μM or 100 μM DAPT for 18–24 h during placode elongation (stages 30–32), analyzed at stage 36 (i.e., immediately post-treatment; E), stage 39 (F; inset shows higher power view of preopercular neuromast line) and stage 43 (G,H). By stage 39 onwards, neuromast lines contain more neuromasts, irregularly spaced and with more hair cells, than seen in stage-matched controls. (I-L) Embryos treated with 50 μM or 100 μM DAPT for 18–24 h from stage 36, when the first ampullary organ primordia are already forming, analyzed immediately post-treatment at stages 37–38 (I), and at stage 39 (J; inset shows higher power view of preopercular neuromast line) and stage 43 (K,L). From stage 39 onwards, embryos have more neuromasts, irregularly spaced and with more hair cells. At stage 43, ampullary organs contain more electroreceptors and are clustered together in places. (M,N) Embryo treated with 50 μM DAPT for 18–24 h from stage 39, when ampullary organs start to erupt, and analyzed at stage 43. Neuromasts contain more hair cells and are irregularly spaced; ampullary organs contain more electroreceptors and are clustered together, forming large patches. Abbreviations: ao, ampullary organs; e, eye; nm, neuromasts; S, stage. Scale bars: 200 µm except for D,H,L,N, 100 µm.

Fig. 4

Fgf signaling pathway gene expression during lateral line organ development in paddlefish. Whole-mount in situ hybridization in paddlefish embryos for the indicated genes and stages. (A-E) Fgfr1 is expressed in the otic, infraorbital and preopercular neuromast lines at stage 36 (A) and at stage 39 (B), expression is also seen in the developing ampullary organ fields flanking the neuromast lines. At both stage 41 (C) and stage 46 (D,E), fgfr1 continues to be expressed in neuromast lines and ampullary organ fields. Panel E shows a higher-power view of the area caudal to the eye at stage 46: dotted lines indicate approximate boundaries of the neuromast lines. (F-H) Fgf3 expression at stage 36 (F) is strong in the midbrain-hindbrain boundary, olfactory system and epibranchial placodes, and weak in the infraorbital, otic and preopercular neuromast lines. By stage 39 (G), fgf3 is more strongly expressed in neuromast lines and ampullary organ fields; expression is also seen in gill filaments and taste buds. This expression pattern persists through stage 46 (H). (I-K) Fgf10 expression is seen at stage 36 (I) in the otic, infraorbital and preopercular neuromast lines, and in all neuromast lines at stage 41 (J) and stage 46 (K). Fgf10 is not expressed in the ampullary organ fields at any stage. (L-N) Fgf20 is expressed in the otic, infraorbital and preopercular neuromast lines at stage 36 (L) and in all neuromast lines at stage 41 (M), when it is also expressed in taste buds and gill filaments. At stage 46 (N), fgf20 continues to be expressed in all neuromast lines and is now also expressed in ampullary organs. Abbreviations: ao, ampullary organ; e, eye; epi, epibranchial placodes, gf, gill filaments; io, infraorbital lateral line; mhb, midbrain-hindbrain boundary; nm, neuromast; ol, otic lateral line; olf, olfactory epithelium; ov, otic vesicle; pop, preopercular neuromast line; S, stage; tb, taste buds. Scale bars: 200 µm except D, 1 mm.

Fig. 5

Differential expression of Fgf pathway genes in mature paddlefish lateral line organs. Skin-mount preparations from the same infraorbital region of stage 46 paddlefish embryos, following whole-mount in situ hybridization for the indicated genes. (A,B) Fgfr1 is expressed more strongly at the periphery of neuromasts and in interneuromast cells than in the central neuromast domain (A), and is excluded from the central domain of ampullary organs (B). (C-D²) Fgf3 is centrally expressed in neuromasts (C) and in smaller (presumably younger) ampullary organs (D¹). However, in larger (presumably mature) ampullary organs, fgf3 is expressed more strongly, and patchily, in a subset of peripheral cells (D²). (E,F) Fgf20 is expressed in the central domain of neuromasts (E), but in ampullary organs, it is expressed much more strongly, and patchily, in a subset of peripheral cells (F). (G,H) Fgf10 is expressed throughout neuromasts and in interneuromast cells (G,H), but not in ampullary organs (H). The dotted line in panel H outlines the approximate boundary of an ampullary organ. Abbreviations: ao, ampullary organ; nm, neuromasts. Scale bar: 20 µm.

Fig. 6

SU5402 treatment during paddlefish lateral line organ development yields contrasting phenotypes in neuromasts and ampullary organs. Whole-mount immunostaining using an antibody raised against bullfrog parvalbumin-3 (Heller et al., 2002), which labels paddlefish hair cells and electroreceptors (Modrell et al., 2011a). Dotted lines in higher-power views indicate approximate boundaries of neuromast lines. (A-D^’) Embryos treated for 18–24 h from stages 30–32 (placode elongation stages, with the first neuromast primordia detectable at stage 32). At both stage 37/8 (A-B′) and stage 41 (C-D′), comparison of 100 μM SU5402-treated embryos with stage-matched DMSO controls reveals fewer neuromasts, some with more hair cells, though no obvious effect on ampullary organs. (E-H′) Embryos treated for 18–24 h from stage 36, when the first ampullary organ primordia are already detectable by histology. At stage 39, when ampullary organs begin to erupt (E-F′), comparison of 100 μM SU5402-treated embryos with stage-matched DMSO controls reveals fewer neuromasts, though with no obvious effect on hair cell number, and the precocious emergence of ampullary organs, some with many more electroreceptors than seen in DMSO controls. At stage 42 (G-H′), fewer neuromasts were seen in 100 μM SU5402-treated embryos than in stage-matched DMSO controls, again with no obvious change in hair cell number, while ampullary organs were present in normal numbers but with more electroreceptors. (I-J′) Embryos treated for 18–24 h from stage 39, when ampullary organs start to erupt. At stage 43, comparison of 50 μM SU5402-treated embryos with stage-matched DMSO controls showed no effect on lateral line organs. Abbreviations: ao, ampullary organ; e, eye; olf, olfactory system; ov, otic vesicle; S, stage. Scale bars: 200 µm.