Transcriptional Regulation of Jaw Osteoblasts: Development to Pathology.

Craniofacial and jaw bones have unique physiological specificities when compared to axial and appendicular bones. However, the molecular profile of the jaw osteoblast (OB) remains incomplete. The present study aimed to decipher the bone site-specific profiles of transcription factors (TFs) expressed in OBs in vivo. Using RNA sequencing analysis, we mapped the transcriptome of confirmed OBs from 2 different skeletal sites: mandible (Md) and tibia (Tb). The OB transcriptome contains 709 TF genes: 608 are similarly expressed in Md-OB and Tb-OB, referred to as "OB-core"; 54 TF genes are upregulated in Md-OB, referred to as "Md-set"; and 18 TF genes are upregulated in Tb-OB, referred to as "Tb-set." Notably, the expression of 29 additional TF genes depends on their RNA transcript variants. TF genes with no previously known role in OBs and bone were identified. Bioinformatics analysis combined with review of genetic disease databases and a comprehensive literature search showed a significant contribution of anatomical origin to the OB signatures. Md-set and Tb-set are enriched with site-specific TF genes associated with development and morphogenesis (neural crest vs. mesoderm), and this developmental imprint persists during growth and homeostasis. Jaw and tibia site-specific OB signatures are associated with craniofacial and appendicular skeletal disorders as well as neurocristopathies, dental disorders, and digit malformations. The present study demonstrates the feasibility of a new method to isolate pure OB populations and map their gene expression signature in the context of OB physiological environment, avoiding in vitro culture and its associated biases. Our results provide insights into the site-specific developmental pathways governing OBs and identify new major OB regulators of bone physiology. We also established the importance of the OB transcriptome as a prognostic tool for human rare bone diseases to explore the hidden pathophysiology of craniofacial malformations, among the most prevalent congenital defects in humans.

In Situ Hybridization in Mineralized Tissues: The Added Value of LNA Probes for RNA Detection.

In situ hybridization (ISH) is one of the fundamental methods in developmental biology and neurobiology. Their first ISH protocols were reported in 1969 (Gall and Pardue, Proc Natl AcadSci USA 63:378-83, 1969). Since several decades, ISH based on the specific hybridization of 100-2000 nucleotides long probes enabled the localization of DNA/RNA sequences in tissues and cells with high cellular resolution. But sometimes a limited sensitivity notably in mineralized tissues (Obernosterer et al., Nature Protocols 2:1508-14, 2007).Here we describe a recent improvement of in situ hybridization efficiency by applying nucleotide locked nucleic acid (LNA)-incorporated oligodeoxynucleotide probes (20 LNA/DNA nucleotide probes) essentially used for noncoding miRNA and messenger RNAs.

An improved method for the recovery of mycoplasmas from the external ear canal of goats.

The efficacy of ear canal flushing and ear canal and mouth swabbing methods for the isolation of mycoplasmas was investigated in 39 goats. Of the 19 goats positive for Mycoplasma spp., 14 (73.7%) were positive with the ear canal flushing method, 4 (21.0%) were positive with both ear canal flushing and mouth swabbing methods, and 1 (5.3%) was positive by the mouth swabbing method. Mycoplasma arginini, M. mycoides subsp. mycoides, and M. mycoides subsp. capri were identified by direct immunofluorescence and growth inhibition tests. Previous reports on the isolation of M. arginini from the ear canal of goats were not found in the literature.