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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

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Asymmetric craniofacial remodeling and lateralized behavior in larval flatfish

Alexander M. Schreiber

Department of Embryology, Carnegie Institution of Washington, 3520 San Martin Drive, Baltimore, MD 21218, USA

View larger version (66K): [in a new window]   Fig. 1. Body swim-posture changes during flounder metamorphosis as one eye migrates to the opposite side of the head. (A) Early pre-metamorphosis, 12 d.p.f.; (B) no eye migration or sustained lateral tilt; late pre-metamorphosis, 16 d.p.f.; no eye migration, but swims with sustained 3-6° right tilt; (C) pro-metamorphosis, 20 d.p.f.; start of right eye migration, swims with sustained 10-15° right tilt; (D) early metamorphic climax, 24 d.p.f.; right eye has migrated half-way to the dorsal mid-line, swims with a sustained 17-22° right tilt; (E) late metamorphic climax, 26 d.p.f.; right eye close to dorsal mid-line, swims with sustained 50-70° right tilt; (F) post-metamorphic juvenile, 30 d.p.f.; right eye has migrated to dorsal mid-line, swims with 80-90° right tilt; (G) settled sub-adult, 120 d.p.f.; right eye (r) is now on the left side of the head and adjacent to the left eye (l), which is now in a more ventral position than in stage I. Arrow in G denotes position of mouth. Bars, 0.2 mm (A-F) and 0.5 cm (G).

View larger version (69K): [in a new window]   Fig. 2. Remodeling of skull bone (Alizarin Red) and cartilage (Alcian Blue) during flounder metamorphosis. Skull symmetry first breaks when the cartilaginous right supraorbital bar degrades before the left in pro-metamorphosis (B,B'), allowing passage for the migrating right eye. Right (A-D) and left (A'-D') sides of larvae in different developmental stages. (A,A') Early pre-metamorphosis, left (green arrow) and right (red arrow) supraorbital bars are intact; (B,B') late pro-metamorphosis, right supraorbital bar is degrading (open red arrow) and frontal bones (orange arrowhead) begin to stain with Alizarin Red; (C,C') early metamorphic climax, further degradation of right supraorbital bar and Alizarin Red staining of frontal bones; (D,D') late metamorphic climax, left supraorbital bar has degraded (open green arrow); (E,E') post-metamorphic juvenile, pseudomesial bar (above broken line) is formed. Bars, 0.3 mm.

View larger version (80K): [in a new window]   Fig. 3. Staining for bone in vivo with calcein is more sensitive than traditional Alizarin Red histology. Note how the frontal bones (arrows), left parietal bone (p), dorsal fin rays (df), and much of the axial skeleton stain clearly with calcein, but remain unstained by traditional Alizarin Red. (A) Bone (Alizarin Red) and cartilage (Alcian Blue); (B) fluorescent calcein stain for bone. Both larvae are late pre-metamorphic (14 d.p.f.). Bars, 0.5 mm.

View larger version (125K): [in a new window]   Fig. 4. Remodeling of skull bone during flounder metamorphosis as visualized by calcein. The most dramatic changes that take place are asymmetric condensation and deformation of the frontal (left frontal bone, closed arrow) and parietal bones (left and right parietal bones, LP, RP) (A-F), and the formation of the pseudomesial bar (above broken line) and elongated right lateral ethmoid. (A-F) Frontal views; (A'-F') right saggital views; (A''-F'') left saggital views; (A,A'') early pre-metamorphosis, 12 d.p.f.; (B,B'') late pre-metamorphosis, 16 d.p.f.; (C,C'') pro-metamorphosis, 20 d.p.f.; (D,D'') early metamorphic climax, 22 d.p.f.; (E,E'') late metamorphic climax, 24 d.p.f.; (F,F'') post-metamorphic juvenile, 26 d.p.f.. Dorsal fin (open arrow), right lateral ethmoid (white arrowhead), anterior parietal barb (black arrowhead) pseudomesial bar (above broken line). Bars, 0.2 mm.

View larger version (67K): [in a new window]   Fig. 5. Head remodeling in dextral flounder produces morphological and behavioral mirror images of their sinistral siblings. Asymmetric frontal bone condensation (A) and deformation (B), lateral ethmoid elongation (C), supraorbital bar resorption (D), pseudomedial bar formation (E), and tilting (F) and settling (H) behaviors are all reversed compared with typical sinistral flounder development (G; also refer to Figs 2, 4). Pro-metamorphosis, frontal (A); early metamorphic climax, frontal (B), left saggital (C), right saggital (D); post-metamorphic juvenile, left saggital (E); pro-metamorphic larva with reversed tilt and eye migration (F); settled post-metamorphic juveniles with left-sided (G) and right-sided (H) morphology and behavior viewed from above. Calcein-stained bone (A-C,E); Alizarin Red (bone) and Alcian Blue (cartilage) (D). Right frontal bone (long white arrow), dorsal fin (short white arrow), left lateral ethmoid (white arrowhead), left anterior parietal barb (black arrowhead), right supraorbital bar (red arrow), left supraorbital bar (open green arrow), pseudomesial bar (above broken line), dorsal (d), ventral (v). Bars, 0.2 mm (A-F) and 1.0 cm (G,H).

View larger version (34K): [in a new window]   Fig. 6. A summary of changes in larval swimming and settling behaviors during spontaneous development, and after metamorphic induction with thyroid hormone (100 nmol l-1T3) or inhibition with methimazol (0.1 mol l-1) treatment.

View larger version (62K): [in a new window]   Fig. 7. Treatment of pre- and pro-metamorphic larvae with exogenous T3 induces lateralized behaviors and symmetric frontal bone development. Although untreated pre- (B) and pro-metamorphic (I) larvae initially have narrower right frontal bones compared with left, T3 treatment induces a bilaterally symmetric condensation of both frontals so they become similarly thick (D,K). T3 treatment of pre-metamorphic larvae induces settling (E) and tilted swimming (F,G) behaviors in the absence of eye migration. (A,B) Late pre-metamorphosis (14 d.p.f.), untreated; (C,D) late pre-metamorphosis after 100 nmol l-1 T3 72 h; (E) settled bilaterally symmetric larvae following T3 treatment (view from above); (F,G) swimming larva after T3 treatment (frontal view). Late pro-metamorphosis (20 d.p.f.), untreated (H,I); late pro-metamorphosis after 100 nmol l-1 T3 72 h (J,K). Cartilage (Alcian Blue) (A,C,H,J); calcein stain for bone (B,D,I,K). Right supraorbital bar (red arrow); frontal bones (white arrow); left and right parietal bones (LP, RP). Red structures in G denote relative position of the labyrinths. Bars, 0.2 mm.

View larger version (76K): [in a new window]   Fig. 8. Metamorphosis is inhibited by treatment with methimazol (a goiterogen), and resumes with subsequent thyroid hormone treatment. Inhibition of endogenous thyroid hormone production with methimazol (0.1 mol l-1) for 4 weeks inhibits eye migration, frontal bone deformation, supraorbital bar (SB) degradation, and pectoral fin remodeling, but not ossification or formation of right lateral ethmoid and parietal barb bones (A,B'). Subsequent T3 treatment (100 nmol l-1, 1 week) results in partial eye migration, SB degradation, and remodeling of pectoral fins to the adult form, but no further frontal bone deformation. (A,A',C,C') Bone (Alizarin Red) and cartilage (Alcian Blue) processed tissues; (B,B',D,D') calcein-stained bone. Boxed regions denote pectoral fin locations; for higher magnifications see Fig. 10. Intact and resorbed right SB (closed and open red arrows, respectively); frontal bones (white arrows); lateral ethmoid (white arrowheads); parietal barb (black arrowheads). Bars, 1.0 mm (A,C); 0.2 mm (A',B,B',C'D,D').

View larger version (61K): [in a new window]   Fig. 9. Symmetrically metamorphosed variants develop normal lateralized behavior. Despite having either no eye migration (right and left sides, A,A') or both eyes migrating (C,C'), both of these fish (each 60 d.p.f.) settle with their right sides on the bottom. Bone (Alizarin Red) and cartilage (Alcian Blue) stains of two variants, aged 63 d.p.f. (B,B') and 52 d.p.f. (D,D'). (E,F) Pro-metamorphic symmetrical variants (20 d.p.f.) with abnormal bilaterally symmetric frontal bone condensation. Frontal bone (orange arrowheads); overhanging dorsal fin ray (white arrow); resorbed right (open red arrows) and left (open green arrows) supraorbital bars; elongated right and left lateral ethmoid bones (white arrows in black arrowheads). Bars, 0.2 mm

View larger version (79K): [in a new window]   Fig. 10. Remodeling of the larval flounder pectoral fin during spontaneous metamorphosis and with altered thyroid status. Changes in fin morphology that normally occur from 20 to 30 d.p.f. (A-G) can be induced with exogenous thyroid hormone treatment from 7-11 d.p.f. (H-J), or inhibited by chemical ablation of thyroid hormone with methimazol (0.1 mol l-1) for 6 weeks after pre-metamorphosis (K). Thyroid hormone induction (100 nmol l-1 T3) for 1 week following 6 weeks of methimazol treatment transforms the enlarged larval pectoral fin into the juvenile form (L). A juvenile bilaterally symmetric variant (60 d.p.f.) has a normally remodeled pectoral fin (M). (A-M) Bone (Alizarin Red) and cartilage (Alcian Blue) processed tissues. Late pre-metamorphosis (A), late pro-metamorphosis (B), early metamorphic climax (C), mid-climax (D), late climax (E), juvenile (F), sub-adult (G). Untreated early pre-metamorphosis (H), 3 day T3-treated (I), 4 day T3-treated (J). fs, foramen scapulae; mdz, zone of matrix decomposition; pop, postcoracoid process; pr, proximal radial; prp, precoracoid process; sco, scapulocoracoid; mr, marginal rays. Bars, 0.2 mm (in A, for A-J), 0.2 mm (in K, for K-M).