alcian-blue and 5-bromo-4-chloro-3-indolyl-beta-galactoside

The emergence of craniofacial skeletal elements, and of the jaw in particular, was a crucial step in the evolution of higher vertebrates. Most facial bones and cartilage are generated during embryonic development by cranial neural crest cells, while an osteochondrogenic fate is suppressed in more posterior neural crest cells. Key players in this process are Hox genes, which suppress osteochondrogenesis in posterior neural crest derivatives. How this specific pattern of osteochondrogenic competence is achieved remains to be elucidated. Here we demonstrate that Hox gene expression and osteochondrogenesis are controlled by epigenetic mechanisms. Ezh2, which is a component of polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 in histone 3 (H3K27me3), thereby functioning as transcriptional repressor of target genes. Conditional inactivation of Ezh2 does not interfere with localization of neural crest cells to their target structures, neural development, cell cycle progression or cell survival. However, loss of Ezh2 results in massive derepression of Hox genes in neural crest cells that are usually devoid of Hox gene expression. Accordingly, craniofacial bone and cartilage formation is fully prevented in Ezh2 conditional knockout mice. Our data indicate that craniofacial skeleton formation in higher vertebrates is crucially dependent on epigenetic regulation that keeps in check inhibitors of an osteochondrogenic differentiation program.

Topics: Alcian Blue; Animals; Anthraquinones; Cartilage; Chondrogenesis; Chromatin Immunoprecipitation; DNA Methylation; Enhancer of Zeste Homolog 2 Protein; Epigenesis, Genetic; Facial Bones; Flow Cytometry; Galactosides; Gene Expression Regulation, Developmental; Histones; Immunohistochemistry; Indoles; Mice; Mice, Transgenic; Microarray Analysis; Neural Crest; Osteogenesis; Polycomb Repressive Complex 2; Real-Time Polymerase Chain Reaction

The cell adhesion molecule N-cadherin is implicated in many morphogenetic processes, including mesenchyme condensation during limb development. To further understand N-cadherin function, we characterized a new N-cadherin allele containing the lacZ reporter gene under the regulation of the mouse N-cadherin promoter. The reporter gene recapitulates the expression pattern of the N-cadherin gene, including expression in heart, neural tube, and somites. In addition, strong expression was observed in areas of active cellular condensation, a prerequisite for chondrogenic differentiation, including the developing mandible, vertebrae, and limbs. Previous studies from our laboratory have shown that limb buds can form in N-cadherin-null embryos expressing a cardiac-specific cadherin transgene, however, these partially rescued embryos do not survive long enough to observe limb development. To overcome the embryonic lethality, we used an organ culture system to examine limb development ex vivo. We demonstrate that N-cadherin-deficient limb buds were capable of mesenchymal condensation and chondrogenesis, resulting in skeletal structures. In contrast to previous studies in chicken using N-cadherin-perturbing antibodies, our organ culture studies with mouse tissue demonstrate that N-cadherin is not essential for limb mesenchymal chondrogenesis. We postulate that another cell adhesion molecule, possibly cadherin-11, is responsible for chondrogenesis in the N-cadherin-deficient limb.

Topics: Alcian Blue; Alleles; Animals; beta-Galactosidase; Cadherins; Cell Adhesion; Chondrogenesis; Crosses, Genetic; Extremities; Fluorescent Antibody Technique, Indirect; Galactosides; Genes, Reporter; Genetic Techniques; Heterozygote; Homozygote; Indoles; Lac Operon; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Fluorescence; Models, Genetic; Organ Culture Techniques; Promoter Regions, Genetic; Retroviridae; Terminal Repeat Sequences; Time Factors; Transgenes