Years ago when I was still working in the lab, I was looking for control RNA options for my experiment in the Ambion catalog when I came across a listing for dinosaur brain RNA and DNA. I had to call their customer service and ask about the items because I could not believe such things existed. The representative I spoke with said I had found their joke listing and sent me a free t-shirt. While we are not quite to the point of selling dinosaur nucleic acid in life science company catalogs, there is some intriguing research in the journal Bone that does suggest that fossilized dinosaur osteocytes and associated proteins may be within our grasp.

Earlier work had found tissue and cells from dinosaur bone fossils that could be identified as blood vessels and osteocytes. Schweitzer et al. chose to focus on osteocytes, cells present in mature bone (1). The first step toward confirming the presence of osteocytes in fossils was examining the cell structures using microscopy and seeing if they resemble the known shape of osteocytes. The putative cell-like structures were derived from demineralized long bones from Tyrannosaurus rex and Brachylophosaurus canadensis and compared with similarly treated ostrich and alligator bones. The dinosaur cell-like structures had a variety of forms in T. rex, with filopodia radiating in three dimensions and a variety of shapes from broad cells to long, narrow cells. B. canadensis cells were closely associated with the matrix fibers but were distinct from them, and the cells included filopodia. Transmission electron micrographs of cells showed the contents varied in amount in T. rex from sparse to dense while the B. canadensis cells tended to be empty except for a single condensed area. For comparison, the ostrich osteocyte had lots of internal contents, and filopodia extensions were seen for all three species of cells.

After visual analysis of the cell structures seemed to indicate these were osteocyte cells from two dinosaurs, immunological techniques were used to probe for the presence of different proteins that are found in osteocytes. Staining with polyclonal antibodies showed that the cytoskeletal proteins actin and tubulin looked the same in dinosaur, alligator and ostrich cells: filamentous pattern for actin and more diffuse binding for tubulin. The authors performed three controls (no secondary antibody, an anti-human testosterone antibody for the primary antibody and staining of biofilm-forming soil isolates of E. coli in place of osteocytes), none of which showed binding in any cells. Monoclonal antibodies for an epitope of chicken phosphoendopeptidase (PHEX), a protein needed for bone mineralization, were used to stain a specific avian-origin protein. Because dinosaurs and birds share a common ancestor, the authors hypothesized that PHEX may be conserved enough to react when testing dinosaur osteocytes. In fact, they discovered both dinosaur cells and the ostrich cells reacted with the PHEX monoclonal antibody, but the nonavian alligator cells did not.

To detemine if cells contained DNA, Schweitzer et al. used an antibody that reacts with the double-stranded DNA (dsDNA) backbone to probe the dinosaur cells. While this antibody cannot determine if any DNA that react is endogenous to the cell, the staining pattern for both T. rex and B. canadensis were faint, not surprising after millions of years, and in a small, localized area inside the cell membrane. A similar small, localized staining site appeared for the ostrich osteocytes. The presence of DNA was further confirmed using propidium iodide, a dye that intercalates in DNA, and 4′,6′-diamidino-2-phenylindoledihydrochloride (DAPI), a stain that preferentially binds dsDNA at A-T residues. In all cases, the dyes stained localized regions similar to those stained by the dsDNA antibody, and the ancient cell structures stained with lower intensity than the modern ostrich cells.

Mass spectrometry was performed on powdered dinosaur bone samples that were digested with trypsin. Peptides that corresponded to actin and tubulin were found in the mass spectrometry analysis as well as histone H4, a protein associated with DNA. T. rex and B. canadensis cells also reacted with anti-histone H4 antibodies. Actin, tubulin and histone H4 are conserved proteins across all Bilateria animals, and the results of the proteomic analysis showed that the sequences of T. rex and B. canadensis peptides detected were identical to the consensus sequences of these three proteins.

The methods used in this article, while limited to a small sample of proteins, did demonstrate evidence of dinosaur cell-like structures that resembled osteocytes, reacted with antibodies that recognize conserved proteins and dsDNA and were stained with DNA dyes. I find it amazing that protein samples millions of years old are resilient enough to both react to antibody epitopes and resemble cells by microscopic analysis. While these results are from only two bone samples, the evidence for osteocytes retaining characteristics that we can probe with our molecular tools is intriguing. While some may view the data generated by Schweitzer et al. with skepticism, I believe with more samples and more proteomic analyses, future work could extend how far into the past we can peer and add to our body of knowledge on ancient animals.

Reference

1. Schweitzer, M.H., Zheng, W., Cleland, T.P. and Bern, M. (2013). Molecular analyses of dinosaur osteocytes support the presence of endogenous molecules, Bone, 52 (1) 414-423. DOI: 10.1016/j.bone.2012.10.010