Post-embryonic remodelling of neurocranial elements: a comparative study of normal versus abnormal eye migration in a flatfish, the Atlantic halibut

Abstract

The process of eye migration in bilaterally symmetrical flatfish larvae starts with asymmetrical growth of the dorsomedial parts of the ethmoid plate together with the frontal bones, structures initially found in a symmetrical position between the eyes. The movement of these structures in the future ocular direction exerts a stretch on the fibroblasts in the connective tissue found between the moving structures and the eye that is to migrate. Secondarily, a dense cell population of fibroblasts ventral to the eye starts to proliferate, possibly cued by the pulling forces exerted by the eye. The increased growth ventral to the eye pushes the eye dorsally. Osteoblasts are deposited in the dense cell layer, forming the dermal part of the lateral ethmoid, and at full eye migration this will cover the area vacated by the migrated eye. When the migrating eye catches up with the previous migrated dermal bones, the frontals, these bones will be remodelled to accommodate the eye. Our findings suggest that a combination of extremely localized signals and more distant factors may impinge upon the outcome of the tissue remodelling. Early normal asymmetry of signalling factors may cascade on a series of events.

Fig. 1

Stage 7, symmetrical larvae. (A) Dorsal view of head double stained for bone (alizarin red) and cartilage (alcian blue). Calcified bone is red and cartilage is blue. (B) Cross-section anterior to eyes (hyaline ethmoid cartilage: pink). (C) Cross-section anterior region of eyes. B and C show sections 5 µm thick, stained with toluidine blue. Scale bar = 100 µm.

Fig. 2

Stage 8, eye index 0. (A) Dorsal view of head double stained for bone (alizarin red) and cartilage (alcian blue). Calcified bone is red and cartilage is blue. (B) Cross-section anterior to eyes (hyaline ethmoid cartilage: pink). (C) Cross-section anterior region of eyes. B and C show sections 5 µm thick, stained with toluidine blue. Scale bar = 100 µm.

Fig. 3

Early stage 9, eye index 2. (A) Dorsal view of head double stained for bone (alizarin red) and cartilage (alcian blue). Calcified bone is red and cartilage is blue. (B) Cross-section anterior to eyes (hyaline ethmoid cartilage: pink). (C) Cross-section anterior region of eyes. B and C show sections 5 µm thick, stained with toluidine blue. Scale bar = 100 µm.

Fig. 4

Late stage 9, eye index 4. (A) Dorsal view of head double stained for bone (alizarin red) and cartilage (alcian blue). Calcified bone is red and cartilage is blue. (B) Cross-section anterior to eyes (hyaline ethmoid cartilage: pink). (C) Cross-section anterior region of eyes. B and C show sections 5 µm thick, stained with toluidine blue. Scale bar = 100 µm.

Fig. 5

Cross-sections of the posterior border of the eyes. (A) Stage 7, eye index 0; (B) stage 7, eye index 0; (C) stage 8, eye index 1; (D) stage 9, eye index 2; (E) stage 9, eye index 3. Black arrows show the zone of proliferating cells on the abocular side of the dorsomedial part of the ethmoid cartilage. White part of arrow indicates inactive part of cartilage with no cell proliferation. Black arrowhead in A and B shows the ocular frontal process that grows parallel to the cartilage situated dorsally to it. Scale bar = 100 µm.

Fig. 6

Cross-sections of the anterior region of the eyes. Stage 9 (myotome height 7.2 mm), eye index 2. Low-power micrograph: scale bar = 500 µm; higher-power micrographs A–E: scale bar = 1 µm. Growth of the dorsomedial part of the ethmoid cartilage moving towards the ocular-side eye (arrow 1). Eye moves closer to the retroorbital vesicle (black triangle), which has a laterally compressed shape. At the same time the collagen fibre-rich fibroblastic mucochondroid is stretched (arrow 2). The dense subdermal fibroblastic layer proliferates, and grows towards the dorsal margin (arrow 3). Asterisk, collagen fibre-rich fibroblastic mucochondroid; #, glycoprotein-rich fibroblastic mucochondroid.

Fig. 7

Mean ± SE TRAP activity in stage 8 normal, stage 9 and juvenile (Juv.) normal (N) and abnormal (A) larvae. Black column = ocular frontal process, white column = abocular frontal process (n = 24). TRAP activity is expressed as stained area as measured by stereology. Different letters above columns denote significant differences (P < 0.05); lower-case letters indicate differences within normal (a–c) or abnormal (i–j) fish. Upper-case letters indicate differences between normal and abnormal juveniles.

Fig. 8

Mean ± SE area of TRAP activity as measured by stereology in stage 9 normal (left) and abnormal larvae (right). Black column = ocular-side frontal process, white column = abocular-side frontal process (n = 9).

Fig. 9

Mean ± SE area of TRAP activity as measured by stereology in juvenile normal (left) and abnormal fish (right). Black column = ocular-side frontal process, white column = abocular-side frontal process (n = 10).