Fig 1.
Ventral views of dissected, skeletally stained viscerocranial skeletons for NF 46–66+.
Blue is cartilage, red is bone; LJ, lower jaw (the small, separate cartilage near the midline in NF 46–62 is the infrarostral, the larger, more lateral one is Meckel’s cartilage); CH, ceratohyal; CB, the first ceratobranchial (the four collectively are the branchial basket): HY; adult hyale; the boxes show the approximate regions selected in frontal sections for quantifying cell features; scale bars are 5 mm and apply to all panels except the second one in which the NF 46 skeleton is expanded to a comparable size with the NF 50 skeleton; arrow indicates the inflection point in lower jaw curvature; NF 46–59 covers tadpole growth, NF 59–66 covers metamorphosis, and the NF 66–66+ series spans approximately 5 days after NF 66. The NF 66–66+ series shows three postmetamorphic changes: loss of LJ cartilage just anterior to the joint, expansion of the adjacent dermal bones, and changes in CH histology. Modified with permission from [42].
Fig 2.
Criteria to score mitosis and nuclear fragmentation.
(a) BrdU-pulsed chondrocytes at different stages of mitosis in phase micrographs. (b) DAPI-stained interphase nuclei, and nuclei showing signs of recent division and nuclear fragmentation in fluorescence micrographs. * indicates a nucleus that appears to have been fragmented by sectioning.
Fig 3.
Morphogenesis of the lower jaw (LJ), ceratohyal (CH) and ceratobranchial (CB) cartilages from NF 41 to 44.
(a and e) NF 41 and 43 frontal, resin-embedded, H&E stained sections. (b-d) NF 41+ transverse, resin-embedded, H&E stained sections. (f-h) NF 44 frontal sections with BrdU label, hematoxylin counterstain. (i-k) NF 44 frontal sections stained with alcian blue for cartilage and direct red for various collagens; IH, infrahyoideus muscle; OH, orbitohyoideus muscle; scale bar is 0.2 mm.
Fig 4.
Representative sections of lower jaw (LJ), ceratohyal (CH) and branchial arch (BA) cartilages showing chondrocyte size, shape, arrangement, and matrix at different stages.
(a-i, l-o) frontal, BrdU-labeled, hematoxylin or alcian blue-counterstained sections through middle portions of left LJ, CH and second ceratobranchial cartilages at NF 47, 54, 58, 64, and 67 (right is to the midline, up is anterior or lateral); arrows in g and l indicate large chondrocytes in the outer (more lateral) half of the cartilage. (j and k) transverse, resin-embedded, H&E-stained sections of branchial arch cartilages at NF 59. J shows an ornate process containing round chondrocytes of different sizes (arrows point to the smallest), capillaries, interstitial space, epithelia, and no perichondrium. K shows the transition from ceratobranchial base (lower right) to vertical rod (upper left), which is marked by a decrease in cell size, change from polygonal to more rounded cell outlines, and absence of perichondral cells (arrows) in the vertical rod. Scale bar is 0.1 mm.
Fig 5.
Percentages of BrdU-labeled chondrocytes for lower jaw, ceratohyal and branchial arch cartilages from NF 47 to 67.
Each stage was scored from 5–9 specimens using 10 X photos of 10 μm-thick sections through central portions of left and right regions at the approximate locations of the boxes in Fig 1 NF 56 and 62, total number of specimens is 155; error bars show standard errors in a and c-d, and minimum and maximum values in b; asterisks indicate a significant difference among cartilages for a stage in a and among regions within a cartilage in c and d; one asterisk means p <0.05, two means p<0.01, and three means p<0.001 using ANOVA in R.
Fig 6.
Percentages of lower jaw and ceratohyal chondrocytes progressing through mitosis in the lower jaw and ceratohyal at NF 55–6 and 58–9.
Each column represents data collected from phase photos of cartilage from a single specimen following a 2-hour pulse of BrdU; see Materials and Methods and Fig 2A for descriptions and images of the six stages of cell division.
Fig 7.
Maximum cell dimensions for lower jaw, ceratohyal and branchial arch cartilages at NF 47–67.
Each stage was scored from 40 X photos of the left and right cartilages of a single specimen using the 30 largest chondrocytes in each photo; error bars show standard deviations as standard errors are generally too small to be visible; asterisks indicate a significant difference for a stage among cartilages in a and among regions within a cartilage in b-d; one asterisk means p <0.05, two means p<0.01, and three means p<0.001 using ANOVA in R.
Fig 8.
Cell shape, orientation, percent matrix and nuclear fragmentation for the three cartilages at NF 47–67.
(a) ratio of cell long axis to short axis. (b) angle of cell long axis to cartilage long axis. (c) percent matrix. (d) percentage of chondrocytes showing signs of recent nuclear division and nuclear fragmentation. Data for a-c are from the same photos and cells used for Fig 7; error bars in a and b are standard errors; asterisks indicate a significant difference among cartilages for a stage; one asterisk means p <0.05, two means p<0.01, and three means p<0.001 using ANOVA in R. Data for d are from 63X DAPI fluorescence photos of a large central portion of each cartilage from 1–2 specimens at each stage; ovals indicate the two highest values; see Materials and Methods and Fig 2B for descriptions and images of nuclear fragmentation and recently divided nuclei.
Fig 9.
Frontal sections of the distal region of left lower jaws showing metamorphic changes in histology, shape and cell features.
Right is to the front of the head and up is to the lateral side. (a) a late larval specimen (NF 58) showing BrdU labeling in transversely aligned cell clusters inside the cartilage, and the jaw joint with the palatoquadrate (arrow), which lies in the plane of the long axis of the cartilage at larval stages. (b) a resin embedded, H&E stained section at NF 59 (the smaller cartilage is the palatoquadrate) showing matrix and clusters of round chondrocytes near the distal end (*) and a layer of flattened cells outside the distal end (arrow). (c-f) a series from NF 61 to 64+ specimens showing the emergence of rows of small chondrocytes aligned with the distal end (arrow in c). BrdU labeling (c, e and f) remains low in perichondral cells, the newly emerging chondrocytes, and the larger, more proximally situated chondrocytes of the original larval cartilage. (g) a NF 67 specimen showing light Alcian blue staining of matrix around the outermost rows of new chondrocytes, strong staining around the larger, more proximally positioned chondrocytes, and moderate staining around the innermost row of new chondrocytes (arrow), which are larger and more circular than in outer rows. (h) the head of a humerus from the same specimen as g showing a similar pattern of chondrocytes and matrix staining. Scale bar is 0.2 mm.
Fig 10.
Frontal sections of the proximal (a-d) and middle (e-f) regions of left lower jaws showing metamorphic changes in Meckel’s cartilage (Mc) and infrarostral (Ir). Right is to the midline of the head in a-d and to the proximal region in e-f. (a) a late larval (NF 58) specimen showing a small condensation (*) posterior to the joint, and flattened cells in the joint space. (b) a NF 63 specimen showing the condensation having fused with both cartilages, and more matrix around cells in the joint space. (c and d) a NF 65/6 specimen showing alcian blue stained matrix, the dentary bone (de) on the outer edge, the angulosplenial bone (as) on the inner edge, and thin columns of small, unchondrified cells (*) in the midline. The absence of matrix in the midline is consistent with this part being bent repeatedly in opposite directions when feeding and ventilating. (E and f) the middle part of a NF 64 left lower jaw at two locations, the inflection point that has just emerged in its outer edge (e, large arrow here and in Fig 1 NF 64), and just proximal to this point (f). E shows large chondrocytes (circle and ellipse) in the outer (more lateral) portion of the cartilage and cluster boundaries running obliquely to its central axis in the inner portion (smaller arrows). F shows a round cell cluster (circle) in the outer portion and more obliquely aligned cluster boundaries in the inner portion (arrows). Scale bars for a-d and e-f are 0.2 and 0.1 mm respectively.
Fig 11.
Sections through the proximal half of left ceratohyals showing metamorphic changes in cartilage histology, shape and cell features.
Left is to the outside of the head and up is anterior in a-c and dorsal in e. (a and b) frontal, BrdU-labeled, hematoxylin-counterstained sections at late larval (NF 58) and late metamorphic (NF 64) stages respectively (arrows in b indicate the edges of the cartilage). (c) a frontal, resin-embedded, H&E stained section through the same region as a and b soon after metamorphosis (NF 66+). (d) a close up of the rectangular region outlined in c. (e) a transverse, BrdU-labeled, hematoxylin-counterstained section through the left lower jaw (LJ) and ceratohyal (CH) at NF 66. The transition from NF 58 to 64 (a to b) is marked by narrowing of the cartilage and most large chondrocytes acquiring more daughter nuclei and cells within their original perimeters. The transition from NF 64 to 66+ (b to c) is marked by the emergence of large, largely empty cell lacunae (*) that are interspersed across the width of the cartilage with discrete, equantly shaped cell clusters, and smaller lacunae that appear to contain cellular debris (arrows). While some lacunae resemble cluster outlines in b, others are transversely elongated with oval or more irregular shapes that generally conform to the curvature of adjacent cell clusters. The cell clusters contain many, small chondrocytes that have spherical nuclei and are separated by more matrix than the chondrocytes at NF 58. E shows high BrdU labeling in the ceratohyal, but not the lower jaw, at NF 66. Scale bars for a-d and e are 0.1 and 0.5 mm respectively.
Fig 12.
Frontal sections through left ceratohyals showing distributions of cell division and death labels.
Anterior is up for a-d, and to the right for e-f. (a) BrdU labeled chondrocytes and alcian blue staining of matrix persist across the width of the cartilage to late metamorphosis (NF 65). (b) a phase micrograph at NF 63 showing the locations of DAPI-stained chondrocyte nuclei that appear to have recently divided (green), have undergone nuclear fragmentation (red), and be in interphase (yellow); see Fig 2B for scoring criteria. Recently divided nuclei are interspersed with ones exhibiting nuclear fragmentation. (c) capase labeling of a few peripheral chondrocytes in the distal tip at NF 63. (d-f) PCNA labeling of chondrocytes is strongest in the cartilage center at NF 62/3 (d) and 65 (e-f); arrow in d indicates the medial edge of the cartilage; scale bars for a, b and c-f are all 0.2 mm.
Fig 13.
Transverse sections through left branchial arch skeletons showing metamorphic changes in cartilage histology, shape and cell features.
Dorsal is up, medial is to the right. (a) Alcian-blue stained second and third partitions at a late larval stage (NF 58) showing ceratobranchial bases (cb2 and 3), the intervening gill slit (gs), and the vertical rods (r) that partition the space into flow chambers. The rods have anterior and posterior extensions that support filter surfaces comprised of ornate processes (op). Dorsal folds of pharyngeal epithelium (*) hang down into each flow chamber to force water to flow through the filter surfaces (arrows), into the spaces between ornate processes and rods, and out the gill slit. (b) a close-up of the rectangular region outlined in a, showing a rod, ornate processes and blood vessels (bv) within the rod. (c) brown E-cadherin staining of the squamous epithelium that lines rods and ornate processes at NF 59, and captures food particles. The columnar, mucous secreting epithelium (mse) that caps the inner walls and partitions transports the food particles to the esophagus. (d and e) progressive collapse of the dorsal portions of the partitions in early metamorphosis (NF 61 and 62), and the peripheral matrix in ceratobranchial bases ceasing to stain blue for chondroitin sulphate. (f) BrdU labeling of epithelial cells, chondrocytes, and perichondral cells in ventral portions at a late larval stage (NF 59). (g) BrdU-labeled chondrocytes at a mid-metamorphic stage (NF 62). (h and close up i) TUNEL labeling of epithelial cells, but not chondrocytes at an early metamorphic stage (NF 60). (j) caspase-3 labeling of epithelial cells, and less frequently and more faintly of chondrocytes at NF 62. Scale bars are 1 mm for a, 0.5 mm for b-e and h, and 0.2 mm for f-g and i-j.
Fig 14.
Effect of T3 on cartilage shape and cell behaviors in a NF 46 lower jaw.
(a) the Meckel’s cartilage (MC) and infrarostral cartilage (IR) of a day 3, T3-treated specimen (upper) and a day 0, control specimen (lower) that were dissected from alcian blue-stained whole mounts of NF 46 specimens immersed in 50 nM T3. (b) ventral views of intact whole mounts treated for 1–4 days. (c) the cartilages after dissection. (d and e) frontal sections of BrdU-labeled lower jaws of day 3 control and day 3 T3-treated specimens respectively. Scale bars for b, c and d-e are 2, 1, and 0.5 mm respectively.