Evidence of proteins, chromosomes and chemical markers of DNA in exceptionally preserved dinosaur cartilage

Abstract

A histological ground-section from a duck-billed dinosaur nestling (Hypacrosaurus stebingeri) revealed microstructures morphologically consistent with nuclei and chromosomes in cells within calcified cartilage. We hypothesized that this exceptional cellular preservation extended to the molecular level and had molecular features in common with extant avian cartilage. Histochemical and immunological evidence supports in situ preservation of extracellular matrix components found in extant cartilage, including glycosaminoglycans and collagen type II. Furthermore, isolated Hypacrosaurus chondrocytes react positively with two DNA intercalating stains. Specific DNA staining is only observed inside the isolated cells, suggesting endogenous nuclear material survived fossilization. Our data support the hypothesis that calcified cartilage is preserved at the molecular level in this Mesozoic material, and suggest that remnants of once-living chondrocytes, including their DNA, may preserve for millions of years.

Keywords: DNA markers; cartilage; chromosomes; collagen II; dinosaur; nuclei.

Figure 1.

Ground section of Hypacrosaurus (MOR 548) supraoccipital shows exceptional histological preservation of calcified cartilage. (A) An isolated supraoccipital (So) of Hypacrosaurus in dorsal view. (B–D) Ground section of another So showing calcified cartilage with hypertrophic chondrocyte lacunae. (C) Some cell doublets appear empty (green arrow), but others (pink arrow) present darker, condensed material consistent in shape and location with a nucleus (white arrows). (D) Dark, condensed, and elongated material with morphological characteristics of metaphase chromosomes. The limit of the cell lacuna is visible (black arrow). (E) Caudal view of a juvenile emu skull (∼8–10 months old) showing the So and exoccipitals (Exo) in articulation. (F, G) Ground section (stained with Toluidine blue) of calcified cartilage from this emu skull showing cell doublets (pink arrows) with remnants of nuclei (white arrows) and others without intracellular content (green arrow).

Figure 2.

Alcian blue histochemical stain capitalized on differential presence of glycosaminoglycans in the calcified cartilage and bone of Hypacrosaurus. Unstained (A, B, E, F) and Alcian-blue stained (C, D, G, H) paraffin sections of Hypacrosaurus and emu cartilage and bone. A strong, positive blue staining is seen in Hypacrosaurus cartilage (C), comparable to the intense, but darker stain found in modern emu cartilage (G). This suggests that glycosaminoglycans are still present in the cartilaginous matrix of this dinosaur. In contrast, the fossil and extant bones show a very light blue stain (D, H). Images are at the same scale.

Figure 3.

Immunohistochemical staining of Hypacrosaurus cartilage. (A, C, E, G, I, K) are overlay images showing cartilage and localized binding; (B, D, F, H, J, L) are fluorescent images using FITC label. Hypacrosaurus calcified cartilage (A, B) shows positive, localized staining when exposed to antibodies (Ab) raised against avian Collagen II, with green fluorescent signal representing antibody-antigen complexes arranged globularly in the extracellular matrix. Immunoreactivity is diminished, as illustrated by longer integration time and fainter signal, when compared to calcified and hyaline cartilage from an emu (E, F). Antibody reactivity was decreased after collagenase II digestion in Hypacrosaurus (C–D) and emu cartilage (G–H), demonstrating that reactivity to Collagen II is specific for epitopes of that protein. Hypacrosaurus (I, J) and emu cartilage (K, L) shows no staining when exposed to antibodies rained against avian Collagen I. Images are at the same scale.

Figure 4.

Isolated chondrocytes of Hypacrosaurus and their positive response to two DNA assays. (A, B, E) Isolated chondrocytes of Hypacrosaurus and emu photographed under transmitted light (green arrows). Hypacrosaurus chondrocytes were successfully isolated as individual cells (A) and cell doublets (B). Hypacrosaurus (C) and emu chondrocytes (F) showing positive response to propidium iodide (PI), a DNA intercalating dye, to a small and circular region that locates intracellularly (white arrows). Hypacrosaurus (D) and emu chondrocytes (G) also show a similar binding when exposed to 4^′,6^′-diamidino-2-phenylindole dihydrochloride (DAPI), another DNA-specific stain (black arrows) although in both cases, emu cell staining is significantly greater than in the dinosaur cells.