FIGURE SUMMARY
Title

In vivo zebrafish morphogenesis shows Cyp26b1 promotes tendon condensation and musculoskeletal patterning in the embryonic jaw

Authors
McGurk, P.D., Swartz, M.E., Chen, J.W., Galloway, J.L., Eberhart, J.K.
Source
Full text @ PLoS Genet.

Homozygous carriers of b1024 display second pharyngeal arch lower jaw muscle defects. (A) Schematic of connective tissues in the larval zebrafish jaw, modified from Chen and Galloway, 2014. (B, C) Ventral craniofacial muscles in wild-type and b1024 embryos. (C) Muscle fibers from an intermandibularis posterior muscle extend past the mandibulohyoid junction and insert alongside the adjacent interhyal muscle or at the hyohyal junction (arrows). Muscle fibers apparently from the sternohyoideus are present in the ventral midline of mutants (arrowhead). Ectopic muscle fibers split off from the intermandibularis anterior muscle in a small percentage of mutants (asterisk). (D) 4x digital zoom of a single Z slice from confocal image in C. Arrows show ectopic paths of intermandibularis posterior muscle fibers. Arrowheads show ectopic midline attachments of interhyal muscle fibers. A three-way junction (red arrowhead in D) forms between ectopic intermandibularis posterior, interhyal, and hyohyal (asterisk in D) muscle fibers. All images ventral view, anterior to the left. Scale bars = 50 μm

The b1024 lesion is a nonsense mutation in cyp26b1 that causes skeletal defects. (A-B) Wild-type head skeleton stained with Alcian Blue and Alizarin Red dyes. (C) In b1024 mutants the ethmoid plate (brackets in A,C) and parasphenoid bone are narrow and the parachordal (arrows in A,C), basicapsular commissure (arrowheads in A,C), and lateral commissure (asterisks in A,C) cartilages of the posterior neurocranium are hypoplastic. (D) The midline symphysis of Meckel’s (red arrowheads and red insets in B,D) and ceratohyal (black arrowheads and black insets in B,D) cartilages are absent in b1024 homozygotes, fusing the left and right cartilage elements, and the basihyal cartilage (arrows in B,D) is hypoplastic. (E-F) A single base substitution in cyp26b1 (arrowhead) results in an in-frame stop codon (box). (G) b1024 causes a later truncation of cyp26b1 than previously characterized alleles, but still eliminates essential components of the cytochrome P450 enzyme domain encoded in exon 6. All images ventral view, anterior to the left. Scale bars = 50 μm

Loss of Cyp26b1 function disrupts morphogenesis of the mandibulohyoid junction. (A-J) Stills from S1 Movie. (A) All first and second pharyngeal arch muscles are present at 53 hpf. (A’) Interhyal muscles (ih) extend toward the second pharyngeal arch midline while the intermandibularis posterior muscles (imp) extend from the first arch to the anterior edge of the second arch. (B,B’) Around 55 hpf, the tips of intermandibularis posterior and interhyal muscles on each side of the head connect (arrows). (C,C’) Before 60 hpf, the left and right sides meet in the midline, connecting the four muscles of the mandibulohyoid junction (arrowhead). (C’) Hyohyal muscles (hh) extend toward the second pharyngeal arch midline while the tips of sternohyoideus muscles pass just dorsally (arrowheads). (D,D’) Around 63 hpf, the hyohyal muscles meet end-to-end in the midline (arrowhead). (E,E’) At 72 hpf, now broader muscles sit end-to-end at the mandibulohyoid and hyohyal junctions. In cyp26b1 mutants, the timing of jaw muscle differentiation is normal (F,F’), but morphogenetic events are altered. (G,G’) Elongation toward the midline by intermandibularis posterior and interhyal muscles is slower (compare arrows to those in B’). (H,H’) The intermandibularis posterior and interhyal muscles connect poorly across the midline (distance between arrows = 46.5 μm, compare between arrows in C’ = 38.2 μm). (I,I’J,J’) Muscle fibers extend into the space bounded by the interhyal and hyohyal muscles (arrowheads). Both aberrant behaviors have phenotypic readouts at 72 hpf. (K) Where the muscles are narrowest at the junction between intermandibularis posterior and interhyal muscles (see arrows in C’,H’), the mandibulohyoid junction is significantly wider in mutants than in wild-type siblings. (L) There is also a significantly larger surface area in which the ends of intermandibularis posterior and interhyal muscles extend ectopically. All images ventral view, anterior to the left. Scale bar = 50 μm.

Cyp26b1 functions in the neural crest to promote craniofacial musculoskeletal patterning. (A) Schematic for transplantation of embryonic cells at 6 hpf. The blue area shown on the right side of A will contribute to the neural crest on one side of the head (B, C). (D-E) Donated neural crest cells improve skeletal phenotypes in a cyp26b1 mutant host. We saw a wider palate with a flared ethmoid plate in mutant hosts (arrow in D). (E) Depletion of Alcian-positive cartilage matrix indicates partial rescue of Meckel’s cartilage fusion (red arrowhead/red inset) and ceratohyal morphogenesis and fusion at the midline is rescued on the donor side (black arrowhead/black inset). (F) On the side of the head without donated neural crest, muscles project ectopically (arrows) and muscle fiber bundles split (arrowhead), compared to normal phenotypes on the donor side. Lateral view in B. All other images ventral view, anterior to the left. Scale bars = 50 μm.

Loss of Cyp26b1 function disrupts cranial tendon differentiation. (A-B) Tsp4b (green) is enriched at the muscle attachment sites in wild-type zebrafish at 4 and 5 dpf. (C) At 4 dpf, cyp26b1 mutants display Tsp4b at jaw muscle attachments, though weakly at the mandibulohyoid junction (arrowheads in A’,C’) and sternohyoideus tendons (arrows in A’,C’). (D) At 5 dpf, punctate deposits of Tsp4b can be seen at all ectopic points of jaw muscle attachment. All images ventral view, anterior to the left. Scale bar = 50 μm.

Patterning of scxa-expressing head tendon progenitors is disrupted in cyp26b1 mutants. (A) At 60 hpf, bright puncta of condensing scxa-positive tenoblasts form in a line between the intermandibularis posterior and interhyal muscles (arrow), and other tenoblasts populate a mass of midline cells from the oral ectoderm to the mandibulohyoid junction. (A’) In the second pharyngeal arch, the ends of the hyohyal (arrowheads) and sternohyoideus (arrows) muscles are labeled by scxa-positive condensations. (B) In cyp26b1 mutants, the pattern of scxa expression suggests less condensation at the mandibulohyoid junction (arrow) and less separation of anterior and posterior tenoblasts. (C) At 4 dpf, the mandibulohyoid (arrow) and hyohyal (arrowhead) junctions express scxa strongly, and most muscle attachment points are scxa-positive. Notably, two spurs extend from the hyohyal junction (C’, outlines) and insert near the anterior edge of each interhyal muscle (E’, outlines). (E,E’) The sternohyoideus tendons insert in the midline, just dorsal to the hyohyal junction structures. (D) Four-day-old mutants display some scxa expression at most muscle attachment points, but expression associated with the intermandibularis muscles (arrow) and the hyohyal junction (arrowhead) is reduced. (D’) The spurs of the hyohyal junction are highly dysmorphic. (F’) The sternohyoideus tendons are rod-like but hypoplastic (arrows). (G,I) Cells expressing xirp2a are present at the tips of all cranial muscles at 60 hpf and 4 dpf. (H,J) Cells at muscle tips are also labeled with xirp2a in cyp26b1 mutants. All images ventral view, anterior to the left. Scale bars = 50 μm

Loss of Cyp26b1 function disrupts tendon morphogenesis in the second pharyngeal arch midline. (A-L) Stills from S2 Movie. (A,A’) The emerging intermandibularis muscles (arrows) are surrounded by tenoblasts at 51 hpf (B,B’). As tenoblasts converge on the midline, intermandibularis muscles point their posterior tips medially (also see S6B Fig). Meanwhile, tenoblast masses (arrowheads) associated with the hyohyal and sternohyoideus muscles on each side have become visible. (C,C’) By 57 hpf, condensing cells are visible at the mandibulohyoid junction (outline), where scxa-positive cells have segregated from the more anterior group. (D,D’) By 60 hpf, condensation of the sternohyoideus tendons is apparent (arrows). (E,E’) By 63 hpf, tenoblasts attached to each hyohyal muscle connect in the midline and a condensation forms (arrowhead). (F,F’) Approaching 3 dpf, tenoblasts at the mandibulohyoid (arrow) and hyohyal (white arrowhead) junctions are highly condensed between the muscle tips, and sternohyoideus tendons are elongated (open arrowheads). (G,G”) In morpholino-injected embryos, intermandibularis muscles (arrows) emerge normally among a field of tenoblasts around 51 hpf. (H,H’) Tenoblasts migrate slowly toward the midline, and intermandibularis posterior muscles point posteriorly at 54 hpf (also see S6D Fig). (I,I’) Tenoblasts at the mandibulohyoid junction are neither condensing nor separating from adjacent tenoblasts at 57 hpf (outline). (J,J’) Despite surrounding abnormalities, sternohyoideus tendons initiate condensation around 60 hpf (arrows). (K,K’) Connections between intermandibularis posterior muscles and sternohyoideus tendon condensations persist, and at 63 hpf the muscle tips (red arrows) now extend past mandibulohyoid tenoblasts. (L) Compared to controls, the sternohyoideus tendons are poorly condensed and elongated by 70 hpf, and both intermandibularis posterior and both hyohyal muscles connect to either end of these tendon rudiments. (L’) Mesenchymal tenoblasts sit between the intermandibularis posterior muscles (arrow) and no condensation has formed connecting the hyohyal muscles (red arrowhead). All images ventral view, anterior to the left. Scale bar = 50 μm

Cyp26b1 function before 60 hpf is necessary for mandibulohyoid junction formation. (A,D) Embryos raised in media containing 0.01% DMSO develop normally. (B) 100% of embryos treated with talarozole between 54 and 60 hpf displayed ectopic muscle projection in the second pharyngeal arch, and the intermandibularis posterior muscles overextended in 58.3% of embryos (n = 64). (C) Among embryos treated from 60 to 72 hpf, 76.9% had mild ectopic muscle projections, but 96.2% developed normal tendon and end-to-end muscle connections at the mandibulohyoid junction (n = 26). (E,F) Talarozole-treated embryos recapitulated cyp26b1 mutant skeletal phenotypes of Meckel’s cartilage fusion (red arrowheads and insets), ceratohyal fusion (black arrowheads and insets), and basihyal reduction (arrows). All images ventral view, anterior to the left. Scale bar = 50 μm

Cells expressing cyp26b1 separate anterior and posterior tenoblast masses between 54 and 60 hpf. (A-D) 54 hpf zebrafish embryo. (B) Tenoblasts of the future mandibulohyoid junction (arrow) display a flattened posterior boundary and maintain a small distance from tenoblasts lining the posterior edge of the second pharyngeal arch (arrowheads). (C) With the shape of the second arch visible (outline) one can see that cells expressing cyp26b1 line the posterior edge of the arch midline and display a flattened anterior boundary abutting the mandibulohyoid tenoblasts (arrow). (D) With channels combined, cyp26b1 expression (blue, in situ hybridization) labels non-tenoblast cells separating tenoblasts (green, anti-mCherry antibody in scxa:mCherry transgenic) and second arch muscles (red, MF20) that are mostly or entirely cyp26b1-negative. (E-H) 60 hpf zebrafish embryo. (F) Tenoblasts are more compact at the forming mandibulohyoid junction (arrow), and further separated from the sternohyoideus tendon condensations (arrowhead). (G) Strong cyp26b1 expression now labels bilateral cell masses of the posterior edge of the second arch (arrows). (H) Non-tenoblast cells expressing cyp26b1 sit anterior to the sternohyoideus tendon condensations on each side, facing the nearby interhyal muscles. All images ventral view, anterior to the left. Scale bar = 50 μm.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ PLoS Genet.