Putting Body Parts in Their Places

How HOX genes run development, on butterfly wings.

I have written about the HOX complex of genes several times, because they constitute a grail of developmental genetics- genes that specify the identity of body parts. They occupy the middle of a body plan cascade of gene regulation, downstream from broader specifiers for anterior/posterior orientation, regional and segment specification, and in turn upstream of many more genes that specify the details of organ and tissue construction. Each of the HOX genes encodes a transcriptional regulator, and the name of one says it all- antennapedia. In fruit flies, where all this was first discovered, loss of antennapedia converts some legs into antennae, and extra expression of antennapedia converts antennae on the head into legs.

The HOX complex (named for the homeobox DNA binding motif of the proteins they encode) is linear, arranged from head-affecting genes (labial, proboscipedia) to abdomen-affecting genes (abdominal A, abdominal B; evidently the geneticist's flair for naming ran out by this point). This arrangement is almost universally conserved, and turns out to reflect molecular mechanisms operating on the complex. That is, it "opens" in a progressive manner during development, on the chromosome. Repression of chromatin is a very common and sturdy way to turn genes off, and tends to affect nearby genes, in a spreading effect. So it turns out to be easy, in some sense, to set up the HOX complex to have this chromatin repression lifted in a segmental fashion, by upstream regulators, whereby only the head sections are allowed to be expressed in head tissues, but all the genes are allowed to be expressed in the final abdominal segment. That is why the unexpected expression of antennapedia, which is the fifth of eight HOX genes, in the head, leads to a thoracic tissue (legs) forming on the head.

A recent paper delved a little more deeply into this story, using butterflies, which have a normal linearly conserved HOX cluster and are easy to diagnose for certain body part transformations (called homeotic) on their beautiful wings. The main thing these researchers were interested in is the genetic elements that separate one part of the HOX cluster from other parts. These are boundary or "insulator" elements that separate topologically associated domains (called TADs). Each HOX gene is surrounded by various regulatory enhancer and inhibitor sites in the DNA that are bound by regulatory proteins. And it is imperative that these sites be directed only to the intended gene, not neighboring genes. That is why such TADs exist, to isolate the regulation of genes from others nearby. There are now a variety of methods to map such TADs, by looking where chromatin (histones) are open or closed, or where DNA can be cut by enzymes in the native chromatin, or where crosslinks can be formed between DNA molecules, and others.

The question posed here was whether a boundary element, if deleted, would cause a homeotic transformation in the butterflies they were studying. They found, unfortunately, that it was impossible to generate whole animals with the deletions and other mutations they were engineering, so they settled for injecting the CRISPER mutational molecules into larval tissues and watching how they affected the adults in mosaic form, with some mutant tissues, some wild-type. The boundary they focused on was between antennapedia (Antp) and ultrabithorax (Ubx), and the tissues the forewings, where Ubx is normally off, and hindwings, where Ubx is normally on. Using methods to look at the open state of chromatin, they found that the Ubx gene is dramatically opened in hindwings, relative to forewings. Nevertheless, the boundary remains in place throughout, showing that there is a pretty strong isolation from Antp to Ubx, though they are next door and a couple hundred thousand basepairs apart. Which in genomic terms is not terribly far, while it leaves plenty of space for enhancers, promotes, introns, boundary elements, and other regulatory paraphernalia.

Analysis of the site-to-site chromosomal closeness and accessibility across the HOX locus of the butterfly Junonia coenia. The genetic loci are noted at the bottom, and the site-to-site hit rates are noted in the top panels, with blue for low rates of contact, and orange/red for high rates of contact. At top is the forewing, and at bottom is the hindwing, where Ubx is expressed, thus the high open-ness and intra-site contact within its topological domain (TAD). Yet the boundary between Ubx and Anp to its left (dotted lines at bottom) remains very strong in both tissues. In green is a measure of transcription from this DNA, in differential terms hindwing minus forewing, showing the strong repression of Ubx in the forewing, top panel.

The researchers naturally wanted to mutate the boundary element, (Antp-Ubx_BE), which they deduced lay at a set of binding sites (featuring CCCTC) for the protein CTCF, a well-known insulating boundary regulator. Note, interestingly, that in the image above, the last exon (blue) of Ubx (transcription goes right to left) lies across the boundary element, and in the topological domain of the Antp gene. This means that while all the regulatory apparatus of Ubx is located in its own domain, on the right side, it is OK for transcription to leak across- that has no regulatory implications. 

Effects of removing the boundary element between Ubx and Antp. Detailed description is in the text below. 

Removal of this boundary element, using CRISPER technology in portions of the larval tissues, had the expected partial effects on the larval, and later adult, wings of this butterfly. First, note that in panel D insets, the wild type larval forewing shows no expression of Ubx, (green), while the wild type hind wing shows wide-spread expression. This is the core role of the HOX locus and the Ubx gene- locate its expression in the correct body parts to then induce the correct tissues to develop. The larval wing tissue of the mosaic mutant, also in D, shows, in the forewing, extensive patchy expression of Ubx. This is then reflected in the adult (different animals) in the upper panels, in the mangled eyespot of the fully formed wing (center panel, compared to wild-type forewing and hindwing to each side). It is a small effect, but then these are small mutations, done in only a fraction of the larval cells, as well.

So here we are, getting into the nuts and bolts of how body parts are positioned and encoded. There are large regions around these genes devoted to regulatory affairs, including the management of chromatin repression, the insulation of one region from another, the enhancer and repressor sites that integrate myriad upstream signals (i.e. other DNA binding proteins) to come up with the detailed pattern of expression of these HOX genes. Which in turn control hundreds of other genes to execute the genetic program. This program can hardly be thought of as a blueprint, nor a "design" in anyone's eye, divine or otherwise. It resembles much more a vast pile of computer code that has accreted over time with occasional additions of subroutines, hacks, duplicated bits, and accidental losses, adding up to a method for making a body that is robust in some respects to the slings and arrows of fortune, but naturally not to mutations in its own code.

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Hungary for Power

Hungary has become a one-party, authoritarian state, not a democracy.

Victor Orban recently paid a visit to Donald Trump in Florida, with glowing photos and pledges of goodwill. Republicans in the US have nurtured a deep fascination and alliance with Orban and his government, holding several CPAC conventions in Hungary, and hosting Orban and his lieutenants at US events. It is clear that they view Hungary as a shining example and template of where they could take the US. Not the shining city on a hill of Reagan's democratic and anti-authoritarian dreams, but a whole other kind of city, one that never will fall into Democratic hands again.

So it is worth looking in detail at what has happened in Hungary, to observe the ideals of our current Republicans and what is in store for the rest of us from a second Trump term. I was, incidentally, beaten to the punch of this analysis by a recent story in the Atlantic. Orban's party, Fidesz, is very similar to the GOP in its mix of business right-wingery and rural values. Its strength is handing out the red meat of traditional, anti-cosmopolitan values to the rural base, along with helpful economic subsidies. In the pivotal 2018 election, it won all the rural districts, even though the opposition bowed to the logic of re-written (winner-take-all) electoral system and tried to join into a unified party. 

Fidesz came to power originally on an anti-socialist platform, vowing to get rid of the remnant bits of the prior communist system, which had settled into the same kind of semi-kleptocratic mode as in most of the former Soviet states and its satellites. That they did, but not to bring an end to corruption, let alone authoritarianism, but rather to partake themselves instead. After coming into power, Fidesz rewrote the constitution, in ways large and small to entrench their own power, and has since continued a campaign of extremely effective, gradual, and often surreptitious legislation to cement its advantages. Gerrymandering is now standard procedure, which when combined with the winner-take-all districts creates the opportunity to win overwhelming majorities in parliament founded on very thin electoral pluralities. Small parties can not win any more, but are also prohibited from combining with other small parties into election list coalitions.

The courts were remade by putting them under the control of a political appointee- the president of the National Judicial Office. This president is appointed by parliament, and in turn appoints, promotes, and runs the operations and budget of the whole judicial system. Needless to say, it is heavily influenced by the now Fidesz-controlled parliament and executive.

The media has been remade by gradual pressure on independent media owners to sell to Fidesz-friendly interests, which now control 90% of the country's media. Government advertising buys were strategically placed with friendly outlets, and government run media was put under direct political control. A Russian inspired "security" law was passed to outlaw ill-defined criticism of the state, public morality, or "imbalance" of coverage, answerable naturally to a parliamentary-appointed body, rather than the courts. Imagine if in the second Trump administration, PBS and NPR were put under political control and given a "FOX" makeover. 

Hungary is now effectively a one-party authoritarian state with managed elections. We are not far off. To see the battle of titanic interests and billionaires now openly showering money on favored candidates, and extending their tentacles down to the school board level, is sickening. The Republican party has partnered with Heritage foundation to offer an openly Orbanist plan for the second Trump administration. The court system has already re-written our constitution in extensive ways over the last four years, without an amendment being passed, or even proposed. The antics of Judge Eileen Cannon show that very little may remain of the rule of law if it is left in the hands of partisan extremists.

And our media is in even more perilous condition, with the relentless lying of FOX, Sinclair, and their ecosystem. The Republican convention just past was a pageant of lies and grift, betokening the criminal enterprise that party has turned into. Headed by their adored, and now divine, leader who is not just a felon and business fraud, but rapist and insurrectionist as well. But no matter. With enough money, and enough shamelessness, anything is possible.

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The Long Tail of Genome Duplication

A new genomic sequence of hagfish tells us a little about our origins.

Hagfish- not a fish, and not very pretty, but it occupies a special place in evolution, as a vertebrate that diverged very early (along with lampreys, forming the cyclostome branch) from the rest of the jawed vertebrates (the gnathostome branch). The lamprey has been central to studies of the blood clotting system, which is a classic story of gradual elaboration over time, with more steps added to the cascade, enabling faster clotting and finer regulation.

A highly schematic portrayal (not to scale!) of the evolutionary history of animal life on earth.

A recent paper reported a full genome sequence of hagfish, and came up with some interesting observations about the history of vertebrate genomes. At about three billion nucleotides, this genome is about as large as ours. (Yet again, size doesn't see, to matter much, when it comes to genomes.) They confirm that lampreys and hagfish make up a single lineage, separate from all other animals and especially from the jawed vertebrates. For example, though lampreys have 84 chromosomes to the hagfish's 17, this resulted from repeated splitting of chromosomes, and each lamprey chromosome can be mostly mapped to one hagfish chromosome, accepting that a lot of other gene movement and change has taken place in the roughly 460 million years since these lineages diverged. 

Hagfish (bottom) and lamprey (top) chromosomes pretty much line up, indicating that despite the splitting of the lamprey genome, there hasn't been a great deal of shuffling over the intervening 460 million years.

The most important parts of this paper are on the history of genome duplications that happened during this early phase of vertebrate evolution. Whole genome duplications are an extremely powerful engine of change, supplying the organism with huge amounts of new genetic material. Over time, most of the duplicated genes are discarded again (in a process they call re-diploidization). But many are not, if they have gained some foothold in providing more of an important product, or differentiated themselves from each other in some other way. Our genomes are full of families, some extremely large, of related genes that have finely differentiated functions. Many of these copies originated in long-ago genome duplications, while others originated in smaller duplication accidents. It is startling to hear from self-labeled scientists in the so-called intelligent design movement that there is some rule or law against such copying of information, by their ridiculous theories of specified information. Hagfish certainly never heard of such a thing.

At any rate, these researchers confirm that the earliest vertebrate lineage, around 530 million years ago, experienced two genome duplications which led to a large increment of new genes and evolutionary innovation. What they find now is that the cyclostome lineage experienced another genome three-fold duplication (near its origin, about 460 million years ago, leading to another round of copies and innovation. And lastly, the gnathostome lineage separately experienced its own genome four-fold duplication around the same time, after it had diverged from the cyclostome lineage. One might say that the gnathostomes made better use of their genomic manna, generating jaws, teeth, ears, thymus, better immune systems, and the other features that led them to win the race of the animal kingdom. But hagfish are still around, showing that primitive forms can find a place in the scheme of things, as the biosphere gets larger and more diverse over time.

A classic example of gene replication is the Hox cluster, which are a set of genes that have the power of dictating what body part occurs where. They are gene regulators that function in the middle of the developmental sequence, after determination of the overall body axis and segmentation, and themselves regulating downstream genes governing features as they occur in different segments, such as limbs, parts of the head, fingers, etc. Flies have one Hox cluster, split into two parts. The extremely primitive chordate amphioxus, which far predates the cyclostomes, also has one complete Hox cluster, as diagrammed below. Most other vertebrates, including us, have four Hox clusters, amounting to over thirty of these transcription regulators. These four clusters arose from the inferred genome duplications very early in the vertebrate lineage, prior to the advent of the cyclostomes. 

Hox clusters and their origins, as inferred by the current authors. The red/blue points at the left mark whole genome duplications (or more) that have been inferred by these or other authors. More description is in the main text below.

The inferred genome duplications during early chordate evolution, noted on the far left of the diagram above, led to duplicated clusters of Hox genes. Amphioxus (top) is the earliest branching chordate, and has only one full Hox cluster of transcription regulators, which, in general terms, control, during development, the expression of body parts along the body axis, with the order of genes in the cluster paralleling expression and action along the body axis. Chicken as a gnathostome has four copies of the cluster, with a few of the component genes lost over time. Hagfish have six copies of this Hox cluster, some rather skeletal, stemming from its genome duplication events. Clearly several whole clusters have also been lost, as in some cases the genome duplications experienced by the cyclostomes resolved back to diploidy without leaving an extra copy of this cluster. The net effect is to allow all these organisms greater options for controlling the identity and form of different parts of the body, particularly, in the case of gnathostomes, the head.

Genome duplications are one of those fast events in evolution that are highly influential, unlike the usual slow and steady selection and optimization that is the rule in the Darwinian theory. Unlike mass extinction, another kind of fast event in evolution, genome duplications are highly constructive, providing fodder on a mass (if microscopic) scale for new functions and specializations that help account for some of the more rapid events in the history of life, such as the rise of chordates and then vertebrates in the wake of the Cambrian explosion.

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Living on the Edge of Chaos

 Consciousness as a physical process, driven by thalamic-cortical communication.

Who is conscious? This is not only a medical and practical question, but a deep philosophical and scientific question. Over the last decades and century, we have become increasingly comfortable assigning consciousness to other animals, in ever-wider circles of understanding and empathy. The complex lives of chimpanzees, as brought into our consciousness by Jane Goodall, are one example. Birds are increasingly appreciated for their intelligence and strategic maneuvering. Insects are another frontier, as we appreciate the complex communication and navigation strategies honeybees, for one example, employ. Where does it end? Are bacteria conscious?

I would define consciousness as responsiveness crosschecked with a rapidly accessible model of the world. Responsiveness alone, such in a thermostat, is not consciousness. But a nest thermostat that checks the weather, knows its occupant's habits and schedules, and prices for gas and electricity ... that might be a little conscious(!) But back to the chain of being- are jellyfish conscious? They are quite responsive, and have a few thousand networked neurons that might well be computing the expected conditions outside, so I would count them as borderline conscious. That is generally where I would put the dividing line, with plants, bacteria, and sponges as not conscious, and organisms with brains, even quite decentralized ones like octopi and snails, as conscious, with jellyfish as slightly conscious. Consciousness is an infinitely graded condition, which in us reaches great heights of richness, but presumably starts at a very simple level.

These classifications imply that consciousness is a property of very primitive brains, and thus in our brains, is likely to be driven by very primitive levels of its anatomy. And that brings us to the two articles for this week, about current theories and work centered on the thalamus as a driver of human consciousness. One paper relates some detailed experimental and modeled tests of information transfer that characterizes a causal back-and-forth between the thalamus and the cortex, in which there is a frequency division between thalamic 10 - 20 Hz oscillations, whose information is then re-encoded and reflected in cortical oscillations of much higher frequency, at about 50 - 150 Hz. It also continues a long-running theme in the field, characterizing the edge-of chaos nature of electrical activity in these thalamus-cortex communications, as being just the kind of signaling suited to consciousness, and tracks the variation of chaoticity during anesthesia, waking, psychedelic drug usage, and seizure. A background paper provides a general review of this field, showing that the thalamus seems to be a central orchestrator of both the activation and maintenance of consciousness, as well as its contents and form.

The thalamus is at the very center of the brain, and, as is typical for primitive parts of the brain, it packs a lot of molecular diversity, cell types, and anatomy in a very small region. More recently evolved areas of the brain tend to be more anatomically and molecularly uniform, while supporting more complexity at the computational level. The thalamus has about thirty "nuclei", or anatomical areas that have distinct patterns of connections and cell types. It is known to relay sensory signals to the cortex, to be central to sleep control and alertness. It sits right over the brain stem, and has radiating connections out to, and back from, the cerebral cortex, suggestive of a hub-like role. 

The thalamus is networked with recurrent connections all over the cortex.

The first paper claims firstly that electrical oscillations in the thalamus and the cortex are interestingly related. Mouse, rats, and humans were all used as subjects and gave consistent results over the testing, supporting the idea that, at very least, we think alike, even if what we think about may differ. What is encoded in the awake brain at 1-13 Hz in the thalamus appears in correlated form (that is, in a transformed way) as 50-100+ Hz in the cortex. They study the statistics of recordings from both areas to claim that there is directional information flow, not just marked by the anatomical connections, but by active, harmonic entrainment and recoding. But this relationship fails to occur in unconscious states, even though the thalamus is at this time (in sleep, or anesthesia) helping to drive slow wave sleep patterns directly in the cortex. This supports the idea that there is a summary function going on, where richer information processed in the cortex is reduced in dimension into the vaguer hunches and impressions that make up our conscious experience. Even when our feelings and impressions are very vivid, they probably do not do justice to the vast processing power operating in the cortex, which is mostly unconscious.

Administering psychedelic drugs to their experimental animals caused greater information transfer backwards from the cortex to the thalamus, suggesting that the animal's conscious experience was being flooded. They also observe that these loops from the thalamus to cortex and back have an edge-of-chaos form. They are complex, ever-shifting, and information-rich. Chaos is an interesting concept in information science, quite distinct from noise. Chaos is deterministic, in that the same starting conditions should always produce the same results. But chaos is non-linear, where small changes to initial conditions can generate large swings in the output. Limited chaos is characteristic of living systems, which have feedback controls to limit the range of activity, but also have high sensitivity to small inputs, new information, etc., and thus are highly responsive. Noise is random changes to a signal that may not be reproducible, and are not part of a control mechanism.

Unfortunately, I don't think the figures from this paper support their claims very well, or at least not clearly, so I won't show them. It is exploratory work, on the whole. At any rate, they are working from, and contributing to, a by now quite well-supported paradigm that puts the thalamus at the center of conscious experience. For example, direct electrical stimulation of the center of the thalamus can bring animals immediately up from unconsciousness induced by anesthesia. Conversely, stimulation at the same place, with a different electrical frequency, (10 Hz, rather than 50 Hz), causes immediate freezing and vacancy of expression of the animal, suggesting interference with consciousness. Secondly, the thalamus is known to be the place which gates what sensory data enters consciousness, based on a long history of attention, ocular rivalry, and blind-sight experiments.

A schematic of how stimulation of the thalamus (in middle) interacts with the overlying cortex. CL stands for the ventral lateral nucleus of thalamus, where these stimulation experiments were targeted. The greek letters alpha and gamma stand for different frequency bands of the neural oscillation.

So, from both anatomical perspectives and functional ones, the thalamus appears at the center of conscious experience. This is a field that is not going to be revolutionized by a lightning insight or a new equation. It is looking very much like a matter of normal science slowly accumulating ever-more refined observations, simulations, better technologies, and theories that gain, piece by piece, on this most curious of mysteries.

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