- Top of page
- AN UNEXPECTED PAIRING
- DIVERSITY AMONG SPECIES
- ANCIENT CONNECTIONS?
- TODAY's RELEVANCE
- A CONVERSATION WITH THE EXPERTS
On the surface, the Hedgehog (Hh) pathway and primary cilia make strange bedfellows. Hh is a dynamic regulator of a myriad of developmental processes, ranging from spinal cord and limb patterning to lung branching morphogenesis. By contrast, immotile primary cilia were long considered ancestral holdovers with no known function. Considering the disparate perceptions of these two phenomena, the relatively recent discovery that there is a symbiotic-like relationship between Hh and cilia was unexpected. This primer covers the basics of primary cilia and Hh signaling, highlighting variations in ways they are connected across species, and also discusses the evolutionary implications of these findings. Roles of cilia in signal transduction are analyzed further in an interview with Søren T. Christensen, PhD, and Andrew S. Peterson, PhD, in the A Conversation With the Experts section. Developmental Dynamics 239:1255–1262, 2010. © 2010 Wiley-Liss, Inc.
A CONVERSATION WITH THE EXPERTS
- Top of page
- AN UNEXPECTED PAIRING
- DIVERSITY AMONG SPECIES
- ANCIENT CONNECTIONS?
- TODAY's RELEVANCE
- A CONVERSATION WITH THE EXPERTS
Featured below is a discussion with Hh/cilia experts Søren T. Christensen, PhD, and Andrew S. Peterson, PhD (Fig. 2) about current topics in the field.
Figure 2. Left: Søren T. Christensen, PhD, Associate Professor, Department of Biology, University of Copenhagen, Denmark. Right: Andrew S. Peterson, PhD, Associate Director, Department of Molecular Biology, Genentech, South San Francisco, CA.
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Developmental Dynamics: What is your lab's research focus?
Andrew Peterson: Our entry point into biological areas has always been the phenotype of a mutant mouse. A number of years ago, we carried out several screens using random mutagenesis and began studying mutants with provocative patterns of developmental defects in the forebrain. One of these turned out to be a mutation in the retrograde motor for cilia transport, Dnchc2. As we worked out the mechanism, we realized that several other mutations that we had in our hands probably affected cilia processes as well. One of those was a mutation in Rfx4, a transcription factor whose role in development is at least in part to regulate cilia proteins. This has provided a very rich source of starting points and provided a way to screen for and analyze protein complexes that are required for signal transduction within the cilia.
Søren Christensen: Our lab investigates the mechanisms by which cilia assemble and disassemble, and how the primary cilium coordinates signaling pathways in cell-cycle entry, migration, and differentiation, which are important in, for example, tumorigenesis. A great deal of our work is based on cell and molecular biological studies of cultures of embryonic and cancer stem cells and a variety of different cell lines from human and mouse tissues. One main line of research is focused on how ciliary signaling pathways, such as the PDGFRα, Hedgehog, and Wnt pathways, coordinate the proliferation, migration, and positioning of cells during development and in tissue regeneration and how these pathways regulate the differentiation of stem cells such as during heart development. My partner, Lotte B. Pedersen, and I are fortunate to have a lab workforce of very talented students and technicians that deliver the goods and bring about new ideas, so we never run out of new projects to pursue.
Dev Dyn: What initially provoked your interest in this field?
AP: Kathryn Anderson published a paper in Nature in 2003 describing the requirement for intraflagellar transport for Hh signaling in the mouse. That sentence sums up the information in the paper pretty well and at the time it wasn't possible to integrate the observation into anything we knew about signal transduction. I put the idea aside but certainly didn't forget it.
SC: I think it all began in 1999 when Peter Satir and I started to look at the sensory function of motile cilia in the protozoan ciliate, Tetrahymena. In these cells, we cloned a ciliary protein kinase that linked extracellular signaling to regulation of cell survival and chemotaxis. We then took this a step further to show that PDGFRα signaling is coordinated by the fibroblast primary cilium to regulate cell-cycle entry and migration. Another person who provoked my interest in primary cilia was Denys Wheatley, who actually was the first to introduce me to primary cilia. Then of course, the seminal work by Joel Rosenbaum and colleagues on IFT was a blockbuster for all in this new and exciting field of cell biology.
Dev Dyn: Which papers have most impacted your research?
AP: Clearly Kathryn's 2003 paper (see Huangfu et al.,2003).
A paper from Jonathan Scholey's lab has been very important in our thinking about how transport of signal transduction complexes to the tip of the cilia might work (see Ou et al.,2005).
Beyond those two it is hard to pick out single papers. The work of Joel Rosenbaum to describe the basic processes of intraflagellar transport was the absolutely critical foundation for the field but they were described in a number of papers.
SC: This is a difficult question to answer. Certainly, the work by Joel Rosenbaum and colleagues on IFT and the follow-up on mechanosensing and polycystic kidney disease by Gregory Pazour, Bradley Yoder, Helle Prætorius, and others were an eye-opener to the entire field on primary cilia.
My initial interest in primary cilia, however, actually came from a series of original, and in some cases speculative, papers before any molecular evidence on the function of primary cilia had emerged. For example, Anthony Poole and co-workers suggested that the primary cilium functions as a “cybernetic probe” that senses chemical and physical changes in the extracellular environment (see Poole et al.,1985), and Denys Wheatley later published a provocative paper on the implications of dysfunction or agenesis of primary cilia, which he predicted to cause major disorders in the body (see Wheatley,1995). These and other similar papers in the field had a great influence on my choice of career.
Dev Dyn: Do you think all Shh signaling is mediated through primary cilia in vertebrates?
AP: The evidence is very strong that proper Shh signal transduction occurs in the cilia. Does that mean that nothing happens if cilia are disrupted or are not present? No, I think that some events, for example processing of Gli3 to its repressor form, can still occur. That by itself does not provide very strong evidence that signal transduction takes place outside of the cilia in any meaningful way during normal development. If you dam a stream, the water will find a way around it but its normal course is straight downhill.
SC: I concur with Andy. All available data strongly support the conclusion that the primary cilium plays a key role in coordination of vertebrate Hh signaling. However, we have just begun to sort out the significance of the cilium in regulating the individual steps in, e.g., Gli processing and other molecular mechanisms that eventually lead to altered gene expression, turning the Hh signaling pathway on and off.
Dev Dyn: Why do you think cilia are used for Hh signaling and other modes of signal transduction?
AP: Long ago, the first sensory systems must have been to find or respond to locally higher concentrations of nutrients. Major sensory systems that are most clearly derived from such a beginning, such as vision and smell, use specialized cilia structures. I would guess that Hedgehog signaling is derived from a nutrient sensing system but was co-opted to respond to the internal rather than the external milieu. That pushes the question back to one of why cilia should be used to sense the external milieu. In C. elegans, cilia are present at “ports” in the cuticle armor that protects the organism from the outside. Olfactory reception is organized at those sites and cilia provide an excellent means of limiting exposure of the cell to the outside environment whilst still sampling it.
To turn the question around, I think it is much more difficult (for me at least) to try to understand what evolutionary forces caused Drosophilia to abandon cilia for signal transduction in the internal milieu. It seems like the ancestral state is one that has worked well for us, so why not for them?
SC: Another intriguing aspect is the potential link between cilia and coordination of extracellular signaling events in the context of evolution. Protozoan cilia have receptors and signal transduction components that perceive environmental stimuli essential for, e.g., mating and motility, and in C. elegans, signaling via dendritic endings of sensory neurons is vital for developmental, physiological, and behavioral responses. Further, in many cases there are strong homologies in ciliary signaling components across the eukaryotic taxa. As an example, Maureen Barr and colleagues originally showed that homologues of the mammalian polycystic kidney-disease genes, polycystins-1 and -2, are part of the signaling machinery of sensory neurons in C. elegans. Further, Huang et al. showed that a flagellar polycystin-2 homologue is involved in the mating process of Chlamydomonas. As far as I know, neither organism has kidneys, and it is therefore tempting to speculate that coordination of signaling pathways, such as polycystin signaling, by ciliated structures was a precondition for the evolution of metazoan life as we know it and ultimately for development of the complexity of tissues and organs in vertebrate organisms, such as the kidneys.
Dev Dyn: Currently there is evidence for relatively few signaling pathways being linked to primary cilia. Do you think there are more connections that have yet to be discovered?
SC: Obviously, a large number of signaling pathways still need to be investigated in relation to primary cilia. However, up to now a panoply of diverse signaling systems have already been linked to primary cilia, including ion channels and osmolyte transporters, receptor tyrosine kinases, Wnt and Hh signaling, purinergic receptor signaling, neurotransmission and neuronal regulation, as well as interactions with extracellular matrix proteins. And the list is growing as we speak because an increasing number of people from different areas of life sciences are now entering the field of primary cilia.
AP: I think that there are undiscovered signal transduction events that occur in cilia but they are likely to be more isolated parts of a signaling network, and therefore more difficult to discern. Hh signaling has a set of core events that take place in cilia and they take place there in all Hh-responsive cells. The developing choroid plexus is responsive to the ionic balance of the cerebrospinal fluid (CSF) in a fashion that is dependent on cilia. That implies that there are critical signal transduction events that take place there but the molecular events remain unexplored. The choroid plexus is a case where the consequences of defective signal transduction, hydrocephaly, are easily apparent but I have little doubt that there are other cell type–specific examples that are not so easily recognized.
Dev Dyn: Do you think cell or tissue type–specific cilia modifications like those found in photoreceptors and olfactory sensory neurons may be more common than we realize?
SC: I think that the composition of ciliary signaling systems in some cases may mirror the functionality of the cell type. For example, primary cilia situated deep inside tissues and organs may in some cases comprise signaling pathways different from those that protrude from the apical surface into a lumen, such as on epithelial cells, to carry out diverse functions at various time points during development and in tissue homeostasis. Further, the composition of ciliary signal systems may fluctuate as part of the dynamic process that controls cell differentiation to determine cell fate and function during development. On top of this, there are several examples of modified primary cilia such as photoreceptors and olfactory sensory neurons that carry out highly specialized functions, which also reflect the diversity of ciliary signaling systems.
AP: Photoreceptors are an example of a highly derived version of a primary cilium. Such an extreme specialization is very unlikely to be found elsewhere but given that we can easily distinguish morphological differences using electron microscopy (EM) between cilia present in different tissues of the body makes it seem certain that there are specializations of cilia in different tissues. The specializations of photoreceptors are easily identifiable but if the specialization is at the molecular level, such as cell-specific IFT components, rather than at the utrastructural level, it will take much more work to ferret out. Given all of that, I would guess that specialization is probably more the rule than the exception.
Dev Dyn: Do you think like primary cilia, motile cilia may similarly be linked to certain signaling pathways?
SC: Absolutely, there is no reason to think otherwise. Evidently, classic type motile cilia with 9+2 microtubules are sensory organelles that control behavioral responses in, e.g., protozoan organisms, and I do not see why sensory capacity of motile cilia should have been lost through evolution; on the contrary! There are already a number of papers showing the unique localization of signaling pathways in motile cilia in mammals. Our group previously identified a number of signaling components in motile cilia lining the epithelium of the mammalian oviduct, including TRP ion channels as well as progesterone and angiopoetin receptors. We do not know yet the precise function of these ciliary ion channels and receptors, but we do know that that the oviduct is subjected to spectacular changes in the physiochemical milieu during the estrous cycle. And since the ciliary localization of the receptors is increased upon ovulation, we suspect that they take part in the coordination of a variety of signaling events that control, e.g., transport of the ovulated oocyte after adhesion to the ciliated infundibulum and priming of the ampulla region for reception and finally fertilization of the oocyte. Further, motile cilia of the airway system sense toxins or noxious compounds that regulate ciliary motility and help protect the lungs. There are other reports on receptors in airway cilia, such as FGF receptors, and I am confident that future research will show that motile cilia are as important sensory organelles as primary cilia when it comes to the detection of the external environment and the coordination of signaling events. And not only in the regulation ciliary beat frequency per se, but also in developmental processes and tissue homeostasis in other organs and tissues such as on the ovarian surface epithelium and in ependymal and choroid plexus epithelia.
AP: I agree with Søren completely and he gave some nice examples. Hard evidence will need to come from a sufficient understanding of the signaling and motility roles of a particular type of motile cilia that they can be disentangled. The example of the choroid plexus that I referred to above may be a particularly amenable situation. The role of cilia in regulating ionic balance in the CSF precedes the acquisition of motility during development and Brad Yoder's group has shown that the signaling mechanism involves regulation of cAMP levels. The role of cilia beating in keeping the CSF moving is straightforward so the situation is pretty ripe for distinguishing the two roles.
Dev Dyn: What are some important questions that remain to be answered?
SC: There are still a million questions to answer and, evidently, more questions will come forward as we learn more about the primary cilium in human health and disease. We have only begun to unravel the mysteries at the tip of the iceberg, and my bet is that we will be surprised to see how big this iceberg actually is. My gut feeling also tells me that many more signaling pathways than those previously described are regulated directly or indirectly by the cilium, and it will be important to understand how these signaling pathways are transmitted via the centrosomal region to change the gene expression profile of the cell. In addition, the monitoring and organization of appropriate signaling events seem to depend highly on appropriate trafficking of the signaling systems to and within the cilium. In a way, the various signaling pathways and their partners in ciliary assembly and trafficking can be regarded as the world-wide web. There are so many signaling pathways translated through the cilium and somehow they must be connected, some kind of network to determine when to turn signals on and off. Ultimately, new information about these events will lead us a step further towards the treatment of ciliopathies, such as cancer, developmental defects, and behavioral disorders.
AP: One of the most exciting areas right now is the regulation of specific cargo transport events in cilia. For example work from CC Hui's, from Kathryn Anderson's, and from my own laboratory identified a role for the Kif7 in Hh signaling. Kif7 is localized to the base of the cilia in the absence of Hh and moves to the tip along with Gli proteins in the presence of Hh. Kif7 seems likely to be an anterograde motor so it is an intriguing question as to whether it functions as a cargo adapter that regulates Gli transport into cilia or perhaps is itself a cilia motor. In either case, it does not appear to be required for other cilia transport events and so is an example of a regulator of molecularly specific transport and signal transduction within the cilia.
The question of how the cilia respond to the cell cycle is also an intriguing one. We know that the cilia are disassembled and centrioles are replicated and do their job of providing a framework for the segregation of chromosome during metaphase. What are the signals that trigger disassembly? What are the underlying molecular events involved in disassembly? How are signaling events that take place in the cilia affected during these events? Is this used as a means of allowing or vetoing certain signaling events during the cell cycle?
In a different vein, the question of how signaling complexes are formed in the cilia is a fascinating one and fundamental to its role in signal transduction. Signal transduction components need to enter the cilia in a regulated fashion and the downstream events need to be communicated to the cytoplasm or nucleus. Could this be as simple as regulating entry and exit of specific molecules into the cilia?