Imaging bone cells in 420-million-year-old jawless fish

The early armored fish Bothriolepis, which Yara Haridy affectionately refers to as a “beetle mermaid”. Art by Brian Engh, dontmesswithdinosaurs.com.

If I had to sum up my main research program over the past 20+ years, it would be, “Why pneumatic bone?” Or as I typically put it in my talks, most bone has marrow inside, so if you find bone with air inside, someone has some explaining to do (f’rinstance).

One of the reasons I like hanging out with Yara Haridy is that she is interested in an even more fundamental question: “Why bone?” And also “How bone?” And she has a paper out today that gives us new insights into the form and function of bone cells — osteocytes — in some of the earliest vertebrates that had them (Haridy et al. 2021; if you’re in TL;DR mode, here’s the link). 

Bones have multiple functions in vertebrate bodies: they’re a mechanical framework for our muscles, and a mineral reservoir, and form armor in many taxa, and are involved in hormone regulation, and doubtless other things that we are still discovering, even now. To fulfill those functions, bone tissue has to be formed in the first place, it has to be maintained, and it has to be able to be reshaped as an individual grows. Derived extant vertebrates, including humans, have an impressive array of cellular machinery to make all those things happen. Central to most of those operations are osteocytes, the cells inside living bone, which maintain intimate connections to extracellular bone tissue and to other osteocytes via fine, tentacle-like processes.

Individual osteocytes look something like the Flying Spaghetti Monster. The central portion of the FSM, with the meatballs, is the osteocyte body, and the noodly appendages are the processes.

Now imagine that you cloned the FSM many, many times, and the resulting array of FSMs stayed in physical contact with each other via their noodly appendates, forming a network.

Then imagine that you entombed all of the clones FSMs in concrete. This is more or less what cellular bone — the kind you find in humans, dinosaurs, and even some jawless fish — looks like on the microscopic scale: osteocytes (the FSMs) and their processes (the noodly appendages) embedded in space-filling stuff (the bone matrix). Some critters, including teleost fish, have acellular bone, but I don’t have time for those unbelievers today.

When an animal dies and decomposes, the osteocytes and their processes decay away, leaving behind the spaces that they used to occupy. The big spaces that hold osteocytes are called lacunae, and the little tunnels that hold the osteocyte processes (noodly appendages, in this metaphor) are canaliculi. Collectively, the lacunae and canaliculi form the lacunocanalicular network or LCN.

Those spaces can then be filled by matrix — not extracellular bone matrix, but future rock matrix, like mud and clay. In point of fact, not all of the spaces are filled with matrix. Even in their 420-million-year-old fish, Yara and colleagues found some osteocyte lacunae that had not been filled with matrix, and were filled by air instead. Whether the lacunocanalicular network is filled with matrix or air, its preservation in fossil bone has turned out to be a boon for paleontologists, because we can ‘see’ the sizes and shapes of osteocytes, and their level of connectivity, by studying the lacunae and canaliculi they left behind. 

Histological thin section of bone in the osteostracan Tremataspis mammillata (MB.f.TS.463), imaged with transmitting light microscopy showing osteocyte lacunae (osl) and canaliculi (ca); scale bar, 100 microns. Haridy et al. (2021: fig 2A).

Traditionally osteocyte lacunae and canaliculi in fossil bone have been imaged by taking thin sections of the specimens and looking at them under microscopes.

Synchrotron tomography of bone of Bothriolepis trautscholdi (MB.f.9188a) with the vasculature and osteocytes segmented; scale bar, 0.4 mm. (D) Close-up of tomography in (C) showing the resolution of the osteocyte lacunae volumes; scale bar, 10 microns. bs, bone sample; vs, vasculature channels. Modified from Haridy et al. (2021: fig. 2C-D).

If you’re fancy, you can also do synchrotron tomography, which is fine enough to show osteocyte lacunae — the colored blobs in the image on the right, above.

Those methods have their limitations. Light microscopy will reveal both lacunae and canaliculi in 2D, but it’s hard to get a 3D understanding of the lacunocanalicular network that way (at least in fossils; in modern samples it can be done with confocal miscoscopy). Synchrotron tomography can resolve lacunae in 3D, but not canaliculi, sort of like a map that shows only cities but not the highways that connect them.

Enter FIB-SEM: focused ion beam scanning electron microscopy. An ion gun blasts the specimen with a beam of gallium ions, which vaporizes a slice of the specimen that is less than 1 micron thick, and an SEM images the freshly exposed face. If you do this over and over again, you can build up a 3D model of the stuff that once occupied the volume that got zapped. 

FIB-SEM tomography imaging and processing of the fossil jawless vertebrate Tremataspis mammillata (MB.f.9025). (A and B) FIB-SEM setup showing the FIB in relation to the SEM both aimed at the region of interest. (C) Bone surface with an excavate area made by the FIB. (D) Internal wall of the excavated area lined with small black dots that are the fossil osteocyte lacunae. (E) Single osteocyte lacuna from the surface that is scanned; the single SEM image shows the lacunae and canaliculi in black and the mineralized bone in gray; scale bar, 5 microns. (F and G) An image stack is obtained, and 3D made of fossil LCN can be made. Haridy et al. (2021: fig 3).

FIB-SEM is fine enough to resolve both osteocyte lacunae and canaliculi — the lacunocanalicular network or LCN — in three dimensions, in fossil specimens where confocal light microscopy doesn’t always work very well. And the resolution is pretty insane. The rough edges on the 3D models of the LCN aren’t sampling artifacts, they’re accurately reflecting the real morphology of the walls of the lacunae and canaliculi as they were preserved in the fossil bone.

But wait — that’s not all! Not only can FIB-SEM show us osteocyte lacunae and canaliculi in incredible detail in three dimensions, it can also help us figure out at least some of what osteocytes were doing. Together, osteocytes and their processes can sense mechanical strain in bone, trigger bone remodeling, and resorb and lay down bone from the walls of the lacunae and canaliculi. That last process starts with osteocytic osteolysis — the resorption of bone matrix (= osteolysis) from the lacunae and canaliculi by the osteocytes themselves (as opposed to the more familiar destruction of bone at a larger spatial scale by osteoclasts), which is typically followed by the replacement of new matrix where the old bone used to be. Lots of extant vertebrates do osteocytic osteolysis, especially those that have a high demand for calcium and phosphorus in physiologically challenging times. Examples including migrating salmon, lactating mice, and lactating humans. But when did that capacity evolve — did the earliest osteocytes already have the ability to resorb and replace bone? As Yara said to me when she was telling me about her new paper, “We think we know how things work by looking at extant animals, but we’re looking at this highly pruned tree, and we can’t just assume that things worked the same way earlier in our evolutionary history.” 

Yara wanted to investigate when osteolytic osteolysis first evolved when she started her dissertation in 2018, but she didn’t know that FIB-SEM existed. Then she was visiting a neutron tomography facility in Berlin and she saw a poster on the wall about people using FIB-SEM to image corrosion in batteries on ultra-fine scales. She thought, “Wow, the corrosion pits in the batteries look like osteocytes!” The rest you probably figured out faster than it’s taking me to write this sentence: together with her collaborators, she got some samples of bone from the jawless fish Tremataspis and Bothriolepis and zapped them with the FIB-SEM.

Osteocytic osteolysis as a mechanism for early mineral metabolism. (A to C) Illustrations depicting the process of osteocytic osteolysis; the phases are stasis phase, dissolution phase, and redeposition phase, respectively. (D) Single SEM image from the FIB-SEM acquisition showing the air-filled osteocyte lacunae and canaliculi of T. mammillata. (E) Same SEM image as in (D) with contrast shifted to show the demineralized zone surrounding the lacunae. (F and G) 3D render of the stack of images from (D and E) T. mammillata (MB.f.9025). The 3D model shows several osteocytes and their canaliculi, with the red areas showing where the “areas of low density” were found. ald, area of low density; os, osteocyte. Scale bar, 5 microns.

And, wonder of wonders, some of the osteocyte lacunae in Tremataspis were surrounded by a halo of less-dense bone, which is evidence for osteocytic osteolysis. Now, Yara and colleagues can’t be sure whether the bone is less dense because it was being resorbed when the animal died — the actual lytic or bone-destructive phase — or because new bone was being laid down after the old bone had been resorbed; naturally the new bone is less dense as it being formed than it will be when it is complete. They also can’t be sure why the process was occurring in that one individual Tremataspis. Mice only do osteocytic osteolysis when they’re lactating, and salmon only do it when they’re migrating, so the presence of osteocytic osteolysis might indicate that the Tremataspis in question was doing something stressful related to its ecology or life history — both topics we know almost nothing about.

Yara and colleagues didn’t find any evidence of osteocytic osteolysis in their Bothriolepis sample, but this is one of those ‘absence of evidence is not evidence of absence’ things — you wouldn’t find evidence of osteocytic osteolysis in my skeleton either, despite a long ancestral history, because I’m skeletally healthy, not fasting or migrating, and not lactating. Possibly other Bothriolepis individuals that were going through a rough patch, metabolically speaking, would show osteocytic osteolysis. So far, as a species, we’ve only looked at the one sample, from the one individual.

I asked Yara what she wanted people to take away from her new paper. Her response:

1. We have technology can image fossil bone cells at the same resolution that we can see modern bone cells.
2. Bone metabolism was going on 420 million years ago, in the earliest osteocytes, the same way it happens in modern mammals, including humans. 

I expect that we will see a lot more FIB-SEM papers on fossils in years to come. That research program started today, with the publication of Haridy et al. (2021). I often sign off posts with “stay tuned”, and this time I really mean it.

Reference

Yara Haridy, Markus Osenberg, André Hilger, Ingo Manke, Donald Davesne, and Florian Witzmann. 2021. Bone metabolism and evolutionary origin of osteocytes: Novel application of FIB-SEM tomography. Science Advances 7(14): eabb9113.
DOI: 10.1126/sciadv.abb9113