Methods 66 (2014) 363–364
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Guest Editor’s Introduction
Developmental Biology
The earliest studies of the developing embryo, such as those carried out by Aristotle and Fabricius, used no tools except the human eye [8]. Then about 350 years ago, the newly-invented compound microscope was used to examine biological material and the study of biology, including embryonic development, was revolutionized. Somewhat less than 50 years ago, the study of embryology underwent another revolution with the advent of recombinant DNA methodology and DNA sequencing. Since this time, the field of developmental biology has been at the cutting edge in application of new technologies to investigate previously unanswerable questions at the level of cellular and molecular mechanisms of embryogenesis. In this volume we will explore twelve different applications of current methodology to study specific aspects of embryonic development. The focus is broadly on application of techniques to non-mammalian model systems. When compared to mammalian models these alternative systems offer reduced expense, generally superior access to the developing embryo for manipulation and visualization and also the ability to collect much greater amounts of embryonic tissue, particularly at early developmental stages. Development of the epicardium of the heart is a fascinating but remarkably complex biological process. Briefly, cells originating near the dorsal mesothelium come in contact with the muscular surface of the embryonic heart and then proliferate and migrate to surround the heart muscle with a new epicardial tissue layer. This tissue then undergoes extensive differentiation into vascular endothelial cells that will form the coronary blood vessels, vascular smooth muscle cells, myocardium and cardiac fibroblasts. Progress towards better understanding of the biology of this process has been hampered by the lack of cell culture conditions to maintain the epicardial precursor cells in an undifferentiated state. The isolation and culture conditions described by Garriock et al. [3] maintain epicardial precursor cells in an undifferentiated state and should greatly improve molecular and cellular analysis of regulation of the different developmental fates. The next seven chapters introduce new methods for embryological experiments using the Xenopus model system. Xenopus has remarkable advantages, particularly for the study of early aspects of embryology, due to external development, the large physical size of the embryo, the large number of embryos that can be generated, the resilience of embryonic tissue to manipulations such as explants and implants, the high quality genomic sequence and finally to the abundance of embryonic RNA or protein that can be obtained. On the other hand, the Xenopus community has been somewhat slow to embrace transgenesis and genetic methods and the high throughput genomics and proteomics techniques that have become common in zebrafish and http://dx.doi.org/10.1016/j.ymeth.2014.03.033 1046-2023/Ó 2014 Elsevier Inc. All rights reserved.
mouse. The first of the Xenopus chapters [10] provides robust methodology for examination of sarcomere formation in skeletal muscle. Although this area of research has been explored for some time by the Ferrari laboratory [2] there are many opportunities for further discoveries in the field. Perhaps one of the most important aspects of the Nworu et al. [10] chapter is the demonstration that so many of the commercial antibodies raised against mammalian muscle proteins specifically detect the homologous Xenopus proteins using immunofluorescent techniques. Additional chapters show that small molecule inhibitors or activators of specific signaling pathways can be very effectively used with Xenopus embryos and that these chemical modulators are often more effective than the protein reagents that have been traditionally used by the field [6,5]. These studies also show that reagents that have been demonstrated to work effectively for cells in culture are not necessarily effective for whole embryo experiments. The next two chapters describe how state-of-the-art nucleic acid sequencing methods may be applied for the study of early Xenopus development, first for examination of gene expression profiles in embryonic tissues [1] and second for analysis of transcription regulatory networks by whole genome chromatin immunoprecipitation (ChIP) studies [15]. The final Xenopus chapter demonstrates how transgenic reporter constructions can be used to observe, in real time, the location of growth factor-mediated signaling events in the live embryo [14]. From the Halpern laboratory we have a description of a new method for regulated gene expression in zebrafish transgenics [13]. The Q transcriptional regulatory system shows promise for tissue specific expression of transgenes, without encountering the methylation mediated silencing reported with the more commonly used GAL4/UAS system. This method will further extend the impressive range of transgenesis approaches available during zebrafish embryogenesis. The final four chapters are devoted to recent methodological advances in the chick model system. Chick has a proud history in the study of embryonic development but in recent years the system has tended to be overshadowed by other model systems more amenable to genetic gain and loss of function studies. On the other hand, transgenic avian lines are now available and these have proven to be wonderful models for live imaging of morphogenetic movements and tissue patterning [11]. The first two chick chapters [4,7] describe improved methods for chick embryo and embryonic tissue culture which further extend the utility of the chick system for examination of extended developmental processes. The final two chapters provide efficient methods for loss of function studies in chick using morpholinos [9] and efficient transgenesis methods
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Guest Editor’s Introduction / Methods 66 (2014) 363–364
using lentivirus vectors [12]. Taken together, these chapters hold promise for the continuation of chick as a central model for developmental biology studies. References [1] N.M. Amin, P. Tandon, E. Osborne Nishimura, F.L. Conlon, Methods 66 (2014) 398–409. [2] M.B. Ferrari, K. Ribbeck, D.J. Hagler, N.C. Spitzer, J. Cell Biol. 141 (6) (1998 Jun 15) 1349–1356. [3] R.J. Garriock, T. Mikawa, T.P. Yamaguchi, Methods 66 (2014) 365–369. [4] A. Honda, S.D. Freeman, X. Sai, R.K. Ladher, P. O’Neill, Methods 66 (2014) 447– 453. [5] C. Lewis, P.A. Krieg, Methods 66 (2014) 390–397. [6] C.T. Myers, S.C. Appleby, P.A. Krieg, Methods 66 (2014) 380–389. [7] H. Nagai, M. Sezaki, H. Nakamura, G. Sheng, Methods 66 (2014) 441–446.
[8] Joseph. Needham, A History of Embryology, Abelard-Schuman, New York, 1959. [9] A. Norris, A. Streit, Methods 66 (2014) 454–465. [10] C.U. Nworu, P.A. Krieg, C.C. Gregorio, Methods 66 (2014) 370–379. [11] Y. Sato, G. Poynter, D. Huss, M.B. Filla, B.J. Rongish, C.D. Little, S.E. Fraser, R. Lansford, PLoS One 5 (2010) 1–12. [12] S.L. Semple-Rowland, J. Berry, Methods 66 (2014) 466–473. [13] A. Subedi, M. Macurak, S.T. Gee, E. Monge, M.G. Goll, C.J. Potter, M.J. Parsons, M.E. Halpern, Methods 66 (2014) 433–440. [14] H.T. Tran, K. Vleminckx, Methods 66 (2014) 422–432. [15] A.E. Wills, R. Gupta, E. Chuong, J.C. Baker, Methods 66 (2014) 410–421.
Paul A. Krieg E-mail address: [email protected]
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Guest Editor’s Introduction
Developmental Biology
The earliest studies of the developing embryo, such as those carried out by Aristotle and Fabricius, used no tools except the human eye [8]. Then about 350 years ago, the newly-invented compound microscope was used to examine biological material and the study of biology, including embryonic development, was revolutionized. Somewhat less than 50 years ago, the study of embryology underwent another revolution with the advent of recombinant DNA methodology and DNA sequencing. Since this time, the field of developmental biology has been at the cutting edge in application of new technologies to investigate previously unanswerable questions at the level of cellular and molecular mechanisms of embryogenesis. In this volume we will explore twelve different applications of current methodology to study specific aspects of embryonic development. The focus is broadly on application of techniques to non-mammalian model systems. When compared to mammalian models these alternative systems offer reduced expense, generally superior access to the developing embryo for manipulation and visualization and also the ability to collect much greater amounts of embryonic tissue, particularly at early developmental stages. Development of the epicardium of the heart is a fascinating but remarkably complex biological process. Briefly, cells originating near the dorsal mesothelium come in contact with the muscular surface of the embryonic heart and then proliferate and migrate to surround the heart muscle with a new epicardial tissue layer. This tissue then undergoes extensive differentiation into vascular endothelial cells that will form the coronary blood vessels, vascular smooth muscle cells, myocardium and cardiac fibroblasts. Progress towards better understanding of the biology of this process has been hampered by the lack of cell culture conditions to maintain the epicardial precursor cells in an undifferentiated state. The isolation and culture conditions described by Garriock et al. [3] maintain epicardial precursor cells in an undifferentiated state and should greatly improve molecular and cellular analysis of regulation of the different developmental fates. The next seven chapters introduce new methods for embryological experiments using the Xenopus model system. Xenopus has remarkable advantages, particularly for the study of early aspects of embryology, due to external development, the large physical size of the embryo, the large number of embryos that can be generated, the resilience of embryonic tissue to manipulations such as explants and implants, the high quality genomic sequence and finally to the abundance of embryonic RNA or protein that can be obtained. On the other hand, the Xenopus community has been somewhat slow to embrace transgenesis and genetic methods and the high throughput genomics and proteomics techniques that have become common in zebrafish and http://dx.doi.org/10.1016/j.ymeth.2014.03.033 1046-2023/Ó 2014 Elsevier Inc. All rights reserved.
mouse. The first of the Xenopus chapters [10] provides robust methodology for examination of sarcomere formation in skeletal muscle. Although this area of research has been explored for some time by the Ferrari laboratory [2] there are many opportunities for further discoveries in the field. Perhaps one of the most important aspects of the Nworu et al. [10] chapter is the demonstration that so many of the commercial antibodies raised against mammalian muscle proteins specifically detect the homologous Xenopus proteins using immunofluorescent techniques. Additional chapters show that small molecule inhibitors or activators of specific signaling pathways can be very effectively used with Xenopus embryos and that these chemical modulators are often more effective than the protein reagents that have been traditionally used by the field [6,5]. These studies also show that reagents that have been demonstrated to work effectively for cells in culture are not necessarily effective for whole embryo experiments. The next two chapters describe how state-of-the-art nucleic acid sequencing methods may be applied for the study of early Xenopus development, first for examination of gene expression profiles in embryonic tissues [1] and second for analysis of transcription regulatory networks by whole genome chromatin immunoprecipitation (ChIP) studies [15]. The final Xenopus chapter demonstrates how transgenic reporter constructions can be used to observe, in real time, the location of growth factor-mediated signaling events in the live embryo [14]. From the Halpern laboratory we have a description of a new method for regulated gene expression in zebrafish transgenics [13]. The Q transcriptional regulatory system shows promise for tissue specific expression of transgenes, without encountering the methylation mediated silencing reported with the more commonly used GAL4/UAS system. This method will further extend the impressive range of transgenesis approaches available during zebrafish embryogenesis. The final four chapters are devoted to recent methodological advances in the chick model system. Chick has a proud history in the study of embryonic development but in recent years the system has tended to be overshadowed by other model systems more amenable to genetic gain and loss of function studies. On the other hand, transgenic avian lines are now available and these have proven to be wonderful models for live imaging of morphogenetic movements and tissue patterning [11]. The first two chick chapters [4,7] describe improved methods for chick embryo and embryonic tissue culture which further extend the utility of the chick system for examination of extended developmental processes. The final two chapters provide efficient methods for loss of function studies in chick using morpholinos [9] and efficient transgenesis methods
364
Guest Editor’s Introduction / Methods 66 (2014) 363–364
using lentivirus vectors [12]. Taken together, these chapters hold promise for the continuation of chick as a central model for developmental biology studies. References [1] N.M. Amin, P. Tandon, E. Osborne Nishimura, F.L. Conlon, Methods 66 (2014) 398–409. [2] M.B. Ferrari, K. Ribbeck, D.J. Hagler, N.C. Spitzer, J. Cell Biol. 141 (6) (1998 Jun 15) 1349–1356. [3] R.J. Garriock, T. Mikawa, T.P. Yamaguchi, Methods 66 (2014) 365–369. [4] A. Honda, S.D. Freeman, X. Sai, R.K. Ladher, P. O’Neill, Methods 66 (2014) 447– 453. [5] C. Lewis, P.A. Krieg, Methods 66 (2014) 390–397. [6] C.T. Myers, S.C. Appleby, P.A. Krieg, Methods 66 (2014) 380–389. [7] H. Nagai, M. Sezaki, H. Nakamura, G. Sheng, Methods 66 (2014) 441–446.
[8] Joseph. Needham, A History of Embryology, Abelard-Schuman, New York, 1959. [9] A. Norris, A. Streit, Methods 66 (2014) 454–465. [10] C.U. Nworu, P.A. Krieg, C.C. Gregorio, Methods 66 (2014) 370–379. [11] Y. Sato, G. Poynter, D. Huss, M.B. Filla, B.J. Rongish, C.D. Little, S.E. Fraser, R. Lansford, PLoS One 5 (2010) 1–12. [12] S.L. Semple-Rowland, J. Berry, Methods 66 (2014) 466–473. [13] A. Subedi, M. Macurak, S.T. Gee, E. Monge, M.G. Goll, C.J. Potter, M.J. Parsons, M.E. Halpern, Methods 66 (2014) 433–440. [14] H.T. Tran, K. Vleminckx, Methods 66 (2014) 422–432. [15] A.E. Wills, R. Gupta, E. Chuong, J.C. Baker, Methods 66 (2014) 410–421.
Paul A. Krieg E-mail address: [email protected]
