Conventional µCT only visualizes the zebrafish mineralized skeleton, while DICE-PPC-SRµCT adds soft-tissue information. (A-C) Volume renderings of 1-3mpf zebrafish scanned at a voxel size of 5.43µm using conventional µCT. (D-F) Volume renderings of 1-3mpf zebrafish stained with iodine and scanned at 0.727µm voxel size (D) and 3µm (E, F) using PPC-SRµCT. (A?-F?) Sagittal virtual thin sections through the midline. (A??-F??) Transverse virtual thin sections through the head (immediately posterior to the eyes). Scale bars, 500µm (A-A??, D-D??) and 1mm (B-B??, C-C??, E-E??, F-F??).

DICE-PPC-SRµCT at submicron resolution provides a detailed histological information. All images represent virtual thin sections from a 1mpf zebrafish scanned at 0.727µm voxel size. (A) Transverse view of the brain and optic nerves. (B) Longitudinal section through the spinal cord. (C) Transverse view of the eye, with the optic nerve and retinal layers visible. (D) Sagittal view of the crista ampullaris in the lateral semicircular canal. (E) Sagittal view of the heart. (F) Longitudinal view of the head region of the kidney. (G) Longitudinal view of the ovary. (H, I) Transverse and sagittal views of the pharyngeal teeth, with dental pulp visible. (J) Sagittal view of the two approximately perpendicular layers of hypaxial trunk musculature. (K) Sagittal view through the neurocranium (top) and hyomandibula (bottom), with visible chondrocyte lacunae and endochondral ossification of the hyomandibula. (L) Sagittal view through the dentary bone, using minimal projection with adjusted contrast to reveal osteocyte lacunae features in the bone. Inset image displays the same view but with comparable visualization settings to previous soft-tissue panels. (M) Sagittal section through the lapillus otolith, again with adjusted contrast to reveal the otolith microstructure showing growth rings. Inset image displays the same view but with comparable visualization settings to previous soft-tissue panels. (N) Section through the pectoral fin cartilaginous endoskeleton. ad, adipocytes; b, brain; ba, bulbus arteriosus; bc, body cavity; cu, cupula; dp, dental pulp; dr, distal radials; e, eye; em, epaxial muscle; gcl, ganglion cell layer; hme, hypaxial muscle (external); hmi, hypaxial muscle (internal); inl, inner nuclear layer; ipl, inner plexiform layer; lac, chondrocyte lacunae; na, neural arches; num, cell nuclei; oc, osteocytes; on, optic nerves; onl, outer nuclear layer; opl, outer plexiform layer; ot , optic tectum; pcl, photoreceptor cell layer; pr, proximal radials; r, rib; rbc, red blood cells; rpe, retinal pigmented epithelium; sv, sinus venosus. Scale bars, 100µm.

nkx3.2 ^-/- zebrafish display the distinctive ?open mouth? phenotype. Anterolateral and anterior volume renderings of DICE-PPC-SRµCT scanned nkx3.2 ^-/- zebrafish at 1mpf (left) and 2mpf (right). Scale bars, 1mm.

nkx3.2 ^-/- zebrafish display defects in lower jaw-associated musculature. (A) Lateral volume rendering of the wild-type zebrafish head skeleton and relevant associated muscles. (A?) Close up of the rendered adductor mandibulae muscle bundles A1-A3. (A??) False-coloured transverse virtual thin section [position indicated by dotted line in (A)] highlighting the protractor hyoideus muscle and distinct bundles of the adductor mandibulae. (B?D) Renderings of the isolated A1, A2, and A3 bundles of the adductor mandibulae, respectively. (E, F) The left and right lateral views of a single nkx3.2 ^-/- zebrafish, with the panels in (F-F??) reversed to facilitate comparison. Arrows in (B, E?, F?) indicate the maxillary tendon of A1. Asterisk in (E?) indicates the unusual anterior attachment site of the A1 bundle to the lower jaw. Arrowheads in (E?) and (F?) indicate the abnormal organization of muscle bundles. (G, H) Dorsal view of the lower jaw skeleton and associated musculature in wild-type and nkx3.2 ^-/- fish, with the basihyal rendered transparent. In (G), the maxilla and premaxilla were also rendered as partially transparent, and the right adductor mandibulae was removed to reveal the quadrate below. In (H), arrowhead indicates protractor hyoideus muscle fibers crossing the midline. Asterisk indicates ectopic ossifications embedded in the right adductor mandibulae. Both zebrafish are 2mpf and were scanned with a voxel size of 3µm using DICE-PPC-SRµCT. aa, anguloarticular; bh, basihyal; ch, ceratohyal; d, dentary; e, eye; en, endopterygoid; hm, hyomandibula; IMA, intermandibularis anterior; m, maxilla; mc, Meckel?s cartilage; mpt, metapterygoid; PH, protractor hyoideus; pm, premaxilla; po, preopercle; q, quadrate. All scale bars, 500µm, except on virtual thin sections (A??, E??, and F??) 300µm.

Late larval nkx3.2 ^-/- zebrafish displays deformed and missing ligaments in the Weberian apparatus. (A, D) Lateral renderings of the occipital bones, Weberian apparatus, and anterior rib-bearing vertebrae (colored as bone) with associated cartilage (blue), ligaments (shades of purple), and swim bladder (yellow) of 1mpf wild-type and nkx3.2 ^-/- zebrafish. Red asterisks in (D) indicate the absence of the anterior interossicular and suspensor ligaments. (B, E) False-coloured sagittal virtual thin sections through the interossicular ligament in wild-type and nkx3.2 ^-/- zebrafish. Red asterisk in (E) indicates the absence of a ligamentous connection between the intercalarium and scaphium, as what may be anterior part of the interossicular ligament appears to be connected to lateral process instead. (C, F) False-coloured virtual thin sections through the suspensor ligament in wild-type and nkx3.2 ^-/- zebrafish. The red asterisk in (F) indicates the absence of the suspensor ligament between the tripus and parapophysis 4. boc, basioccipital; cl, claustrum; eoc, exoccipital; ic, intercalarium; IOSa, anterior interossicular ligament; IOSp, posterior interossicular ligament; lp2, lateral process 2; n, neural complex cartilage; oss, os suspensorium; pop4, parapophysis 4; r4, rib 4; sb, swim bladder; sc, scaphium; SUS, suspensor ligament; t, tripus; V5, vertebrae 5. Scale bars, 100µm.

Juvenile nkx3.2 ^-/- zebrafish displays missing ligaments in the Weberian apparatus. (A, D) Lateral renderings of the occipital bones, Weberian apparatus, and anterior rib-bearing vertebrae with associated cartilage (blue), ligaments (shades of purple), and swim bladder (yellow) of wild-type and nkx3.2 ^-/- juvenile zebrafish. Red asterisks in (D) indicate the absence of the interossicular and suspensor ligaments. (B, E) False-coloured sagittal virtual thin sections through the interossicular ligament in wild-type and nkx3.2 ^-/- zebrafish. Red asterisks in (E) indicate the absence of the interossicular ligament. (C, F) False-coloured virtual thin sections through the suspensor ligament in wild-type and nkx3.2 ^-/- zebrafish. The red asterisk in (F) indicates the absence of the suspensor ligament between the tripus and parapophysis 4. boc, basioccipital; cl, claustrum; eoc, exoccipital; ic, intercalarium; IOSa, anterior interossicular ligament; IOSp, posterior interossicular ligament; lp2, lateral process 2; n, neural complex cartilage; oss, os suspensorium; pop4, parapophysis 4; r4, rib 4; sb, swim bladder; sc, scaphium; SUS, suspensor ligament; t, tripus; V5, vertebrae 5. Scale bars, 300µm.

Late larval nkx3.2 ^-/- zebrafish displays severe reduction of the sinus perilymphaticus. (A, F) Lateral rendering of the sinus endolymphaticus (green) and sinus perilymphaticus (magenta) in the context of the occiput and Weberian apparatus (white, transparent) in 1mpf wild-type and nkx3.2 ^-/- zebrafish. (A?, F?) Anterodorsal renderings of the sinus endolymphaticus and sinus perilymphaticus (anterior to the left). False coloured transverse (B, G), longitudinal (C, H), and sagittal (D, I) virtual thin sections through the middle of the sinus endolymphaticus. Red asterisk in (I) indicates the blind posterior termination of the sinus impar. (E, J) False coloured transverse virtual thin sections through the atria of the sinus perilymphaticus. Red asterisks in (J) indicate the absence of the atria of the sinus perilymphaticus. Virtual thin section positions are indicated by labelled dotted lines in (A, A?). a, atrium; ast, asteriscus; cl, claustrum; csi, cavum sinus impar; dsu, ductus sacculo-utricularis; lag, lagena; sac, saccule; sc, scaphium; se, sinus endolymphaticus; sgt, sagitta; sp, sinus perilymphaticus; tc, transverse canal. Scale bars, 100µm.

Juvenile nkx3.2 ^-/- zebrafish displays complete absence of the sinus perilymphaticus. (A, F) Lateral rendering of the sinus endolymphaticus (green) and sinus perilymphaticus (magenta) in the context of the occiput and Weberian apparatus (white, transparent) in 2mpf wild-type and nkx3.2 ^-/- zebrafish. (A?, F?) Anterodorsal renderings of the sinus endolymphaticus and sinus perilymphaticus (anterior to the left). (B-D, G?I) False coloured transverse, longitudinal, and sagittal virtual thin sections through the middle of the sinus endolymphaticus. Red asterisk in (I) indicates the blind posterior termination of the sinus impar. (E, J) False coloured transverse virtual thin sections through the atria of the sinus perilymphaticus. Red asterisks in (J) indicate the absence of the atria of the sinus perilymphaticus. Virtual thin section positions are indicated by labelled dotted lines in (A, A?). a, atrium; ast, asteriscus; cl, claustrum; dsu, ductus sacculo-utricularis; lag, lagena; sac, saccule; sc, scaphium; se, sinus endolymphaticus; sgt, sagitta; sp, sinus perilymphaticus; tc, transverse canal. Scale bars, 300µm.

Volume measurements of 3D segmented soft tissues. Volume measurements of muscles (A), Weberian ligaments (B), and inner ear sacs (C). Circles indicate wild-type tissues, while triangles indicate nkx3.2 ^-/- tissues. Each point represents a single tissue volume measurement. Dotted lines connect corresponding tissues from 1mpf and 2mpf zebrafish of the same genotype to approximate growth trajectories. Different tissues are indicated by individual colors and line styles in agreement with the color codes used in the previous figures. Y-axes are log- or pseudolog-scaled. X-axes display standard length (mm). AM, adductor mandibulae; PH, protractor hyoideus; IMA, intermandibularis anterior; IOS, interossicular; SUS, suspensor; se, sinus endolymphaticus; sp, sinus perilymphaticus.