DNA, proteins and dinosaurs: another brick in the wall

Updated: Jul 29, 2020

    A recent paper of Bailleul et al. [1] made some noise in the palaeontologic field. Indeed, protein residuals, chromosomes and DNA markers (broken molecules related to DNA but not complete DNA molecular structure) comparable to emu have been spotted in a dinosaur thanks to histologic, immunologic and histochemical methods. Let's have a deeper look into this exciting discovery!



Time portal: when molecules survive taphonomy


    The very first discovery related to this study is that what we took for granted about biomolecule preservation was kind of wrong. A threshold of 1 My was expected for proteins, and DNA was thought to withstand around 100.000 years only. The oldest genome known would be around 700.000 years old [2-4]. Several studies in the past already challenged these assumptions in other fossils from the Mesozoic (the second era of the Phanerozoic, from ca. 252 to 66 My) still showing proteins, pigments but also DNA-related molecular remnants (e.g. [5-8]). So, the work of Bailleul et al. did not reinvent fossil molecular research, but definitely framed what organics can be expected in the fossil record, even after dozens of My, and break the assumption that such molecules would not survive the effect of time, which basically stopped molecular research on fossils older than 1 My.


Ok... but what are the evidence? Is is THAT hard to retrieve such molecules from fossils?


   The finer, the more difficult to preserve. Fossilisation is a process that records dead organisms in sedimentary rocks (rocks that are formed through debris accumulation, in lakes or the sea for instance). Even though we still discover lots of fossil worldwide a year, that does not mean that such recorded organisms represent their environment and their actual abundance when they were still alive. Actually, the primary palaeobiologic signal, the biocenosis (the actual diversity at a given time and space) (Fig. 1a), is not perfectly represented in the dead assemblage called the thanathocenosis (Fig. 1b). Moreover, the thanatocenosis is not perfectly represented by what is preserved and reached us as well, called the taphocenosis (Fig. 1c). Many filters are involved in these successive, unfortunate, yet inevitable loss of information (Fig. 1d) [9].


Figure 1. Illustration of the difference between the real palaeobiologic signal, and what we collect as fossils. (a) Cambrian sea (ca. 500 My) with abundant life here and there, in and on the sea bottom, and in the water column (biocenosis). (b) Dead assemblage (thanathocenosis) that already does not genuinely represent the life assemblage in (a). (c) Burial and fossilisation, with differential preservation depending on the organism and the surrounding environment (taphocenosis). (d) Weathering acting on fossiliferous sedimentary rocks. (e) Fossil collection. Modified, from [9].

    Most of the primary signal (the biocenosis - Fig. 2.1) is lost through corpse scavenging or destruction by other animals (predation, stomping, etc.) but also by natural forces (from the streams and landslides to tectonics), or chemical conditions prevailing in the direct environment (including acidity and the presence of oxygen). Even if the dead body (or its remains) survived until there (waow), it has to be recovered or buried (Fig. 2.2) prior to mineralising (original material of the organism replaced by minerals, 'rocky' materials); in other words it has to be preserved under favourable conditions to lose the less possible information in the rocks themselves (Fig. 2.3). Finally, the dead body becomes a fossil, and thanks to tectonics, a fossil buried deep can be brought again to the surface where it can be finally collected, conserved and studied (Fig. 2.4) [9].

     Bone morphology is the best preserved component of a fossil bone, however, finer biologic features can be less properly represented. Bone histology can be completely altered during taphonomic process (fossilisation process applied on a remnant), apatitic crystals making up the original bone are rearranged [10;11], proteins at the base of these apatitic crystals are most of the times lost, and I don't even talk about biomolecules like DNA (Fig. 2.5). Every palaeontologist knows that a good fossil is rare, no matter what. So retaining biomolecules at this point is almost a miracle.

    And you thought it was over? NOOOO! After all of these steps 'isolated from outer factors', if the fossil outcrops where there it has no possibility to be reported, even a wonderful fossil could be degraded because of rain, oxygen, bird guano, and so on! Hard day to be a fossil, huh? So yes, this is THAT hard to retrieve such molecules in the fossil record. And this is why this study brings very relevant information about it.


Figure 2. Fossilsation steps. (1) the organism (here, an individual of Tyrannosaurus rex) which represents the primary signal dies and falls to the ground, along the shore or a river bank for instance. Scavenging and predative behaviours can alter this primary signal. (2) After weeks, months and years after the death of the organism, most of the organic matter has rotten or had been the target of predators. The skeleton - if not stomped or disambled by streams, rain, animals, etc. - is getting recovered by sediments (sand, mud) and is retrieved for its initial environement of deposition. (3) Decades or hundreds of years after the death of the organism, its remains are getting buried for good. At this point, the bones are getting mineralised through taphonomic process, and depending on chemical conditions, surrounding fluids, etc., the fossil in creation can be altered further. (4) Millions of years later, the remains are now fossils, contained within sedimentary rocks. Depending on the outcrop conditions, the fossil can be altered further, again. (5) Like in the study of Bailleul et al. [1], some fossils are so well preserved that they can hide organics, like blood cells, proteins, or even DNA markers. Surprise, mothafuck*! © Raúl Martin

The work that changed the game: Hypacrosaurus stebingeri from the Rockies, Montana


    Hypacrosaurus is a hadrosaurian genus (the famous 'duck-billed' dinosaurs) from the Upper Cretaceous (ca. 100-66 My) of the Rockies, Montana, USA, discovered during the 80's [12;13]. Many disarticulated bones were found together in nesting grounds, and some of them were destroyed for histologic purposes [12;14]. One highly preserved supraoccipital (one of the bones at the base of the cranium - Fig. 3A) retained some calcified cartilage containing some 'amorphous extracellular matrix' (simpler, some organic matter at the cellular level) and peculiar chondrocyte lacunae (spaces previously occupied by specific cells of the cartilage, left empty after the death of the animal) (Fig. 3B-D). These lacunae are highly suggestive of the metaphasis of the mitosis (a stage during the cell 'clonal' reproduction, called mitosis) or what is referred to chondroptosis (programmed death of the chondrocyte). Some of the lacunae did show some kind of preserved cells, with what could be remnants of nuclear content resembling chromosomes. The impressive thing is that all of these features are really close to that is seen in the extant emu (a living dinosaur by the way) (Fig. 3E-G).


Figure 3. Thin section of a supraoccipital of Hypacrosaurus stebingeri showing a wonderful histologic preservation of calcified cartilage. A-D: Hypacrosaurus stebingeri. E-G: Extant emu. (A) Isolated supraoccipital of Hypacrosaurus in dorsal view. (B) General view of bone histology of the supraoccipital of Hypacrosaurus. (C) Zoom (red square of (B)) showing chondrocyte lacunae. Green arrow: empty cell doublets. Pink arrow: cell doublets with appearing dark, condensed material around the expected nucleus location (white arrows). (D) Zoom (red square in (C) showing dark, condensed and elongated organic material consistent with chromosomes in metaphasis or chondroptosis. (E) Caudal view of an extant emu skull showing the supraoccipital (So) and both exoccipitals (Exo) besides, in articulation. (F) General view of bone histology of the supraoccipital from an extant emu. (G) Zoom (red square in (F)) showing cell doublets (pink arrows), remnants of nuclei (white arrows) and other intracellular content (green arrow). From [1].

    To have a fresher look at these cellular remnants, a new sample from the same nestings and comparable in size of the one discussed above has been sectionned and prepared for immunologic and histochemical analyses. To make it simple, histochemical analyses are techniques of tissue staining to spot biologic compounds like acidic materials, proteins, etc. in order to highlight specific tissues like cartilage (Fig. 4A-B against C-D; Fig. 4E-F against G-H). Immunologic techniques encompass antigen-antibody relationships in a sample to identify among others specific proteins (Fig. 5). Thanks to those approaches, identification of preserved organic matter in Hypacrosaurus was made possible and directly compared to an extant emu supraoccipital sample. It turned out that firstly that chemical differentiation in extinct and extant dinosaurs are similar in such tissue (Fig. 4C-D,G-H), and second that collagen II (the most important proteinic building block of cartilage) (Fig. 5) have been preserved through dozens of millions of years! As expected, they are concentration differences between these bones separated by ca. 75 My [1], reflecting preservation biases. However, it does not change the game that much.


Figure 4. Histochemical analysis based on Alcian blue stain colouring proteins in both the bone and cartilage of Hypacrosaurus and from an extant emu. Unstained (A-B,E-F) and stained (C-D,G-H) thin sections. In (C), there is a strong positive staining in the cartilage of Hypacrosaurus, comparable to what is seen in the emu in (G), albeit the response for the emu is more intense. On the contrary, fossil and modern bones do not react much to Alcian blue. From [1].
Figure 5. Immunohistochemical staining of the cartilage of Hypacrosaurus and from an extant emu. (A,C,E,G,I,K) are images showing cartilage and localised bindings. (B,D,F,H,J,L) are images showing fluorescence enhancing what is seen in the other images. To make it simpler, (A-B,E-F) are pictures taken before retrieving the collagen II, and (C-D,G-H) are pictures taken after retrieving collagen II from the same thin sections. Everything that is stained in green is cartilage proteins including collagen II among others. (I-L) show the absence of collagen I binding in both Hypacrosaurus and the emu as that protein is absent from cartilage. From [1].

    Even more, further analyses of isolated chondrocytes (Fig. 6A-B,E) revealed the presence of DNA markers (broken fragments of DNA) within (Fig. 6C-D,F-G)! Reactants used in this study were properly chosen to make sure experimentally that specific staining could not be the result of contamination, reinforcing genuine dinosaur DNA interpretation. Again, as expected, concentrations are lower than in the emu sample, reflecting preservation biases just like the proteins preserved in the cartilage. Next to histologic evidence of condensed nuclear organic matter previously seen under the microscope, observed local DNA staining could also point towards such condensed matter within the nucleus. Even though the metaphasis is morphologically similar to the chondroptosis from a chromosomal point of view, the chondroptotic hypothesis is preferred: in this type of bone, there is a high chrondroptose rate in regions where bone and cartilage meet, exactly where chondrocytic lacunae were studied in Hypacrosaurus.


Figure 6. Isolated chondrocytes of Hypacrosaurus and from an extant emu, and their positive response to two assays (PI and DAPI stains). Unstained (A-B,E) and stained (C-D,F-G) thin sections. (A-B,E) show isolated chondrocytes under transmitted light (green arrows). Isolated single cell (A) and cell doublet (B) of Hypacrosaurus. (C,F) Strong responses to PI (propidium iodide) located inside the cells (white arrows) in Hypacrosaurus and the emu. (F,G) Similar response to DAPI (4',6'-diamidino-2-phenylindole dihydrochloride) located as well within the cells (dark arrows). In both cases, the overall response is greater in the extant emu. From [1].

We don't need no thought control


     So what? Are we cloning dinosaurs for Easter?


   Well, no. Definitely. DNA markers are only small fragments that survived long enough to allow scientists to discover them. They will not give access to even a fragmentary genome that would be filled with reptile, snake, spider, parrot or whatever genes in order to 'make it work'. The most important conclusion of this work is that we have underestimated for a long time the preservation potentials of biomolecules in the fossil record. Even though it requires a high preservation degree and that such favourable conditions are not met for maybe even 1% of all fossils ever, more 'high grade' fossils could open the highway heading to new, unknown palaeontologic boulevards. Moreover, the deposition settings of the studied bones do not match the high degree of preservation encountered at the molecular level, leaving still some mysteries behind yet to resolve.

     Maybe that such biomolecules are off the radar but preserved in many fossil throughout the whole tree of life. Maybe not. Anyway if only one or two molecules could be identified regularly across species and genera with enough accuracy to read molecular changes spatio-temporally, just that would already be a gigantic step towards the big scheme of evolution. All in all it's just another brick in the wall.


DNA markers: another step towards Jurassic Park-like fantasy? © Adobe Stock


References


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