mages are dorsal views, except (R-U), which are ventral views. Higher magnification views of places boxed in (G,K,O) are shown in accompanying images (I, M and Q, white boxes) and (H, L and P, black boxes). Note ectopic crestin positive cells in dorsal neuroepithelium (K,L,O,P) and lowered migratory CNCCs streams (K,M,O,Q) in lrp5 CRISPR injected embryos.
Postmigratory CNCCs, after they reach their final destinations, differentiate to establish the head skeleton. We tested no matter whether the observed defects in CNCC proliferation and migration lead to decreased numbers of postmigratory CNCCs inside the pharyngeal arches. For this, we knocked-down lrp5 in fli1:EGFP transgenic zebrafish that express GFP in CNCC derivatives also as vascular endothelial cells [27]. At 30 hpf, the mandibular (md), hyoid (hy) and 3 branchial (br) patches of postmigratory CNCCs show distinct GFP expression in wild-type embryos (Fig 7A). In contrast, 65% from the lrp5 morphants (n = 32) showed disturbed Tetracosactide organization in this area (Fig 7B). We followed development in the affected embryos over time and analyzed morphogenesis and position of GFP constructive CNCC derivatives. At 48 hpf, pharyngeal arches had been effectively established inside the caudal head area of handle embryos and visible as five clearly distinguishable columns of GFP good cells (Fig 7C and 7E). In contrast, lrp5 morphants failed to establish suitable pharyngeal arch morphology. Only a single group of migratory
Proliferation of premigratory CNCCs is impacted by knock-down of lrp5. (A,B) 20 ss embryos stained for pH3 cells in M-phase. (A) Wild-type embryo, (B) lrp5 morphant. Frames demarcate region of cell count (roi, area of interest) and are magnified in (A’,B’) (counted nuclei marked by asterisks). Note that in lrp5 morphants pH3 optimistic cells are lowered in number. (C) Quantification of pH3 cell numbers within the neuroepithelium of rhombomeres four. N = 9/11 (wild-type/lrp5 morphant). P 10-6, t-test. (D,E) 20 ss embryos stained for BrdU incorporation. (D) Wild-type embryo, (E) lrp5 morphant. Frames demarcate area of cell count (roi) and are shown with larger magnification in (D’,E’). Note that in lrp5 morphants, BrdU labeled cells are decreased in number. (F) Quantification of BrdU cell numbers in 1 unilateral neuroepithelium of rhombomeres 4. N = 9/11 (wild-type/lrp5 morphant). P = 1.05×10-6, t-test. (G-J) ccnd1 expression in 20 ss embryos. (G,H) Wild-type embryo, (I,J) lrp5 morphant. Note that ccnd1 expression levels are improved in lrp5 morphants. Anterior would be to the left in all images.
GFP-positive cells might be identified inside the posterior hindbrain and most likely represented the 5th branchial arch (ba5; Fig 7D and 7F). At 72 hpf, the majority of CNCC derivatives have reached their final destinations and 17764671 the distribution of GFP-positive cells reflects the main architecture in the mature ventral cranial skeleton. Structures including Meckel’s cartilage (mc), ceratohyal (ch) as well as the five ceratobranchials (cb) are distinguishable (Fig 7G and 7I). In lrp5 morphants, around the other hand, pharyngeal arches are absent and serious malformations are observed inside the cranial skeleton. Only rudiments on the caudal pharyngeal arches stay (Fig 7H). Although most components on the anterior head skeleton are visible in ventral views (mc; Fig 7J), a lot more posterior structures are morphologically not distinguishable (ch, cb; Fig 7J). With each other, this suggests that a lrp5 knock-down initially results in proliferation defects 
in premigratory CNCCs, conse