Fascinated by nature to become a curiosity driven scientist
As told by Christine Holt

My upbringing, family and education
My interest in science stemmed from my rural roots in Northumberland, northern England, where I spent my early days roaming around the fields, woods and streams with my older brother, Stephen. In many ways it was an idyllic childhood. My parents were caring and full of fun, they supported and encouraged me in whatever I wanted to do. I went to a small traditional primary school in a nearby village for children and at age 10, I was sent away to Harrogate College, an all-girl boarding school.

At Harrogate, I enjoyed Nature Studies and Art and I remember being inspired by a teacher, Mrs. Smales, who decorated the classroom with her skilful artwork of flora and fauna and took us for walks in the woods. At age 16, I moved to a sixth form college, St. Clare’s College, in Oxford, where I studied a mix of arts and biology at A-level. This exposed me to the possibility of going to university, which up until then I had not considered.

In 1973, I went to Newcastle University to study Zoology, however, I was not keen on what I viewed as an ‘old-fashioned’ biology curriculum based on classification systems and Latin words. The following year, I switched to Sussex University as it offered what seemed to me a much more exciting modern biology course. I had teachers such as John Maynard Smith, who used to join us in the students’ Common room for coffee sitting cross-legged on the floor after his Lectures. It was a revelation to learn that there were many interesting and unanswered questions in biology.

I had teachers such as John Maynard Smith (1920-2004), who used to join us in the students’ Common room for coffee sitting cross-legged on the floor after his Lectures

In my final undergraduate year, I studied developmental biology and neuroscience and became fascinated by the emerging work on how nerves make connections in the brain. It was a particular wonder to me how the retina makes an accurate ‘map’ of the outside world in the brain. At the suggestion of Mike Land, my personal tutor, I applied for a PhD with John Scholes in the Biophysics Unit at King’s College, London, who was doing elegant work on how retinal-topographic order is precisely arrayed in the ribbon-shaped optic nerve of cichlid fish.

My scientific career
John, my supervisor, encouraged me to do my PhD work with very little interference and direction. I chose to focus on the initial events during the formation of the visual system of the frog, Xenopus. I wanted to understand how the embryonic eye transforms into the mature eye during development, and to do this, we thought it could be possible to trace the cell movements by surgically removing tiny pieces of embryonic eye tissue, incubating them for a few minutes in a tiny droplet of radioactive nucleotide, and then replacing them in the eye. Amazingly, the technique worked well, enabling me to discover the cell migrations occurring during eye formation which helped to resolve a major discrepancy in the literature regarding retinal axis specification.

A breakthrough moment in my scientific career happened when I was cycling to work along Oxford Street one morning

A breakthrough moment in my scientific career happened when I was cycling to work along Oxford Street one morning. I was thinking about how to label growing axons (techniques were not available then) when the idea struck me that if I used a radioactive amino acid (H3-proline) to label proteins instead of a DNA nucleotide, I could visualise axons from different eye bits of the eye and trace their journey as they first grew out and navigated their way through the brain. Work in other labs had shown that regenerating optic nerves are initially quite disordered and only sort out later. In the embryo, however, I found that the axons from the eye grew unerringly along the pathway to their targets in the midbrain where they made a clear topographic map, right from the start.

Science as a courtship strategy
Towards the end of my PhD, in 1981, I was considering doing a postdoc and I visited Bill Harris (my future husband as it turned out), who had recently set up his own lab at the University of California San Diego (UCSD) and was studying similar questions in the Mexican axolotl. During this short visit, Bill set up a recording chamber to map the visual responses of tiny tadpole brains. Together, in just one week, we had gathered electrophysiological data that revealed the existence of a tiny functional topographic map in the initial visual projection which supported my anatomical map data. The next year, I took up an MRC postdoctoral fellowship to work in the Physiology Department at Oxford University with the Colin Blakemore and Ian Thompson, both inspirational neuroscientists who made doing science enjoyable and fun.

I married Bill Harris in 1983 in Hexham, Northumberland, and we returned to UCSD together to live in La Jolla.In the next few years, we had two beautiful children, Julia and Jake, who have both followed careers in science. They are the joy of my life, along with our five grandchildren.

As an Assistant-Professor-in-Residence, I had free-rein to do my research in Bill’s lab. I made some progress, for example showing reversing the order of axon arrival in the tectum does not alter the topographic map. However, the combined pressures of motherhood and teaching a large embryology course, slowed me down, as I had no research assistant or research group of my own. This changed when I was awarded an NIH Grant, which enabled me to hire a technician. In 1992, I gained a tenure-track position in the Biology Department at UCSD and I was able, then, to start building my own research group.

The combined pressures of motherhood and teaching a large embryology course, slowed me down. I had no research assistant or research group of my own, but this changed when I was awarded an NIH Grant

My Science

The Research Leading-up to the Discovery
In the pre-molecular era (1980s), our experimental approach was largely to use surgical embryological techniques to challenge the growth of axons.For example, I transplanted eyes into very young embryos, forcing axons to grow into immature brains, and Bill grafted eyes to ectopic locations forcing axons to enter the brain in unusual places - they almost always grew directly to their targets. But what was guiding these axons? How did they know how to navigate?The molecular mechanisms involved in guiding axon growth began to be uncovered in the late 1980s/early 1990s, with discoveries in many other labs of guidance molecules and their receptors. In 1990, we developed a lipofection technique that enabled us to introduce genetically engineered molecules (such as dominant negative N-cadherin and FGF receptor) into developing neurons in vivo and used it over the next decade to test the roles of specific molecules in axon guidance at a single cell level.

In 1997, after fifteen years at UCSD, Bill and I moved to the University of Cambridge, UK. This was an exciting move as Developmental Neurobiology as a field was strong in the UK in the late 1990s with many labs doing ground-breaking work on axon guidance, such as Andrew Lumsden and colleagues at King’s, London, and Michael Bate at Cambridge.

With many collaborators over several years, we discovered some of the key steps involved in the navigation of growing axons in the visual system. For example, Shin-ichi Nakagawa and our colleague Carol Mason found that a molecule, Ephrin-B, pops-up at the optic chiasm where it helps to direct the selective divergence of retinal axons to the correct side of the brain. With Mu-Ming Poo (who developed the powerful turning assay) and Marc Tessier-Lavigne (who discovered Netrin-1) we showed that a gradient of Netrin-1 or BDNF attracts the growth of retinal axons whereas other molecules, such as Sema3A and Slit2, repel their growth. These findings were in-line with others in the field and fitted nicely with the view that attractive and repulsive cues in the environment activate receptors in the growth cone, triggering signalling cascades that affect cytoskeletal changes and lead to directional turning.

Milestones in the Road to Discovery

The Autonomous Growth Cone
In 1986, Bill and I did 9-month sabbatical at the Max Planck Institute in Tubingen, Germany, working with Friedrich Bonhoeffer who had discovered that EphrinA, a repulsive guidance factor for retinal axons, was involved in topographic map formation. In Bonhoeffer’s lab, we made live-movies of axons growing in the vertebrate brain using his new fluorescent-dye (DiI) tracing method and state-of-the-art time-lapse microscopes.

This was a most exciting and memorable time, being able to see the tip of a growing axon, the growth cone, moving in the living brain for the first time! These live recordings would often go through the night and Bill would trudge through the snow into the lab at 3.00am or 4.00am to adjust the focus settings (I had a second baby on-the-way). One time, as I was preparing a sample for the recording dish, the eye accidentally became detached. Thinking that the axons would die without their cell bodies, I was about to throw it away and try again but Friedrich said “No! Let’s see what happens to the axons without the eye.” Remarkably, we found that the soma-less growth cones grew and navigated normally through the brain. This gave me a key insight: growth cones are autonomous little subcellular compartments that do not need to consult their cell bodies (where the nucleus is located) to make pathfinding decisions.

I wondered: Could mRNAs be present and translated in growing axons?

mRNA in Axons?
Soon after we moved to Cambridge, UK, a collaborator Roger Bradley at the Salk Institute sent me some images of an in situ hybridization showing the mRNA of a cell adhesion molecule (NF-protocadherin) in the Xenopus larval retina. As expected, the signal was high in the cell bodies of the retinal ganglion cells (which give rise to the axons comprising the optic nerve) but what I found particularly puzzling was that it was also very high in the optic nerve itself. At that time, mRNAs were not known to be present in axons, so my first thought was that he had mixed up the images and sent me an antibody-stained section instead. However, he was adamant he had sent the correct images. So, I wondered: “Could mRNAs be present and translated in growing axons?”.

Fast Protein Synthesis
In 2000, an issue of the journal Science landed on my desk with a paper from Mark Bear’s lab reporting that synaptic activation triggers rapid protein synthesis that modifies synaptic transmission in dendrites, confirming Erin Schuman’s pioneering 1996 work showing that local protein synthesis is required for immediate synaptic plasticity. If new proteins could be synthesized locally at synapses within minutes and affect synaptic plasticity, I wondered if new protein synthesis might also be involved in the directional guidance of axons. The experiment to test this was simple: add an inhibitor of protein synthesis in a growth cone guidance assay. I tried to interest people in the lab to do this experiment for many months. Finally, my PhD-student, Douglas Campbell, tried it, though years later he revealed that he only did so to stop me from repeatedly asking!

In 2001, we published our local protein synthesis results in Neuron, after rejections from Nature and Science

I found Doug in a heightened state of excitement in the lab one morning in 2000 – his first set of experiments the previous evening had shown that inhibition of protein synthesis completely blocked the guidance responses of axons - a stunning result! The collapse guidance assays were short (10 minutes), so we thought it unlikely the effect was via the distant cell body. To test this directly, Doug cut the axons and conducted turning guidance assays on sawn-off axons. Amazingly these sawn-off growth cones exhibited normal cue-induced turning, however, this was abolished in the presence of a protein synthesis inhibitor demonstrating that new protein synthesis, localised to the axon, was critical for axon guidance. This exciting finding jived well with the highly autonomous behaviour of growth cones that Bill, Friedrich and I had seen previously and indicated that growing axons have their own machinery for translating mRNAs and for supplying new proteins.

In 2001, we published our local protein synthesis results in Neuron (after rejections from Nature and Science). Our results were greeted with a mixture of excitement and scepticism.

Pioneering ultrastructural work by Oswald Steward and colleagues had revealed translation machinery, such as polyribosomes, in dendrites, but no evidence for their presence in mature axons.

Inspired by Gary Bassell, Ken Kosik and Rob Singer’s finding of beta-actin mRNA in the growth cones of cortical neurons, we found that beta-actin mRNA was also abundant in retinal axon growth cones and, importantly, was translated rapidly in response to attractive cues such as Netrin-1. Kimmy Leung, Trina Lu Bo and Andrew Lin devised ways to visualise beta-actin mRNA dynamics and its translation using live fluorescent microscopy and found that local protein synthesis was spatially precise, and dynamic.

An attractive cue-gradient triggered polarised translation of beta-actin mRNA across the growth cone within minutes, sometimes in a single filopodium on the side nearest the cue. Repulsive cues triggered the translation of different mRNAs that disassembled the cytoskeleton on the near-side, thus giving rise to the differential translation model of turning. Later in vivo work using live imaging by Hovy Wong and Julie Lin showed that new axonal branches emerge at sites where beta-actin mRNA granules dock and that arbor architecture is impoverished when local translation of beta-actin is disrupted. Sparse neuronal branching is often associated with neurodevelopmental disorders suggesting a link with dysfunctional local protein synthesis.

Pioneers in the field
Over the next 10 years, our work focussed on the isolation and identification of the full repertoire of mRNAs present in axons and the investigation of their functional roles. Krishna Zivraj and colleagues in my lab spent hundreds of hours using laser capture microscopy to collect single retinal growth cones, one-by-one, and subjected the isolated mRNA to microarray analyses. To our astonishment, she found over 1000 different mRNAs! This paralleled Kelsey Martin’s pioneering microarray analysis on hippocampal neuronal processes and Erin Schuman’s RNA sequencing work, showing 100s-1000s of mRNAs present in neuronal processes. In 2016, Hosung Jung together with Toshiaki Shigeoka in my lab found a genetic way to label the translating ribosomes inside the growing retinal axons of mice and were able to show a changing composition of 1000s of mRNAs being translated in developing axons as well as mature axons.

My journey from a child who was fascinated by nature to a scientist driven by curiosity would not have been possible without the many talented co-workers and collaborators along the way

Another unexpected discovery: local translation keeps axons alive
In 2012, using Erin Schuman’s proteomics labelling technique (BONCAT), Jason Yoon, Hosung Jung and Asha Dwivedy and other colleagues in my lab identified an intermediate filament protein whose mRNA was locally translated in retinal axons. When Jason inhibited the axonal synthesis of this protein, he found that the axons navigated normally. Disappointed that there was no guidance defect he went home, dejected, thinking he had wasted two years of work on getting ‘no phenotype’.Two days later, however, when he checked his embryos again, he found, remarkably, that the axons had degenerated. This was a turning point in our understanding of the role of local translation in axon survival. Our more recent research, and that of others, has provided further compelling evidence of an important link between local translation neurodegenerative disorders such as FUS-related ALS.

My journey from a child who was fascinated by nature to a scientist driven by curiosity would not have been possible without the many talented co-workers and collaborators along the way and the support I received for my blue-skies research from agencies like the Pew Charitable Trusts, McKnight Foundation, the Wellcome Trust, MRC, NIH and the ERC.

I've been very fortunate to share this journey with Bill whose clarity of thought, deep scientific knowledge and extraordinary positivity have guided every project. My advice to young scientists would be to always keep an open mind to new ideas, listen to what the data are telling you, and do not give up asking bold questions and looking for answers!