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I came across this post on The Node with some fantastic imaging from Zelzer and colleagues at the Weizmann of the cells that connect tendon to bone.  The paper sheds light on the developmental origins of the cells that form the eminences; the units that connect tendon to bone. As it turns out, these do not originate from cartilage (chondrocytes), as previously thought, but instead from a separate lineage specified slightly later in development.  These progenitor cells can be identified by the genes they express.  In the image below, the arrows point to these cells, represented in green [Sox9-positive (green), Col2a1-negative (red)].

Blitz et al. 2013 Development 10.1242/dev.093906

Brains are made up of many different types of cells that come about through common stem cells called neuroblast progenitors. These progenitors can produce the number and diversity of cell types necessary to form a central nervous system. As neuroblasts divide to make more cells, they can potentially specify different types of cells with each round of division, and depending upon the stage of development. 

Lineage development from larva to adult. Cartoon diagram highlighting a single neural lineage in the larval brain (left) and in the adult brain (right). Note the decreased contribution of primary neurons in the adult brain compared to the larval brain. GMC ganglion mother cell. Spindler and Hartenstein, Dev Genes Evol. 2010 220: 1–10. doi: 10.1007/s00427-010-0323-7

In the fruit fly, most of the cells in the adult central nervous system have been mapped back to their embyonic neuroblast progenitor, of which there are about 100 or so. As such, the fruit fly brain is a popular model for understanding neural circuitry and development. One of the better-studied neuroblast lineages, referred to as NB7-1, gives rise to seven progenitor cells, the first five of which give rise to lineages of motorneurons that will wire the nervous system to the muscles. The remaining two neuroblasts give rise to interneurons; the cells that connect neurons within the nervous system.

Though not innervated by NB7-1 motorneurons, these are  developing flight muscles of the fruit fly (visualised in GREEN), which are remodeled from larval muscle templates by the fusion of adult muscle precursors (visualised in BLUE). The motor neurons (visualised in RED) of the larval muscles are re-specified to innervate the flight muscles. These muscles and motor neurons are a unique example of how adult structures are refashioned from embryonic counterparts during metamorphosis in animals. Imaged by: S. Roy (Institute of Molecular and Cell Biology, Singapore; Cover image, EMBO J, 08.08.2007)

Each of the five motorneuron-specifying neuroblasts gives rise to a different lineage of cells. The lineages specified in the first two neuroblasts, called U1 and U2, are governed by a gene called hunchback (hb), which is normally only active in the first two progenitors. The hb gene codes for a protein that regulates the activity of other genes called a transcription factorTranscription factors work by recognising DNA sequences in a gene’s promoter; the bit immediately preceding the coding sequence to a gene. Once bound to the promoter, they summon the molecular machinery (or are sometimes already pre-assembled to it) necessary to decode that gene into a template to make a protein. In some cases, like with hb, transcription factors (the proteins) bind to their own gene promoters. This is usually done to ensure that a lot of that particular transcription factor is produced, as it may be necessary to activate a great number of downstream genes. In fact, some transcription factors are responsible for turning on a whole cascades of gene activity; in some cases altering the identity of a lineage of cells.

In fact, if hb is experimentally turned on in any of the first five progenitors, it will turn on the same U1/U2 motorneuron identity in these cells. Curiously though, hb’s affect is limited to those cells as the remaining two (interneuron-specifying) progenitors and their resulting neurons do not respond to the hb signal. When a sub-group of cells is capable of responding to an inductive signal like this, it is said to be competent.

Chris Doe’s group at the Howard Hughes Medical Institute utilises this predictable progression of neuronal specification to understand how competence is regulated in cells. As the identity of each cell has been mapped from the embryo through larval stages and on to the adult, NB7-1 gives us a system to understand how cellular identity is fundamentally achieved at the genetic level. So the next time someone flips off fly research within earshot, enlighten them.

So if hb‘s job is to turn on the genes involved in the U1/U2 motorneuron program, why can the first five cells respond, but not the other two? Remember that the DNA is very highly packaged and organised within the cell. There are a number of ways that the cell can organise a specific region of DNA on a chromosome in order to ensure it is ready for activity when necessary. One of the mechanisms involves specifically positioning different regions of chromosomes in different parts of the nucleus. This filing system is not entirely different than that used by most first year university students in determining if clothes are still wearable (I’ll speak for the men here):  the pile of clothes on the floor in the middle of the room (though subject to a smell-test) are fair game. The clothes on the edges of the room; still on their shelves and hung up in the closet, like that sweater your lovely Aunt [so-and-so] knitted you for Christmas last year, are out of the way and generally not (read: never, in the case of the sweater) used. In a similar way, by tethering regions of a chromosome to the edge of the nucleus, to a structure called the nuclear lamina, they’re effectively inaccessible, no matter what transcription factor proteins are available to decode those regions.

Chris Doe’s group recently published findings that show that in the first two progenitors, the hb gene is physically positioned in the interior of the nucleus, a region that promotes its activity. In the 3rd, 4th and 5th progenitors, the gene is silent but remains poised in the interior (ie. it failed the smell-test; it remains on the dorm floor) and responsive to hb-induction. After the 5th division, hb gets repositioned to the edge of the nucleus (right next to the sweater) where it is silenced. This positioning is inherited by any of the future cells in that neuroblast’s lineage, ensuring that they hb protein is no longer produced in these cells. Experimentally interfering with this repositioning enables more cells to respond to the hb signal, resulting in the production in a greater number of U1/U2 motorneurons. This adds to the growing body of examples of genes that are regulated in a developmentally-relevant and heritable manner by tethering to the nuclear lamina. This also provides a mechanistic frame work to understand developmental competence at the molecular level in cells. 

We should have this whole biological-ball-of-wax figured out in about 100 years, or so (that bit is generally always implied in these sorts of assertions). At least that was a brief point that Professor Adrian Bird made at the start of his lecture at the Royal Society before accepting the RSGSK Prize last Tuesday night. It’s an intriguing prospect, but one that largely fell out of the scope of his talk. That said though, if biologists do find themselves out of work a century from now, the blame can be pointed squarely at the likes of Professor Bird. His research has contributed a significant amount to our understanding of one of the mechanisms involved in silencing the activity of genes across the genome.  In the process, he worked out the cause to a severe autism-spectrum disorder affecting one in 10,000 female births and developed a mouse model to help understand it, and potentially reverse it.

Twelve years ago the first draft sequence of the human genome was published and we are only just scratching the surface of understanding all of the information encoded within it. The genome’s publication and the tools born of the genomic era changed the way we approached molecular biology; we turned to more targeted approaches. However it quickly became apparent that there was a lot more information there and a lot fewer genes than we anticipated. The 21,000 (or so) genes that code for proteins proved to be just as numerous as animals we used to stare down our supposedly evolved noses at (read: flatworms).  These, while central as the blueprints to the various parts that make up the cell, are only 1% of the story.

The other 99% is composed of swathes of genetic information strewn throughout the genome that carry instructions on how and when to use the 1%.  It’s a shame economics couldn’t take a page from this arrangement, right? Consortium projects like ENCODE have significantly furthered our understanding how some rest of this genomic DNA works, but large gaps remain in our understanding of how all of this information is organised and utilised inside cells.If you could take all of the DNA that is inside a single human cell, strip it of its protein packaging and arrange the bits from the 23-pairs of chromosomes end-to-end, you would have something in the neighbourhood of two metres in length. All of that fits into a cell nucleus that has a diameter in the order of 10s of microns (that’s one-one hundred thousandth of a metre). Needless to say, one incredible folding act needs to take place to get all of it in there. DNA wraps around a spindle of proteins called histones, forming the repeating unit of DNA architecture that packages those two-ish meters of double helix into chromatin. This nucleosomal structure coils back on itself almost the same way a phone cord will if you start to twist it, packing all of that chromatin in the nucleus rather tightly. The big question is then, how does the cell access the right gene, at the right time, because the process of turning on genes (aka. transcription) requires that they are physically read, and therefore accessible amongst all of that coiling.

Part of this is explained by a field called Epigenetics that really took off in the post-genome era. Its origins go back at least as far as the 1940s-50s, when scientists like Barbara McClintock were discovering that the story of how genes worked didn’t stop at the DNA sequence itself.  She went on to win a Nobel Prize in 1983 for her work on mobile DNA elements that regulate gene expression, even though at the time her work was regarded by some as an eccentricity of Maize genetics. But that’s a whole other post!

Colour variation in maize is regulated by mobile DNA elements in their genome called transposons.

The part of the DNA sequence that immediately precedes that of a gene is called the promoter.  Think of this as a staging ground for the molecular machines that read the genes. In short, the promoter promotes gene activity, thus some or all of the following information can be found there:

  • The When – During what developmental stage should gene-X be active; embryo? child? adult?
  • The Where – In what ‘type’ of cell should gene-X be active; muscle? bone? brain?
  • and The How Much – Is a lot or only a little of gene-product-X needed?

This is nowhere near the whole story at the sequence level, but for now let’s assume it is, as I’m about to add a layer of complexity to it. The prefix epi- in epigenetics comes from Greek, meaning over or on top of as it represents the genetic regulation that supersedes that which is imparted by the DNA sequence, itself. Put another way, epigenetics is like a very elaborate filing system for genetic information in a cell.

Think of it this way, if all cells contain essentially the same set of genetic blueprints, then there have to be ways to mark the genes pertinent to a neuron’s function, for example, as active, while leaving the others who could be either irrelevant or potentially detrimental to neuronal function marked as silent. Epigenetics can be thought of as the collective set of molecular sign posts that index an epigenome, ensuring that only those genes are available to that cell for use when and where they are promoted to do so.

There are principally two ways that these  marks work.  One set involves the marking the histones, the proteins that package DNA into chromatin. These are chemically modified in a number of ways, facilitating a rich code of information that marks genes, where their promoters start, where their coding sequences start/end and in what degree of  readiness for transcription genes should adopt for that particular cell type.  How this code is read by the cell and how the cell organises this information in a functional way inside cells are still very actively researched topics.

The other type involves the direct chemical modification of DNA, called DNA methylation, which is generally associated with gene silencing.  It was this, and the mechanisms that the cell uses to detect this modification that Professor Bird’s group works on.  Certain DNA sequences can be chemically modified by the addition of methyl groups, and these tend to accumulate near the start of gene promoters. As is often the case with biology, when molecules get chemically modified, there’s usually another molecule that is able to read  or recognise this modification.  This, in essence, is how biological information flows in the cell.  One molecule is modified, another binds to it, and that binding effects and outcome.  In this case, methylating the DNA in promoters suddenly makes promoters a target for a protein called MeCP2, which leads to a cascade of events that results in the silencing of that gene. Mutations in MeCP2, as it turns out, are the major cause of Rett Syndrome.  During the lecture, professor Bird showed encouraging results that Rett Syndrome symptoms could be reversed with gene therapy treatment in the animal models within four weeks of treatment (and yes, that’s the same mouse).

Growing up in a university biology programme, things had a certain order: we biologists kept to our corner of the sandbox, playing only occasionally with the chemists and even less with the physicists.  Evenings were another matter, but that falls within the remit of other sites on these here internets. This pedagogically imposed segregation may have been a necessary evil to ensure that burgeoning scientists remained focused while learning the fundamentals to their respective disciplines. And while I’m sure we could argue the pros and cons to this approach in a teaching-then-and-now sense, I can attest to the fact that it has created cohort after cohort of scientists who still adhere to these artificial boundaries, often to the detriment of progress.

So people eventually start to think next-gen; realising that working together helps, and not just for technological progression. INTERDISCIPLINARY becomes a buzz word and suddenly university departments start springing up all over the place; the bastard children of tawdry scientific consorts between pairs of specialties; Bio-Engineers, Molecular-Anthropologists, Mecahno-Compu-Socio-Biologists—oh my! And yes, I know those were all bio-heavy. But come on, this is real science I’m talking about here! [Oh how we still play with each other!]  Most major granting bodies have special categories for partnerships between biology and other facets of science. There I go again. It’s an artificial distinction.

Think of it this way: it’s all about scale.  Physics generally governs the realms that study the interactions of the individual components of matter and energy at the subatomic level. Astrophysicist try to understand how these same principles then apply across scales that describe the cosmos.  As subatomic particles associate to generate atoms of the various elements, we have chemistry.  Atoms begin to interact with each other, interact with energy, and organise into complex structures.  After a sufficient amount of certain types of complexity, you’ll enter the realm of living things; biology. At the moment, I find myself at the nexus of mechanics (physics and engineering) and biology, or mechanobiology.  More specifically, mechanobiology deals with how living systems respond to mechanical forces.

I remember sitting in a talk at Wayne State University given by Neal Pellis when he still worked for NASA about biological experiments being carried out in zero gravity by NASA astronauts thinking, If embryos don’t have gravity, how do they orient themselves; head vs. feet, front vs. back?  The merits of the question itself are less important, but it was probably one of the first times I can remember trying to reconcile biology in the larger context of physics. [SCIENCE BOUNDARY KLAXON!]  Positional information in embryonic development is crucial. This is how embryos can start to build themselves from a single cell.  They form a basic pattern, based often on spacial cues from their environment, in our case a uterus, and build from that basic pattern.  Through migration and interactions with other layers of cells and tissues, cells begin to specify, axes form; front becomes different from back.  But there it is: migration. Cells move. Cells don’t just react to forces, cells generate forces.  [SCIENCE BOUNDARY KLAXON!] Dammit, wish I’d listened more during physics!

My brain began to dole out interdisciplinary olive-branches in ernest when I was in Cambridge, as our group often waxed biophysical, trying to understand how physical forces impacted developing embryos. A friend of mine worked on understanding the rhythm of flows in fluid inside the a fertilised egg. It turns out that these rhythms are established from the moment of fertilisation.  Sperm entry into the egg triggers calcium fluctuations that cause contractions in a network of proteins that force the fluid in the cell, the cytoplasm, to start to flow in sync with the calcium spikes. Moreover, these flows can serve as good predictors of embryo health for mammalian IVF embryos.

But it wasn’t until I came to QM that I started to address some of these questions myself.  I am, at heart, a developmental biologist, so one of the questions I was curious about was how it is that stem cells respond to mechanical forces such as gravity or the pull from a neighbouring cell?  Also, at what point do those mechanical signals start to play a role  in the journey from stem cell to bone, our chief load-bearing tissue in the body, for example.  This is a particularly important question when you consider it in the context of regenerative medicine, where the goal is ultimately starting with a patient cell, reprogramming it into an embryonic-like state, and using that as a therapeutic base to treat disease.  If the patient donates their own cells, there are no issues with rejection.  Currently, though we have the technology to reprogram adult cells into a more embryonic state, theoretically making it possible to create every cell type in the body, we lack the fine control in necessary to coax stem cells into certain fates, such that if we have millions of cells in a petri dish, for example, only a relatively small fraction do what we tell them to do, which raises all sorts of flags about their safety.  This is where understanding how cells respond to all sorts of signals, whether they’re mechanical or chemical, becomes important.

Ultimately what defines one cell type from another is down to its function; usually dictated by the sorts of genes that are turned on, or expressed,  in a cell.  Genes live in the nucleus of the cell. So the general path of information starts with an external (extra-cellular) signal that is transmitted (transduced), that results in some sort of change in what the cell is doing genetically, in the nucleus. A lot of attention has been directed at understanding how this process works biochemically for decades; with molecules binding receptors on the external side of the cell membrane that then trigger signalling cascades that result in a change it he pattern of genes a cell expresses.  As it turns out, mechanical forces may be processed in a similar manner; originating on the outside of the cell and being transduced through the cytoplasm to the nucleus, altering gene expression. This was the subject matter of a recent review that I had the pleasure of co-authoring.

Annual Reviews Biomedical Engineering 14: Mechanical Regulation of Nuclear Structure and Function.

And as a bit of shamless self promotion, what follows are excerpts from the historical section, and one of my figures of the nucleus.  Anyone who’d like a full reprint, please contact me.

Conceptually, the idea that development and differentiation are shaped by extrinsic (mechanical) forces is ancient. Aristotle thought that embryonic development was in part directed by the locomotive component of the soul. This so-called Aristotelian soul differed from the more traditional or spiritual concept of a soul in that it is generated through the (physical) mixing of male and female semen (9), producing a teleological driving force that gradually gestates that individual organism to its final form (10). Teleological reasoning such as this persisted for millennia. However, over time, the notion of the Aristotelian soul as a driving force was either replaced by a divine, perfecting force (11) or obviated altogether by the notion that organisms were preformed and grew with no need for ontological development or differentiation (10). In the mid-1800s, Darwin published his theory describing evolution as a slow and gradual process of descent with modification, resulting in stochastic variability capable of conferring selectable (morphological) advantages to promote survival or increased reproductive fitness of an individual. He included many observations of embryos, as he saw them as “picture[s], more or less obscured, of the common parent-form of each great class of animals” (12, p. 450). Along with this, and more central to this work, he was also among the first to promote a movement away from teleological thinking, suggesting that evolution (and embryology) need not be a process seeking to perfect a morphology, per se (11). This point was later elaborated on by Wilhelm Roux and Julius Wolff (13), in Germany, toward the end of the century.

Wolff was an orthopedic surgeon who conducted research into how the structure of bones changed with alteration of function owing to either growth or pathology. His ideas followed from those of Roux; namely, he maintained that life has two periods, an embryonic one characterized by trophic organ growth and differentiation, and an adult one in which growth and remodeling occurs only when stimulated by healing or (cellular) turnover (13). These stimuli, which Roux collectively referred to as developmental mechanics (11), had an overall effect on tissues irrespective of life period, resulting in their remodeling. Wolff postulated that there was a causal relationship between the physical forces acting on bones and the observed changes to both gross morphology and internal architecture. In particular, he predicted that remodeling of bone trabeculae followed the mathematical trajectories of the forces acting on them. He also went a step further, suggesting that these same mechanical signals could provide one plausible mechanism for Darwin’s theory of natural selection (13). Since the publication of Wolff ’s work, the accuracy of some of his mathematical predictions has been called into question (reviewed in 14). Another common criticism is that his theory cannot be considered a law because other bones and tissues do not exhibit the universality sufficient to explain the phenomena he described in cancellous bone. Despite these detractions, his observations were made well in advance of the discovery of radiography and modern tissue biology techniques. Wolff ’s work is thus deservedly recognized for its pioneering contributions to the burgeoning fields of orthopedics and tissue mechanobiology.

Figure 1, Mechanical Regulation of Nuclear Structure and Function, Annual Reviews Biomedical Engineering 14

Figure 1: The archetypal SUN-KASH domain association in the LINC complex. (a) The LINC complex is typified by an association between SUN-domain proteins ( yellow) on the inner nuclear membrane and KASH-domain proteins ( green, blue, and orange) on the outer nuclear membrane. KASH-domain proteins form a functional link with the networks of cytoplasmic intermediate filaments, microtubules, and actin microfilaments, which compose the cytoskeleton. SUN-domain proteins bind to the nuclear lamina, a network of intermediate filaments composed of varying isoforms of A- and B-type lamins, LAPs, and LRs. The lamina also serves as a tethering point for the genome, with associations reported among various lamins, LAPs, and LRs. (b) KASH domains associate with SUN domains in the perinuclear space, and this association maintains the architecture of the nuclear envelope. SUN proteins can associate promiscuously with KASH proteins and can also form homo- and heterodimers with other SUN proteins. (c) Two competing models explain the 3D organization of CTs in the interphase nucleus. The CT-IC model posits that a largely DNA-free compartment of contiguous spaces between adjacent CTs exists. The second model, known as the intermingling model, maintains that whereas CTs occupy nonrandom spaces in the interphase nucleus, there is a large amount of intermingling of the chromatin between adjacent CTs. Abbreviations: CT, chromosome territory; IC, interchromosomal compartment; KASH, Klarsicht/ANC-1/Syne-1 homology; LAP, lamin-associated protein; LINC, linker of the nucleoskeleton and cytoskeleton; LR, lamin receptor; NPC, nuclear pore complex;SUN, Sad1 and UNC84.

June 2017
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