Locally released retinoic acid repatterns the first branchial arch cartilages in vivo.

The fates of cranial neural crest cells are unique compared to trunk neural crest. Cranial neural crest cells form bone and cartilage and ultimately these cells make up the entire facial skeleton. Previous studies had established that exogenous retinoic acid has effects on neurogenic derivatives of cranial neural crest cells and on segmentation of the hindbrain. In the present study we investigated the role of retinoic acid on the skeletal derivatives of migrating cranial neural crest cells. We wanted to test whether low doses of locally applied retinoic acid could respecify the neural crest-derived, skeletal components of the beak in a reproducible manner. Retinoic acid-soaked beads were positioned at the presumptive mid-hindbrain junction in stage 9 chicken embryos. Two ectopic cartilage elements were induced, the first a sheet of cartilage ventral and lateral to the quadrate and the second an accessory cartilage rod branching from Meckel's cartilage. The accessory rod resembled a retroarticular process that had formed within the first branchial arch domain. In addition the quadrate was often displaced laterally and fused to the retroarticular process. The next day following bead implantation, expression domains of Hoxa2 and Hoxb1 were shifted in an anterior direction up to the mesencephalon and Msx-2 was slightly down-regulated in the hindbrain. Despite down-regulation in neural crest cells, the onset of Msx-2 expression in the facial prominences at stage 18-20 was normal. This correlates with normal distal beak morphology. Focal labeling of neural crest with DiI showed that instead of migrating in a neat group toward the second branchial arch, a cohort of labeled cells from r4 spread anteriorly toward the proximal first arch region. AP-2 expression data confirmed the uninterrupted presence of AP-2-expressing cells from the anterior mesencephalon to r4. The morphological changes can be explained by mismigration of r4 neural crest into the first arch, but at the same time maintenance of their identity. Up-regulation of the Hoxa2 gene in the first branchial arch may have encouraged r4 cells to move in the anterior direction. This combination of events leads to the first branchial arch assuming some of the characteristics of the second branchial arch.