Publisher Summary This chapter focuses on the cellular and molecular mechanisms that regulate emb... more Publisher Summary This chapter focuses on the cellular and molecular mechanisms that regulate embryonic vascular development. Cellular and molecular characterization of embryonic endothelial cells has significantly improved the understanding of blood vessel development and behavior. Together, classical observations and more recent molecular and genetic approaches have helped to elucidate many aspects of vascular specification, differentiation, and remodeling. The cardiovascular system is the first functional organ formed during embryogenesis, arising long before other organs and tissues. Abnormalities in its assembly or function almost invariably lead to embryonic lethality. The reason that cardiovascular function is critical to the survival of higher organisms lies in the fact that every cell must receive nutrition and eliminate wastes via blood vessels. Studies have demonstrated that the cells in complex tissues are generally located within about 100–200 μm of an endothelial-lined blood vessel, which is the diffusion limit for oxygen, otherwise they perish from starvation and/or asphyxiation. During formation, the general pattern of the embryonic vascular system is strikingly stereotyped within a species, and highly conserved between different vertebrate species. The defining cell type of the vascular system is the endothelial cell, which forms the seamless lining of the entire circulatory system, including the heart and all arteries and veins, large and small. Given the architectural conservation of the vasculature between species, it is likely that endothelial cells arise and assemble following genetically hard-wired molecular cues that are conserved across vertebrates.
Abstract A Xenopus laevis gastrula cDNA library has been screened in order to identify sequences ... more Abstract A Xenopus laevis gastrula cDNA library has been screened in order to identify sequences that are expressed early in development. We find that the mRNA encoding translation elongation factor 1-α (EF-1α) is synthesized in very large amounts in the early embryo. Transcription of EF-1α mRNA commences at the midblastula transition (MBT), and new EF-1α protein is synthesized very soon after this, as determined by the association of EF-1α mRNA with polysomes. The nucleotide sequence of a full-length EF-1α cDNA clone and the deduced amino acid sequence of Xenopus EF-1α protein are presented.
RNA-binding proteins are known to play an important role in a number of aspects of development, a... more RNA-binding proteins are known to play an important role in a number of aspects of development, although in most cases the precise mechanism of action remains unknown. We have previously described the isolation of an RNA-binding protein, hermes, that is expressed at very high levels in the differentiating myocardium. Here, we report experiments aimed at elucidating the functional role of hermes in development. Utilizing the Xenopus oocyte, we show that hermes is localized primarily to the cytoplasm, can associate in a multiprotein complex, and is able to bind to mature RNA transcripts in vivo. Overexpression of hermes in the developing embryo dramatically and specifically inhibits heart development. In particular, transcripts encoding the myocardial differentiation markers, cardiac troponin I and cardiac alpha-actin, are absent, and overall morphological development of the heart is eliminated. Examination of markers of precardiac tissue showed that expression of GATA-4 is normal, while the levels of Nkx2-5 mRNA are strongly reduced. Overall, these studies suggest that hermes plays a role in the regulation of mature transcripts required for myocardial differentiation. To our knowledge, this is the first evidence for an RNA-binding protein playing a direct role in regulation of vertebrate heart development.
All creatures from flies to humans possess a heart-like contractile organ responsible for movemen... more All creatures from flies to humans possess a heart-like contractile organ responsible for movement of fluid through the body. Molecular studies have revealed a remarkable degree of evolutionary conservation in the genetic pathways underlying heart development. In Drosophila, the transcription regulatory proteins TINMAN, PANNIER, MEF2, plus members of the TBX and HAND families are required for regulation of heart muscle differentiation and correct morphogenesis of the heart. Expression of the cardiac transcription factors is regulated by growth factor signalling from adjacent tissues. In vertebrates, members of precisely the same transcription factor families are required for heart development and their expression is also subject to growth factor regulation. One major difference is that higher organisms usually express several members of each protein family, leading to at least partial functional redundancy. In both flies and mammals, several distinct transcription regulatory proteins interact both genetically and physically to precisely regulate transcription of the structural, morphological and physiological genes required for establishment of correct heart structure and function. Key Concepts: The transcription regulatory system required for development of the heart involves members of several distinct transcription factor families. Interactions between a number of distinct transcription factors are essential for gene regulatory function. Genetic regulation of heart development is evolutionarily conserved. Keywords: Tinman; Nkx2-5; GATA factors; MEF2; Tbx; Hand; Isl1
Single-stranded RNA molecules of defined sequence are useful as substrates for the investigation ... more Single-stranded RNA molecules of defined sequence are useful as substrates for the investigation of such cellular processes as RNA processing and translation, and when highly labeled they serve as extremely efficient hybridization probes. An in vitro transcription system based on the unusual properties of SP6 RNA polymerase facilitates the synthesis of homogeneous single-stranded RNA molecules of any desired sequence. In this chapter we describe the SP6 transcription reaction in detail and point out the advantages and disadvantages of the use of single-stranded RNA molecules as substrates and as hybridization probes. In addition, we review recent studies on the use of SP6 transcripts as anti-sense RNAs that can hybridize to mRNAs in vivo and thereby block translation of a particular gene product.
Proceedings of the National Academy of Sciences, 1991
We have studied by in situ hybridization the expression of the genes encoding the somatic form an... more We have studied by in situ hybridization the expression of the genes encoding the somatic form and the oocyte form of Xenopus laevis eEF-1 alpha. The somatic form of eEF-1 alpha (eEF-1 alpha S) mRNA is virtually undetectable in male and female germ cells of the adult gonad but is very abundant in embryonic cells after the neurula stage. In contrast, another form of eEF-1 alpha (eEF-1 alpha O) mRNA is highly concentrated in oogonia and in previtellogenic oocytes but is undetectable in eggs and embryos. eEF-1 alpha O mRNA is also present in spermatogonia and spermatocytes of adult testis. The latter finding identifies eEF-1 alpha O mRNA as a germ cell-specific gene product. Although germ cells contain very little eEF-1 alpha S mRNA, several eEF-1 alpha S retropseudogenes exist in X. laevis chromosomes. These genes are thought to arise in germ cells from reverse transcription of mRNA and subsequent integration of the cDNA copies into chromosomal DNA. We suggest that eEF-1 alpha S pseud...
Proceedings of the National Academy of Sciences, 1983
A cDNA clone bank has been constructed from chicken embryonic RNA. Clones hybridizing poorly to e... more A cDNA clone bank has been constructed from chicken embryonic RNA. Clones hybridizing poorly to embryonic histone gene probes were selected as possible variant gene transcripts. The DNA sequence of one cDNA predicts an extremely variant H2A protein (H2A.F), which is 40% divergent from the most abundant H2A protein in chicken erythrocyte chromatin. The H2A.F gene is not highly conserved across large species barriers, but in the chicken there may be a family of linked genes. The H2A.F mRNA is approximately equal to 820 base pairs in length and, unlike most other histone mRNAs, is polyadenylylated. Significantly, the H2A.F transcript shows a limited tissue distribution in the chicken embryo.
Publisher Summary This chapter discusses the in vitro ribonucleic acid (RNA) synthesis with SP6 R... more Publisher Summary This chapter discusses the in vitro ribonucleic acid (RNA) synthesis with SP6 RNA polymerase. The chapter describes the use of in vitro transcription systems for the synthesis of RNAs for use as substrates and hybridization probes. The chapter discusses the advantages of the technique and several associated problems. The system of SP6 RNA polymerase is used as an example throughout the chapter, but the observations are stated to be general and can be applied to any of the phage polymerase and promoter systems, including T3 and T7. The chapter discusses the in vitro transcription with bacteriophage RNA polymerase, vectors containing bacteriophage promoters, RNA synthesis reaction, and several related concepts. The optimum conditions for in vitro transcription of deoxyribonucleic acid (DNA) cloned into plasmids containing an SP6 promoter are described in the chapter. The normal product of a transcription reaction contains a triphosphate group at the 5' end of the molecule. RNA is very stable when stored as an ethanol suspension and unlike DNA it does not seem to aggregate. As a result, very reproducible samples of RNA can be taken from an ethanol suspension providing that it is vortexed prior to setting up the reaction. The chapter reviews some of the problems most commonly encountered when using in vitro transcription systems and some potential solutions.
To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated... more To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these ...
A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structu... more A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).
Publisher Summary This chapter focuses on the cellular and molecular mechanisms that regulate emb... more Publisher Summary This chapter focuses on the cellular and molecular mechanisms that regulate embryonic vascular development. Cellular and molecular characterization of embryonic endothelial cells has significantly improved the understanding of blood vessel development and behavior. Together, classical observations and more recent molecular and genetic approaches have helped to elucidate many aspects of vascular specification, differentiation, and remodeling. The cardiovascular system is the first functional organ formed during embryogenesis, arising long before other organs and tissues. Abnormalities in its assembly or function almost invariably lead to embryonic lethality. The reason that cardiovascular function is critical to the survival of higher organisms lies in the fact that every cell must receive nutrition and eliminate wastes via blood vessels. Studies have demonstrated that the cells in complex tissues are generally located within about 100–200 μm of an endothelial-lined blood vessel, which is the diffusion limit for oxygen, otherwise they perish from starvation and/or asphyxiation. During formation, the general pattern of the embryonic vascular system is strikingly stereotyped within a species, and highly conserved between different vertebrate species. The defining cell type of the vascular system is the endothelial cell, which forms the seamless lining of the entire circulatory system, including the heart and all arteries and veins, large and small. Given the architectural conservation of the vasculature between species, it is likely that endothelial cells arise and assemble following genetically hard-wired molecular cues that are conserved across vertebrates.
Abstract A Xenopus laevis gastrula cDNA library has been screened in order to identify sequences ... more Abstract A Xenopus laevis gastrula cDNA library has been screened in order to identify sequences that are expressed early in development. We find that the mRNA encoding translation elongation factor 1-α (EF-1α) is synthesized in very large amounts in the early embryo. Transcription of EF-1α mRNA commences at the midblastula transition (MBT), and new EF-1α protein is synthesized very soon after this, as determined by the association of EF-1α mRNA with polysomes. The nucleotide sequence of a full-length EF-1α cDNA clone and the deduced amino acid sequence of Xenopus EF-1α protein are presented.
RNA-binding proteins are known to play an important role in a number of aspects of development, a... more RNA-binding proteins are known to play an important role in a number of aspects of development, although in most cases the precise mechanism of action remains unknown. We have previously described the isolation of an RNA-binding protein, hermes, that is expressed at very high levels in the differentiating myocardium. Here, we report experiments aimed at elucidating the functional role of hermes in development. Utilizing the Xenopus oocyte, we show that hermes is localized primarily to the cytoplasm, can associate in a multiprotein complex, and is able to bind to mature RNA transcripts in vivo. Overexpression of hermes in the developing embryo dramatically and specifically inhibits heart development. In particular, transcripts encoding the myocardial differentiation markers, cardiac troponin I and cardiac alpha-actin, are absent, and overall morphological development of the heart is eliminated. Examination of markers of precardiac tissue showed that expression of GATA-4 is normal, while the levels of Nkx2-5 mRNA are strongly reduced. Overall, these studies suggest that hermes plays a role in the regulation of mature transcripts required for myocardial differentiation. To our knowledge, this is the first evidence for an RNA-binding protein playing a direct role in regulation of vertebrate heart development.
All creatures from flies to humans possess a heart-like contractile organ responsible for movemen... more All creatures from flies to humans possess a heart-like contractile organ responsible for movement of fluid through the body. Molecular studies have revealed a remarkable degree of evolutionary conservation in the genetic pathways underlying heart development. In Drosophila, the transcription regulatory proteins TINMAN, PANNIER, MEF2, plus members of the TBX and HAND families are required for regulation of heart muscle differentiation and correct morphogenesis of the heart. Expression of the cardiac transcription factors is regulated by growth factor signalling from adjacent tissues. In vertebrates, members of precisely the same transcription factor families are required for heart development and their expression is also subject to growth factor regulation. One major difference is that higher organisms usually express several members of each protein family, leading to at least partial functional redundancy. In both flies and mammals, several distinct transcription regulatory proteins interact both genetically and physically to precisely regulate transcription of the structural, morphological and physiological genes required for establishment of correct heart structure and function. Key Concepts: The transcription regulatory system required for development of the heart involves members of several distinct transcription factor families. Interactions between a number of distinct transcription factors are essential for gene regulatory function. Genetic regulation of heart development is evolutionarily conserved. Keywords: Tinman; Nkx2-5; GATA factors; MEF2; Tbx; Hand; Isl1
Single-stranded RNA molecules of defined sequence are useful as substrates for the investigation ... more Single-stranded RNA molecules of defined sequence are useful as substrates for the investigation of such cellular processes as RNA processing and translation, and when highly labeled they serve as extremely efficient hybridization probes. An in vitro transcription system based on the unusual properties of SP6 RNA polymerase facilitates the synthesis of homogeneous single-stranded RNA molecules of any desired sequence. In this chapter we describe the SP6 transcription reaction in detail and point out the advantages and disadvantages of the use of single-stranded RNA molecules as substrates and as hybridization probes. In addition, we review recent studies on the use of SP6 transcripts as anti-sense RNAs that can hybridize to mRNAs in vivo and thereby block translation of a particular gene product.
Proceedings of the National Academy of Sciences, 1991
We have studied by in situ hybridization the expression of the genes encoding the somatic form an... more We have studied by in situ hybridization the expression of the genes encoding the somatic form and the oocyte form of Xenopus laevis eEF-1 alpha. The somatic form of eEF-1 alpha (eEF-1 alpha S) mRNA is virtually undetectable in male and female germ cells of the adult gonad but is very abundant in embryonic cells after the neurula stage. In contrast, another form of eEF-1 alpha (eEF-1 alpha O) mRNA is highly concentrated in oogonia and in previtellogenic oocytes but is undetectable in eggs and embryos. eEF-1 alpha O mRNA is also present in spermatogonia and spermatocytes of adult testis. The latter finding identifies eEF-1 alpha O mRNA as a germ cell-specific gene product. Although germ cells contain very little eEF-1 alpha S mRNA, several eEF-1 alpha S retropseudogenes exist in X. laevis chromosomes. These genes are thought to arise in germ cells from reverse transcription of mRNA and subsequent integration of the cDNA copies into chromosomal DNA. We suggest that eEF-1 alpha S pseud...
Proceedings of the National Academy of Sciences, 1983
A cDNA clone bank has been constructed from chicken embryonic RNA. Clones hybridizing poorly to e... more A cDNA clone bank has been constructed from chicken embryonic RNA. Clones hybridizing poorly to embryonic histone gene probes were selected as possible variant gene transcripts. The DNA sequence of one cDNA predicts an extremely variant H2A protein (H2A.F), which is 40% divergent from the most abundant H2A protein in chicken erythrocyte chromatin. The H2A.F gene is not highly conserved across large species barriers, but in the chicken there may be a family of linked genes. The H2A.F mRNA is approximately equal to 820 base pairs in length and, unlike most other histone mRNAs, is polyadenylylated. Significantly, the H2A.F transcript shows a limited tissue distribution in the chicken embryo.
Publisher Summary This chapter discusses the in vitro ribonucleic acid (RNA) synthesis with SP6 R... more Publisher Summary This chapter discusses the in vitro ribonucleic acid (RNA) synthesis with SP6 RNA polymerase. The chapter describes the use of in vitro transcription systems for the synthesis of RNAs for use as substrates and hybridization probes. The chapter discusses the advantages of the technique and several associated problems. The system of SP6 RNA polymerase is used as an example throughout the chapter, but the observations are stated to be general and can be applied to any of the phage polymerase and promoter systems, including T3 and T7. The chapter discusses the in vitro transcription with bacteriophage RNA polymerase, vectors containing bacteriophage promoters, RNA synthesis reaction, and several related concepts. The optimum conditions for in vitro transcription of deoxyribonucleic acid (DNA) cloned into plasmids containing an SP6 promoter are described in the chapter. The normal product of a transcription reaction contains a triphosphate group at the 5' end of the molecule. RNA is very stable when stored as an ethanol suspension and unlike DNA it does not seem to aggregate. As a result, very reproducible samples of RNA can be taken from an ethanol suspension providing that it is vortexed prior to setting up the reaction. The chapter reviews some of the problems most commonly encountered when using in vitro transcription systems and some potential solutions.
To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated... more To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these ...
A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structu... more A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).
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