- Email: [email protected]
Unlocking mechanisms of development through advances in tools
Unlocking mechanisms of development through advances in tools David McClay* Duke University, Durham, NC, United States *Corresponding author: e-mail address: [email protected]
Abstract This perspective describes how our understanding of sea urchin development has been enabled by advances in technology. The early conceptual discoveries that put the sea urchin embryo on the research map had to wait until technologies were available to explain how those concepts worked. The explanatory phase continues as a number of mechanisms continue to be understood in ever greater detail, all made possible by further technical advances.
This book is the third in a Methods series on Echinoderms. It is timely in that new technologies treat our appetites for discovery by providing us with tools to dig ever deeper into an understanding of how the embryo works. In my own career new technologies have been profound in their ability to enable discovery. When I entered graduate school, I was interested in morphogenesis and marveled at the changes in cell arrangements that occurred at gastrulation. At the time one could see them and take time lapse films of them. Cut and Paste experiments were possible. But imagine, at the time there were no molecules identified that participated in those movements. Our understanding of adhesion, motility, signaling, pattern formation, and cell biology were rudimentary at best. So, how was I going to begin to understand how morphogenesis actually worked? Over the next decades, by adapting one new approach after another an understanding of that process began to emerge. That quest for understanding continues to this day and I look forward to the ability to look ever deeper into the process. And while my focus has been on morphogenesis, everyone who started graduate school at that time had the same opportunities to adapt emerging technologies to look deeper into their areas of interest. What has emerged is a remarkable depth of understanding on how embryos are built, on how the cells of those embryos are specified, on how patterning unfolds, on the nature of evolutionary change, and on many more topics.
Methods in Cell Biology, Volume 151, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.03.005 © 2019 Elsevier Inc. All rights reserved.
37
38
Unlocking mechanisms of development through advances in tools
After being asked to provide a perspective on the occasion of this volume, I thought it might be of interest to review where we’ve been as a field and how our current understanding of the echinoderm embryo has been driven by technological advances over the past decades. As part of that, at the request of the editors, I’ll also try to convey the some of the major impacts made through research with echinoderm embryos, and in particular the sea urchin, and these discoveries have been many, over the last 100+ years. Importantly, along the way, the new methods were absolutely essential for that growth in our fundamental understanding of developmental biology. My doctoral mentor, Gene Lehman, took the Woods Hole Embryology course late in the 1940s. As he later related, many of the pioneering experiments in molecular biology going on at that time were still obscure to most scientists, and the connections between genes, DNA, RNA and proteins were yet to be made, so the embryology course was long on phenomenology and short on molecular understanding. The great German embryologists had discovered many of the principles that are found currently in the first chapter of developmental biology textbooks – the properties of induction, signaling, gradients, adhesion changes, etc., but none of these was explained at a molecular level, and worse, there were few ideas or technologies available to address those phenomena. So, the embryologists at the time had to be satisfied largely with discovering new phenomena even though they had no way to explore how those phenomena worked, how they were programmed to occur in embryos of every generation, or how the information directing those phenomena was stored. While the power of today’s many tools was lacking, the ingenuity of the embryologists at the time was not. Their intellectual power should not be underestimated. Experimental embryology had engaged investigators from the late 1800s and a number of tools for “cut and paste” experiments, along with advances in microscopy and development of dyes provided the resources for a number of important discoveries and insights. Nevertheless, it wasn’t until there was an ability to work with DNA, RNA and proteins that the big advances began to explain how all of these phenomena worked. In those decades leading up to the revolution in molecular biology the sea urchin became one of the leading model organisms in this quest for understanding. Many embryologists and cell biologists headed to marine laboratories to escape the Winter or to enjoy science during the Summer months where some of the early phenomena were identified, especially at the Stazione Zoologica in Naples Italy, the Marine Biological Laboratory at Woods Hole in the United States, and the Misaki Marine Station in Japan. Some of the discoveries made at those places were transformative. Boveri used the sea urchin to deduce that chromosomes contained the units of heredity. Driesch used the sea urchin to demonstrate that preformation was wrong and that regulative development somehow worked such that complete embryos would result if blastomeres were isolated at the two- or four-cell stage. Horstadius used the sea urchin to demonstrate a number of phenomena, including induction, involved in setting up germ layers, indicating that frogs were not the only game in town. There were many other early discoveries made using sea urchin embryos during the decades leading to the 1950s but as we look back on those, they were all phenomenologically based.
Unlocking mechanisms of development through advances in tools
Watson and Crick’s working understanding of DNA structure and function launched intense work on DNA in the 1950s, and the function of RNA was uncovered in the 1960s. These advances plus the advances made by phage geneticists provided the intellectual fuel for the molecular approaches that took off. We shouldn’t forget the importance of Sputnik in these advances because it stimulated the US government to vastly increase its funding for research of all kinds, including basic research in biology. In Developmental Biology (the new name for the field that had formerly been called Embryology, driven in part by the merging with geneticists), the arrival of new tools for discovery began to be introduced with remarkable speed. Investigators who had focused on their favorite model organism, now worked to adapt the flood of technologies to the many unanswered questions they had previously been unable to approach. For some scientists, the sea urchin embryo became a favorite model for the simple reason that a huge number of embryos could be grown synchronously such that at any stage the embryos could be treated as one for biochemical purposes and explored for molecular function. Roy Britten and Eric Davidson took advantage of this property to study quantitative properties of DNA using the newly discovered reassociation kinetics of DNA after melting the double strand and recording the rate of reassociation via Cot curves. They discovered that a large portion of the sea urchin DNA was repetitive, and assuming that the non-repetitive DNA represented genes, and assuming a rough average size for each gene they estimated that the sea urchin genome contained 20–25,000 genes, a pretty good estimate as it turned out. The 1960’s also produced enormous advances in protein identification and analysis. As one example, Laemmli’s introduction of SDS gel electrophoresis expanded the ability to visualize individual proteins in a complex mix of proteins. This technical advance was introduced to students in the Physiology and Embryology courses at Woods Hole in the Summer of 1969 and as a consequence the technology rapidly spread to Developmental Biologists everywhere. This discovery was later used by Tim Hunt and others at Woods Hole to discover the cyclins, a discovery partly made using sea urchin material, and that seminal discovery led to his Nobel Prize in 2001. Many other technologies were introduced during the 1960s that greatly aided investigators in their challenge to understand protein synthesis. Ultracentrifugation was introduced allowing purification of ribosomes, identification of polysomes and for isolation of organelles. Electron microscopy, introduced in the 1950’s made it possible to see the structures that govern the cellular activities. Introduction of pharmacological inhibitors such as actinomyosin D, puromycin, and others enabled investigators to inhibit RNA synthesis or protein synthesis thereby leading to a growing understanding of how the central dogma worked and was controlled. Many of the seminal experiments with these new technologies were performed using sea urchin material. By the beginning of the 1970s this basic understanding of the gene and the means by which proteins were encoded now launched a vast undertaking to identify specific genes and proteins involved in developmental processes. The technologies of the 1970s and 1980s provided the access to specific genes and for tools to discover their function. The discoveries that had the greatest impact
39
40
Unlocking mechanisms of development through advances in tools
on understanding how development worked were of two sorts. First, the discoveries we refer to as molecular biology provided many tools. These were the now familiar approaches that enabled scientists to clone and sequence genes and express those genes both in vivo and in vitro. The sea urchin was a major beneficiary of those technologies and Larry Kedes, working with sea urchin histones, cloned the first eukaryotic gene, a histone gene. At the same time the field was looking for molecular markers of embryonic territories, and there were few approaches available to identify such molecules. A major advance came with the development of monoclonal antibodies by Caesar Milstein. My lab was attempting to learn how cells of the different tissues in the embryo organized themselves and we were looking for molecular markers to help with those experiments. We took advantage of the monoclonal technology and developed antibodies to sea urchin germ layers and territories. These tools, developed by us, and by others, provided approaches, and molecules to begin to understand how cell diversification leads to the cellular rearrangements that occur at morphogenesis. We had ideas about how the system worked but until we had the technologies to actually test those ideas, the systems remained frustratingly phenomenological. While the antibodies were valuable to us, another advance I would consider as the second major advance during this time was the famous genetic screen in Drosophila by Jani Nusslein-Volhard and Eric Wieschaus. It was a huge game changer. Not only was it now possible to clone genes and produce reagents for testing those genes for function, but the genes obtained in the Drosophila screen provided a rich group of transcription factors and signals that led to an explosion of discovery. As we now know, most of those genes were found to be expressed not only in Drosophila but also by many other embryos, including the sea urchin. This advance and others like it rapidly expanded the list of genes that contribute to the specification and patterning of embryos. To get at those molecules and understand their function further technical advances were necessary, some for all model embryos, but specific tools had to be discovered for work in each system, including the sea urchin. Andy McMahon, when in Eric Davidson’s lab, provided an important step in overcoming that challenge by developing methods for injecting the egg of the sea urchin. This made it possible to directly perturb expression of a molecule in vivo. Although antibodies could detect and localize proteins it was also important to visualize expression and localization of mRNAs. The development of in situ hybridization by Lynne Angerer was most important in this regard. At first that technology was crude, using radioactivity, but within several years the current approaches for localizing RNA expression using colorimetric in situ hybridization approaches helped transform the field. By the end of the 1990s every one of the major model embryos had advanced to the point where hundreds of RNAs and proteins were known to be involved in specification and diversification of the increasingly complex developmental states. The ease and speed of experimental perturbation in the sea urchin embryo allowed Eric Davidson and colleagues to begin assembling working models of gene regulatory
Unlocking mechanisms of development through advances in tools
networks that governed early specification of the embryo. I still remember the day in 1998 that I received a call from Eric inviting my lab to participate in the identification of the signals and transcription factors that specify cell identities during early development. Led by the Davidson lab, we and others in the community produced the first models of regulatory states during early development. In that search for understanding, additional technologies were demanded. Automated sequencing technologies and the rapid introduction of new genomics technologies had a huge impact and provided the momentum for publication of the sea urchin genome in 2006. This vastly expanded the number of genes available for discovery of structure and function, and investigators soon took advantage of an ever-expanding repertoire of genomic technologies, each of which enabled developmental biologists to pry open new areas of discovery. As I look back, I never dreamed that the humble questions we tried to address in the 1970s could now be understood at a level of mechanistic detail beyond our imagination. Of course, this story could be told in a similar way for each of a number of embryos that constitute the major model systems. Some of these have utilized the power of genetics to explore frontiers of understanding. Drosophila and C. elegans both have forward genetics as an exceptional asset and in each many other technologies expanded the versatility of those genetic approaches. Zebrafish was adapted as the most ideal vertebrate system for forward genetics. Technology provided ideal advantages for other model systems as well. In mice, for example, gene knockout approaches gave that model system a valuable tool for advancing an understanding of how mammalian development works. In tunicates, an electroporation method allowed for easy introduction of molecular constructs into cells for experimentation and perturbation. As with the other model systems, an understanding of how development works in the sea urchin has been advanced through new technical applications. Thus, the publication of this third Methods Book includes new and updated technologies that surely will enable new discoveries in the sea urchin embryo. The sea urchin has provided crucial discoveries in each of the decades preceding this and those have advanced our understanding of cell biology, developmental biology, molecular biology, evolution, physiology, and biochemistry. There is every reason to believe that in the coming decades this flow of new information will continue, and, as before that flow will be possible through adaptation of new technologies. Consequently, I thank the tireless efforts of the editors in compiling these tools all of us can use for our research. Our field has made remarkable inroads toward understanding how the classic phenomena, identified by our predecessors, actually work at a molecular level. I look forward to the discoveries made possible by these methods and yet newer methods that certainly will allow us to continue exploring how development works.
41
Abstract This perspective describes how our understanding of sea urchin development has been enabled by advances in technology. The early conceptual discoveries that put the sea urchin embryo on the research map had to wait until technologies were available to explain how those concepts worked. The explanatory phase continues as a number of mechanisms continue to be understood in ever greater detail, all made possible by further technical advances.
This book is the third in a Methods series on Echinoderms. It is timely in that new technologies treat our appetites for discovery by providing us with tools to dig ever deeper into an understanding of how the embryo works. In my own career new technologies have been profound in their ability to enable discovery. When I entered graduate school, I was interested in morphogenesis and marveled at the changes in cell arrangements that occurred at gastrulation. At the time one could see them and take time lapse films of them. Cut and Paste experiments were possible. But imagine, at the time there were no molecules identified that participated in those movements. Our understanding of adhesion, motility, signaling, pattern formation, and cell biology were rudimentary at best. So, how was I going to begin to understand how morphogenesis actually worked? Over the next decades, by adapting one new approach after another an understanding of that process began to emerge. That quest for understanding continues to this day and I look forward to the ability to look ever deeper into the process. And while my focus has been on morphogenesis, everyone who started graduate school at that time had the same opportunities to adapt emerging technologies to look deeper into their areas of interest. What has emerged is a remarkable depth of understanding on how embryos are built, on how the cells of those embryos are specified, on how patterning unfolds, on the nature of evolutionary change, and on many more topics.
Methods in Cell Biology, Volume 151, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.03.005 © 2019 Elsevier Inc. All rights reserved.
37
38
Unlocking mechanisms of development through advances in tools
After being asked to provide a perspective on the occasion of this volume, I thought it might be of interest to review where we’ve been as a field and how our current understanding of the echinoderm embryo has been driven by technological advances over the past decades. As part of that, at the request of the editors, I’ll also try to convey the some of the major impacts made through research with echinoderm embryos, and in particular the sea urchin, and these discoveries have been many, over the last 100+ years. Importantly, along the way, the new methods were absolutely essential for that growth in our fundamental understanding of developmental biology. My doctoral mentor, Gene Lehman, took the Woods Hole Embryology course late in the 1940s. As he later related, many of the pioneering experiments in molecular biology going on at that time were still obscure to most scientists, and the connections between genes, DNA, RNA and proteins were yet to be made, so the embryology course was long on phenomenology and short on molecular understanding. The great German embryologists had discovered many of the principles that are found currently in the first chapter of developmental biology textbooks – the properties of induction, signaling, gradients, adhesion changes, etc., but none of these was explained at a molecular level, and worse, there were few ideas or technologies available to address those phenomena. So, the embryologists at the time had to be satisfied largely with discovering new phenomena even though they had no way to explore how those phenomena worked, how they were programmed to occur in embryos of every generation, or how the information directing those phenomena was stored. While the power of today’s many tools was lacking, the ingenuity of the embryologists at the time was not. Their intellectual power should not be underestimated. Experimental embryology had engaged investigators from the late 1800s and a number of tools for “cut and paste” experiments, along with advances in microscopy and development of dyes provided the resources for a number of important discoveries and insights. Nevertheless, it wasn’t until there was an ability to work with DNA, RNA and proteins that the big advances began to explain how all of these phenomena worked. In those decades leading up to the revolution in molecular biology the sea urchin became one of the leading model organisms in this quest for understanding. Many embryologists and cell biologists headed to marine laboratories to escape the Winter or to enjoy science during the Summer months where some of the early phenomena were identified, especially at the Stazione Zoologica in Naples Italy, the Marine Biological Laboratory at Woods Hole in the United States, and the Misaki Marine Station in Japan. Some of the discoveries made at those places were transformative. Boveri used the sea urchin to deduce that chromosomes contained the units of heredity. Driesch used the sea urchin to demonstrate that preformation was wrong and that regulative development somehow worked such that complete embryos would result if blastomeres were isolated at the two- or four-cell stage. Horstadius used the sea urchin to demonstrate a number of phenomena, including induction, involved in setting up germ layers, indicating that frogs were not the only game in town. There were many other early discoveries made using sea urchin embryos during the decades leading to the 1950s but as we look back on those, they were all phenomenologically based.
Unlocking mechanisms of development through advances in tools
Watson and Crick’s working understanding of DNA structure and function launched intense work on DNA in the 1950s, and the function of RNA was uncovered in the 1960s. These advances plus the advances made by phage geneticists provided the intellectual fuel for the molecular approaches that took off. We shouldn’t forget the importance of Sputnik in these advances because it stimulated the US government to vastly increase its funding for research of all kinds, including basic research in biology. In Developmental Biology (the new name for the field that had formerly been called Embryology, driven in part by the merging with geneticists), the arrival of new tools for discovery began to be introduced with remarkable speed. Investigators who had focused on their favorite model organism, now worked to adapt the flood of technologies to the many unanswered questions they had previously been unable to approach. For some scientists, the sea urchin embryo became a favorite model for the simple reason that a huge number of embryos could be grown synchronously such that at any stage the embryos could be treated as one for biochemical purposes and explored for molecular function. Roy Britten and Eric Davidson took advantage of this property to study quantitative properties of DNA using the newly discovered reassociation kinetics of DNA after melting the double strand and recording the rate of reassociation via Cot curves. They discovered that a large portion of the sea urchin DNA was repetitive, and assuming that the non-repetitive DNA represented genes, and assuming a rough average size for each gene they estimated that the sea urchin genome contained 20–25,000 genes, a pretty good estimate as it turned out. The 1960’s also produced enormous advances in protein identification and analysis. As one example, Laemmli’s introduction of SDS gel electrophoresis expanded the ability to visualize individual proteins in a complex mix of proteins. This technical advance was introduced to students in the Physiology and Embryology courses at Woods Hole in the Summer of 1969 and as a consequence the technology rapidly spread to Developmental Biologists everywhere. This discovery was later used by Tim Hunt and others at Woods Hole to discover the cyclins, a discovery partly made using sea urchin material, and that seminal discovery led to his Nobel Prize in 2001. Many other technologies were introduced during the 1960s that greatly aided investigators in their challenge to understand protein synthesis. Ultracentrifugation was introduced allowing purification of ribosomes, identification of polysomes and for isolation of organelles. Electron microscopy, introduced in the 1950’s made it possible to see the structures that govern the cellular activities. Introduction of pharmacological inhibitors such as actinomyosin D, puromycin, and others enabled investigators to inhibit RNA synthesis or protein synthesis thereby leading to a growing understanding of how the central dogma worked and was controlled. Many of the seminal experiments with these new technologies were performed using sea urchin material. By the beginning of the 1970s this basic understanding of the gene and the means by which proteins were encoded now launched a vast undertaking to identify specific genes and proteins involved in developmental processes. The technologies of the 1970s and 1980s provided the access to specific genes and for tools to discover their function. The discoveries that had the greatest impact
39
40
Unlocking mechanisms of development through advances in tools
on understanding how development worked were of two sorts. First, the discoveries we refer to as molecular biology provided many tools. These were the now familiar approaches that enabled scientists to clone and sequence genes and express those genes both in vivo and in vitro. The sea urchin was a major beneficiary of those technologies and Larry Kedes, working with sea urchin histones, cloned the first eukaryotic gene, a histone gene. At the same time the field was looking for molecular markers of embryonic territories, and there were few approaches available to identify such molecules. A major advance came with the development of monoclonal antibodies by Caesar Milstein. My lab was attempting to learn how cells of the different tissues in the embryo organized themselves and we were looking for molecular markers to help with those experiments. We took advantage of the monoclonal technology and developed antibodies to sea urchin germ layers and territories. These tools, developed by us, and by others, provided approaches, and molecules to begin to understand how cell diversification leads to the cellular rearrangements that occur at morphogenesis. We had ideas about how the system worked but until we had the technologies to actually test those ideas, the systems remained frustratingly phenomenological. While the antibodies were valuable to us, another advance I would consider as the second major advance during this time was the famous genetic screen in Drosophila by Jani Nusslein-Volhard and Eric Wieschaus. It was a huge game changer. Not only was it now possible to clone genes and produce reagents for testing those genes for function, but the genes obtained in the Drosophila screen provided a rich group of transcription factors and signals that led to an explosion of discovery. As we now know, most of those genes were found to be expressed not only in Drosophila but also by many other embryos, including the sea urchin. This advance and others like it rapidly expanded the list of genes that contribute to the specification and patterning of embryos. To get at those molecules and understand their function further technical advances were necessary, some for all model embryos, but specific tools had to be discovered for work in each system, including the sea urchin. Andy McMahon, when in Eric Davidson’s lab, provided an important step in overcoming that challenge by developing methods for injecting the egg of the sea urchin. This made it possible to directly perturb expression of a molecule in vivo. Although antibodies could detect and localize proteins it was also important to visualize expression and localization of mRNAs. The development of in situ hybridization by Lynne Angerer was most important in this regard. At first that technology was crude, using radioactivity, but within several years the current approaches for localizing RNA expression using colorimetric in situ hybridization approaches helped transform the field. By the end of the 1990s every one of the major model embryos had advanced to the point where hundreds of RNAs and proteins were known to be involved in specification and diversification of the increasingly complex developmental states. The ease and speed of experimental perturbation in the sea urchin embryo allowed Eric Davidson and colleagues to begin assembling working models of gene regulatory
Unlocking mechanisms of development through advances in tools
networks that governed early specification of the embryo. I still remember the day in 1998 that I received a call from Eric inviting my lab to participate in the identification of the signals and transcription factors that specify cell identities during early development. Led by the Davidson lab, we and others in the community produced the first models of regulatory states during early development. In that search for understanding, additional technologies were demanded. Automated sequencing technologies and the rapid introduction of new genomics technologies had a huge impact and provided the momentum for publication of the sea urchin genome in 2006. This vastly expanded the number of genes available for discovery of structure and function, and investigators soon took advantage of an ever-expanding repertoire of genomic technologies, each of which enabled developmental biologists to pry open new areas of discovery. As I look back, I never dreamed that the humble questions we tried to address in the 1970s could now be understood at a level of mechanistic detail beyond our imagination. Of course, this story could be told in a similar way for each of a number of embryos that constitute the major model systems. Some of these have utilized the power of genetics to explore frontiers of understanding. Drosophila and C. elegans both have forward genetics as an exceptional asset and in each many other technologies expanded the versatility of those genetic approaches. Zebrafish was adapted as the most ideal vertebrate system for forward genetics. Technology provided ideal advantages for other model systems as well. In mice, for example, gene knockout approaches gave that model system a valuable tool for advancing an understanding of how mammalian development works. In tunicates, an electroporation method allowed for easy introduction of molecular constructs into cells for experimentation and perturbation. As with the other model systems, an understanding of how development works in the sea urchin has been advanced through new technical applications. Thus, the publication of this third Methods Book includes new and updated technologies that surely will enable new discoveries in the sea urchin embryo. The sea urchin has provided crucial discoveries in each of the decades preceding this and those have advanced our understanding of cell biology, developmental biology, molecular biology, evolution, physiology, and biochemistry. There is every reason to believe that in the coming decades this flow of new information will continue, and, as before that flow will be possible through adaptation of new technologies. Consequently, I thank the tireless efforts of the editors in compiling these tools all of us can use for our research. Our field has made remarkable inroads toward understanding how the classic phenomena, identified by our predecessors, actually work at a molecular level. I look forward to the discoveries made possible by these methods and yet newer methods that certainly will allow us to continue exploring how development works.
41