Rose CS .

             metamorphosis
             cartilage development

	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 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 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 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.
	S1 Fig. Views of the branchial basket flow chambers and cartilage skeleton in a mature, NF 58 larva. (a and b) anterior and posterior views into an E-cadherin-labeled basket at the levels of the otic (inner ear) capsule (oc in a, lower arrow in c) and posterior edge of the eye (b, upper arrow in c). Long arrows in a-b indicate the epithelium-lined anterior and posterior filter surfaces of the first partition; short arrows indicate folds of dorsal epithelium; white arrows indicate mucus secreting columnar epithelium. (c) a ventral view of a dissected, alcian blue-stained basket showing the thickened ceratobranchials (cb1-4) that delimit the “gill” slits (gs, as Xenopus tadpoles atypically lack gill filaments, the term is a misnomer for this species). (d) a dorsal view of the region outlined in c showing the vertical arrays of ornate processes in the wall of the basket (long arrows) and the large ornate processes around gill slits (short arrows). (e) a close-up of the part of the partition outlined in d showing adjacent rods (arrows) lined with ornate processes that become smaller dorsally towards their tips. Scale bars for a-c and d-e are 2 and 1 mm respectively. The branchial basket arises in embryogeny from four ceratobranchials laterally and two hypobranchials medially. The cartilages fuse to each other and extend dorsally to form walls and two partitions that enclose three flow chambers. Water enters a flow chamber from the mouth cavity, passes through filter surfaces and exits ventrally via the slit between ceratobranchial bases. The walls of the partitions and inner walls of the basket are lined by thin epithelia that define complex filter surfaces (a-b). The filter surfaces are raised as a result of many, small, polygonally shaped, closely spaced ornate processes of cartilage (or “arboresecent growths” [109]) that lift the epithelium off the supporting cartilage (see also Fig 13A–13C). The ornate processes of arches 1 and 4 are arranged in vertical arrays along the inner walls of the branchial basket (long arrows in d). The vertically aligned ornate processes of arches 2 and 3 are supported by larger cartilages that branch off from each of many thin rods that extend in a line along each ceratobranchial base (arrows in e). The anterior and posterior branches of each rod contribute to the filter surfaces of the flow chambers anterior and posterior to it (Fig 13A–13C). We consider each vertical assemblage of ornate processes and its supporting rod or wall cartilage as a highly modified gill raker (see [108] for more specific terminology and [78] for SEM micrographs of the vertical arrays of ornate processes). The ornate processes decrease in size dorsally and the largest, near the ceratobranchial bases, form a meshwork above each gill slit (small arrows in d). The dorsal edges of walls and partitions are capped with a continuous tract of columnar, mucous secreting epithelium (white arrows in b, mse in Fig 13C) that collects food particles from the ornate processes and transports them to the esophagus [51].
	S3 Fig. Using CellProfiler to map the spatial distributions of chondrocyte size, shape, and orientation across entire sections of the NF 58/9 ceratohyal. This involved merging multiple 10 X phase photographs into a composite that captured an entire frontal or transverse section of the ceratohyal. (a) frontal and transverse sections for a left ceratohyal; the arrow shows the approximate level of the transverse section. Each composite was then used to create two images, an inverse, gray scale image of chondrocyte outlines (b) and one of white dots on a black background to indicate the locations of the cell nuclei within the chondrocytes (not shown). All cells had to be given a dot regardless of whether the nucleus was visible in the section. Any slide debris in the gray scale image was removed with Photoshop and any incomplete chondrocyte borders were closed to ensure that regions within the cartilage would be treated as separate cells. CellProfiler, which is free open source software [110], was used to process the two images to digitally outline all cells, and calculate the X, Y coordinates of their centers, the lengths of the long and short dimensions of best fitting ellipses, and the angles of the long dimensions. These data were then used to create a map with each cell represented by an ellipse that conveys its general size, shape, and orientation, and is colored to indicate its cell size-shape class (c). Any ellipse with an axis ratio greater than two is considered an artefact of having to treat all irregular spaces within the cartilage as potential cells. Yellow dots were added to show the location of BrdU-labelled nuclei. Only the ceratobranchial met the CellProfiler requirements that a large central portion could be reliably captured in a single section and that cell outlines are largely contiguous due to minimal matrix accumulation. The results from doing this on NF 47, 53 and 58/9 specimens agree with the quantitative and qualitative results already described.

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