Making a grade: Sonic Hedgehog signalling and the control of neural cell fate

Medal Review5 February 2009free access Making a grade: Sonic Hedgehog signalling and the control of neural cell fate James Briscoe Corresponding Author James Briscoe Developmental Neurobiology, National Institute for Medical Research, London, UK Search for more papers by this author James Briscoe Corresponding Author James Briscoe Developmental Neurobiology, National Institute for Medical Research, London, UK Search for more papers by this author Author Information James Briscoe 1 1Developmental Neurobiology, National Institute for Medical Research, London, UK *Corresponding author. Developmental Neurobiology, National Institute for Medical Research (NIMR), MRC, The Ridgeway, Mill Hill, London NW7 1AA, UK. Tel.: +44 20 8816 2559; Fax: +44 20 8816 2523; E-mail: [email protected] The EMBO Journal (2009)28:457-465https://doi.org/10.1038/emboj.2009.12 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info My grandfather once told me that you should never do anything that might win you a medal. This advice came from his time in the military and although he was a man whose opinion I respected greatly, this was one piece of advice that I never thought would be relevant to me as a scientist. In my defence, it was never my intention or ambition to win a medal: no one could have been more surprised and delighted than I was when I received the email from EMBO informing me that I had won the Gold Medal. Indeed, looking back, I can honestly say it was never my intention or ambition to become a research scientist. Although one or two of my current colleagues appear to have had well-structured career plans before they could walk, read or say 'tenure committee decision' I did not. Similar to many scientists I suspect, I accidentally fell into research. I enjoyed and did reasonably well in science classes at school, so it was obvious to choose a science subject when thinking about university. I ended up at Warwick University studying Microbiology and Virology, a course that involved a great deal of molecular biology. The elegance and complexity of bacterial and viral life and the way in which studying these organisms at a molecular level seemed to resolve the complexity to simple, straightforward explanations of function captivated me. An intercalated year spent working at Amersham International in Cardiff (now GE Healthcare) reinforced this view and whetted my appetite for some proper research. Back at Warwick for my final year, I was particularly struck by how much had been learned about the mechanism at work in eukaryotic cells from the study of viruses and viral infections. Signalling, the start With this in mind, I decided to look for a suitable PhD position. Certainly my university studies and experience at Amersham contributed to this decision, but it is also important to note that this was 1992, the depth of the recession in the UK. Seeing my friends lose their jobs and struggle to make ends meet was also a strong motivator. A safe academic job seemed preferable to the uncertainties of life in the 'real world'. I was fortunate to gain a place in Ian Kerr's lab at the Lincolns Inn Fields laboratories of the Imperial Cancer Research Fund (now Cancer Research UK). I was doubly fortunate to arrive in the lab and institute at just the right time. Ian had a long-standing interest in unravelling how interferon (IFN) initiated an antiviral state in cells. Together with George Stark's group, which had occupied an adjoining space in the institute, they had embarked on a somatic cell genetic approach to identify genes required for the IFN response (Stark, 1997). This was beginning to come to fruition as I joined the lab. Between them, the labs had screened for and isolated several mutant cell lines that were unresponsive to IFNα/β and/or IFNγ. By identifying the mutated gene in one of these lines, Sandra Pellegrini had already shown that the tyrosine kinase Tyk2 was a component of the IFN signal-transduction pathway (Pellegrini et al, 1989; Velazquez et al, 1992). When I joined the lab, I was set to work with an experienced postdoc, Mathias Müller, to help find the defective genes in two further mutants. One of the cell lines turned out to be deficient in a STAT transcription factor that forms part of the IFN-regulated transcriptional complex (Müller et al, 1993a). For the other cell line, I was given the job of testing a series of candidate genes. On the basis of our hunches, one candidate was the Tyk2-related kinase JAK1. Meanwhile, Mathias was busy with more complicated experiments using cDNA libraries to screen for clones that restored IFN responses in the mutant cells, in case none of our guesses paid off. My task was simple. It involved transfecting mutant cells with candidate genes, exposing them to IFN, preparing RNA and then using RNase protection assays to test whether the induction of IFN-dependent gene expression had been re-established. The assay was usually finished late on the fifth day of the experiment and ordinarily the assays would be exposed to autoradiographic film overnight, ready to be analysed the next morning. But we were always impatient. So instead of overnight, we would set up the exposure and then nip around the corner to the George pub for an hour. After a pint or two, Mathias and I would return and run the film through the developer. After such a short exposure, the bands on the film were very faint, but if you held it up to the light at just the right angle you could make them out. I remember doing this with the assay containing Jak1 and hardly believing my eyes when I saw weak bands indicating that the mutants had indeed regained an IFN response (Müller et al, 1993b). The excitement and exhilaration of this experience hooked me into research. These rare moments when you see a result that nobody else has seen before, when for a short period you are the only person to have this knowledge, still make science rewarding and compelling. These studies, together with elegant biochemical and molecular experiments from the labs of Darnell, Ihle and co-workers in the USA, were the foundation of what is now referred to as the JAK/STAT signalling pathway (Darnell et al, 1994; Ihle, 1995). It was an exciting time. Hardly a week seemed to pass without the JAKs and STATs being implicated in another signal-transduction pathway and the mutant cell lines proved an invaluable resource. We were able to use them to show that the cytokine IL-6 used the JAK/STAT pathway. They were the ideal tools for molecular structural studies of the JAK and STAT proteins and for dissecting the mechanism of signal transduction (Guschin et al, 1995; Briscoe et al, 1996; Kohlhuber et al, 1997). Ian was a challenging but purposeful supervisor, always focused and supportive and I could not have asked for more generous and helpful colleagues in the lab. Moreover, the entire atmosphere at ICRF was vibrant and stimulating. To me it seemed like the centre of the signal-transduction world; down the corridor Richard Treisman's lab was dissecting the MAP kinase pathway, whereas a floor below Julian Downward's lab was identifying new Ras effectors and Peter Parker's lab was analysing PKC signalling. This atmosphere was further enhanced when Paul Nurse and a group of developmental biologists, including Phil Ingham, David Ish-Horowitz and Julian Lewis, moved to the institute. The seminars from these groups were my first exposure to embryology, introducing me to the type of questions that developmental biologists would like to tackle. The images of embryos were more aesthetically pleasing than the graphs and blots I was accustomed to and the questions the researchers asked seemed to me to be more profound. I began to realize that this was a subject I wanted to pursue. Specifically, having spent the previous few years studying the mechanisms of signal transduction in tissue culture cells, I decided I wanted to analyse a signal-transduction mechanism in vivo during embryogenesis. Moving into morphogens Once again, having made a decision, I found myself in a fortunate situation when Tom Jessell offered me a postdoc position in his lab at Columbia Medical School in New York City. Tom is interested in understanding the molecular mechanisms that direct the assembly of neuronal circuits in the spinal cord and how the organization of these circuits coordinates muscle movement and behaviour (Jessell, 2000). A first step in this process is the production of the appropriate neuronal subtypes in the right place and time in the forming neural tube. The question of how different types of neurons are produced in their correct positions in the spinal cord is a specific example of a general and fundamental problem in developmental biology. How do cells know where they are within a tissue and how is this information translated so that they form the appropriate structures for their positions? The framework that has emerged over the last century to answer this question involves the concept of positional information and signalling gradients. Lewis Wolpert best encapsulated this idea in a highly influential paper in 1969 (Wolpert 1969, 1996) in which he introduced what is now called the French Flag Model. This is a general mechanism to divide a field of cells into three equal partitions such as the red, white and blue of the French tricolour. In the model, a fixed part of the tissue is postulated to correspond to an organizer that produces a signal. The signal propagates through the rest of the tissue to establish a gradient. Cells within the tissue respond and interpret the graded signal in a quantitative manner. Cells therefore ascertain their distance from the organizer according to the concentration of the signal (Figure 1A). The beauty of this model is that the abstract concept of positional information is replaced by a tangible biochemical coordinate system that defines the location of a cell with respect to a fixed point within the tissue. The term morphogen was adopted to define signals that function in this way. This definition emphasizes two characteristics of a morphogen: it must function in a concentration-dependent manner to induce different responses at different thresholds and it must spread through a tissue to act at a distance from its source. The model also raises several additional questions that have to be answered for a complete understanding of how a morphogen works. How does the signal spread through the tissue to establish a gradient? How is the extracellular gradient perceived by responding cells, specifically how is the quantitative information transduced across the membrane and through the signalling pathway to control differential gene expression? How is the continuous graded information interpreted to generate discrete, all-or-none changes in gene expression that must underpin the switches in cell type produced at different concentrations of signal? Figure 1.Sonic Hedgehog acts in a graded manner to pattern the ventral neural tube. (A) A morphogen can pattern a developing tissue. A secreted signal (blue) is produced from a localized source (S) and spreads through the tissue to establish a gradient. Cells respond to different concentrations of the signal by regulating different sets of genes (red, orange and yellow). This induces distinct cell fates (A, B and C) at different distances from S (adapted from the French Flag Model in Wolpert, 1969). (B) Shh protein (brown) is produced from the notochord (n) and floor plate at the ventral midline of the neural tube. Shh spreads dorsally establishing a gradient that controls the generation of distinct neuronal subtypes (interneurons (V0–V3) and motor neurons (MN)). In vitro, different concentrations of Shh are sufficient to induce the distinct neuronal subtypes. The concentration of Shh necessary to induce a specific subtype corresponds to its position of generation in vivo. Download figure Download PowerPoint These issues are of particular relevance in the spinal cord where several distinct classes of neurons including motor neurons (MNs) and a number of interneurons involved in relaying sensory information and coordinating motor output are generated (Jessell, 2000). Each neuronal population arises from blocks of proliferating progenitors that are arrayed in a stereotypic order along the dorsal–ventral (DV) axis of the neural tube. Experiments that dated back to the 1920s indicated that in the ventral half of the spinal cord the pattern of neuronal generation is directed by cues emanating from the floor plate, a population of cells that reside at the ventral midline of the neural tube, and the notochord (reviewed in Placzek et al, 1991). A couple of years before I arrived in New York, the Jessell lab and several other labs cloned vertebrate homologues of the Drosophila gene Hedgehog, which encode secreted signalling proteins (Echelard et al, 1993; Krauss et al, 1993; Riddle et al, 1993; Chang et al, 1994; Roelink et al, 1994). It turned out that one of them, Sonic Hedgehog (Shh), was expressed in the notochord and floor plate and corresponds to the instructive cue that provides DV polarity to the ventral neural tube (Figure 1B). Moreover, the initial studies strongly hinted that Shh patterned the neural tube in a concentration-dependent manner, as predicted if it was a morphogen (Marti et al, 1995; Roelink et al, 1995). Whether Shh satisfied the exacting criteria necessary to qualify it as a morphogen and how cells interpreted the signal to specify distinct neuronal cell types remained to be determined. These were the questions that excited me on arriving in the Jessell lab. However, having no previous experience in developmental biology and never having laid hands on an embryo before, I teamed up with a skilled developmental biologist, Johan Ericson. We worked closely together. Johan showed extreme patience during the first few months as I tried to learn the experimental techniques that were much more delicate than ones I used previously. He also showed a similar level of patience, but for a much longer period of time, on the Bronx public golf course where in response to his drives straight up the middle of the fairway, I would inevitably hack my way through the rough, zigzagging slowly towards the green. My golf never improved, but nonetheless, the scientific partnership with Johan continued with long discussions about which experiments to do and arguments over what the results meant. These were interspersed by sage and timely advice from Tom offering piercing insight and ensuring we kept our focus. The idea that Shh was necessary for correct formation of MNs and other ventral neuronal subtypes in vivo was supported by loss-of-function studies (Chiang et al, 1996; Ericson et al, 1996). To further understand how Shh controlled neuronal subtype identity, we carried out experiments using explants of naïve neural tissue. The results indicated that exposure to varying concentrations of Shh protein induced distinct neuronal subtypes characteristic of the ventral neural tube (Ericson et al, 1997a, 1997b). Progressive 2- to 3-fold changes in Shh concentration led to the generation of the different neuronal subtypes and there was a good correlation between the concentration of Shh necessary to induce each neuronal subtype and their position of generation in vivo (Figure 1B). Thus, the induction of neurons generated in more ventral regions of the neural tube required correspondingly higher Shh concentrations. In addition to being necessary for their development then, Shh was sufficient to induce ventral interneurons and MNs in neural tissue. Furthermore, these experiments supported the idea that Shh functions as a morphogen with cells in the ventral neural tube being exposed to ventralHIGH–dorsalLOW gradient of Shh, emanating from the ventrally located notochord and floor plate. Coding neural cell fate These findings highlighted the question of how positional identity is imposed on progenitor cells and how this determines neuronal subtype identity. A series of studies over the next few years suggested that a group of transcription factors, predominately homeodomain proteins, were important intermediaries in the process (Ericson et al, 1997a, 1997b; Briscoe et al, 1999, 2000; Sander et al, 2000; Novitch et al, 2001; Vallstedt et al, 2001). These transcription factors exhibit distinct patterns of expression along the DV axis of the neural tube (Figure 2). On the basis of their mode of regulation by Shh signalling, we subdivided them into two groups, termed I and The responses of the proteins to graded Shh signalling were using of This that the expression of each I protein is at distinct thresholds of Shh their ventral of expression are by Shh signalling. neural expression of the proteins on Shh signalling, so their of expression are by graded Shh The expression of classes of proteins of progenitors within the ventral neural tube, each of which generated a distinct neuronal Thus, the of homeodomain protein expression to correspond to a transcriptional that positional identity to progenitors that the neuronal subtype generated by each Figure Shh signalling acts by the expression of a set of transcription factors in neural (A) Shh signalling results in the of I proteins such as and and is required for the of proteins such as and between of I and proteins generate switches in gene The expression of I and proteins defines of progenitors in the ventral neural tube. These progenitor are arrayed along the dorsal–ventral axis and each one of the distinct neuronal subtypes characteristic of the ventral neural tube. (B) The expression of the transcription factors and of the expression patterns that distinct progenitor in the neural tube. Download figure Download PowerPoint The idea that the progenitor transcription factor the fate of neurons was supported by and loss-of-function experiments in and embryos (Ericson et al, 1997a, 1997b; Briscoe et al, 1999, 2000; Sander et al, 2000; Vallstedt et al, 2001). expression of a I or protein in the neural tube the position in which neuronal subtypes were generated in a manner predicted by the expression of the I and the in of progenitor transcription factors in switches of neuronal In these we were by a generous of mutant embryos from several in particular and in and and in These experiments also the of between of I and proteins expressed in progenitor (Figure This first for the protein and the I protein (Ericson et al, 1997a, 1997b; Briscoe et al, 2000). The that dorsally in embryos in another of but exciting scientific It suggested how the system could be further when experiments indicated a between and similar were to other of I and proteins et al, 2000; Vallstedt et al, 2001). the indicated that between of I and proteins the DV of gene the positions at which distinct neuronal subtypes are The also a for the response of genes to the gradient of a mechanism for the of a graded signal into or none changes in gene expression is for the function of a Moreover, the of in the neural tube mechanisms involved in other developing such as the of the Drosophila embryo and 1991). Thus, it a general for the of cell fate in response to graded a direct Although the of Shh to induce distinct neuronal subtypes in a concentration-dependent manner suggested that Shh at long to control gene no direct in vivo of a gradient of Shh protein had been It was that Shh its by an signal to positional information to the neural tube. To test the of Shh signalling, I worked with in lab, located on the floor the Jessell lab. They had with a series of genetic experiments in that was the for and of to the movement of through tissue and 1996). They on to mutated forms of that as of signalling in Drosophila and expression of the vertebrate of the mutant in the neural tube Shh signalling in cells and in the of the cell types found in the ventral neural tube et al, 2001). This indicated that Shh at long to control gene expression and cell fate in the neural tube. Moreover, I that the of signalling in more dorsally cells responding as if exposed to a higher concentration of This indicated similar to in a mechanism the spread of Shh in the neural signalling the of These were further supported by work from lab, including the direct of a gradient of Shh in the neural tube et al, 2001; and et al, the studies strongly signal and indicated that Shh functions over a distance in the neural tube. Together with of its concentration-dependent this that Shh signalling, in the neural tube, the definition of a These studies graded Shh signalling in the neural tube as an system for understanding the molecular mechanism of morphogen This the question of how cells the concentration of Shh through the signal-transduction pathway to differential gene expression in the It is this question that I was to as I set up my lab several and this is still the main of the lab. In another fortunate of as I was looking for an I that wanted to several new group The atmosphere and the of of the institute me and I at the when offered a position. However, what was not to me the day I was that the space I had been was at the very of the the by and Although I was used to the from in New York, it was a to my first arrived in my lab, during a straight from on the of In of and within a few months we moved into new lab space on a more floor of the institute complete with its then the and my colleagues have been the of the during seminars and the generous of and make a and place to The of Shh signalling We focused on the question of how graded information from Shh signalling is perceived and in responding cells. Shh signalling on two already the which proteins, and which is for signals see and 2001; and In the of Shh, and of Shh to this signal The mechanism of signal of and is the subject of much However, the that the signal with the regulation of a of containing transcriptional effectors as proteins and three genes are expressed in the neural tube and several studies had to the functions they have in neural tube and et al, in lab had shown that a of in embryos et al, led to a in the generation of the floor plate and the of these are the cell types induced by concentrations of with the of these cell there was a ventral in the production of whereas neuronal classes located to MNs were A mutant and had more than mutants et al, these embryos still produced MNs and interneurons to This suggested that with from is required for the cell types that the of Shh signalling. The the question of the of and whether was involved in responses to the PhD from having to the with Johan Ericson (now back in and his lab to test the of and the function of in neural used a of that the transcriptional but its function to transcriptional in the neural tube. Similar to the results with the mutant that signalling, the a in progenitor cell identity and a to generate MNs and ventral interneurons et al, and This the of in the of positional information to cells responding to graded Shh signalling. had been to function as an of Shh signalling in the neural tube. this in the of progenitor located in the of the neural tube with a in the identity of the neurons generated in this This was in a of made in lab. This only a of et al, to Moreover, function in embryos restored the expression of several ventral progenitor transcription factors that are in Shh mutant embryos and 2000). This supported the idea that only of is required in the neural tube. Although ventral cell types were generated in the the is in particular the DV organization characteristic of the neural tube is and 2000). This suggested two the induction of ventral cell types can place in the of Shh signalling, as long as the of is other signals might positional to the ventral neural tube when Shh signalling is Thus, without transcriptional from proteins, cells the positional information by Shh signalling, the between progenitor transcription factors within or other signals might gene In this the or the of other signals that cells could different positional in a neural tube of cell In this it is to note that