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First published online January 31, 2006
Journal of Experimental Biology 209, 610-621 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02056
Asymmetric craniofacial remodeling and lateralized behavior in larval flatfish
Department of Embryology, Carnegie Institution of Washington, 3520 San Martin Drive, Baltimore, MD 21218, USA
e-mail: Schreiber{at}ciwemb.edu
Accepted 21 December 2005
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: thyroid hormone, metamorphosis, flounder, Paralichthys lethostigma, skull, remodeling
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Miwa et al., 1988;
Schreiber and Specker,
1998).
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). The
dorsal light reflex, which causes a typical upright swimming fish to tilt
towards a lateral light source until equilibrium is established between visual
and gravitational sensory input, may play a role in the development of
flatfish lateralized behaviors as the migrating eye changes the perceived
angle of light incidence (Graf and Baker,
1990; Neave,
1985). In adult flatfish, however, there is also evidence for
asymmetry in the hindbrain (Meyer et al.,
1981) and otoliths of the inner ear
(Helling et al., 2005),
central and peripheral vestibular regions well known to mediate vertebrate
postural control. The first part of this study dissociates eye migration from
tilted swimming and settling to show that these adult behaviors result from a
response to thyroid hormone that is independent of eye migration in the
southern flounder (Paralichthys lethostigma).
All vertebrates develop lateralized visceral organs during embryogenesis
(Yost, 1995), but flatfish are
virtually the only vertebrates that also undergo significant asymmetric
post-embryonic development. It has generally been assumed that the unique
asymmetries characteristic of adult flatfish develop during metamorphosis
(Youson, 1988), and the
phenomenology of asymmetric flatfish skull development and eye migration
during this period has been widely documented
(Brewster, 1987;
Okada et al., 2001;
Okada et al., 2003a;
Okada et al., 2003b;
Saele et al., 2004;
Wagemans et al., 1998).
However, the possibility that metamorphic laterality is preceded by subtle
larval asymmetry has not been explored. Flatfish species are described as
either `sinistral' (both eyes are on the left side of the adult head) or
`dextral' (both eyes on the right side). Within a species some fish may have
`reversed' asymmetry and appear as morphological and behavioral mirror images
of their siblings (Hubbs and Hubbs,
1945; Norman,
1930; Policansky,
1982). The ontogeny of behavioral and craniofacial reversal has
not been described, due in part to the difficulty of identifying reversed
larvae before substantial eye migration has taken place. The second part of
this study analyzes swimming and feeding behaviors of pre-metamorphic southern
flounder larvae to predict post-metamorphic sidedness in this predominantly
sinistral species. This approach, combined with a sensitive in vivo
bone stain (calcein), shows that behavior and skull asymmetries present in
larvae before the start of eye migration correspond with ensuing
post-metamorphic laterality.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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salinity). After
hatching (48 h post-fertilization), larvae were fed live zooplankton
(Brachionus plicatilis) and brine shrimp (Artemia) nauplii
through the end of metamorphosis (approximately 31 days post-fertilization;
d.p.f.) according to methods described
(Daniels, 2000;
Daniels and Watanabe, 2003).
Stages of larval development were adapted from the alphabetical description
(Minami, 1982) of the position
of the migrating eye for the congeneric Japanese flounder (P.
olivaceus): early (6-13 d.p.f.) and late (14-19 d.p.f.) pre-metamorphosis
(D-, D+; no eye migration); pro-metamorphosis (20-23 d.p.f., E-, E, E+; start
of eye migration); early, mid and late metamorphic climax (24-30 d.p.f., F, G,
H; eye migrates towards dorsal midline); post-metamorphic juvenile (31 d.p.f.,
I; migrating eye has crossed the dorsal mid-line); sub-adult (migrating eye is
located entirely on the left side of the head, and the left eye is in a more
ventral position than in stage I). The development of flounder pectoral fin
skeletal morphology has not been systematically described and was also used to
define developmental stage in manner that was independent of eye position (see
Results).
Metamorphic variants
Wild southern flounder are typically a sinistral (left-sided) species
(Daniels, 2000). In the present
study, three metamorphic variants of laboratory raised flounder are described:
(1) sinistral (left-sided), (2) dextral (right-sided), and (3) bilaterally
symmetric forms with either no eye migration, or migration of both eyes to the
dorsal midline. Variant frequencies were determined using 3000 yolk-sac larvae
(3 d.p.f.) distributed into six 120 l aquaria (500 larvae/aquaria); after
completing metamorphosis the variants were counted and their settling side
noted.
Screening for lateralized behavior
Though not previously reported, larval southern flounder exhibit tilted
swimming and lateralized feeding behaviors before and during metamorphosis,
most swimming with their left side tilted towards the water surface and a
minority with their right side tilted upwards. To determine if pre-metamorphic
tilted swimming and feeding behaviors of individual larvae are lateralized
(unidirectional) and if larval laterality corresponds with post-metamorphic
laterality (tilted swimming, settling) and morphological asymmetry (eye
migration, skull development), a simple procedure was developed to
differentiate between left- and right-tilters. A bright light is placed above
the larval aquarium and live brine shrimp are added to prompt feeding
behaviors. Since brine shrimp are phototropic, they concentrate at the water
surface where the larvae are now forced to feed. In the presence of brine
shrimp, larvae with tilted swimming exhibit two characteristic behaviors: (1)
skimming the water surface with the future eyed side (supplementary material
Movie 1: Sinistral surface skimming), and (2) swimming several centimeters
below the surface with the non-migratory eye facing upwards, apparently
tracking brine shrimp before darting upwards to feed (supplementary material
Movie 2: Sinistral tracking, and Movie 3: Dextral tracking). Because flatfish
larvae have unusually narrow bodies with long dorsal-ventral profiles, these
tilted feeding behaviors are easily identified when viewed from above. Younger
larvae without tilted swimming do not exhibit these behaviors in the presence
of brine shrimp and always swim and feed upright (supplementary material Movie
4: Upright feeding). After screening larvae for surface skimming and tracking
behaviors, individuals are isolated and behavior examined in the absence of
brine shrimp to estimate the default angle of swimming tilt at different
developmental stages (see below).
To determine when tilted swimming begins, and to see if this behavior corresponds with post-metamorphic sidedness, the swimming behaviors of 1000 larvae living in two 120 l aquaria were observed daily from 7 d.p.f. through metamorphosis. When larvae first exhibited tilted swimming, left and right tilters (N=20) were collected for developmental staging and histological examination, and a larger number (N=50) were transferred to four behavior-segregated 40 laquaria and raised through metamorphosis. Larvae remaining in the 120 l aquaria were collected at different stages for behavioral and histological examination.
To estimate the degree of tilt from the upright (vertical) during ordinary (non-feeding) swimming, frontal photographs were taken (n=6-10 photographs/fish, and N=3-6 fish/stage) of larvae at different developmental stages in the absence of brine shrimp. A straight line from the anteriormost tip of the jaw to the ventralmost region of the gut defined the vertical axis of the fish; these are two anatomical points that remain medial during metamorphosis. The horizontal axis of reference was a line parallel to the water surface, and the angle of tilt defined as the angle by which the fish's vertical axis deviates from a line perpendicular to the horizontal reference. Differences in degree of tilt were analyzed using a nested factorial design (SuperANOVA, Abacus Concepts, Berkeley, CA, USA) consisting of two factors: developmental stage and individual fish. Significance was accepted when P<0.05. Fisher's PLSD post hoc test was performed when appropriate, as indicated by significance using analysis of variance (ANOVA).
Thyroid hormone/methimazol treatment, histology and digital morphing
Larval fish at early pre-metamorphosis (7 d.p.f.), late pre-metamorphosis
(14 d.p.f.) and pro-metamorphosis (20 d.p.f.) were induced to metamorphose by
adding 3,5,3'-triiodothyronone (T3) (Sigma Chemical Co., St Louis, MO,
USA) to the water (T3 final concentration, 100 nmol l-1) for 3-8
days. Methimazol (0.1 mol l-1; Sigma), an inhibitor of endogenous
thyroid hormone (TH) production (Brown,
1997), was dissolved directly into water and administered for up
to 6 weeks (starting at late pre-metamorphosis) to inhibit metamorphosis.
In vivo bone staining was modified from the technique described
for zebrafish by Du et al.
(2001): 10 mg calcein powder
(Sigma Chemical Co.) was dissolved directly into 100 ml saltwater to make a
0.01% solution, then passed through a 5 µm filter to remove insoluble
particles; live larvae were incubated in the solution (10-20 min), followed by
three rinses (15 min each) in calcein-free saltwater. Larvae were observed
live, or euthanized in tricaine-methanesulfonate (MS 222) and mounted in
liquid OCT embedding compound (Electron Microscopy Sciences, Hatfield, PA,
USA) for orientation and photography. Specimens were photographed using a Spot
RT camera mounted onto a Leica (Deerfield, IL, USA) MZ12 fluorescent
stereomicroscope using a GFP filter. At least 20 fish per developmental stage
were analyzed.
For histological identification of bone and cartilage, whole-body larvae
were stained with Alcian Blue and Alizarin Red as described
(Klymkowsky and Hanken, 1991)
with the following modifications: larvae were fixed in 4% neutral buffered
paraformaldehyde for 2 days at room temperature, rinsed in water, and
dehydrated through graded alcohols. Prior to staining, samples were
photo-bleached for 3-12 h in 0.3% H2O2 in 5% KOH at room
temperature.
The skull morphing movie (supplementary material Movie 7) was derived from still photographs of calcein-stained larvae at the different developmental stages depicted in Fig. 4. Images were cropped using Adobe Photoshop, aligned using Image J software for UNIX, morphed with Morph Age software for Mac OSX, and assembled using Final Cut Pro.
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Results |
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Development of lateralized behavior
Prior to metamorphosis no eye migration is observable. From hatching (2
d.p.f.) through to the end of yolk resorption (4-5 d.p.f.) the larvae did not
maintain an upright swimming posture, but rather swam with random orientation
in the presence of water currents caused by aeration, or drifted in the water
column perpendicular to the bottom with head facing down in the absence of
water flow, with occasional spontaneous bursts of non-directional swimming
(not shown). Larvae began to swim with the sustained upright posture typical
of most fish by 5 d.p.f. Early pre-metamorphic larvae (6-13 d.p.f.) swim with
an upright posture (Fig. 1A),
and late pre-metamorphic larvae (14-19 d.p.f.) swim with a 3-6° sustained
right tilt (Fig. 1B). By the
start of eye migration, pro-metamorphosis (20 d.p.f.), larvae swim with a
10-20° right tilt (Fig.
1C). The degree of tilt increases to 17-26° by early climax
(24 d.p.f.) (Fig. 1D and
supplementary material Movie 6: Climax tilt), shifts abruptly to a 50-80°
tilt by late climax (26 d.p.f.; Fig.
1E), and the fish swim virtually parallel to the bottom
(80-90°) by the juvenile stage (30 d.p.f.;
Fig. 1F). Changes in degree of
tilt per developmental stage are summarized in
Table 1. Eye migration
continues after metamorphosis during the juvenile stage, and by the time the
fish is a sub-adult (120 d.p.f.) the right eye is located fully on the left
side of the head and the left eye has moved to a more ventral position
(Fig. 1G; also see
Fig. 5G-H). Larvae in late
pre-metamorphosis swim predominantly in the water column of the aquarium, but
also occasionally settle to the bottom on their future `blind' side. As
metamorphosis proceeds the fish spend less time swimming and more time
settled, and by mid-climax only rarely swim. Lateralized feeding and sustained
tilted swimming behaviors were first evident at 14 d.p.f. at the start of late
pre-metamorphosis. Larvae with these lateralized actions also displayed
lateralized hiding and escape behavior: when chased with a pipet they often
rapidly swim to the tank bottom where they settle with their non-migrating eye
facing up (not shown). When left- and right-tilters were screened at 14 d.p.f.
and raised in separate aquaria through the end of metamorphosis
(N=50), 100% of left-tilters settled on their left side and 100% of
right-tilters on their right side.
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Craniofacial remodeling
Craniofacial morphology visualized with traditional bone (Alizarin Red) and
cartilage (Alcian Blue) histology is bilaterally symmetrical through late
pre-metamorphosis (Fig.
2A,A'). Craniofacial symmetry first breaks as the right (but
not left) cartilaginous supraorbital bar (SB) becomes thinner during early
pro-metamorphosis (not shown), and is degraded by late pro-metamorphosis
(Fig. 2B,B'). The left SB
appears thinner by early climax (Fig.
2C'), and by late climax
(Fig. 2D') has completely
degraded. The frontal bones stain progressively with Alizarin Red in a
proximal-distal manner from pro-metamorphosis through late climax
(Figs 2B-D). The pseudomesial
bar, a bone that forms only under the migrated eye, is visible by Alizarin Red
stain in the juvenile stage 10 days after metamorphosis is completed
(Fig. 2E).
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Dissociation of lateralized behavior from eye migration in thyroid hormone-treated larvae and bilaterally symmetrical metamorphic variants
A summary of results for TH induction and methimazol treatment on larval
swimming and settling behaviors is shown in
Fig. 6. Treatment of early
pre-metamorphic larvae (7 d.p.f.) with 100 nmol l-1 T3 for 96 h
induced settling behavior, but individuals settled alternately between sides
and displayed no side preference (not shown). Swimming behavior was not
evaluated for these fish. T3 induction of late pre-metamorphic larvae (14
d.p.f.) for 72 h, however, was sufficient to induce lateralized settling
(Fig. 7E) and tilted swimming
(60-85°angle; Fig. 7F,G)
(supplementary material Movie 8: TH-induced swimming and settling behaviors).
Individuals consistently settled onto the same side; though they were only
rarely observed swimming in the water column, they did so with their settled
side facing down. The percent of larvae with reversed tilted swimming and
settling behavior following T3 treatment (15%) was similar to that for
spontaneous metamorphosis (16%). Interestingly, these T3-induced behavioral
changes were accompanied by little to no eye migration
(Fig. 7C,D). T3 treated
pre-metamorphic larvae experienced a dramatic and symmetric condensation of
both frontal bones without lateral deformation
(Fig. 7D). Retention of the
cartilaginous right supraorbital bar (SB) after 72 h T3 treatment is likely
not responsible for inhibition of eye migration, as both SBs had degraded in a
bilaterally symmetric manner after 6 days of treatment (not shown), yet the
eyes still remained symmetrically placed with no frontal bending even after 8
days treatment (not shown). T3-treatment of pro-metamorphic larvae (which at
the start of treatment already displayed some eye migration, asymmetric
frontal bone condensation and bending, and right lateral ethmoid formation)
induced the same behavioral changes, as well as bilaterally symmetric frontal
bone condensation and eye-migration roughly commensurate with the amount of
frontal bending (Fig.
7H-K).
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Treatment of late pre-metamorphic larvae with 0.1 mol l-1 methimazol (an inhibitor of endogenous TH production) for 6 weeks inhibited eye migration (Fig. 8B'), resorption of the right SB (Fig. 8A'), frontal bone bending (Fig. 8B) and pectoral fin remodeling (Fig. 8A), but did not inhibit bone mineralization (broad Alizarin Red staining in Fig. 8A), bilateral frontal bone condensation (Fig. 8B), or development of right parietal barb and lateral ethmoid bones (Fig. 8B'). After 4 weeks of methimazol treatment these fish still swim with a 5-15° tilt typical of larvae in late pre-/early climax (not shown). Subsequent treatment with 100 nmol l-1 T3 for 1 week induced the fish to settle to the bottom of the tank. T3 treatment also induced pectoral fin remodeling (Fig. 8C; also see Fig. 10K,L) and right SB resorption (Fig. 8C'). The right eye migrated further than in fish treated only with methimazol (Fig. 8C',D'), though the frontals did not appear to bend further (Fig. 8D). Therefore, elongation of the right lateral ethmoid in the absence of eye migration (Fig. 8B') and subsequent induction of eye migration with thyroid hormone treatment in the absence of significant frontal bone deformation (Fig. 8D,D') together suggest that asymmetric skull development alone is insufficient for eye migration.
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Symmetrically metamorphosed variants all displayed normal 85-90° tilted swimming and normal settling behavior after metamorphosis (supplementary material Movie 9: Settled sinistral and symmetrical variants), despite either the lack of eye migration (Fig. 9A,A') or having both eyes located at the dorsal mid-line (Fig. 9C,C'). In symmetrical juveniles both left and right supraorbital bar cartilages resorb normally (Fig. 9B,B',D,D') and pectoral fins have remodeled to their adult form (see Fig. 10M), suggesting that these variants respond to increased endogenous TH production and are not hypothyroid. Bone mineralization is not inhibited, but the frontal bones are abnormally symmetrical in shape compared with other variants: both frontals have condensed to approximately the same thickness, although the frontal bones remain medially positioned. In further contrast with the other two more common variants, both left and right lateral ethmoids appear similarly elongated in length, and the pseudomesial bar has not formed. When pro-metamorphic tilting larvae (10-15° right or left tilt) were screened and stained with calcein, some larvae displayed bilateral frontal bone condensation with no lateralized bend and bilaterally symmetric eye position (Fig. 9E,F). Although most of these do not survive through metamorphosis, the survivors likely develop into symmetrically metamorphosed variants.
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Pectoral fin remodeling
Surprisingly, the extensive flatfish pectoral fin remodeling that takes
place during metamorphosis has not been systematically documented. Since the
pectoral fins develop autonomously in response to TH, they are useful markers
for classifying larval developmental stages independent of eye migration and
skull morphology. Pre-metamorphic larvae have large paddle-like pectoral fins
(Fig. 10A) that shrink in size
during pro-metamorphosis (Fig.
10B). The long postcoracoid process is almost entirely resorbed by
early climax (Fig. 10C), and
four proximal radials and a distinct scapulocoracoid are formed by late climax
(Fig. 10D-F). In a sub-adult,
the pectoral fin marginal rays have ossified
(Fig. 10G). These changes,
which normally take place during metamorphic climax, can be induced in early
pre-metamorphic larvae treated with 100 nmol l-1 T3 in as little as
3-4 days (Fig. 10H-J). In
contrast, the pectoral fins of larvae raised in 0.1 mol l-1
methimazol for 6 weeks starting at late pre-metamorphosis grow abnormally
large and retain their larval form entirely
(Fig. 10K; also see
Fig. 8A). Interestingly, the
pectoral fins of these methimazol treated larvae can be induced to remodel to
their juvenile form when 100 nmol l-1 T3 is administered for an
extra week in lieu of methimazol (Fig.
10L; also see Fig.
8B). The progression of pectoral fin development and responses to
thyroid hormone follow the typical teleost patterns described for zebrafish
(Grandel and Schulte-Merker,
1998; Brown, 1997).
Although bilaterally symmetric flounder variants may exhibit no eye migration
or frontal bone bending (see Fig.
9), their pectoral fins always remodel to the juvenile form
(Fig. 10M), suggesting that
these fish are not hypothyroid.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Miwa et al.,
1988; Schreiber and Specker,
1998). However, the specific mechanisms responsible for inducing
tilted swimming and settling behaviors in flatfish remain essentially unknown.
In principle, the abrupt transition to lateralized behavior during
metamorphosis could be influenced by asymmetric changes in eye position, inner
ear morphology, and/or central changes in vestibular connectivity or activity.
It is known that balance in many upright swimming fish is sustained through
interactions between the directions of light and gravity such that a typical
fish will tilt towards a lateral light source until a new balance with its
gravistatic senses is reached (Graf and
Meyer, 1983; von Holst,
1935). Indeed, pre-metamorphic larval flatfish exhibit a dorsal
light response similar to typical upright fish
(Neave, 1985), and as one eye
begins to migrate during metamorphosis the larvae tilt towards their future
blind side when illuminated from above. This tilting could be attributable to
a change in the angle of perceived light due to eye migration
(Graf and Baker, 1990;
Neave, 1985). Three findings
from the present study, however, suggest that tilting and settling behaviors
can be dissociated from - and thus occur independently of - asymmetric eye
position: (1) lateralized feeding and sustained tilted swimming behaviors are
apparent prior to eye migration as early as the start of late
pre-metamorphosis (Fig. 1B) and
correspond with post-metamorphic sidedness, (2) treatment of pre-metamorphic
larvae with thyroid hormone induces acute tilting and settling with little to
no eye migration (Fig. 7E-G), and (3) symmetrically metamorphosing flounder variants also display normal
lateralized swimming and settling behaviors
(Fig. 9). Interestingly, early
pre-metamorphic larvae (7 d.p.f.) treated with thyroid hormone are induced to
settle at the bottom of the tank, but do so without displaying a side
preference. This suggests that lateralized behavioral competence is
established by late pre-metamorphosis (14 d.p.f.) when larvae do respond to
thyroid hormone treatment with unilateral settling. The ability to dissociate
settling behaviors, skull remodeling and pectoral fin development
(Fig. 10) from each other in
TH-induced larvae and symmetrical variants suggests these are each autonomous
developmental programs mediated by TH, similar to the autonomy of
developmental programs displayed in metamorphosing frogs
(Brown et al., 2005;
Das et al., 2002;
Schreiber and Brown, 2003;
Schreiber et al., 2001). From
a practical perspective, the ability to predict post-metamorphic sidedness
based upon pre-metamorphic feeding and swimming behavior is a useful tool for
screening dextral-from sinistral-behaving larvae for analysis during
metamorphosis.
Since eye position does not influence the development of adult lateralized
behavior in flatfish, changes in the peripheral or central vestibular systems
must be responsible for these metamorphic changes. However, the gross anatomy
and bilateral symmetry of the labyrinths in flatfish
(Jacob, 1928), the
distribution of the vestibular nuclear complex in the hindbrain of larval and
juvenile turbot Scopthalmus maximus
(Jansen and Enger, 1996), and
the peripheral and central oculomotor apparatus
(Graf and Baker, 1985) are
bilaterally symmetrical and similar to those of other teleosts. Interestingly,
all flatfish except for the most primitive
(Platt, 1983) display nearly
omni-directional hair-cell polarization of the inner ear's saccular and
lagenar otoliths (Jorgensen,
1976; Platt,
1973). This pattern, which is unique amongst vertebrates, is
thought to accommodate, but not necessarily induce, the adult flatfish
posture. Other unusual features of flatfish sacculae are accessory growth
centers that form at the start of metamorphosis
(Jearld et al., 1990). A
central regulatory feature unique to flatfish is the significant
reorganization of the vestibular-ocular pathways required to stabilize eye
position during head movements (Graf and
Baker, 1983). These aforementioned flatfish peculiarities,
however, are bilaterally symmetric phenomena and alone do not explain the
development of lateralized behavior. Some evidence for asymmetric peripheral
and central postural control in adults does exist, though. 2-deoxyglucose is
taken up differentially by the bilateral vestibular nuclei of adult flatfish
(Meyer et al., 1981),
suggesting that lateralized behavior may be due to a `permanent imbalance in
vestibular neuron activity'. Morphological asymmetries in the otoliths of
flatfish have also been reported
(Lychakov, 1996;
Sogard, 1991), and mass
asymmetries, specifically in the utricles and saccules of adult flatfish, such
that the heavier otoliths are located on the bottom side
(Helling et al., 2005). These
asymmetries are interesting, especially considering the observation
(Graf and Baker, 1990) that
adult flounder exhibit different postural responses of left side vs
right side utricular neurectomy or hemilabyrinthectomy. However, it remains to
be seen if these adult flatfish asymmetries actually develop during
metamorphosis and induce postural change, or if they develop only after
behavioral change has already been established.
Historically, bilateral symmetry in the flatfish skull has been thought to
first break at the start of eye migration during metamorphosis
(Youson, 1988), the earliest
reported asymmetry being differential resorption of the left and right
cartilaginous supraorbital bars (SB)
(Wagemans et al., 1998;
Williams, 1901). The present
study uses an in vivo bone labeling technique
(Du et al., 2001) to show that
skull asymmetry is already established by the start of late pre-metamorphosis,
and possibly earlier. Specifically, sinistral southern flounder have a
noticeably smaller right parietal and thinner right frontal bone compared with
the left side during pre-metamorphosis (Figs
4B,
7B), and these asymmetries are
further exaggerated during metamorphic climax
(Fig. 4C-F). In contrast,
pre-metamorphic larvae with reversed parietal and frontal asymmetry
metamorphose with dextral morphology (Fig.
5). These asymmetries have not been previously described, and the
asymmetric architecture of the frontals before metamorphosis may facilitate
their characteristic bending at climax. The cause of frontal bone bending, a
unique feature of flatfish development, remains unknown. Interestingly, in
bilaterally symmetric metamorphic variants both frontals are symmetrically
shaped and do not develop a frontal bend
(Fig. 9). The ability to
dissociate eye migration from asymmetric development of the lateral ethmoid
bones or frontal bone deformation in methimazol and thyroid hormone-treated
larvae (Fig. 9) suggests that
asymmetric skull development alone is inadequate for eye migration. A possible
role for ocular muscle and orbit remodeling in facilitating eye migration
cannot be ruled out.
The proportion of flatfish displaying reversed morphology and behavior in a
natural population varies among species from virtually none in the
tonguefishes (Cynoglosidae) (Munroe,
1996) to as high as 100% in some starry flounder (Platichthys
stellatus) populations (Policansky,
1982). There are no reports of reversed southern flounder in
nature, though we show 16% reversal when this species is raised in the
laboratory. This suggests that reversed (dextral) southern flounder have low
survival in nature, but enhanced survival in the laboratory when they are
identified early and segregated from their sinistral siblings. A fundamental
aberration in skull morphogenesis of dextral compared with sinistral flounders
could explain their differential survival. However, this study shows that in
the southern flounder, dextral skull ontogeny (SB degradation, frontal bone
asymmetry, and formation of lateral ethmoid and pseudomesial bars on the blind
side) proceeds as a mirror image reversal of their sinistral siblings,
suggesting that factors other than skull morphology account for differential
survival. For example, the optic chiasm of all dextral southern flounder and
congeneric summer flounder (P. dentatus) that we have observed
display double-crossed optic nerves (which could impair vision) compared with
their partially uncrossed sinistral siblings (A.M.S., unpublished data), an
observation previously noted in other flatfish species
(Parker, 1903).
The genetics of flatfish reversal are not well understood. Hashimoto et al.
(2002) have isolated a
Japanese flounder (Paralichthys olivaceus) clonal line (reversed,
`rev') whose offspring display a relatively high (20-30%) frequency of
reversal. They have proposed that the directionality of metamorphic asymmetry
is determined by the rev locus in a manner independent from the
control of visceral asymmetry. Although the factors that mediate sinistral
vs dextral skull development are not known, a differential bilateral
sensitivity to thyroid hormone is likely involved. Treatment of
pre-metamorphic larvae with triiodothyronine (T3) induced bilaterally
symmetrical condensation of both frontal bones
(Fig. 7D) and symmetrical
resorption of both SBs (not shown). Eye migration in these larvae is
inhibited, possibly due to failure of the symmetrical frontals to bend and
allow passage for the migrating eye, as may be the case for the aforementioned
symmetrical metamorphic variants. Interestingly, the occurrence of bilaterally
symmetrical flatfish variants in aquaculture has been reported
(Okada et al., 2003b;
Pittman et al., 1998), and
symmetrical variants appear similar to symmetrical spotted halibut
(Verasper variegatus) juveniles that were artificially produced by
treating early pre-metamorphic larvae with thyroxin
(Tagawa and Aritaki, 2005).
Therefore, a symmetrical condensation of the frontals due either to early TH
treatment or abnormal bilateral sensitivity to endogenous TH during
metamorphosis may explain this unusual phenotype. TH receptors are expressed
in cartilage and presumptive osteoblasts of Japanese flounder larvae
(Yamano and Miwa, 1998), and
asymmetric sensitivity to TH in flatfish could, in principle, be regulated by
differential expression of thyroid hormone receptor levels or isoforms,
transcriptional coregulators, or types I, II or III deiodinase activity.
In summary, the main findings of this study are that (1) lateralized swimming behaviors and eye migration in larval flatfish both develop in response to thyroid hormone during metamorphosis, but are independent of each other, and (2) behavioral and craniofacial asymmetries are present before metamorphosis and can be used to predict post-metamorphic sidedness. Therefore, the abrupt 90° change in body orientation with respect to gravity during metamorphosis most likely results from asymmetric remodeling of central vestibular connectivity (hindbrain) and/or peripheral sensory organs (inner ear) in response to thyroid hormone. The thyroid hormone-responsive genes that ultimately mediate asymmetric skull and vestibular/inner ear remodeling remain to be identified.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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