Differences in Lateral Line Patterns Between Medaka and Zebrafish
Spatial distributions of neuromasts on the caudal fin were compared between adult medaka and zebrafish using the hair cell marker DiAsp (Collazo et al.,1994). In medaka, up to 10 neuromasts constitute a single, closely packed cluster in the medial inter-ray region of the caudal fin (Fig. 1A,B; Ishikawa,1990). We refer to the cluster of neuromasts on the caudal fin of medaka as the caudal neuromast cluster (CNC) in the following experiments. In contrast, four lines of neuromasts extend along inter-ray spaces in zebrafish (Fig. 1C,D). The set of neuromasts that form on the caudal fin is referred to as the caudal lateral line (CLL, Dufourcq et al.,2006). To understand the mechanisms that regulate the differences between caudal neuromasts in medaka and zebrafish, we examined the embryonic and postembryonic development of neuromasts in both species.
The teleost caudal fin originates from a ventral region of the embryonic fin fold that will subsequently form the hypural bones and fin rays (Goodrich,1930; Ishikawa,1990; Ledent,2002). In medaka, a single pair of neuromasts was located symmetrically on the left and right sides of the prospective caudal fin region at 8.5 days postfertilization (dpf; Fig. 1E, difference in position of neuromasts between both sides of each embryo was less than 10 μm in length along the axis of the tail, n = 42). The neuromasts on the prospective caudal fin region are referred to as “terminal neuromasts” (Gompel et al.,2001; Ledent,2002; Pichon and Ghysen,2004). The neuromasts on the remaining part of the trunk and tail region are designated as “trunk neuromasts” in the following description. The terminal neuromast was precisely located in medaka (317 ± 12.8 μm from tip of the notochord, n = 84, Fig. 1F). These results contrast with the variable and asymmetrical distribution of the trunk neuromasts (Fig. 1E).
In zebrafish, on the other hand, two to four terminal neuromasts were observed on each side of the body (Fig. 1G, 2. 83 ± 0.57 on average at 5 dpf, n = 236; as previously reported, Gompel et al.,2001). In most cases (65%), there were three neuromasts on one side of the body, but a significant proportion had two or four neuromasts on one side (26% and 9%, respectively, n = 236). There was no correlation between the number of terminal neuromasts on the left and right sides of an individual (Fig. 1H, κ2 = 9.26, P > 0.05). The position of the most rostral terminal neuromast was more variable in zebrafish than that of the terminal neuromast in medaka (Fig. 1I, 249 ± 41.0 μm from tip of the notochord in embryos with three neuromasts, n = 131). Moreover, even when an embryo had the same number of terminal neuromasts on both sides, most of these neuromasts were located asymmetrically (Fig. 1J), resembling the pattern of distribution of trunk neuromasts in terms of asymmetrical localization (Fig. 1G). These results demonstrate that the terminal neuromasts in zebrafish are variable both in number and position, in contrast to the single pair of neuromasts that are located symmetrically in medaka.
Terminal Neuromast Is Established Prior to the Caudal Fin Formation in Medaka
We then examined the timing of determination of the position of the terminal neuromast during medaka development. The eye absent 1 (eya1) gene was used as a marker of proneuromasts before differentiation of hair cells. In zebrafish, eya1 mRNA is expressed in neuromasts and in their precursors (Sahly et al.,1999; Dufourcq et al.,2006). A partial cDNA fragment of the medaka eya1 gene (Koster et al.,2000) was used to isolate the remaining coding region, and we confirmed that this gene is orthologous to zebrafish eya1 (Fig. 2A). As predicted, eya1 mRNA was strongly expressed in medaka neuromast cells (Fig. 2B,C).
Figure 2. Positioning of the terminal neuromast in medaka. A–C: Characterization of the medaka eya1 gene. A: Schematic drawings and phylogenetic tree of Eya proteins. Amino acid sequence similarity (%) is shown for each domain. dEya, mEya, zEya and olEya indicate Eya in Drosophila, mouse, zebrafish and medaka, respectively. B: Lateral view of a 9 days postfertilization (dpf) medaka embryo labeled with the eya1 RNA probe. C: Higher magnification for the boxed region in B (left panel). Lateral view of a neuromast, showing strong expression of eya1 mRNA in the neuromast cells (right panel). D–J: Displacement of the terminal neuromast during caudal fin formation. The terminal neuromast and its precursor at 3 dpf (D,D′), 4 dpf (E,E′,F,F′), 6 dpf (G,H,H′), 9 dpf (I), and 3 weeks (J) are identified by eya1 mRNA expression (D′,E′) and DiAsp staining (G,I,J). The boxed regions in E and G are viewed under a differential interference contrast (DIC) microscope (F,H;F′,H′ show apical surfaces of the same region). Ventral loop of the caudal vein is indicated by dotted lines (F) and arrows (H). H1 and H2 indicate hypural plates. K: Morphology of the caudal fin in the adult Da mutant. K′: Position of the caudal neuromast cluster (CNC; the boxed region in K). Inset shows the terminal neuromast in an 8.5 dpf fry of the Da mutant. Position of the neuromast and its precursor is indicated by arrowheads in each panel. Lateral views except for the lower panel in D′, which shows a ventral view. Scale bars = 500 μm in B–G,I–K,K′.
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eya1 mRNA was symmetrically expressed on the left and right sides of the prospective caudal fin region at 3 dpf, before the morphological caudal fin structures became visible (Fig. 2D,D′). A ventral loop of the caudal vein was observed in the prospective caudal fin region at 4 dpf (Fig. 2E,F). The proneuromast, recognized as a spherical aggregate of eya1-expressing cells, was located near the ventral loop of the caudal vein (Fig. 2E′,F′). At 6 dpf, hair cells had differentiated and had become DiAsp-positive (Fig. 2G). The differentiated neuromast remained near the ventral loop of the caudal vein, which was encompassed by developing hypural plates H1 and H2 (Fig. 2H,H′; Ishikawa,1990). These results demonstrate that the terminal neuromast in medaka is precisely located before caudal fin structures, such as the vein and the hypural plates, become apparent.
Displacement of the Terminal Neuromast During Caudal Fin Formation in Medaka
At 9 dpf, shortly after hatching, the terminal neuromast moved dorsally, maintaining a consistent position relative to the hypural plates and the fin rays during caudal fin formation (Fig. 2I,J). The terminal neuromast retained the same position relative to caudal fin structures in the dorsoventrally symmetrical (homocercal) adult caudal fin (Fig. 1B), suggesting that the dorsal movement of the neuromast may be associated with morphological changes of the caudal fin structures. In the mutant Da (double anal fin), the epural bone adopts a hypural-like morphology (Ishikawa,1990), and the notochord fails to bend up (Fig. 2K, Ishikawa,1990). In the adult Da mutant, the terminal neuromast remains ventral to the medial region of the caudal fin, keeping its relative position to the hypural plates (Fig. 2K′; Ishikawa,1990). These results support our interpretation that the dorsal movement of the terminal neuromast is caused by the displacement of morphological structures during caudal fin formation.
Formation of the Accessory Neuromasts in Medaka
In adult medaka, the terminal neuromast developed into the CNC (Fig. 3A,A′), and the number of neuromasts per cluster was correlated with increasing body size (Fig. 3B). In amphibians and zebrafish, founder neuromasts give rise to rows of accessory neuromasts through a budding process (Stone,1933; Ledent,2002; Ghysen and Dambly-Chaudière,2005). The CNC in medaka may, therefore, also originate from the founder neuromast on the caudal fin. The neuromasts comprising the CNC were innervated by collaterals of the same axonal fascicle (Fig. 3A′), supporting this idea. In normal development, however, addition of new neuromasts is a relatively slow process, and is difficult to study. In the following experiments, we studied regeneration of the CNCs because the developmental process can be accelerated by eliminating the neuromasts (Dufourcq et al.,2006; López-Schier and Hudspeth,2006).
Figure 3. Accessory neuromast formation of the terminal neuromast in medaka. A,A′: Caudal fin of adult medaka. A: Scales develop in the proximal region of the caudal fin near the hypural plates. A′: The same sample observed under fluorescent illumination. The caudal neuromast cluster (CNC) is innervated by a single lateral line nerve fascicle indicated by arrows. Inset shows a differential interference contrast (DIC) image of the nerve projecting to the CNC. B: Number of neuromasts increases in a cluster during postembryonic development. C–H,H′: Budding of the founder neuromast during regeneration. C: Embryos were analyzed in 4 days after removal of the CNC. D–F: The boxed region in C is also shown under DIC optics (D) and fluorescent illumination (F). The same sample was analyzed in situ hybridization using eya1 RNA probe (E). G: Another example shows the large founder neuromast associated with the smaller accessory neuromast (arrowhead). Eya1 mRNA is strongly expressed in the area surrounding the two neuromasts. Inset shows the same sample stained with DiAsp. H,H′: Embryo 12 days after ablation. Two small neuromasts have arisen near the presumptive founder neuromast. Higher magnification of the boxed region is shown in H′. Arrows indicate the root of the lateral line nerve innervating the CNC retrograde labeled with DiAsp. Arrowhead indicates the collateral axonal branch innervating the presumptive accessory neuromast. I: Schematic drawing of the medaka caudal fin. The CNC is composed of a founder neuromast (black dot) associated with its descendant accessory neuromasts (gray dots). All of the neuromasts in the CNC are innervated by nerve branches originating from a single root of the lateral line nerve (gray line). Positions of the scales and hypurals are indicated by lines and dotted lines, respectively. Scale bars = 500 μm in A,C,H.
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We removed the CNCs mechanically as described previously (Baker and Montgomery,1999; Fig. 3C). At 4 days after ablation, a spherical aggregate of cells appeared near the ablated site of the axonal fascicle (Fig. 3D, n = 9 of 12 samples). The cell aggregate resembled a proneuromast and expressed eya1 mRNA (Fig. 3E). Moreover, some of the cells in the aggregate were DiAsp-positive (Fig. 3F), confirming that the cell aggregate was a regenerated neuromast that contained differentiating hair cells. In some cases, a large neuromast was associated with a smaller neuromast carrying fewer hair cells, and eya1 mRNA was expressed in the area surrounding both neuromasts (Fig. 3G, n = 3 of 12 samples), suggesting that the smaller neuromast may be derived by budding of the founder neuromast. At 12 days after ablation, two to three small neuromasts emerged in close proximity to the presumptive founder neuromast, and these small neuromasts were innervated by collateral branches which bifurcated from the root of the axonal fascicle (Fig. 3H,H′, n = 8 of 8 samples). These results suggest that the CNC is composed of accessory neuromasts that originate from a single founder neuromast through a budding process. Further experiments are required to elucidate whether the same process underlies normal development of the CNC.
In adult medaka, 6–8 scales (7.43 ± 0.63 on average, n = 84) developed in the proximal region of the caudal fin (Fig. 3A) and the CNC was located close to the caudal margin of these scales (Fig. 3A′, see Fig. 3I for a schematic drawing).
Migration of the Terminal Neuromasts During Caudal Fin Formation in Zebrafish
In zebrafish, the terminal neuromasts migrate and repattern in the early stages of caudal fin formation (Ledent,2002). We performed a more detailed analysis of the final destinations of the terminal neuromasts, in experiments with embryos that had three terminal neuromasts on each side (designated T1–3, Fig. 1I), because this type was most frequently observed (Fig. 1H).
The earliest morphological sign of the caudal fin structure was the dispersal of melanocytes (Ledent,2002), which was followed by the formation of a ventral loop of the caudal vein at 9 dpf, similar to that in medaka at 4 dpf (Fig. 4A,A′, compare with Fig. 2F). The terminal neuromasts remained asymmetrically positioned between the left and right sides of the body at this stage (Fig. 4A). At a few weeks, when they reached approximately 4.4 mm in length, the hypural structures became visible as condensations of mesenchymal cells (Bird and Mabee,2003; Fig. 4B,B′). Intriguingly, the positions of the terminal neuromasts became more symmetrical as the caudal fin structures formed (Fig. 4C,D). Zebrafish develop five hypural bones (Bird and Mabee,2003; Fig. 4B′–D′); the anterior three (hy1–3) and the posterior two (hy4 and hy5) correspond to the H1 and H2 hypural plates in medaka, respectively (Ishikawa,1990). The ventral loop of the caudal vein was surrounded by hy3 and hy4, and this position coincided with the medial region of the adult caudal fin (Fig. 4E), as has been described in medaka (Fig. 2G–J; Ishikawa,1990). In approximately 5.0-mm fry, the middle of the three terminal neuromasts (T2) was located near the loop of the vein, at the prospective medial region of the caudal fin (Fig. 4E′). We quantified the position of the terminal neuromasts after caudal fin formation, and showed that T2 was located precisely in the medial region of the caudal fin (86%, n = 130), while the remaining two neuromasts (T1 and T3) were less precisely located in dorsal and ventral regions of the caudal fin (Fig. 4G,J). Thus, the positions of terminal neuromasts become determined during caudal fin formation at least partly by neuromast migration in zebrafish, in contrast to the displacement of the terminal neuromast in medaka (Fig. 2G–J).
Figure 4. Positioning of the terminal neuromasts in zebrafish. A–E: Repatterning of the terminal neuromasts during caudal fin formation. Stage or body length of the fry is indicated in each panel. Terminal neuromasts (T1-3) are identified by DiAsp staining and morphological structures (hy1-5 indicate hypural bones 1–5; A–D) visualized under differential interference contrast (DIC) optics (A′–E′,E). Position of the ventral loop of the caudal vein is indicated by the dotted line (A′) and arrows (B′–D′,E). Asterisks in E and E′ indicate positions of fin rays encompassing the middle neuromast T2. Inset in E′ indicates higher magnification of the boxed region. All panels are lateral views except for insets in B–D, which show ventral views. nc, notochord. F–J: Terminal neuromasts and the nerve innervations after the caudal fin formation. F: Confocal images of a neuromast in an Isl1-green fluorescent protein (GFP) transgenic fish stained with DiAsp. Isl1-GFP (green) is expressed in the cells surrounding the DiAsp-positive hair cells (red) and in the apical region of the neuromast. Lateral views; arrow indicates kinocilia; arrowheads indicate the presumptive support cells (see text for details). G: Confocal image of the caudal fin. Two images with different focal planes are shown. Position of the fin rays is indicated as d (dorsal) and v (ventral). H,I: Higher magnifications of the boxed regions in G for the terminal neuromast (H) and the caudal lateral line (CLL) neuromast (I). Isl1-GFP expressing axons bifurcate from the root of the lateral line nerve innervating T1 (H). Arrowheads and arrows indicate the collateral branches projecting to C1-1 and C2-1, respectively. J,K: Positions of the terminal neuromasts (J) and the CLL neuromasts (K) were quantified relative to the position of fin rays (ray position is described in G). Scale bars = 100 μm in A–E, 500 μm in G, 50 μm in F,H,I.
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Accessory Neuromast Formation of Terminal Neuromasts in Zebrafish
Soon after caudal fin formation, when zebrafish fry were approximately 7.0 mm in length, additional neuromasts appeared in distal regions of the caudal fin to form the caudal lateral line system (CLL, Fig. 4G). CLL neuromasts form lines that extend in inter-rays spaces (Fig. 1C, D, Dufourcq et al.,2006). In the adult caudal fin, there were two to four CLL lines on each side (3.85 ± 0.53 on average, n = 708). In the majority of the cases (83%), four lines were present at stereotypic positions on the caudal fin (designated as C1–4, Fig. 4G,K); thus subsequent experiments were conducted on animals with four lines of the CLLs. The four lines were called C1 to C4, from dorsal to ventral.
To identify the origin of CLL neuromasts, we first observed axonal projections. The Isl1-GFP transgenic line expresses green fluorescent protein (GFP) in the efferent lateral line nerve (Higashijima et al.,2000). In adult Isl1-GFP fish, GFP is also expressed in neuromast cells (Dufourcq et al.,2006). We observed that some of the cells surrounding the DiAsp-positive hair cells also expressed Isl1-GFP, and that the signal was mainly restricted to the apical region of the neuromast (Fig. 4F). Support cells possess fine processes that are associated with the apical surface of the skin, and these processes are attached to the hair cells (Owens et al.,2007). Thus, GFP appears to be expressed in support cells, and possibly in hair cells as well (Dufourcq et al.,2006). Isl1-GFP expression in neuromast cells started when the fish were approximately 7.0 mm in length. However, isl1 mRNA expression in the neuromast was not detected by in situ hybridization (data not shown). Thus, it is likely that Isl1-GFP expression in the neuromast does not recapitulate endogenous isl1 gene expression, but rather reflects an artifactual effect of the transgene.
We demonstrated that the efferent nerve innervating the dorsal neuromast T1 extended two collateral axons to the first neuromasts of the C1 and C2 lines (designated as C1-1 and C2-1, respectively, Fig. 4H,I). Similarly, the efferent nerve innervating the ventral neuromast T3 extended two collateral axons to the C3 and C4 neuromasts, whereas the medial neuromast T2 did not extend any axonal collaterals (data not shown). These results suggested that the C1/2 and C3/4 lines may be derived from T1 and T3, respectively. To test this hypothesis, we analyzed the behaviors of T1 and T2 after removal of the CLL neuromasts. When the caudal fin is amputated, the most distal remaining CLL neuromast gives rise to new neuromasts on the regenerated fin, recapitulating normal development (Dufourcq et al.,2006). We amputated the caudal fin distal to the T1-T3 neuromasts when the CLL neuromasts appeared (Fig. 5A). At 3 days after amputation, the caudal fin regenerated (Fig. 5A). In the region distal to T1 and T3, an aggregate of cells resembling an enlarged proneuromast emerged (Fig. 5B,B′, n = 12 of 12 samples). Some of the cells in the aggregate were DiAsp-positive (Fig. 5C), and the cell aggregate was followed by an axonal process of the lateral line efferent nerve (Fig. 5C′). Confocal microscopy revealed that the DiAsp-positive cells were centrally located within the cell aggregate (Fig. 5D). Moreover, the eya1 mRNA was expressed in the T1 and T3 neuromasts and the presumptive proneuromasts (Fig. 5E). As expected, some of the T1 or T3 neuromasts restored two proneuromasts (Fig. 5F). All of these results suggest that the newly formed neuromasts of the CLLs are accessory neuromasts originated from the T1 and T3 neuromasts by a budding process.
Figure 5. Accessory neuromast formation of the terminal neuromasts in zebrafish. A: Embryos were analyzed 3 days after amputation of the caudal fin. B,B′: The boxed region of the lower panel in A is shown under differential interference contrast (DIC) optics. Higher magnification of the regenerated caudal lateral line system (CLL) neuromast is shown in B′. C,C′: The same region as B is shown under fluorescent illumination (C) with a deeper focal plane (C′). Positions of the founder neuromast and the budding neuromast are indicated by dotted lines. Arrowhead indicates DiAsp-positive hair cells. Arrows indicate the extending lateral line efferent nerve. D: Confocal image of a budding neuromast. The fluorescent image is superimposed over the DIC image. E: The same sample as A–C was analyzed by in situ hybridization using eya1 RNA probe. Eya1 mRNA is expressed in the cells between the founder neuromast and the regenerated neuromast (arrows). F: Example of a single terminal neuromast (T3) extending two prospective CLL neuromasts (C3-1 and C4-1) during the regeneration process. Scale bars = 500 μm in A, 50 μm in B–F.
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In adult zebrafish, three scales are vertically aligned in the caudalmost part of the body, covering the joint of the body and the caudal fin at the hypural region. Scales in the dorsal and ventral regions are much larger than that in the medial region (Fig. 6A, n = 90). The position of T1-T3 neuromasts coincided with the proximal region of each scale (Fig. 6A). T1-T3 neuromasts on the scale gave rise to rows of neuromasts that elongated dorsoventrally in the proximal region of the scale (Fig. 6B). These vertical rows of neuromasts closely resembled the rows of neuromasts on the body surface (stitches; Ledent,2002), where each stitch corresponds to each scale (Fig. 6C; Metcalfe,1989). The number of neuromasts per row increased on the scale of the proximal region of the caudal fin (Fig. 6D) by addition of neuromasts at the distal end of the row (Fig. 6B), suggesting that these were also accessory neuromasts formed by a budding process as described in the trunk region (Ledent,2002).
Figure 6. Development of neuromast patterns in adult zebrafish. A: Scales develop in the region corresponding to the three terminal neuromasts. Each terminal neuromast gives rise to a row of accessory neuromasts on the scale. Scales are outlined by dotted lines. B: The accessory neuromasts propagate dorsoventrally on the scale. C: Rows of accessory neuromasts in the tail region (stitches). D: Increase in number of neuromasts per row on the scale (circles, T1-associated scale), and number of neuromasts per line of the caudal lateral line system (CLL; triangles, C1) during postembryonic development. E,F: Examples of neuromast patterns in zebrafish that possess two (E) or four (F) terminal neuromasts on one side of the caudal fin. E: Both terminal neuromasts (indicated by arrow) are colocalized on the dorsal scale. In these cases, the C3 and C4-CLL neuromasts are absent in the ventral region. In this sample, the terminal neuromast is absent in the ventral region on both sides of the caudal fin (2 out of 65 fish showed this phenotype). F: Two of the four terminal neuromasts (indicated by arrow) are colocalized on the middle scale. In these cases, neither neuromast extended beyond the CLL, resulting in four lines of the CLLs on the caudal fin. G: Newly developing CLL neuromast (indicated by arrowhead) in the distal region of the caudal fin. G′: Differential interference contrast (DIC) image of the boxed region in G, showing that the newly differentiated CLL neuromast, which carries two kinocillia (arrowhead). H,H′:Eya1 mRNA expression identifies a budding structure (arrowhead) from the distal-most CLL neuromast (arrow). Higher magnification of the boxed region is shown in H′. I: Schematic drawing of the zebrafish caudal fin. Terminal neuromasts (black dots) generate two types of accessory neuromasts; lines on the caudal fin (CLL neuromasts, gray dots) and rows on scales (white dots). Projection of the lateral line nerve is shown by gray lines. Positions of the scales and caudal margin of hypural bones are indicated by lines and dotted lines, respectively. Scale bars = 500 μm in A–C,E–H, 50 μm in G′,H′.
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As we described, a significant proportion of embryos possessed two (26%) or four (9%) terminal neuromasts (Fig. 1H), and in these cases each neuromast was also in a position that corresponded to one of the three scales in adult fish (Fig. 6E,F). This suggests that the scales or the scale precursors may play an important role in the positioning of terminal neuromasts during postembryonic development, or that both neuromasts and scales depend on a common patterning factor. Moreover, the caudal fin in which the neuromast was absent at the ventral T3 position did not have the C3 or C4-CLL neuromasts (n = 12 of 130 fin sides, Fig. 6E), consistent with our results showing that C3 and C4-CLL neuromasts originated from T3. As described above, the majority (83%) of adult caudal fins had four lines of the CLLs on one side, although 26% of the prospective caudal fins had only two terminal neuromasts. This apparent inconsistency is due to the fact that one of the two terminal neuromasts became always located at the dorsal T1 position after caudal fin formation, and that in a majority of these cases the second terminal neuromast is found at the T3 position. Out of 25 fin sides with two terminal neuromasts, 10 sides had neuromasts at the T1/T2 positions, and 13 sides had neuromasts at the T1/T3 positions.
CLL neuromasts increased in number in a growth-dependent manner (Fig. 6D) by serial addition of neuromasts, with each newly formed neuromast being generated by budding from the distal-most CLL neuromast (Fig. 6G,G′,H,H′). In summary, both the lines of CLL neuromasts and the vertical rows of neuromasts on the scales of the caudal fin originate from the terminal neuromasts in a growth-dependent manner (Fig. 6I for a schematic drawing) as seen with CNC formation on the medaka caudal fin (Fig. 3I).
Diverse Neuromast Patterns on the Caudal Fin in Various Teleost Species
Finally, we asked whether the CLL patterns seen in medaka and zebrafish were also observed in other teleost species. We showed that seven related species of medaka (belongs to the genus Oryzias) had a CNC similar to that in medaka (Oryzias dancena, Fig. 7A, O. luzonensis, O. mekongensis, O. celebensis, O. marmoratus, O. minutillus, and O. javanicus, data not shown), indicating that this pattern is conserved in this taxonomic group. Dermogenys pusillus (wrestling halfbeak), which belongs to the same order Beloniformes as Oryzias, possessed a single line of the CLL neuromasts in the center of the caudal fin (Fig. 7B). Newly born D. pusillus fry had an embryonic pattern identical to that of Oryzias (Fig. 7B), suggesting that the CNC in Oryzias and the medial line of CLL neuromasts in other species may be homologous structures.
Figure 7. A–D: Diversity in neuromast patterns on the caudal fin of teleost species. Distribution of neuromasts is shown for O. dancena (A), D. pusillus (B), P. reticuata (C), and T. niphobles (D). Right panels show representative schematic drawings of caudal fin morphology. Dotted lines indicate the caudal margin of the hypural bones. Insets in A and B, and lower inset in C show the hatched or newly born fry of each species. Arrowheads indicate the terminal neuromasts. Arrows indicate the neuromasts on the opposite side of the fin. Upper inset in C indicates the trunk region of P. reticulata. See text for details. Scale bars = 500 μm.
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In two species from different taxonomic groups, Poecilia reticulata (Cyprinodontiformes, guppy, Fig. 7C) and Takifugu niphobles (Tetraodontiformes, puffer fish, Fig. 7D), there was no CNC or CLL on the caudal fin. In P. retuculata, we showed that each of the two terminal neuromasts remained as a single neuromast through postembryonic development (Fig. 7C). Interestingly, the same is true for the trunk neuromasts: in P. reticulata, no accessory neuromast formation (stitch) is observed anywhere on the body surface (Fig. 7C,D). The same is true in T. niphobles, where neuromasts remain single both on the caudal fin and on the body.
Among the species that present CLL lines on their caudal fin, the number of lines was variable among species: one in D. pusillus (Fig. 7B), two in Tridentiger trigonocephalus (Perciformes) and Misgurnus anguillicaudatus (Cypriniformes, Fig. 8A,C), four in zebrafish, and eight in Leucopsarion petersi (Perciformes) and Carassius gibelio (Cypriniformes, Fig. 8B,D). The number of CLL lines may be determined by the number of terminal neuromasts in embryonic stages and/or by the number of buds originating from each terminal neuromast. Intriguingly, the number of lines of the CLLs does not always reflect phylogeny, but rather appears to reflect ecology. T. trigonocephalus (chameleon goby), which resides in ocean shallows, possessed only two lines of the CLL neuromasts (Fig. 8A). In contrast, the closely related species L. petersi (ice goby, referred to as a neotenic goby; Kon and Yoshino,2002), which belongs to the same family Gobiidae as T. trigonocephalus, is an active swimmer and possessed eight lines of the CLL neuromasts (Fig. 8B). Similarly, M. anguillicaudatus (weather loach, Fig. 8C), a bottom-dwelling fish in rice fields, had fewer lines of the CLLs than the related active swimmer C. gibelio (goldfish, Fig. 8D).
Figure 8. Variation in number of the caudal lateral lines between related species. A–D: Distribution of neuromasts is shown for T. trigonocephalus (A), L. petersi (B), M. anguillicaudatus (C), and C. gibelio (D). Right panels show representative schematic drawings of caudal fin morphology. Dotted lines indicate the caudal margin of the hypural bones. See text for details. Scale bars = 500 μm.
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