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From hemoglobin to urchin spicules
From hemoglobin to urchin spicules Fred Wilt* Department of Cell and Developmental Biology, University of California, Berkeley, CA, United States *Corresponding author: e-mail address: [email protected]
Abstract A retrospective of an academic career. The article poses the question about the values involved in a life in the academy, starting with the role of hemoglobin in the chick embryo and ending with the role of calcium in the sea urchin spine.
What a lucky guy. I was 24 years old, in Paris, on a fellowship of the Carnegie Institution of Washington. I’d just finished a Ph.D. degree at Johns Hopkins, and now I was magically transformed to a postdoctoral scholar in the embryology department of the Universite de Paris. The home base of the department was a small institute near the Bois de Vincenne, across a ring road. I was, however, completely discouraged. My immediate goal was to discover whether or not galactose could serve as a substrate for the earliest stages of chick embryo development. It had been supposed that galactose would be a substrate for the excised chick embryo raised in vitro (This was for a donor embryo of 1–2 somites in age, raised on a bed of agar). It should have been a no brainer, but I couldn’t get the embryos to differentiate under these conditions. Fortunately, during a visit to the lab of Jacques Monod, someone mentioned to me that galactose was notorious for being contaminated with glucose. I immediately recrystallized the galactose, and it failed completely to sustain embryos. The report of galactose was undone, and I had no project. I decided to lick my wounds and strike out in another direction. I set off to find out when hemoglobin was first detectable in the chick embryo. If I could do that, it might be interesting to do something with it. It turned out to be a good hunch, and I spent 4 years in my first job at Purdue chasing hemoglobin. I followed it up by more hemoglobin work for a few years in Berkeley.
Methods in Cell Biology, Volume 151, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.03.006 © 2019 Elsevier Inc. All rights reserved.
43
44
From hemoglobin to urchin spicules
I had been reading a lot during my time in Paris and at Purdue. I remember clearly the rainy afternoon in Paris when I read an article by Tore Hultin. Tore was one of the European scientists who was trying to characterize how proteins were actually made. Among the extracts he tried, he looked at sea urchin eggs and early embryos. He found that soon after insemination, the eggs burst forth with a blossoming of protein synthesis. I remembered being struck by the results. There was a chance of getting in the ground floor of how proteins are made, and relating these early events to early development. So, I made a trip to Stockholm the summer of 1962 to visit Hultin for several weeks; I thought I could learn how sea urchin eggs were turned in to protein synthesizing machines. By chance, Marshall Nirenberg at NIH had just discovered that extracts of E. coli could be programmed with polyuridylic (poly U) acid to synthesize poly phenylalanine (which eventually led to Nirenberg getting the Nobel prize). So I packed a small amount of poly (U) in my kit to bring along to Stockholm. Hultin had previously discovered that within a few minutes after fertilization the urchin egg turned on a prodigious amount of protein synthesis. We repeated these results at Kristineberg, a small marine station about 50 miles north of Goteberg. We got results almost at once. When we added poly (U) to extracts of eggs, they were very accomplished in making poly phenylalanine. I had been struck by the ease with which one could make progress in this field, and I resolved to continue the work. After 4 years at Purdue, I took a job at Berkeley. We worked both on hemoglobin synthesis in chick embryos, and on polypeptide assembly in sea urchin eggs. The major discovery of those first years was the finding that the urchin eggs were fueled by a storehouse of RNAs that were mobilized for protein synthesis. The egg simply had to realize its potential. I should mention my debt to Don Brown, ****(Carnegie Institute in Baltimore), who was discovering the roles of proteins in Xenopus eggs, a truly momentous task. Also my colleague at Berkeley, Dan Mazia, for his support and faith in the sea urchin system. What followed was an exploration of DNA, RNA, and protein synthesis in the development of sea urchins. This effort was primarily due to the late Eric Davidson at Cal Tech. He fearlessly worked on urchins, and he made many important discoveries. This resulted in discovering the method of protein synthesis regulated by a complex system of enhancers. To this day his work (and many colleagues and many others who have graced this book) stands the test of time. We had been working on histone mRNA synthesis with Robert Maxson and Scott Goustin. I suggested to Goustin that the skeleton of the pluteus larva might be an interesting object for study. We knew almost nothing about it; mostly it turned into a time burner. The addition of a visitor, Steve Benson, soon turned our luck around. Benson and others began to unravel the mRNA’s involved in urchin skeleton assembly. This enterprise was initially a joint effort between Eric Davidson and my lab. This blossomed into a major effort on our part. It didn’t take long to see that the major proteins of the urchin could be studied, their own mRNAs isolated, and a whole new way of understanding how the derivatives of the PMC (primary mesenchyme cells)
From hemoglobin to urchin spicules
could be assembled into a skeleton-forming matrix. I think our major accomplishment of this period was the catalog of proteins involved in the synthesis of the urchin skeleton, and the beginnings of the assembly of these proteins into a true skeleton. The enumeration of the constituents that comprised the spicules of the sea urchin skeleton, still an unfinished business, occupied us for over 10 years. We believe that the pay-off has been considerable. The first two members of the protein family are still an important and unsolved mystery. SM50 and SM30 are both quantitatively the back bone of the assemblage. We are still perplexed as to the exact roles of these two members and a host of other proteins, many of them dedicated to spicule assembly. We have also found transition stages in the assembly, but we do not understand the exact roles of SM30 and 50. The discovery of the first such transition stage was made by Beniash, who discovered that the calcium first formed as an amorphous state before calcite was formed. Pupa Gilbert and her collaborators have found that there is a second transition stage in the formation of the final formation of calcite. Another post-doc, Mira Peled, showed that SM50 was essential for spicule formation, but we still don’t know precisely what SM50 does. We have made much progress in the study of the skeleton, but there is a lot more to be done. We probably have a more complete understanding of how the sea urchin larval skeleton is calcified than any other such object. Even so, the entire study of how massive amounts of calcium can be organized into hard parts is still very much a mystery. This would include studies of shells, microscopic plankton, and other related objects. Original research is difficult. To copy someone else is trivial. It doesn’t matter if the stakes are small or large. And what seems to open up a matter often leads to another question that is even more difficult. Thus, even though we thought that finding out the names of the proteins involved in building the skeleton of sea urchin embryos would lead to solving the whole question of how massive amounts of calcium can be assembled into a skeleton, that feat only led to more substantial questions. We’re not even certain what role the SM30 plays. While we know that SM50 is important, it can still be synthesized and no spicules appear. How does this relate to formation of the shells of other marine inverterbrates such as oysters? Do the same players get involved? Present research suggests that there is no commonality between oyster shells (or any other kind of shells) and urchin skeletons. What are we missing?
45
Abstract A retrospective of an academic career. The article poses the question about the values involved in a life in the academy, starting with the role of hemoglobin in the chick embryo and ending with the role of calcium in the sea urchin spine.
What a lucky guy. I was 24 years old, in Paris, on a fellowship of the Carnegie Institution of Washington. I’d just finished a Ph.D. degree at Johns Hopkins, and now I was magically transformed to a postdoctoral scholar in the embryology department of the Universite de Paris. The home base of the department was a small institute near the Bois de Vincenne, across a ring road. I was, however, completely discouraged. My immediate goal was to discover whether or not galactose could serve as a substrate for the earliest stages of chick embryo development. It had been supposed that galactose would be a substrate for the excised chick embryo raised in vitro (This was for a donor embryo of 1–2 somites in age, raised on a bed of agar). It should have been a no brainer, but I couldn’t get the embryos to differentiate under these conditions. Fortunately, during a visit to the lab of Jacques Monod, someone mentioned to me that galactose was notorious for being contaminated with glucose. I immediately recrystallized the galactose, and it failed completely to sustain embryos. The report of galactose was undone, and I had no project. I decided to lick my wounds and strike out in another direction. I set off to find out when hemoglobin was first detectable in the chick embryo. If I could do that, it might be interesting to do something with it. It turned out to be a good hunch, and I spent 4 years in my first job at Purdue chasing hemoglobin. I followed it up by more hemoglobin work for a few years in Berkeley.
Methods in Cell Biology, Volume 151, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.03.006 © 2019 Elsevier Inc. All rights reserved.
43
44
From hemoglobin to urchin spicules
I had been reading a lot during my time in Paris and at Purdue. I remember clearly the rainy afternoon in Paris when I read an article by Tore Hultin. Tore was one of the European scientists who was trying to characterize how proteins were actually made. Among the extracts he tried, he looked at sea urchin eggs and early embryos. He found that soon after insemination, the eggs burst forth with a blossoming of protein synthesis. I remembered being struck by the results. There was a chance of getting in the ground floor of how proteins are made, and relating these early events to early development. So, I made a trip to Stockholm the summer of 1962 to visit Hultin for several weeks; I thought I could learn how sea urchin eggs were turned in to protein synthesizing machines. By chance, Marshall Nirenberg at NIH had just discovered that extracts of E. coli could be programmed with polyuridylic (poly U) acid to synthesize poly phenylalanine (which eventually led to Nirenberg getting the Nobel prize). So I packed a small amount of poly (U) in my kit to bring along to Stockholm. Hultin had previously discovered that within a few minutes after fertilization the urchin egg turned on a prodigious amount of protein synthesis. We repeated these results at Kristineberg, a small marine station about 50 miles north of Goteberg. We got results almost at once. When we added poly (U) to extracts of eggs, they were very accomplished in making poly phenylalanine. I had been struck by the ease with which one could make progress in this field, and I resolved to continue the work. After 4 years at Purdue, I took a job at Berkeley. We worked both on hemoglobin synthesis in chick embryos, and on polypeptide assembly in sea urchin eggs. The major discovery of those first years was the finding that the urchin eggs were fueled by a storehouse of RNAs that were mobilized for protein synthesis. The egg simply had to realize its potential. I should mention my debt to Don Brown, ****(Carnegie Institute in Baltimore), who was discovering the roles of proteins in Xenopus eggs, a truly momentous task. Also my colleague at Berkeley, Dan Mazia, for his support and faith in the sea urchin system. What followed was an exploration of DNA, RNA, and protein synthesis in the development of sea urchins. This effort was primarily due to the late Eric Davidson at Cal Tech. He fearlessly worked on urchins, and he made many important discoveries. This resulted in discovering the method of protein synthesis regulated by a complex system of enhancers. To this day his work (and many colleagues and many others who have graced this book) stands the test of time. We had been working on histone mRNA synthesis with Robert Maxson and Scott Goustin. I suggested to Goustin that the skeleton of the pluteus larva might be an interesting object for study. We knew almost nothing about it; mostly it turned into a time burner. The addition of a visitor, Steve Benson, soon turned our luck around. Benson and others began to unravel the mRNA’s involved in urchin skeleton assembly. This enterprise was initially a joint effort between Eric Davidson and my lab. This blossomed into a major effort on our part. It didn’t take long to see that the major proteins of the urchin could be studied, their own mRNAs isolated, and a whole new way of understanding how the derivatives of the PMC (primary mesenchyme cells)
From hemoglobin to urchin spicules
could be assembled into a skeleton-forming matrix. I think our major accomplishment of this period was the catalog of proteins involved in the synthesis of the urchin skeleton, and the beginnings of the assembly of these proteins into a true skeleton. The enumeration of the constituents that comprised the spicules of the sea urchin skeleton, still an unfinished business, occupied us for over 10 years. We believe that the pay-off has been considerable. The first two members of the protein family are still an important and unsolved mystery. SM50 and SM30 are both quantitatively the back bone of the assemblage. We are still perplexed as to the exact roles of these two members and a host of other proteins, many of them dedicated to spicule assembly. We have also found transition stages in the assembly, but we do not understand the exact roles of SM30 and 50. The discovery of the first such transition stage was made by Beniash, who discovered that the calcium first formed as an amorphous state before calcite was formed. Pupa Gilbert and her collaborators have found that there is a second transition stage in the formation of the final formation of calcite. Another post-doc, Mira Peled, showed that SM50 was essential for spicule formation, but we still don’t know precisely what SM50 does. We have made much progress in the study of the skeleton, but there is a lot more to be done. We probably have a more complete understanding of how the sea urchin larval skeleton is calcified than any other such object. Even so, the entire study of how massive amounts of calcium can be organized into hard parts is still very much a mystery. This would include studies of shells, microscopic plankton, and other related objects. Original research is difficult. To copy someone else is trivial. It doesn’t matter if the stakes are small or large. And what seems to open up a matter often leads to another question that is even more difficult. Thus, even though we thought that finding out the names of the proteins involved in building the skeleton of sea urchin embryos would lead to solving the whole question of how massive amounts of calcium can be assembled into a skeleton, that feat only led to more substantial questions. We’re not even certain what role the SM30 plays. While we know that SM50 is important, it can still be synthesized and no spicules appear. How does this relate to formation of the shells of other marine inverterbrates such as oysters? Do the same players get involved? Present research suggests that there is no commonality between oyster shells (or any other kind of shells) and urchin skeletons. What are we missing?
45