Author Archives: natselrox

Building Brains: Symmetry, Synapses, and Shakepeare

This column will be about the brain, the gooey three pound jelly-like substance inside our skulls. Appearances can be deceptive, for this is quite possibly one of the most complex structures you will ever come across. All our memories, our knowledge, our hopes, dreams, aspirations, our beliefs, our likings, dislikings, our passions, our love, our hatred, almost everything that make us who we are are but activities in this lump of jelly. The billions of cells and trillions of connections that make up this structure are buzzing with activity throughout our lives. In fact, their activities are manifested as what we call Life. As one of my favourite bloggers put it, “”All life is here” in those tangled little fibres.”[1]. To understand life, we must understand the brain. In this column, I will try to pick up one fascinating story from neuroscience research every month and I will try and elaborate on it. My primary aim will be to present the research in a broader context and explain in the process why this matters in the bigger scheme of things, how the research is going to be useful in expanding our knowledge about how the brain (and, in a way, the universe) works. I will try and convey the sense of wonder and beauty that drives most of scientific research and I hope they will be contagious enough to inspire the reader to pursue science, if not as a career then at least as more than a passing interest.

 

This article is about how brains are built. The science that studies it is called Developmental Neurobiology and it is one of the most popular and active disciplines right now. Thousands of peer-reviewed articles are published each year[2]. So you can guess that it is impossible to give a flavour of the entire discipline in this one article. But every single discovery, every single article is actually fascinating in its own way and as is always the case in science, intricately intertwined with the bigger tapestry of our understanding. In this article, we will focus on one single aspect of neuroembryology (that’s another name of this subject) called Neurogenesis. As I mentioned in the introduction, I’ll try to pass on the enthusiasm that makes Developmental Neurobiology my favourite subject!

All of life starts from a single cell. From the humblest of unicellular creatures to the gigantic blue whale, each one of us started our lives as a single cell. So how is it that the single cell could divide itself in such an orchestrated manner to give rise to something so wonderful and complex? Shouldn’t all the daughter cells arising from that be identical copies of each other? How is this information regarding the fate of individual daughter cells transmitted? Turns out that the answer lies in asymmetry. If all the cell divisions, distribution of intracellular products and the extracellular environmental parameters were symmetrical, there would be absolutely no way to differentiate between the daughter cells. Symmetry has a lower information content than asymmetry. Once you develop a gradient of asymmetry, you can work on it and amplify it to regulate the flow of information in a very specific way. The initial bootstrapping through asymmetry is thus key to all of life.

 

Most of the studies in this field have been done in fruit flies, transparent nematode worms and vertebrates like xenopus and zebra-fish. But remarkably, the basic developmental processes in these organisms seem to be highly conserved throughout evolution. We have homologues of most of fundamental processes found in these simple creatures in the higher vertebrates. So although I will chiefly be discussing Drosophila research today, the conclusions we draw can be useful in understanding how the human nervous system is formed.

 

The initial asymmetry in Drosophila embryogenesis is established even before the fertilisation of the oocyte (the fruit-fly equivalent of egg) occurs. The nurse cells that surround the egg secrete substances that are imbibed by the oocyte and asymmetrically distributed in it. Among these substances are genes called bicoid (bcd) and oskar (osk) that establish the antero-posterior axis through their concentration gradients. Other genes called dorsal, cactus and toll establish the dorso-ventral polarity. Now after fertilisation the first genes that are expressed in the zygote (members of a class called the gap genes) arrange themselves in a pattern along this pre-established antero-posterior axis. We have the start of asymmetric life with coded spatial patterns. This anteroposterior patterning is then further reinforced by the expression of pair-rule genes, Hox genes and segment-polarity genes.

 

The development of the nervous system proper starts much later in the life cycle of an organism. But the same basic principle of asymmetric cell division plays a pivotal role in there as well.

 

The majority of functions of the nervous system are controlled at the most basic level by highly specialized cells called Neurons. Now, in order to generate the enormous diversity of function and connectivity in the nervous system, it is imperative that each neuron must be specialized to carry out a specific task. As a result, the neurons show tremendous variety in cellular structure, physiological functions, chemical properties and connectivity. Even the cells from a single region in the brain vary from each other in different aspects. For example, the granule cells of the cerebellum (the part of the brain that plays an important role in motor control) vary significantly in morphology and chemistry from the Purkinje cells of the same region. Similarly, the motor neurons, despite their structural and chemical similarities, have different molecular attributes that make them connect with specific muscle fibers resulting in the precision of movements that we see. This process of developing highly specialized neurons from comparatively similar precursor cells is called Neurogenesis. Question is, how is it done? What determines the fates of neurons?

Sydney Brenner once jokingly said that neurons are either European or American. The fate of an European neuron is mostly determined by its family lineage whereas that of an American neuron is largely shaped by the surroundings in which it grows up! As a general rule it is often said invertebrate neurons mostly belong to the former class whereas the ones in vertebrates fall in the later (any inference drawn herefrom is purely coincidental!). However, a close inspection tells us that the situation is not as binary as it looks. A cell is projected into a definite developmental trajectory by the environmental conditions that it is subjected to. So by default its daughter cells are only allowed to maneuver within that trajectory but with an enormous amount of variability (both reversible and irreversible) provided by the environment that they are now subjected to. More technically, this is controlled by two main processes called spatial patterning and temporal regulation of birth-dates. That is, the spatially coded patterns that surround a cell and the time of the final division that gives rise to it are most pivotal in determining its fate. These result in the cells expressing different transcription factors (regulatory proteins that cause expression of different genes) which ultimately determine their fates.

Spatial control of cell fate determination is all about making the right neurons at the right place. As we saw earlier, the Drosophila embryo is finely subdivided in the anterior to posterior axis into stripes of expressions of gap genes, pair-rule genes, Hox genes and segment-polarity genes. Now the neuroblasts (cells that eventually give rise to the neurons and all other classes of cells in the nervous system) are provided with intrinsic positional information by these genes. These anterio-posterior positional identity genes play important role in determining the identity of the neuroblasts.

 

The first of these spatially coded domains are initially specified by a gradient of the signaling molecule Sonic hedgehog (Shh). Now there’s an interesting story behind the nomenclature of this molecule. For those of you who were avid video-game fans in the 1990’s, must remember Sonic, the hedgehog. The gene was indeed named after the same character. The hedgehog genes were initially called so because mutations in them caused bristled appearances of the Drosophila larvae. The custom was to name the genes after different species of hedgehogs but when this gene was discovered, one of the postdocs in Clifford Tabin’s lab requested to name it after the popular Sega video-game character. Some had reservations about it but the naming was carried out anyway. There are criticisms for naming it thus, as mutations in the gene (actually, its homologue in humans) causes a serious condition known as holoprosencephaly. It seems rather cruel to name a gene after a video game character when mutations of it can cause such devastating conditions in children. But we are digressing from the main story. For a lively discussion of this through models and live demonstrations, watch the Howard Hughes Medical Institute Lecture on the basics of neuroembryology.

 

The progenitors of the neural tube are highly sensitive to the concentration of Shh, and this results in the graded expression of a group of transcription factors. We have a cascade of transcription factors being expressed in a directional manner being started by the initial asymmetry in concentration gradient like we mentioned earlier. Similarly there are other set of genes that divide the embryo and the nervous system along the dorsoventral axis. So you can imagine a sort of grid system being established where a neuroblast in any position can be uniquely identified by expression of these spatial coordinate markers of latitude and longitude. Genes specifying positional information along these two axes confer a positional identity to each of the neuroblasts in the developing nervous system. Once thus being expressed in a neuroblast, the spatial coordinate genes are inherited by all its progenies and they bear the indelible stamp of their birthplaces.

Having thus acquired their positional identities, each neuroblast divides asymmetrically to produce a copy of itself and a cell called the Ganglion Mother Cell (GMC). The neurobalst then goes on to divide further to give rise to a set of GMCs. But the really cool part is that each GMC can be identified not only by the positional information that it inherits from the neuroblast, but also from the order of its generation (that is, whether it was the first, second, or third GMC to be arising from a neuroblast). This temporal coding of information is carried out through a program of transcription factor expression. When the first GMCs are generated, the neuroblasts express a transcription factor called hunchback (hb). Later, they turn off the expression of this gene and turn on another one called Krueppel (kr). GMCs formed at the respective stages thus acquire these transcription factors. The expression of these transcription factors are linked to the cell cycle which functions as a kind of clock. Thus, in addition to the information about the place of their origins, the GMCs also carry with them information about the time of their birth. But it gets more interesting from here.

 

Typically, the progeny of a neuroblast inherits the temporal and spatial coordinates expressed by the parent at its time of birth. However, often the parent divides asymmetrically to give the intrinsic determinants to one of the daughter and not the other. How does a cell accomplish this feat of partitioning information asymmetrically among the daughter cells? The answer, it turns out, has a touch of Shakespeare to it!

 

Two factors, Numb and Prospero (Pro) play a pivotal role in the asymmetric distribution of determinants of cell identity. During the time of division, these tow factors are concentrated in the smaller daughter cell, the GMC, where Prospero enters the nucleus and determine the fate of the GMC. Numb, on the other hand, blocks a signaling pathway that renders the GMC free to move down the determination pathway. But how do Numb and Prospero get asymmetrically distributed in the cell in the first place? Two proteins called Inscuteable (Insc) and Bazooka (Baz) form a complex known as the Insc complex which binds to the apical membrane of the neuroblast and this complex causes the mitotic spindle (that’s like the apparatus that pulls different substances into the daughter cells during cell division) to be arranged vertically. In conjunction with the actin-based cytoskeleton (that’s like the transporter system of cells!) mechanism, this complex drives the distribution of several proteins along the vertical axis so that they are asymmetrically distributed. In particular, a cytoplasmic protein called Miranda is enriched at the basal neuroblast pole and it binds with Numb and Prospero to result in their asymmetric distribution. Wait a second! Miranda binds Prospero? What’s going on in here?

 

When I first read about these proteins, I was pleasantly surprised to find the Shakespeare reference. What on earth is Tempest doing in the middle of a Developmental Genetics textbook? I mailed  Prof. Chris Doe, the guy behind this fascinating nomenclature and asked him about the inspiration behind it. He said something wonderful, “You are right, they are from the Tempest:  Prospero the magician = the controller of fates!”[3] In a befitting tribute to The Bard, we have named the determiner of the fate of a neuroblast, which in a way is key to determining the fate of most of life, after the magician in The Tempest.

 

We are nearly at the end of our story. We have seen how information is passed on to the developing nervous system in a wonderfully coordinated manner. We have seen how neurons that will eventually define who we are become who they are. We have seen how their fates are determined by their origins and the environment they grow up in. We have seen temporal and spatial patterns giving rise to diversity in the nervous system. We have glimpsed into the most magnificent process in the universe where the most complicated computational device in the whole universe is being formed through self-assembly.

 

It has often been asked, what is the utility of studying this? We have our defences ready and we say that an understanding of neural cell fate determination will be important in understanding and treatment of neural diseases and injuries. In the future, we might be able to develop molecular therapies to repair damages in the nervous system. We might even be able to develop specific neurons from stem cells to use them in transplantation therapy.  More importantly, an understanding of this will be able to help us understand neurodevelopmental disorders like the autistic spectrum disorders better.

 

But I think there is a better reason to do all these. Science is in her uninhibited best when she is curiosity-driven. What better reason to pursue a career in research than to be able to name an all-powerful gene after the name of your favourite literary (or video-game) character? What better thing to study than the making of the mind? What better way to understand life than to watch it being formed? We don’t always to need to justify our passions in an utilitarian framework. Science is our most reliable probe into the nature of reality and pursuing it is, in my opinion, one of the best ways to spend the brief amount of time we spend on this planet.

 

Hope you will like this column. More interesting stories about the three pound jelly next month. Till then, have a great time!

References:

 

1. From Neuroskeptic’s wonderful blogpost on the Morgellon’s disease: http://neuroskeptic.blogspot.com/2011/02/web-of-morgellons.html

 

2. About 25,000 articles were published on Developmental Neurobiology in the years between 2000 and 2004 (source: Development of the Nervous System by Sanes and Reh). We can only expect the number to be much higher than that in the last five years or so.

 

3. From the same email conversation with Prof. Doe, I also learnt that now there is a Caliban as well (PMID: 16103875). Shakespearem, it seems, is quite popular among developmental biologists!

 

Most of the other information in the article are from the following books:

 

Principles of Developmetal Genetics (edited by Sally A. Moody)

Development of the Nervous System (by Sanes and Reh)

Developmental Neurobiology (by Rao and Jacobson)

Principles of Neural Development (by Purves and Lichtman) (This book is currently out of print but you can download a copy for free from Purves’ website here)

 

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Large scale synchronisation of biosensors using synergistic coupling

One of the major hurdles in synthetic biology has been the construction of bio-sensors that are accurate over a large scale and that can be monitored without the aid of sophisticated microscopes. Getting a bacteria to signal in one way or another when it encounters an environmental toxin (or something else) is one thing but getting a colony of bacterial cells to signal together in synchrony is a whole lot more difficult. Orchestrating thousands of colonies of bacterial cell is an entirely different ball-game. Yet this is exactly what a group of scientists from the University of California, San Diego have achieved. They report their findings in an article published in the Advanced Online Publications section of Nature. And this is the article that I want to briefly review over here.

To synchronise the bacterial cells within a colony they tapped into a natural mechanism used by bacteria to communicate among themselves, quorum sensing. However, quorum sensing is only effective over a  range of a few micrometers. So they used a different communication molecule, one that is in the gaseous phase and hence can diffuse quickly to longer distances. But gaseous  molecules are weak and short-lived, being in the vapour phase, they disperse a lot quicker. However when these two communicating mechanisms were synergistically coupled, the results were quite surprising.

To achieve the intracellular coupling they used quorum sensing involving acyl-homoserine lactone (AHL). The principle here is quite simple, each bacterial cell synthesises a certain amount of AHL which acts as an inducer. When this inducer binds with a receptor it causes the expression of certain genes which among other products also include the genes for synthesizing the inducer itself. Thus the inducer is technically inducing its own production, rendering it an autoinducer. However the amount of this inducer molecule, AHL in this case, produced by any bacterial cell is not enough to initiate this positive feedback loop. It is only when that the colony reaches a certain size and when a certain critical concentration of the autoinducer has been achieved, that the expression of the downstream proteins occur. As a result, if you insert fluorescent proteins among them, you get a colony of bacterial cells fluorescing in unison. But mind you, this only gets you a coordinated bacterial colony over a very small range. You need something else to scale this up.

So they put a copy of a gene coding for NADH dehydrogenase ll (ndh) under the control of another lux promoter. Now NADH dehydrogenase II is a respiratory enzyme that produces low levels of H2O2   and superoxide. Now H2O being a in the vapour state is able to pass between the 25 micron thick PDMS walls that is used to separate the colonies. This H2O, being a reactive oxygen species, initiates a defence mechanism in bacterial cells once it enters them. Among the different global regulatory systems that mediate this defence mechanism is one called the ArcAB system, which (lo and behold!) has a binding site in the lux promoter region. Under normal conditions, ArcAB is partially active, rendering the lux promoter partially repressed. But once H2O enters the cell, ArcAB swings into action, thus activating the lux promoter and hence initiating the same quorum sensing mediated communication system and hence synchronising this new colony. Now the researchers did a lot of tests to confirm that it was the external H2O, and not endogenously produced molecules, which brought about the desired effects. Describing them is out of scope here but the results confirmed their hypothesis.

So what it is that they finally achieved? Synchronisation of approximately 2.5 million cells over a distance of about 5 mm, exhibiting consistent oscillation with a temporal accuracy of 2 minute compared to the 5-10 minute accuracy of a single oscillator!

The paper includes a bit more about the construction of an arsenic detecting biosensor using this technique but I don’t think that’s entirely relevant here. For a brief description of the experiment and the potential applications of it, watch the video:

 

 

Merry Mythmas and Happy New Year to all our readers!

– Debayan

 

Optic-cup morphogenesis in vitro

One of the more landmark papers in Stem Cell Biology/Regenerative Medicine was published in Nature last week. A group of Japanese scientists managed to take Embryonic Stem cells (ES cells) in a three-dimensional culture medium and managed to produce an almost entire mouse retina (the optic cup). I’d like to provide a basic overview of the research and its potential implications.

The retina is the photosensitive tissue which lines the inner surface of your eye. When light falls on the retina, it initiates a cascade of reactions in the appropriate cells which result in nerve impulses that travel via the optic nerve to the brain and create the visual experiences that we have and mediate other light responses. The layers of the retina are shown in this diagram. Notice how the light enters from below and has to pass through a jungle of neurons before they actually reach the rods and the cones which are the actual photosensitive components of the retina. This sloppy design actually betrays the humble evolutionary origin of the structure where nature never had the chance to go back to the drawing board and redesign it altogether but had to build up on whatever inefficient infrastructure it had.

 

The complexity of the structure, as evident from the picture has always been of considerable interest to biologists. The phylogenetic roots of the structure has been a hot topic in creationist circles and it is probably due to the most popular quote-mine of all time in biology. Charles Darwin wrote in 1872,

To suppose that the eye, with all its inimitable contrivances for adjusting the focus to different distances, for admitting different amounts of light, and for the correction of spherical and chromatic aberration, could have been formed by natural selection, seems, I freely confess, absurd in the highest possible degree.

However, the part that follows soon is often left out!

Yet reason tells me, that if numerous gradations from a perfect and complex eye to one very imperfect and simple, each grade being useful to its possessor, can be shown to exist; if further, the eye does vary ever so slightly, and the variations be inherited, which is certainly the case; and if any variation or modification in the organ be ever useful to an animal under changing conditions of life, then the difficulty of believing that a perfect and complex eye could be formed by natural selection, though insuperable by our imagination, can hardly be considered real. How a nerve comes to be sensitive to light, hardly concerns us more than how life itself first originated; but I may remark that several facts make me suspect that any sensitive nerve may be rendered sensitive to light, and likewise to those coarser vibrations of the air which produce sound.

The phylogeny of the eye has now been researched in quite detail and no sane person now denies the claim that the eye evolved (a number of times) in different species across the animal kingdom. The ontogeny is still pretty much in the dark but in the aforementioned experiment, scientists have, for the first time, demonstrated that the organogenesis is more-or-less a self-organising process.

So how did they do it?

ES cells are pleuripotent cells meaning that they have the capacity to form almost any structure in the body. Under proper circumstances, these cells differentiate into progenitor cells appropriate for the development pathway involved. This is achieved by a complicated interplay of gene-expression and shaping of the epigenetic landscape. The process was previously thought irreversible but more recent experiments suggest that it can be reversed under certain experimental conditions. The gene-expression profile of the progenitor cells provide us with a tool to identify them. In this experiment, they tagged the genes which are specifically expressed in the retina with green fluorescent protein in order to track them.

So they took a culture medium (called the Serum free floating culture of the SFEB-q type) and cultured the ES cells in them. With the treatment of activin, retinal differentiation could be induced in this culture sans the epithelium. Further treatment with basement membrane matrix components led to the epithelial formation. A high percentage of the total cells also became positive for the retinal cell markers (as evident from their glowing due to the green-fluorescent protein tagging). A similar induction if retinal cells could also be achieved by treating the culture with a concoction of purified laminin and entactin.

What followed from there was more or less on autopilot mode with a few tweaking involved here and there. By the sixth day, the culture had separated into regions of retinal and other cell types. On day 7, the retinal cell aggregates showed formation of hemospherical epithelial vesicles evaginating from the main body with upto four vesicles per aggregrate. On days 8-10, the vesicles underwent a dynamic shape change and formed a two-walled cup-like structure. The distal portion of the epithelium progressively folded to give rise to something very similar to the optic cup in the embryo. It then exhibited interkinetic nuclear migration and subsequently generated stratified neural retinal tissue as seen in the live organism! The proximal portion differentiated into mechanically rigid pigment epithelium with a marker profile reminiscent of that of the retinal pigment epithelium progenitors.

The process is absolutely fantabulous to watch and you can do so by going to the Nature website and clicking on the “Movies” tab of the “Supplementary Information” page of the article. If not anything else, green fluorescent structures folding onto themselves is a pretty sight!

I have summarised the experiment but it hardly captures the awesomeness of the actual process. Interested readers are requested to read the original article for a more detailed account.

Now why is this experiment so important? Firstly, it greatly reduces the complexity of the organogenesis of a complicated structure like the eye. Secondly, it has a huge implication in the fields of regenerative medicine and tissue engineering where we might soon be able to grow human retinas in vitro for therapeutic and research purposes. It also demonstrates the power of self-directed processes in biology which often result in the emergence of unfathomable complexity and thus it instills the confidence in us to try and grow more complicated structures in the laboratory. Last but not the least, the process is damn interesting and if you don’t agree you f*** off!

 

– Debayan

(PS. This post was typed in haste and hence I have skipped over most of the important aspects of the research. If anyone has a query, drop by a comment and I’ll be more than happy to reply. )