Isolation of Osteoblast RNA from Danio rerio larvae – presenting a novel method or an update to the existing one !

The arrangement and distribution of cartilages, dermal bones, and muscles that organize the musculoskeletal system takes place during the embryonic development. Bones are of great significance for the human body, which is known to provide skeletal support, form, strength, movement to the body, it serves as a home for hematopoiesis, reservoiring calcium and other important minerals. While intensely investigated in mammals, zebrafish or Danio rerio has been somewhat a late entrant as a model organism in the area of bone research.
In Developmental biology, zebrafish, along with another small teleost species medaka, have long been used as model organisms. Their popularity results from the rapid external development of their larvae, their amenability to genetic manipulation and also, importantly, to the translucency of the larvae, which allows detailed observation of organogenesis in the living fish. The craniofacial skeleton of zebrafish is of comparable complexity to that of terrestrial vertebrates, and is known to contain bones of both dermal and chondral origins, which form from the neural crest-derived cells relatively early in the course of development. Also, of crucial importance is that both the key regulators of skeletal development and the control of the major signaling pathways are known to be highly conserved between mammals and teleosts. Therefore, findings in fish are likely to have an application in mammalian osteogenesis or bone development studies.
Bone Morphogenic Proteins (BMPs) have been widely studied for their important role in many developmental and physiological processes, including skeleton formation and homeostasis. Former studies in zebrafish have highlighted the crucial role of BMP signaling before 48 h post-fertilization (hpf) for cartilage formation in the skull, while further results from our lab revealed that for the onset of bone formation, proper BMP signaling between 48 and 72 hpf is crucial to ensure osteoblast function and ossification.
Transgenic lines such as the Tg(osterix:mCherry) allow not only to observe bone-forming cells (osteoblasts) in vivo with live imaging, along with several staining procedure, we are now interested in isolating RNA from osteoblasts at 5 dpf from zebrafish using FACS and look at the transcriptome of these cells in the presence or absence of BMP inhibitors. In this study we show new method to isolate mRNA from these bone forming cells.