This is 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). Shakespeare, 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)

The cover image is from here: http://pixabay.com/p-440660/?no_redirect