John Sulston: Biologist, Geneticist and Guardian of our Heritage

 

Sir John Sulston died on 6 March 2018, an event reported world-wide by the press, radio and television. Having studied in Cambridge and then worked at the Salk Institute in La Jolla, California, he joined the Laboratory of Molecular Biology in Cambridge to investigate how genes control development and behaviour, using as a ‘model organism’ the roundworm Caenorhabditis elegans. This tiny creature, 1 mm long, was appealing because it is transparent and most adult worms are made up of precisely 959 cells. Simple it may be but this worm has all the bits required for to live, feed and reproduce (i.e. a gut, a nervous system, gonads, intestine, etc.). For his incredibly painstaking efforts in mapping from fertilized egg to mature animal how one cell becomes two, two becomes four and so on to complete the first ‘cell-lineage tree’ of a multicellular organism, Sulston shared the 2002 Nobel Prize in Physiology or Medicine with Bob Horvitz and Sydney Brenner.

Sir John Sulston

It became clear to Sulston that the picture of how genes control development could not be complete without the corresponding sequence of DNA, the genetic material. The worm genome is made up of 100 million base-pairs and in 1983 Sulston set out to sequence the whole thing, in collaboration with Robert Waterston, then at the University of Washington in St. Louis. This was a huge task with the technology available but their success indicated that the much greater prize of sequencing of the human genome — ten times as much DNA as in the worm — might be attainable.

In 1992 Sulston became head of a new sequencing facility, the Sanger Centre (now the Sanger Institute), in Hinxton, Cambridgeshire that was the British component of the Human Genome Project, one of the largest international scientific operations ever undertaken. Astonishingly, the complete human genome sequence, finished to a standard of 99.99% accuracy, was published in Nature in October 2004.

As the Human Genome Project gained momentum it found itself in competition with a private venture aimed at securing the sequence of human DNA for commercial profit — i.e., the research community would be charged for access to the data. Sulston was adamant that our genome belonged to us all and with Francis Collins — then head of the US National Human Genome Research Institute — he played a key role in establishing the principle of open access to such data, preventing the patenting of genes and ensuring that the human genome was placed in the public domain.

One clear statement of this intent was that, on entering the Sanger Centre, you were met by a continuously scrolling read-out of human DNA sequence as it emerged from the sequencers.

In collaboration with Georgina Ferry, Sulston wrote The Common Thread, a compelling account of an extraordinary project that has, arguably, had a greater impact than any other scientific endeavour.

For me and my family John’s death was a heavy blow. My wife, Jane, had worked closely with him since inception of the Sanger Centre and not only had his scientific influence been immense but he had also become a staunch friend and source of wisdom. At the invitation of John’s wife Daphne, a group of friends and relatives gathered at their house after the funeral. As darkness fell we went into the garden and once again it rang to the sound of chatter and laughter from young and old as we enjoyed one of John’s favourite party pastimes — making hot-air lanterns and launching them to drift, flickering to oblivion, across the Cambridgeshire countryside. John would have loved it and it was a perfect way to remember him.

Then …

When John Sulston set out to ‘map the worm’ the tools he used could not have been more basic: a microscope — with pencil and paper to sketch what he saw as the animal developed. His hundreds of drawings tracked the choreography of the worm to its final 959 cells and showed that, along the way, 131 cells die in a precisely orchestrated programme of cell death. The photomontage and sketch below are from his 1977 paper with Bob Horvitz and give some idea of the effort involved.

Photomontage of a microscope image (top) and (lower) sketch of the worm Caenorhabditis elegans showing cell nuclei. From Sulston and Horvitz, 1977.

 … and forty years on

It so happened that within a few days of John’s death Achim Trubiroha and colleagues at the Université Libre de Bruxelles published a remarkable piece of work that is really a descendant of his pioneering studies. They mapped the development of cells from egg fertilization to maturity in a much bigger animal than John’s worms — the zebrafish. They focused on one group of cells in the early embryo (the endoderm) that develop into various organs including the thyroid. Specificially they tracked the formation of the thyroid gland that sits at the front of the neck wrapped around part of the larynx and the windpipe (trachea). The thyroid can be affected by several diseases, e.g., hyperthyroidism, and in about 5% of people the thyroid enlarges to form a goitre — usually caused by iodine deficiency. It’s essential to determine the genes and signalling pathways that control thyroid development if we are to control these conditions.

For this mapping Trubiroha’s group used the CRISPR method of gene editing to mutate or knock out specific targets and to tag cells with fluorescent labels — that we described in Re-writing the Manual of Life.

A flavor of their results is given by the two sets of fluorescent images below. These show in real time the formation of the thyroid after egg fertilization and the effect of a drug that causes thyroid enlargement.

Live imaging of transgenic zebrafish to follow thyroid development in real-time (left). Arrows mark chord-like cell clusters that form hormone-secreting follicles (arrowheads) during normal development. The right hand three images show normal development (-) and goiter formation (+) induced by a drug. From Trubiroha et al. 2018.

John would have been thrilled by this wonderful work and, with a chuckle, I suspect he’d have said something like “Gosh! If we’d had gene editing back in the 70s we’d have mapped the worm in a couple of weeks!”

References

International Human Genome Sequencing Consortium Nature 431, 931–945; 2004.

John Sulston and Georgina Ferry The Common Thread: A Story of Science, Politics, Ethics and the Human Genome (Bantam Press, 2002).

Sulston, J.E. and Horvitz, H.R. (1977). Post-embryonic Cell Lineages of the Nematode, Caenorhabitis elegans. Development Biology 56, 110-156.

Trubiroha, A. et al. (2018). A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function. Scientific Reports 8, Article number: 5647.

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Transparently Obvious

 

Scientists have a well-earned reputation for doing odd things – by which I mean coming up with a ‘finding’ that leaves me, at least, wondering how, in the name of all things wonderful, they ever got money to do their study. To be fair, it’s the ‘social scientists’ – rather than the ‘real’ lot – that excel in this field. An example? Take your pick. They crop up pretty well weekly in the press. I liked the one on how something called ‘personal congruence’ affects marriage survival. The more congruence you and your partner have the better your chances: if, over time, your congruence goes down the tubes, your relationship will surely follow. But what on earth is congruence? Seemingly it’s a ‘state of agreeing.’ Lots of it equals harmony, loss of it = discord. So, it is what you remember from school geometry: it means more or less equal. Wow! Now I’ve grasped the upshot of this ‘study’: agreeably happy couples tend to make it: pairings based on whacking each other with frying pans tend to end in tears. Why didn’t they tell us earlier!!

Axolotl

   Axolotl

Fortunately, in my world, even the weirdies usually turn out to be quite sensible, once you know what’s going on. Many moons ago a girl-friend asked me if I’d like to see her collection of axolotls. Not having a clue what she was on about I gave it an excited ‘yes please’. Whilst it mayn’t have been what I was hoping for (I was very young back then), I immediately fell in love with these wonderful amphibians that I’d never heard of as she explained what I should have known: these ‘Mexican walking fish’ have very large embryos which makes them particularly useful for studying development. These sensational salamanders really are amazing, not least because they can regenerate entire limbs after they’ve been chopped off.

More recently there’s been another unlikely recruit to the scientific armoury: the zebrafish – a tropical freshwater fish from the Himalayas. This mighty minnow was the first vertebrate to be cloned which led to its being genetically modified to give a transparent variety. That’s all good fun but what on earth is the point of a see-through fish? Well, in Betrayed by Nature we pointed out that you can actually watch tumours growing in transparent zebrafish and we got so excited by that we even included a photo – kindly provided by Richard White of the Dana Farber Cancer Institute in Boston. The cancer was a melanoma which had grown into a black mass about 1 cm in diameter in the fish’s body after a small number of tumour cells had been injected a couple of weeks earlier.

And the driver is …

Nearly 15 years ago, just as the first complete sequence of human DNA was being unveiled, Mike Stratton and his colleagues at the Sanger Centre in Cambridge discovered a mutation that arises in about two-thirds of all malignant melanomas. It’s in a gene called BRAF. The protein made by the gene is an enzyme that’s part of a signalling pathway that pushes cells to divide. The mutation changes the shape of BRAF protein so it works 24/7 as an enzyme: the pathway is no longer controlled by a message from the world beyond the cell. It’s a ‘molecular switch’ that’s been flipped by mutation to act as a cancer ‘driver.’

Richard White and his colleagues showed that the same mutation drove melanoma development in zebrafish and that when it did so something remarkable happened. As the tumours got going they turned on a gene that is normally only required during the first 72 hours after fertilization. The gene’s called crestin – because it’s switched on in a tissue called the neural crest where crestin protein helps to form the bony support for the gills. After that it’s switched off and crestin protein never appears again. Except in the pigment-containing cells called melanocytes when they are turning into a tumour.

Seeing the problem

In a great example of how science can work, Charles Kaufman, Leonard Zon and colleagues in Boston and other centres took this finding and made another transgenic variant of the transparent zebrafish. They cut out the stretch of DNA that controls whether the crestin gene is ‘on’ or ‘off’ and hooked it up to a gene that makes a green fluorescent protein (GFP). Result: when the machinery of a cell turns crestin on, GFP is also made – and the cell glows green under the appropriate light. Hence you would expect to see a glowing neural crest early in development but thereafter a non-glowing fish. Unless it has a melanoma. And Zon & Co saw exactly that. Because green fluorescent protein glows so brightly, a single cell shows up and it turned out that whenever one green cell was detected it always went on to expand and grow into a large melanoma tumour.

1 cell to mel

Tracking a single cell turning into a tumour over 6, 9, 11.5 and 17 weeks. The green fluorescence marks an early developmental gene (crestin) being re-activated in a melanoma tumour (from Kaufman et al., 2016).

But why might it be useful to ‘see’ single cells?

Since the original finding by Stratton & Co more detailed studies have confirmed that mutated BRAF is indeed an important ‘driver’ in about two-thirds of malignant melanoma. But here’s the odd thing: lots of melanocytes (the cells that can turn into melanomas) have mutated BRAF – but they don’t become cancerous. Why not? And there’s something else: it’s well-known that ultraviolet radiation in sunlight causes many melanomas and they do indeed often arise on exposed skin – but they can also crop up in places where, as they say, the sun doesn’t shine. So clearly, important though mutated BRAF and sunlight are, there’s something else that’s critical for malignant melanoma.

The Kaufman experiment was remarkable, not least because it offers a way of getting at this key question of what happens in a cell to kick it off as a tumour, by comparison with a near neighbour that remains ‘normal.’

The tumour cells used in this model carry mutated BRAF and another gene, P53, was knocked out. This gives two major genetic drivers and it may be that further genetic changes aren’t needed. If that’s the case, then the decisive push must come either from epigenetic changes (that affect gene expression without change in DNA sequence) or from adaptations of the tumour microenvironment to provide an optimal niche for expansion. At the moment we don’t know very much about these critical areas of cancer biology. Being able to follow single cells may lead us to the answers.

Keep your eye on the transparent minnows!

Reference

Kaufman, C.K., Zon, L.I. et al. (2016). A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science 351, Issue 6272, pp. DOI: 10.1126/science.aad2197