Paul A Krieg
- Professor, Cellular and Molecular Medicine
- Research Scientist, Sarver Heart Center
- Professor, Molecular and Cellular Biology
PhD studies at Adelaide University in Australia in the Department of Biochemistry. Postdoctoral research at Warwick University in Coventry, UK and at Harvard University. Appointed Assistant professor at the University of Texas at Austin in 1988 and was promoted through the ranks to Full Professor in 1997. Moved to the University of Arizona College of Medicine in 1999.
Teach Molecular Genetics and Developmental Biology to Medical students in the College of Medicine.
Basic research into developmental mechanisms of the vertebrate embryo. Focused particularly on development of the heart and vascular system at the level of cell signaling and transcriptional regulation. Most of the studies are carried out using frog and chick embryos.
Departmental SeminarCMM 696A (Fall 2016)
Student SeminarCMM 696B (Fall 2016)
ThesisCMM 910 (Fall 2016)
Cell Biology of DiseaseCMM 404 (Summer I 2016)
Cell Biology of DiseaseCMM 504 (Summer I 2016)
Human HistologyCMM 410 (Summer I 2016)
Human HistologyCMM 510 (Summer I 2016)
ThesisCMM 910 (Summer I 2016)
Departmental SeminarCMM 696A (Spring 2016)
Student SeminarCMM 696B (Spring 2016)
ThesisCMM 910 (Spring 2016)
Topic Molec BiologyMCB 595A (Spring 2016)
ThesisCMM 910 (Winter 2015)
Departmental SeminarCMM 696A (Fall 2015)
ThesisCMM 910 (Fall 2015)
Cell Biology of DiseaseCMM 504 (Summer I 2015)
Departmental SeminarCMM 696A (Spring 2015)
Student SeminarCMM 696B (Spring 2015)
Topic Molec BiologyMCB 595A (Spring 2015)
Departmental SeminarCMM 696A (Fall 2014)
Introductory Biology IMCB 181R (Fall 2014)
ThesisCMM 910 (Summer I 2014)
Departmental SeminarCMM 696A (Spring 2014)
Student SeminarCMM 696B (Spring 2014)
Topic Molec BiologyMCB 595A (Spring 2014)
Departmental SeminarCMM 696A (Fall 2013)
Topic Molec BiologyMCB 595A (Fall 2013)
- Krieg, P., Krieg, P., Lewis, C., Lewis, C., Krieg, P. A., & Krieg, P. A. (2013). Reagents for developmental regulation of Hedgehog signaling. Methods (San Diego, Calif.).More infoWe have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor, cyclopamine, and the activators, Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes.
- Krieg, P., Krieg, P., Myers, C. T., Myers, C. T., Appleby, S. C., Appleby, S. C., Krieg, P. A., & Krieg, P. A. (2013). Use of small molecule inhibitors of the Wnt and Notch signaling pathways during Xenopus development. Methods (San Diego, Calif.).More infoSmall molecule inhibitors of growth factor signaling pathways are extremely convenient reagents for investigation of embryonic development. The chemical may be introduced at a precise time, the dose can be altered over a large range and the chemical may be removed simply by replacing the medium surrounding the embryo. Because small molecule modulators are designed to target conserved features of a protein, they are usually effective across species. Ideally the chemicals offer remarkable specificity for a particular signaling pathway and exhibit negligible off-target effects. In this study we examine the use of small molecules to modulate the Wnt and Notch signaling pathways in the Xenopus embryo. We find that IWR-1 and XAV939 are effective inhibitors of the canonical Wnt signaling pathway while BIO is an excellent activator. For Notch signaling, we find that both DAPT and RO4929097 are effective inhibitors, but that RO4929097 is the more potent reagent. This report provides researchers with useful working concentrations of reagents and a small series of genetic and biological assays that may be used to characterize the role of Wnt and Notch signaling during embryonic development.
- Krieg, P., Krieg, P., Myers, C. T., Myers, C. T., Krieg, P. A., & Krieg, P. A. (2013). BMP-mediated specification of the erythroid lineage suppresses endothelial development in blood island precursors. Blood.More infoThe developmental relationship between the blood and endothelial cell (EC) lineages remains unclear. In the extra-embryonic blood islands of birds and mammals, ECs and blood cells are closely intermixed and blood island precursor cells in the primitive streak express many of the same molecular markers, leading to the suggestion that both lineages arise from a common precursor, called the hemangioblast. Cells within the blood island of Xenopus also co-express predifferentiation markers of the blood and EC lineages. However, using multiple assays we find that precursor cells in the Xenopus blood island do not normally differentiate into ECs, suggesting that classic hemangioblasts are rare or non-existent in Xenopus. What prevents these precursor cells from developing into mature endothelial cells? We have found that BMP signaling is essential for erythroid differentiation and in the absence of BMP signaling precursor cells adopt an EC fate. Furthermore, inhibition of the erythroid transcription pathway leads to endothelial differentiation. Our results indicate that bipotential endothelial/erythroid precursor cells do indeed exist in the Xenopus blood island, but BMP signaling normally acts to constrain EC fate. More generally, these results provide evidence that commitment to the erythroid lineage limits development of bipotential precursors towards an endothelial fate.
- Krieg, P., Krieg, P., Moran, C. M., Moran, C. M., Myers, C. T., Myers, C. T., Lewis, C. M., Lewis, C. M., Krieg, P. A., & Krieg, P. A. (2012). Hedgehog regulates angiogenesis of intersegmental vessels through the VEGF signaling pathway. Developmental dynamics : an official publication of the American Association of Anatomists, 241(6).More infoThe cellular mechanisms regulating branching and growth of the intersegmental vessels (ISVs) are not well understood. We have carried out studies demonstrating that Hedgehog (Hh) signaling is a major regulator of intersomitic vessel growth.
- Krieg, P., Krieg, P., Warkman, A. S., Warkman, A. S., Whitman, S. A., Whitman, S. A., Miller, M. K., Miller, M. K., Garriock, R. J., Garriock, R. J., Schwach, C. M., Schwach, C. M., Gregorio, C. C., Gregorio, C. C., Krieg, P. A., & Krieg, P. A. (2012). Developmental expression and cardiac transcriptional regulation of Myh7b, a third myosin heavy chain in the vertebrate heart. Cytoskeleton (Hoboken, N.J.), 69(5).More infoThe mammalian heart expresses two myosin heavy chain (MYH) genes (Myh6 and Myh7), which are major components of the thick filaments of the sarcomere. We have determined that a third MYH, MYH7B, is also expressed in the myocardium. Developmental analysis shows Myh7b expression in cardiac and skeletal muscle of Xenopus, chick and mouse embryos, and in smooth muscle tissues during later stages of mouse embryogenesis. Myh7b is also expressed in the adult human heart. The promoter region of the Myh7b gene shows remarkable similarity between diverse species, suggesting that transcriptional control mechanisms have been conserved. Using luciferase reporter analysis in rat cardiomyocytes, it can be shown that MEF2, GATA, and E-box regulatory elements are essential for efficient expression of the Myh7b gene. In addition two conserved elements that do not correspond to consensus binding sites for known transcription factors are also essential for full transcriptional activity of the Myh7b reporter. Finally, the Myh7b gene shows a transcriptional response similar to Myh6 in response to cardiac hypertrophy.
- Krieg, P., Krieg, P., Moran, C. M., Moran, C. M., Salanga, M. C., Salanga, M. C., Krieg, P. A., & Krieg, P. A. (2011). Hedgehog signaling regulates size of the dorsal aortae and density of the plexus during avian vascular development. Developmental dynamics : an official publication of the American Association of Anatomists, 240(6).More infoSignaling by the hedgehog (Hh) family of secreted growth factors is essential for development of embryonic blood vessels. Embryos lacking Hh function have abundant endothelial cells but fail to assemble vascular cords or lumenized endothelial tubes. However, the role of Hh signaling during later aspects of vascular patterning and morphogenesis is largely unexplored. We have used small molecule inhibitors and agonists to alter activity of the Hh signaling pathway in the chick embryo. When cyclopamine is added after cord formation, aortal cells form tubes, but these are small and disorganized and the density of the adjacent vascular plexus is reduced. Activation of the Hh pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. The number of endothelial cell filopodia is found to correlate with Hh signaling levels. These studies show that Hh signaling levels must be tightly regulated for normal vascular patterning to be achieved.
- Krieg, P., Krieg, P., Salanga, M. C., Salanga, M. C., Meadows, S. M., Meadows, S. M., Myers, C. T., Myers, C. T., Krieg, P. A., & Krieg, P. A. (2010). ETS family protein ETV2 is required for initiation of the endothelial lineage but not the hematopoietic lineage in the Xenopus embryo. Developmental dynamics : an official publication of the American Association of Anatomists, 239(4).More infoTranscription factors of the ETS family are important regulators of endothelial and hematopoietic development. We have characterized the Xenopus orthologue of the ETS transcription factor, ETV2. Expression analysis shows that etv2 is highly expressed in hematopoietic and endothelial precursor cells in the Xenopus embryo. In gain-of-function experiments, ETV2 is sufficient to activate ectopic expression of vascular endothelial markers. In addition, ETV2 activated expression of hematopoietic genes representing the myeloid but not the erythroid lineage. Loss-of-function studies indicate that ETV2 is required for expression of all endothelial markers examined. However, knockdown of ETV2 has no detectable effects on expression of either myeloid or erythroid markers. This contrasts with studies in mouse and zebrafish where ETV2 is required for development of the myeloid lineage. Our studies confirm an essential role for ETV2 in endothelial development, but also reveal important differences in hematopoietic development between organisms.
- Krieg, P., Krieg, P., Meadows, S. M., Meadows, S. M., Salanga, M. C., Salanga, M. C., Krieg, P. A., & Krieg, P. A. (2009). Kruppel-like factor 2 cooperates with the ETS family protein ERG to activate Flk1 expression during vascular development. Development (Cambridge, England), 136(7).More infoThe VEGF receptor, FLK1, is essential for differentiation of the endothelial lineage and for embryonic vascular development. Using comparative genomics, we have identified conserved ETS and Krüppel-like factor (KLF) binding sites within the Flk1 enhancer. In transgenic studies, mutation of either site results in dramatic reduction of Flk1 reporter expression. Overexpression of KLF2 or the ETS transcription factor ERG is sufficient to induce ectopic Flk1 expression in the Xenopus embryo. Inhibition of KLF2 function in the Xenopus embryo results in a dramatic reduction in Flk1 transcript levels. Furthermore, we show that KLF2 and ERG associate in a physical complex and that the two proteins synergistically activate transcription of Flk1. Since the ETS and KLF protein families have independently been recognized as important regulators of endothelial gene expression, cooperation between the two families has broad implications for gene regulation during development, normal physiology and vascular disease.
- Krieg, P., Krieg, P., Meadows, S. M., Meadows, S. M., Warkman, A. S., Warkman, A. S., Salanga, M. C., Salanga, M. C., Small, E. M., Small, E. M., Krieg, P. A., & Krieg, P. A. (2008). The myocardin-related transcription factor, MASTR, cooperates with MyoD to activate skeletal muscle gene expression. Proceedings of the National Academy of Sciences of the United States of America, 105(5).More infoThe myocardin family proteins (myocardin, MRTF-A, and MRTF-B) are serum response factor (SRF) cofactors and potent transcription activators. Gene-ablation studies have indicated important developmental functions for myocardin family proteins primarily in regulation of cardiac and smooth muscle development. Using Xenopus genome and cDNA databases, we identified a myocardin-related transcription factor expressed specifically in the skeletal muscle lineage. Synteny and sequence alignments indicate that this gene is the frog orthologue of mouse MASTR [Creemers EE, Sutherland LB, Oh J, Barbosa AC, Olson EN (2006) Coactivation of MEF2 by the SAP domain proteins myocardin and MASTR. Mol Cell 23:83-96]. Inhibition of MASTR function in the Xenopus embryo by using dominant-negative constructions or morpholino knockdown results in a dramatic reduction in expression of skeletal muscle marker genes. Overexpression of MASTR in whole embryos or embryonic tissue explants induces ectopic expression of muscle marker genes. Furthermore, MASTR cooperates with the myogenic regulatory factors MyoD and Myf5 to activate transcription of skeletal muscle genes. An essential function for MASTR in regulation of myogenic development in the vertebrate embryo has not been previously indicated.
- Krieg, P., Krieg, P., Moran, C. M., Moran, C. M., Garriock, R. J., Garriock, R. J., Miller, M. K., Miller, M. K., Heimark, R. L., Heimark, R. L., Gregorio, C. C., Gregorio, C. C., Krieg, P. A., & Krieg, P. A. (2008). Expression of the fast twitch troponin complex, fTnT, fTnI and fTnC, in vascular smooth muscle. Cell motility and the cytoskeleton, 65(8).More infoIt is generally believed that proteins of the troponin complex are not expressed in smooth muscle. We have directly assayed for expression of troponin transcripts in mouse vascular smooth muscle and found that troponin sequences normally associated with fast twitch skeletal muscle (fTnT, fTnI, fTnC) were present at significant levels in the thoracic aorta. In situ hybridization experiments demonstrated that fTnT, fTnI and fTnC transcripts were expressed in the smooth muscle layer of mouse blood vessels of all sizes. Protein blot analysis using rat tissue showed that at least two members of the troponin complex, Troponin T and Troponin I, were translated in vascular smooth muscle of the aorta. Finally, immuno-fluorescence microscopy of rat aortic smooth muscle revealed that TnT and TnI are localized in a unique pattern, coincident with the distribution of tropomyosin. It seems likely therefore, that a complete troponin complex is expressed in vascular smooth muscle and is associated with the contractile machinery of the cell. These observations raise the possibility that troponins play a role in regulation of smooth muscle function.
- Krieg, P., Krieg, P., Garriock, R. J., Garriock, R. J., Krieg, P. A., & Krieg, P. A. (2007). Wnt11-R signaling regulates a calcium sensitive EMT event essential for dorsal fin development of Xenopus. Developmental biology, 304(1).More infoIn the frog embryo, a sub-population of trunk neural crest (NC) cells undergoes a dorsal route of migration to contribute to the mesenchyme in the core of the dorsal fin. Here we show that a second population of cells, originally located in the dorsomedial region of the somite, also contributes to the fin mesenchyme. We find that the frog orthologue of Wnt11 (Wnt11-R) is expressed in both the NC and somite cell populations that migrate into the fin matrix. Wnt11-R is expressed prior to migration and persists in the mesenchymal cells after they have distributed throughout the fin. Loss of function studies demonstrate that Wnt11-R activity is required for an epithelial to mesenchymal transformation (EMT) event that precedes migration of cells into the fin matrix. In Wnt11-R depleted embryos, the absence of fin core cells leads to defective dorsal fin development and to collapse of the fin structure. Experiments using small molecule inhibitors indicate that dorsal migration of fin core cells depends on calcium signaling through calcium/calmodulin-dependent kinase II (CaMKII). In Wnt11-R depleted embryos, normal migration of NC cells and dorsal somite cells into the fin and normal fin development can be rescued by stimulation of calcium release. These studies are consistent with a model in which Wnt11-R signaling, via a downstream calcium pathway, regulates fin cell migration and, more generally, indicates a role for non-canonical Wnt signaling in regulation of EMT.
- Krieg, P., Krieg, P., Garriock, R. J., Garriock, R. J., Warkman, A. S., Warkman, A. S., Meadows, S. M., Meadows, S. M., D'Agostino, S., D'Agostino, S., Krieg, P. A., & Krieg, P. A. (2007). Census of vertebrate Wnt genes: isolation and developmental expression of Xenopus Wnt2, Wnt3, Wnt9a, Wnt9b, Wnt10a, and Wnt16. Developmental dynamics : an official publication of the American Association of Anatomists, 236(5).More infoThe Wnt family of growth factors regulate many different aspects of embryonic development. Assembly of the complete mouse and human genome sequences, plus expressed sequence tag surveys have established the existence of 19 Wnt genes in mammalian genomes. However, despite the importance of model vertebrates for studies in developmental biology, the complete complement of Wnt genes has not been established for nonmammalian genomes. Using genome sequences for chicken (Gallus gallus), frog (Xenopus tropicalis), and fish (Danio rerio and Tetraodon nigroviridis), we have analyzed gene synteny to identify the orthologues of all 19 human Wnt genes in these species. We find that, in addition to the 19 Wnts observed in humans, chicken contained an additional Wnt gene, Wnt11b, which is orthologous to frog and zebrafish Wnt11 (silberblick). Frog and fish genomes contained orthologues of the 19 mammalian Wnt genes, plus Wnt11b and several duplicated Wnt genes. Specifically, the Xenopus tropicalis genome contained 24 Wnt genes, including additional copies of Wnt7-related genes (Wnt7c) and 3 recent Wnt duplications (Wnt3, Wnt9b, and Wnt11). The Danio rerio genome contained 27 Wnt genes with additional copies of Wnt2, Wnt2b, Wnt4b, Wnt6, Wnt7a, and Wnt8a. The presence of the additional Wnt11 sequence (Wnt11b) in the genomes of all ancestral vertebrates suggests that this gene has been lost during mammalian evolution. Through these studies, we identified the frog orthologues of the previously uncharacterized Wnt2, Wnt3, Wnt9a, Wnt9b, Wnt10a, and Wnt16 genes and their expression has been characterized during early Xenopus development.
- Krieg, P., Krieg, P., Warkman, A. S., Warkman, A. S., Krieg, P. A., & Krieg, P. A. (2007). Xenopus as a model system for vertebrate heart development. Seminars in cell & developmental biology, 18(1).More infoThe African clawed frog, Xenopus laevis, is a valuable model system for studies of vertebrate heart development. In the following review, we describe a range of embryological and molecular methodologies that are used in Xenopus research and discuss key discoveries relating to heart development that have been made using this model system. We also discuss how the sequence of the Xenopus tropicalis genome provides a valuable tool for identification of orthologous genes and for identification of evolutionarily conserved promoter elements. Finally, both forward and reverse genetic approaches are currently being applied to Xenopus for the study of vertebrate heart development.
- Krieg, P., Krieg, P., Cox, C. M., Cox, C. M., D'Agostino, S. L., D'Agostino, S. L., Miller, M. K., Miller, M. K., Heimark, R. L., Heimark, R. L., Krieg, P. A., & Krieg, P. A. (2006). Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo. Developmental biology, 296(1).More infoThe peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.
- Krieg, P., Krieg, P., Garriock, R. J., Garriock, R. J., D'Agostino, S. L., D'Agostino, S. L., Pilcher, K. C., Pilcher, K. C., Krieg, P. A., & Krieg, P. A. (2005). Wnt11-R, a protein closely related to mammalian Wnt11, is required for heart morphogenesis in Xenopus. Developmental biology, 279(1).More infoWnt11 is a secreted protein that signals through the non-canonical planar cell polarity pathway and is a potent modulator of cell behavior and movement. In human, mouse, and chicken, there is a single Wnt11 gene, but in zebrafish and Xenopus, there are two genes related to Wnt11. The originally characterized Xenopus Wnt11 gene is expressed during early embryonic development and has a critical role in regulation of gastrulation movements. We have identified a second Xenopus Wnt11-Related gene (Wnt11-R) that is expressed after gastrulation. Sequence comparison suggests that Xenopus Wnt11-R, not Wnt11, is the ortholog of mammalian and chicken Wnt11. Xenopus Wnt11-R is expressed in neural tissue, dorsal mesenchyme derived from the dermatome region of the somites, the brachial arches, and the muscle layer of the heart, similar to the expression patterns reported for mouse and chicken Wnt11. Xenopus Wnt11-R exhibits biological properties similar to those previously described for Xenopus Wnt11, in particular the ability to activate Jun-N-terminal kinase (JNK) and to induce myocardial marker expression in ventral marginal zone (VMZ) explants. Morpholino inhibition experiments demonstrate, however, that Wnt11-R is not required for cardiac differentiation, but functions in regulation of cardiac morphogenesis. Embryos with reduced Wnt11-R activity exhibit aberrant cell-cell contacts within the myocardial wall and defects in fusion of the nascent heart tube.
- Krieg, P., Krieg, P., Garriock, R. J., Garriock, R. J., Meadows, S. M., Meadows, S. M., Krieg, P. A., & Krieg, P. A. (2005). Developmental expression and comparative genomic analysis of Xenopus cardiac myosin heavy chain genes. Developmental dynamics : an official publication of the American Association of Anatomists, 233(4).More infoMyosin heavy chains (MHC) are cytoskeletal motor proteins essential to the process of muscle contraction. We have determined the complete sequences of the Xenopus cardiac MHC genes, alpha-MHC and ventricular MHC (vMHC), and have characterized their developmental expression profiles. Whereas alpha-MHC is expressed from the earliest stages of cardiac differentiation, vMHC transcripts are not detected until the heart has undergone chamber formation. Early expression of vMHC appears to mark the cardiac conduction system, but expression expands to include the ventricle and outflow tract myocardium during subsequent development. Sequence comparisons, transgenic expression analysis, and comparative genomic studies indicate that Xenopus alpha-MHC is the true orthologue of the mammalian alpha-MHC gene. On the other hand, we show that the Xenopus vMHC gene is most closely related to chicken ventricular MHC (vMHC1) not the mammalian beta-MHC. Comparative genomic analysis has allowed the detection of a mammalian MHC gene (MyH15) that appears to be the orthologue of vMHC, but evidence suggests that this gene is no longer active.
- Krieg, P., Krieg, P., Small, E. M., Small, E. M., Warkman, A. S., Warkman, A. S., Wang, D., Wang, D., Sutherland, L. B., Sutherland, L. B., Olson, E. N., Olson, E. N., Krieg, P. A., & Krieg, P. A. (2005). Myocardin is sufficient and necessary for cardiac gene expression in Xenopus. Development (Cambridge, England), 132(5).More infoMyocardin is a cardiac- and smooth muscle-specific cofactor for the ubiquitous transcription factor serum response factor (SRF). Using gain-of-function approaches in the Xenopus embryo, we show that myocardin is sufficient to activate transcription of a wide range of cardiac and smooth muscle differentiation markers in non-muscle cell types. We also demonstrate that, for the myosin light chain 2 gene (MLC2), myocardin cooperates with the zinc-finger transcription factor Gata4 to activate expression. Inhibition of myocardin activity in Xenopus embryos using morpholino knockdown methods results in inhibition of cardiac development and the absence of expression of cardiac differentiation markers and severe disruption of cardiac morphological processes. We conclude that myocardin is an essential component of the regulatory pathway for myocardial differentiation.
- Krieg, P., Krieg, P., Small, E. M., Small, E. M., Krieg, P. A., & Krieg, P. A. (2004). Molecular regulation of cardiac chamber-specific gene expression. Trends in cardiovascular medicine, 14(1).More infoThis review focuses on recent studies investigating the genetic regulatory mechanisms leading to formation of morphologically, functionally, and molecularly distinct cardiac chambers. The regulation of four representative chamber-specific genes that have been studied in detail is reviewed. These genes include the atrial-specific genes, myosin light chain-2a (MLC2a), slow myosin heavy chain-3 (slow MyHC3), and atrial natriuretic factor (ANF) and the ventricular specific gene, myosin light chain-2v (MLC2v). Comparison of these promoters reveals some generalizations about the regulatory mechanisms involved in chamber-specific gene expression but, equally, indicates the large gaps in the knowledge concerning this intriguing genetic program.
- Krieg, P., Krieg, P., Vokes, S. A., Vokes, S. A., Yatskievych, T. A., Yatskievych, T. A., Heimark, R. L., Heimark, R. L., McMahon, J., McMahon, J., McMahon, A. P., McMahon, A. P., Antin, P. B., Antin, P. B., Krieg, P. A., & Krieg, P. A. (2004). Hedgehog signaling is essential for endothelial tube formation during vasculogenesis. Development (Cambridge, England), 131(17).More infoDuring embryonic development, the first blood vessels are formed through the aggregation and subsequent assembly of angioblasts (endothelial precursors) into a network of endothelial tubes, a process known as vasculogenesis. These first vessels generally form in mesoderm that is adjacent to endodermal tissue. Although specification of the angioblast lineage is independent of endoderm interactions, a signal from the endoderm is necessary for angioblasts to assemble into a vascular network and to undergo vascular tube formation. In this study, we show that endodermally derived sonic hedgehog is both necessary and sufficient for vascular tube formation in avian embryos. We also show that Hedgehog signaling is required for vascular tube formation in mouse embryos, and for vascular cord formation in cultured mouse endothelial cells. These results demonstrate a previously uncharacterized role for Hedgehog signaling in vascular development, and identify Hedgehog signaling as an important component of the molecular pathway leading to vascular tube formation.
- Krieg, P., Krieg, P., Small, E. M., Small, E. M., Krieg, P. A., & Krieg, P. A. (2003). Transgenic analysis of the atrialnatriuretic factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial specific expression of ANF. Developmental biology, 261(1).More infoThe atrial natriuretic factor (ANF) gene is initially expressed throughout the myocardial layer of the heart, but during subsequent development, expression becomes limited to the atrial chambers. Mouse knockout and mammalian cell culture studies have shown that the ANF gene is regulated by combinatorial interactions between Nkx2-5, GATA-4, Tbx5, and SRF; however, the molecular mechanisms leading to chamber-specific expression are currently unknown. We have isolated the Xenopus ANF promoter in order to examine the temporal and spatial regulation of the ANF gene in vivo using transgenic embryos. The mammalian and Xenopus ANF promoters show remarkable sequence similarity, including an Nkx2-5 binding site (NKE), two GATA sites, a T-box binding site (TBE), and two SRF binding sites (SREs). Our transgenic studies show that mutation of either SRE, the TBE or the distal GATA element, strongly reduces expression from the ANF promoter. However, mutations of the NKE, the proximal GATA, or both elements together, result in relatively minor reductions in transgene expression within the myocardium. Surprisingly, mutation of these elements results in ectopic ANF promoter activity in the kidneys, facial muscles, and aortic arch artery-associated muscles, and causes persistent expression in the ventricle and outflow tract of the heart. We propose that the NKE and proximal GATA elements serve as crucial binding sites for assembly of a repressor complex that is required for atrial-specific expression of the ANF gene.
- Krieg, P., Krieg, P., Gerber, W. V., Gerber, W. V., Vokes, S. A., Vokes, S. A., Zearfoss, N. R., Zearfoss, N. R., Krieg, P. A., & Krieg, P. A. (2002). A role for the RNA-binding protein, hermes, in the regulation of heart development. Developmental biology, 247(1).More infoRNA-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.
- Krieg, P., Krieg, P., Newman, C. S., Newman, C. S., Krieg, P. A., & Krieg, P. A. (2002). Xenopus bagpipe-related gene, koza, may play a role in regulation of cell proliferation. Developmental dynamics : an official publication of the American Association of Anatomists, 225(4).More infoThe homeobox gene koza is a new member of the vertebrate bagpipe-related gene family. Embryonic expression of koza is observed at highest levels in the muscle layer of the somites and, during later development, is restricted to the lateral somitic cells, which correspond to slow twitch muscle tissue. Expression of koza is also observed in the myocardial layer of the heart and in the cement gland. In each of these tissues, koza transcription commences only after the expression of terminal differentiation markers. By injection of synthetic mRNA, we show that overexpression of koza leads to an apparent decrease in the number of cells in the somites. No reduction in cell number is observed when koza is present in neural tissues, suggesting that koza exhibits some tissue specificity in regulation of cell proliferation. Embryonic manipulations show that restriction of koza expression to the slow twitch muscle layer is independent of axial structures but is, at least partly, regulated by signals arising in ectodermal tissue. Finally, in Drosophila, bagpipe expression is regulated by the hedgehog signaling pathway. By using ectopic expression, we show that koza transcription is positively regulated by banded hedgehog. This result indicates that regulation of bagpipe expression by hedgehog signaling is evolutionarily conserved.
- Krieg, P., Krieg, P., Pilcher, K. E., Pilcher, K. E., Krieg, P. A., & Krieg, P. A. (2002). Expression of the Wnt inhibitor, sFRP5, in the gut endoderm of Xenopus. Gene expression patterns : GEP, 2(3-4).More infoSignaling by the Wnt family of secreted growth factors is involved in numerous different aspects of embryonic development and also for maintenance of cellular function in adult tissues. In addition to regulation at the transcriptional level, Wnt activity is modulated by a number of different Wnt-binding proteins. Here we report the cloning and developmental expression pattern of the Xenopus orthologue of secreted Frizzled-related protein 5 (sFRP5), an endogenous inhibitor of Wnt signaling. At early stages of endodermal differentiation, sFRP5 is expressed in the developing liver. At later stages however, sFRP5 expression is down-regulated in the liver and becomes strongly expressed in the region corresponding to the junction between the posterior portion of the stomach and the anterior intestines.
- Krieg, P., Krieg, P., Vokes, S. A., Vokes, S. A., Krieg, P. A., & Krieg, P. A. (2002). Endoderm is required for vascular endothelial tube formation, but not for angioblast specification. Development (Cambridge, England), 129(3).More infoAngioblasts, the precursor cells that comprise the endothelial layer of blood vessels, arise from a purely mesodermal population. Individual angioblasts coalesce to form the primary vascular plexus through a process called vasculogenesis. A number of reports in the literature suggest that signals from the adjacent endoderm are necessary to induce angioblast specification within the mesoderm. We present evidence, using both embryological and molecular techniques, indicating that endoderm is not necessary for the induction of angioblasts. Xenopus embryos that had endoderm physically removed at the onset of gastrulation still express vascular markers. Furthermore, animal caps stimulated with bFGF form angioblasts in the absence of any detectable endodermal markers. These results show that endoderm is not required for the initial formation of angioblasts. While Xenopus embryos lacking endoderm contain aggregates of angioblasts, these angioblasts fail to assemble into endothelial tubes. Endothelial tube formation can be rescued, however, by implantation of endodermal tissue from sibling embryos. Based on these studies in Xenopus, and corroborating experiments using the quail embryo, we conclude that endoderm is not required for angioblast specification, but does play an essential role in the formation of vascular tubes.