Fig. 2

Fig. 2 Loss of PRDM16 causes craniofacial defects in zebrafish and mice. (A–E) Wildtype and prdm16−/− zebrafish embryos were collected at 6 dpf and stained for Alcian blue and Alizarin red. (A) Images of dissected viscerocranium and neurocranium of wildtype (wt) or prdm16−/− embryos. Prdm16−/− embryos and their cartilage phenotypes were present in Mendalian ratios (B). (C–F) Quantification of cartilage elements, Meckel’s cartilage to palatoquadrate (C), distance between trabeculae (D), length of parasphenoid tissue (E), and area of mineralized parasphenoid (F). ceratobranchial arches (cbs), ceratohyal (ch), Meckel’s cartilage (m), palatoquadrate (pq), parasphenoid (ps), trabeculae (tr), (n ​= ​6 for each group measured). Scale bar, 100 ​μm. (G–H) Ventral views of in situ hybridization for col2a1 (G) and dorsal views of in situ hybridization for runx2b (H) in prdm16 mutants or wildtype controls at 48 hpf. eye (e), ceratobranchial arch 5 (cb5), cleithrum (cl), opercle (op). Black arrow heads denote areas of reduced expression of these markers in the developing cartilaginous structures. Scale bar, 250 ​μm. (I–O) Prdm16fl/fl females were bred for timed matings to Prdm16Δ/+;Sox2+/Tg males and embryos were collected at the indicated timepoints. (I) Lateral images of the head show snout defects (white arrowhead) and hypoplasia of the mandible (white line) in Prdm16 mutant animals at E18.5 (top). Dissected palates reveal clefting in a subset of Prdm16 mutants (black arrowheads). (J–M) Alcian blue and Alizarin red stained whole mounts of control and mutants at E18.5. (J) Lateral views of the cranial skeleton. (J’) Ventral views of the cranial skeleton with the mandible removed with higher magnification images of the palantine bone shown below (black star indicates clefting in mutant). Scale bars, 1 ​mm. (K) High magnification lateral views of the middle ear and squamosal bone structures. (L) Middle ear ossicles were dissected and removed from control or Prdm16 mutant mice to reveal severe hypoplasia of the malleus, incus and tympanic ring in mutant animals. Scale bar, 0.5 ​mm. (M-M″) Mandibles were dissected and removed from control or mutant animals. (M’) Lateral views of the right half of the mandible. (M″) Quantification of anterior mandible hypoplasia by measuring the ratio between the anterior and posterior portions of the mandible (P:A). (N) Safranin O and Fast Green stained coronal sections through the anterior-posterior axis of the developing palate at E15.5 in control or mutant mice. Scale bars, 250 ​μm. (O) Dissected and removed laryngeal cartilages from control and Prdm16 mutant embryos at E17.5 Abbreviations: angular process (ap), basisphenoid (bs), condylar process (cd), coronoid process (cp), cricoid cartilage (cc), gonial (gn), hyoid bone (hb), incus (i), lamina obturans (lo), malleus (m), mandible (md), manubrium of the malleus (mm), maxilla (mx), Meckel’s cartilage (mc), nasal septum (ns), palantine (p), palatal shelf (ps), processus brevis (pb), retroarticular process of the squamosal bone (ra), stapes (st), squamosal bone (sq), thyroid cartilage (tc), tongue (t), tracheal rings (tr), tympanic ring (tyr), zygomatic process of the squamosal bone (zps). *p ​≤ ​0.05, **p ​≤ ​0.005, #p ​= ​0.189, Student’s t-test.