L. Solnica-krezel - Academia.edu (original) (raw)
Papers by L. Solnica-krezel
Advances in Microbial Physiology, 1993
Publisher Summary This chapter summarizes the biology of Physarum polycephalum and gives examples... more Publisher Summary This chapter summarizes the biology of Physarum polycephalum and gives examples of how the organism has been utilized for analysis of patterns of inheritance, development, and the mitotic cycle. The chapter introduces recent advances in DNA transformation and gene targeting in the organism, and points to definitive experiments now possible using the new technology with the inveterate biology of this plasmodial slime mould. The phases of life cycle of P. polycephalum are amoeba1 phase, plasmodial phase, the sexual cycle, and inheritance. The chapter focuses on genome organization and cytoskeletal organization. The plasmodial mitotic cycle differs from the amoebal cycle in its absence of cytokinesis, and occurrence of mitosis within the nuclear membrane, orchestrated by intranuclear microtubule-organizing centers (MTOCs). This arrangement prevents nuclear fusion during mitosis that occurs in multinucleate amoebae. The molecular analysis in the plasmodium of P. polycephalum can be achieved by introduction of exogenous molecules. The expression of introduced molecules involves diffusion uptake, macroinjection, and DNA transformation. The sophisticated molecular approaches to cell-biological problems are possible in P. polycephalum and in other protists. Further studies will inevitably reveal more knowledge that has been awaiting discovery in these organisms for so many millennia.
Developmental Biology, 1998
Vertebrate eye development in the anterior region of the neural plate involves a series of induct... more Vertebrate eye development in the anterior region of the neural plate involves a series of inductive interactions dependent on the underlying prechordal plate and signals from the midline of the neural plate, including Hedgehog. The mechanisms controlling the spatiotemporal expression pattern of hedgehog genes are currently not understood. Cyclopia is observed in trilobite (tri) and knypek (kny) mutants with affected convergent extension of the embryonic axis during gastrulation. Here, we demonstrate that tri mutants show a high frequency of partial or complete cyclopia, kny mutants exhibit cyclopia infrequently, while kny m119 tri m209 double-mutant embryos have dramatically reduced convergent extension and are completely cyclopic. We analyzed the relationships between the convergent extension defect, the expression of hedgehog and prechordal plate genes, and the formation of cyclopia in kny m119 and tri m209 mutants. Our results correlate the cyclopia phenotype with the abnormal location of hh-expressing cells with respect to the optic primordium. We show that cyclopia in these mutants is not due to an incompetence of tri and kny cells to respond to Hedgehog signaling. Rather, it is a consequence of exceeding a critical distance (>40-50 m) between hedgehog-expressing cells and the prospective eye field. We hypothesize that at this distance, midline cells are not in an appropriate position to physically separate the eye field and that HH and other signals do not reach the appropriate target cells. Furthermore, tri and kny have overlapping functions in establishing proper alignment of the anterior neural plate and midline cells expressing shh and twhh genes when the partitioning of the eye primordium takes place.
Development, 2011
During vertebrate gastrulation, convergence and extension cell movements are coordinated with the... more During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane o...
Journal of Cell Biology, 2008
During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the... more During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the embryonic tissues, respectively. In zebrafish, regional differences of C&E movements have been observed; however, the underlying cell behaviors are poorly understood. Using time-lapse analyses and computational modeling, we demonstrate that C&E of the medial presomitic mesoderm is achieved by cooperation of planar and radial cell intercalations. Radial intercalations preferentially separate anterior and posterior neighbors to promote extension. In knypek;trilobite noncanonical Wnt mutants, the frequencies of cell intercalations are altered and the anteroposterior bias of radial intercalations is lost. This provides evidence for noncanonical Wnt signaling polarizing cell movements between different mesodermal cell layers. We further show using fluorescent fusion proteins that during dorsal mesoderm C&E, the noncanonical Wnt component Prickle localizes at the anterior cell edge, whereas D...
Journal of Cell Science
The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the ... more The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cytoskeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodium-specific beta 2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit beta 2-tubulin at earlier or only at later stages of development. Amoe...
Genes & Development, 2001
Previous studies identified zebrafish foxc1a andfoxc1b as homologs of the mouse forkhead gene, Fo... more Previous studies identified zebrafish foxc1a andfoxc1b as homologs of the mouse forkhead gene, Foxc1. Both genes are transcribed in the unsegmented presomitic mesoderm (PSM), newly formed somites, adaxial cells, and head mesoderm. Here, we show that inhibiting synthesis of Foxc1a (but not Foxc1b) protein with two different morpholino antisense oligonucleotides blocks formation of morphological somites, segment boundaries, and segmented expression of genes normally transcribed in anterior and posterior somites and expression of paraxis implicated in somite epithelialization. Patterning of the anterior PSM is also affected, as judged by the absence of mesp-b, ephrinB2, and ephA4expression, and the down-regulation of notch5 andnotch6. In contrast, the expression of other genes, includingmesp-a and papc, in the anterior of somite primordia, and the oscillating expression of deltaC and deltaD in the PSM appear normal. Nevertheless, this expression is apparently insufficient for the matur...
Development, 2010
Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation... more Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation entails a variety of cell behaviors, including directed cell migration and cell delamination, which are also involved in other physiological and pathological processes, such as cancer metastasis. Decreased Prostaglandin E2 (PGE2) synthesis due to interference with the Cyclooxygenase (Cox) and Prostaglandin E synthase (Ptges) enzymes halts gastrulation and limits cancer cell invasiveness, but how PGE2 regulates cell motility remains unclear. Here we show that PGE2-deficient zebrafish embryos, impaired in the epiboly, internalization, convergence and extension gastrulation movements, exhibit markedly increased cell-cell adhesion, which contributes to defective cell movements in the gastrula. Our analyses reveal that PGE2 promotes cell protrusive activity and limits cell adhesion by modulating E-cadherin transcript and protein, in part through stabilization of the Snai1a (also known as Sna...
Genes & Development, 2000
Spatial variations in the levels of bone morphogenetic protein (BMP) signaling are a critical det... more Spatial variations in the levels of bone morphogenetic protein (BMP) signaling are a critical determinant of dorsoanterior-ventroposterior pattern in vertebrate embryos. Whereas BMP overexpression abolishes both head and trunk development, known single and double loss-of-function mutations in BMP inhibitors have less dramatic effects. We report that combining mutations in the zebrafish genes bozozok and chordino causes a synergistic loss of head and trunk, whereas most cells express ventro-posterior markers and develop into a tail. Genetic inactivation of BMP signaling fully suppresses these defects. Thus, a remarkably simple genetic mechanism, involving a coinhibition of BMP function by the partially overlapping bozozok and chordino pathways is used to specify vertebrate head and trunk.
Current Topics in Developmental Biology, 1999
Vertebrate embryos, despite quite diverse early morphologies, appear to employ similar cellular s... more Vertebrate embryos, despite quite diverse early morphologies, appear to employ similar cellular strategies and conserved biochemical pathways in their development (Eyal-Giladi, 1997). In the past decade, a small tropical teleost, zebrafish (Danio rerio), became an important model system in which to study development (Streisinger et al., 1981). By combining embryology with molecular and classical genetic methods, our understanding of early inductive and morphogenetic events during vertebrate embryogenesis significantly advanced. In zebrafish, dorsal-ventral polarity is established during early cleavage and is dependent on microtubular transport of determinants from the vegetal pole to the blastomeres positioned on top of the yolk cell. The syncytium forming from these marginal blastomeres in the early blastula exhibits dorsal-ventral asymmetry with beta-catenin localized to the nuclei on the presumptive dorsal side of the syncytium. The yolk cell is a source of signals that induce an...
Development, 1996
In a large-scale screen for mutations affecting embryogenesis in zebrafish, we identified 48 muta... more In a large-scale screen for mutations affecting embryogenesis in zebrafish, we identified 48 mutations in 34 genetic loci specifically affecting craniofacial development. Mutants were analyzed for abnormalities in the cartilaginous head skeleton. Further, the expression of marker genes was studied to investigate potential abnormalities in mutant rhombencephalon, neural crest, and pharyngeal endoderm. The results suggest that the identified mutations affect three distinct aspects of craniofacial development. In one group, mutations affect the overall pattern of the craniofacial skeleton, suggesting that the genes are involved in the specification of these elements. Another large group of mutations affects differentiation and morphogenesis of cartilage, and may provide insight into the genetic control of chondrogenesis. The last group of mutations leads to the abnormal arrangement of skeletal elements and may uncover important tissue-tissue interactions underlying jaw development.
Development, 1996
The notochord is critical for the normal development of vertebrate embryos. It serves both as the... more The notochord is critical for the normal development of vertebrate embryos. It serves both as the major skeletal element of the embryo and as a signaling source for the establishment of pattern within the neurectoderm, the paraxial mesoderm and other tissues. In a large-scale systematic screen of mutations affecting embryogenesis in zebrafish we identified 65 mutations that fall into 29 complementation groups, each leading to a defect in the formation and/or maintenance of the notochord. These mutations produce phenotypic abnormalities at numerous stages of notochord development, thereby establishing a phenotypic pathway, which in turn suggests a genetic pathway for the development of the notochord. Perturbations within adjacent tissues in mutant embryos further indicate the importance of notochord-derived signals for patterning within the embryo and suggest that these mutations will yield additional insight into the cues that regulate these patterning processes.
Development, 1996
One of the major challenges of developmental biology is understanding the inductive and morphogen... more One of the major challenges of developmental biology is understanding the inductive and morphogenetic processes that shape the vertebrate embryo. In a large-scale genetic screen for zygotic effect, embryonic lethal mutations in zebrafish we have identified 25 mutations that affect specification of cell fates and/or cellular rearrangements during gastrulation. These mutations define at least 14 complementation groups, four of which correspond to previously identified genes. Phenotypic analysis of the ten novel loci revealed three groups of mutations causing distinct effects on cell fates in the gastrula. One group comprises mutations that lead to deficiencies in dorsal mesodermal fates and affect central nervous system patterning. Mutations from the second group affect formation of ventroposterior embryonic structures. We suggest that mutations in these two groups identify genes necessary for the formation, maintenance or function of the dorsal organizer and the ventral signaling pat...
Development, 1996
Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the... more Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development. Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for developmental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according ...
Development, 1994
In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover t... more In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500-1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer. At the onset of epiboly, the ex...
In a large scale screen for genetic defects in zebrafish embryogenesis we identified 49 mutations... more In a large scale screen for genetic defects in zebrafish embryogenesis we identified 49 mutations affecting development of the retina. Based on analysis of living embryos as well as histological sections, we grouped the isolated mutations into six phenotypic categories. (1) Mutations in three loci result in a loss of wild-type laminar pattern of the neural retina. (2) Defects in four loci lead to an abnormal specification of the eye anlagen. Only one eye frequently forms in this class of mutants. (3) Seven loci predominantly affect development of the outer retinal layers. Mutants in this category display cell loss mainly in the photoreceptor cell layer. (4) Nine mutations cause retardation of eye growth without any other obvious abnormalities in the retina. (5) A group of twelve mutations is characterized by nonspecific retinal degeneration. (6) Four mutations display retinal degeneration associated with a pigmentation defect. Finally, two mutations, one with absence of the ventral ...
The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence ... more The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence with the constitutively expressed beta 1B-tubulin and the myxamoeba-specific beta 1A-tubulin isotypes. A polyclonal antibody specific for beta 2-tubulin was used to monitor the subcellular distribution of the beta 2-tubulin antigen in the mitotic spindle of the mature plasmodium - the sole microtubular array in that stage of Physarum. By immunofluorescence, the beta 2-tubulin antigen was detected throughout this anastral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No beta 2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the beta 2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial beta 2-tubulin isotype in a mitotic spindle does not determine a closed, anastra...
Encyclopedia of Molecular Biology, 2002
Proceedings of the …, 2000
During zebrafish development, zygotic gene expression initiated at the midblastula transition con... more During zebrafish development, zygotic gene expression initiated at the midblastula transition converts maternal information on embryo polarity into a transcriptional read-out. Expression of a homeobox gene, vega1, is activated at midblastula transition in all blastomeres, but is down-regulated dorsally before gastrulation. Ubiquitous expression of vega1 is maintained in bozozok mutants, in which the dorsal-specific homeobox gene bozozok͞dharma (boz͞dha) is disrupted and organizer formation is impaired. Vega1 inhibits expression of boz͞dha and organizer-specific genes, and causes ventralization resulting in a headless phenotype. In contrast, VP16-vega1, a fusion including the Vega1 homeodomain and VP16 activation domain, elicits ectopic expression of organizer genes and suppresses several aspects of the boz mutant phenotype. We propose that boz͞dha-dependent down-regulation of vega1 in the dorsal region is an early essential step in organizer formation in zebrafish.
…, 2007
Human ARHGEF11, a PDZ-domain-containing Rho guanine nucleotide exchange factor (RhoGEF), has been... more Human ARHGEF11, a PDZ-domain-containing Rho guanine nucleotide exchange factor (RhoGEF), has been studied primarily in tissue culture, where it exhibits transforming ability, associates with and modulates the actin cytoskeleton, regulates neurite outgrowth, and mediates activation of Rho in response to stimulation by activated G␣12/13 or Plexin B1. The fruit fly homolog, RhoGEF2, interacts with heterotrimeric G protein subunits to activate Rho, associates with microtubules, and is required during gastrulation for cell shape changes that mediate epithelial folding. Here, we report functional characterization of a zebrafish homolog of ARHGEF11 that is expressed ubiquitously at blastula and gastrula stages and is enriched in neural tissues and the pronephros during later embryogenesis. Similar to its human homolog, zebrafish Arhgef11 stimulated actin stress fiber formation in cultured cells, whereas overexpression in the embryo of either the zebrafish or human protein impaired gastrulation movements. Loss-of-function experiments utilizing a chromosomal deletion that encompasses the arhgef11 locus, and antisense morpholino oligonucleotides designed to block either translation or splicing, produced embryos with ventrally-curved axes and a number of other phenotypes associated with ciliated epithelia. Arhgef11-deficient embryos often exhibited altered expression of laterality markers, enlarged brain ventricles, kidney cysts, and an excess number of otoliths in the otic vesicles. Although cilia formed and were motile in these embryos, polarized distribution of F-actin and Na + /K +-ATPase in the pronephric ducts was disturbed. Our studies in zebrafish embryos have identified new, essential roles for this RhoGEF in ciliated epithelia during vertebrate development.
Developmental …, 1999
The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone mo... more The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone morphogenetic protein (BMP) activity, which, in part, arises through the interaction of dorsally expressed antagonists Chordin and Noggin with the ventralizing BMPs. The zebrafish mercedes tm305 , ogon m60 , and short tail b180 mutations produce ventralized phenotypes, including expanded bmp2b/4 expression domains. We find that the three mutations are allelic and that the locus they define, renamed ogon (ogo), maps to linkage group 25. The ogo m60 and ogo b180 mutations are deficiencies and thus represent null alleles, whereas the ENU-induced allele ogo tm305 retains partial function. Aspects of the ogo m60 and ogo tm305 mutant phenotypes are fully suppressed by overexpression of BMP antagonists. Moreover, swirl tc300 , a null mutation in bmp2b, is epistatic to ogo m60 mutation, providing further evidence that ogo normally functions in a BMP-dependent manner. Embryonic patterning is highly sensitive to maternal and zygotic ogo gene dosage, especially when the level of zygotic chordin activity is also reduced. However, elimination of the zygotic activity of both genes does not result in a completely ventralized embryo. Thus, while ogo and chordin are required to limit activity of BMPs, additional mechanisms must exist to block these ventralizing signals. We have ruled out zebrafish noggin homologues as candidates for the ogo gene, including a newly identified gene, nog1, which is specifically expressed in the gastrula organizer. The results suggest that ogo encodes an as yet unidentified dorsalizing factor that mediates dorsoventral patterning by directly or indirectly antagonizing BMP activity.
Advances in Microbial Physiology, 1993
Publisher Summary This chapter summarizes the biology of Physarum polycephalum and gives examples... more Publisher Summary This chapter summarizes the biology of Physarum polycephalum and gives examples of how the organism has been utilized for analysis of patterns of inheritance, development, and the mitotic cycle. The chapter introduces recent advances in DNA transformation and gene targeting in the organism, and points to definitive experiments now possible using the new technology with the inveterate biology of this plasmodial slime mould. The phases of life cycle of P. polycephalum are amoeba1 phase, plasmodial phase, the sexual cycle, and inheritance. The chapter focuses on genome organization and cytoskeletal organization. The plasmodial mitotic cycle differs from the amoebal cycle in its absence of cytokinesis, and occurrence of mitosis within the nuclear membrane, orchestrated by intranuclear microtubule-organizing centers (MTOCs). This arrangement prevents nuclear fusion during mitosis that occurs in multinucleate amoebae. The molecular analysis in the plasmodium of P. polycephalum can be achieved by introduction of exogenous molecules. The expression of introduced molecules involves diffusion uptake, macroinjection, and DNA transformation. The sophisticated molecular approaches to cell-biological problems are possible in P. polycephalum and in other protists. Further studies will inevitably reveal more knowledge that has been awaiting discovery in these organisms for so many millennia.
Developmental Biology, 1998
Vertebrate eye development in the anterior region of the neural plate involves a series of induct... more Vertebrate eye development in the anterior region of the neural plate involves a series of inductive interactions dependent on the underlying prechordal plate and signals from the midline of the neural plate, including Hedgehog. The mechanisms controlling the spatiotemporal expression pattern of hedgehog genes are currently not understood. Cyclopia is observed in trilobite (tri) and knypek (kny) mutants with affected convergent extension of the embryonic axis during gastrulation. Here, we demonstrate that tri mutants show a high frequency of partial or complete cyclopia, kny mutants exhibit cyclopia infrequently, while kny m119 tri m209 double-mutant embryos have dramatically reduced convergent extension and are completely cyclopic. We analyzed the relationships between the convergent extension defect, the expression of hedgehog and prechordal plate genes, and the formation of cyclopia in kny m119 and tri m209 mutants. Our results correlate the cyclopia phenotype with the abnormal location of hh-expressing cells with respect to the optic primordium. We show that cyclopia in these mutants is not due to an incompetence of tri and kny cells to respond to Hedgehog signaling. Rather, it is a consequence of exceeding a critical distance (>40-50 m) between hedgehog-expressing cells and the prospective eye field. We hypothesize that at this distance, midline cells are not in an appropriate position to physically separate the eye field and that HH and other signals do not reach the appropriate target cells. Furthermore, tri and kny have overlapping functions in establishing proper alignment of the anterior neural plate and midline cells expressing shh and twhh genes when the partitioning of the eye primordium takes place.
Development, 2011
During vertebrate gastrulation, convergence and extension cell movements are coordinated with the... more During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane o...
Journal of Cell Biology, 2008
During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the... more During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the embryonic tissues, respectively. In zebrafish, regional differences of C&E movements have been observed; however, the underlying cell behaviors are poorly understood. Using time-lapse analyses and computational modeling, we demonstrate that C&E of the medial presomitic mesoderm is achieved by cooperation of planar and radial cell intercalations. Radial intercalations preferentially separate anterior and posterior neighbors to promote extension. In knypek;trilobite noncanonical Wnt mutants, the frequencies of cell intercalations are altered and the anteroposterior bias of radial intercalations is lost. This provides evidence for noncanonical Wnt signaling polarizing cell movements between different mesodermal cell layers. We further show using fluorescent fusion proteins that during dorsal mesoderm C&E, the noncanonical Wnt component Prickle localizes at the anterior cell edge, whereas D...
Journal of Cell Science
The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the ... more The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cytoskeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodium-specific beta 2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit beta 2-tubulin at earlier or only at later stages of development. Amoe...
Genes & Development, 2001
Previous studies identified zebrafish foxc1a andfoxc1b as homologs of the mouse forkhead gene, Fo... more Previous studies identified zebrafish foxc1a andfoxc1b as homologs of the mouse forkhead gene, Foxc1. Both genes are transcribed in the unsegmented presomitic mesoderm (PSM), newly formed somites, adaxial cells, and head mesoderm. Here, we show that inhibiting synthesis of Foxc1a (but not Foxc1b) protein with two different morpholino antisense oligonucleotides blocks formation of morphological somites, segment boundaries, and segmented expression of genes normally transcribed in anterior and posterior somites and expression of paraxis implicated in somite epithelialization. Patterning of the anterior PSM is also affected, as judged by the absence of mesp-b, ephrinB2, and ephA4expression, and the down-regulation of notch5 andnotch6. In contrast, the expression of other genes, includingmesp-a and papc, in the anterior of somite primordia, and the oscillating expression of deltaC and deltaD in the PSM appear normal. Nevertheless, this expression is apparently insufficient for the matur...
Development, 2010
Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation... more Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation entails a variety of cell behaviors, including directed cell migration and cell delamination, which are also involved in other physiological and pathological processes, such as cancer metastasis. Decreased Prostaglandin E2 (PGE2) synthesis due to interference with the Cyclooxygenase (Cox) and Prostaglandin E synthase (Ptges) enzymes halts gastrulation and limits cancer cell invasiveness, but how PGE2 regulates cell motility remains unclear. Here we show that PGE2-deficient zebrafish embryos, impaired in the epiboly, internalization, convergence and extension gastrulation movements, exhibit markedly increased cell-cell adhesion, which contributes to defective cell movements in the gastrula. Our analyses reveal that PGE2 promotes cell protrusive activity and limits cell adhesion by modulating E-cadherin transcript and protein, in part through stabilization of the Snai1a (also known as Sna...
Genes & Development, 2000
Spatial variations in the levels of bone morphogenetic protein (BMP) signaling are a critical det... more Spatial variations in the levels of bone morphogenetic protein (BMP) signaling are a critical determinant of dorsoanterior-ventroposterior pattern in vertebrate embryos. Whereas BMP overexpression abolishes both head and trunk development, known single and double loss-of-function mutations in BMP inhibitors have less dramatic effects. We report that combining mutations in the zebrafish genes bozozok and chordino causes a synergistic loss of head and trunk, whereas most cells express ventro-posterior markers and develop into a tail. Genetic inactivation of BMP signaling fully suppresses these defects. Thus, a remarkably simple genetic mechanism, involving a coinhibition of BMP function by the partially overlapping bozozok and chordino pathways is used to specify vertebrate head and trunk.
Current Topics in Developmental Biology, 1999
Vertebrate embryos, despite quite diverse early morphologies, appear to employ similar cellular s... more Vertebrate embryos, despite quite diverse early morphologies, appear to employ similar cellular strategies and conserved biochemical pathways in their development (Eyal-Giladi, 1997). In the past decade, a small tropical teleost, zebrafish (Danio rerio), became an important model system in which to study development (Streisinger et al., 1981). By combining embryology with molecular and classical genetic methods, our understanding of early inductive and morphogenetic events during vertebrate embryogenesis significantly advanced. In zebrafish, dorsal-ventral polarity is established during early cleavage and is dependent on microtubular transport of determinants from the vegetal pole to the blastomeres positioned on top of the yolk cell. The syncytium forming from these marginal blastomeres in the early blastula exhibits dorsal-ventral asymmetry with beta-catenin localized to the nuclei on the presumptive dorsal side of the syncytium. The yolk cell is a source of signals that induce an...
Development, 1996
In a large-scale screen for mutations affecting embryogenesis in zebrafish, we identified 48 muta... more In a large-scale screen for mutations affecting embryogenesis in zebrafish, we identified 48 mutations in 34 genetic loci specifically affecting craniofacial development. Mutants were analyzed for abnormalities in the cartilaginous head skeleton. Further, the expression of marker genes was studied to investigate potential abnormalities in mutant rhombencephalon, neural crest, and pharyngeal endoderm. The results suggest that the identified mutations affect three distinct aspects of craniofacial development. In one group, mutations affect the overall pattern of the craniofacial skeleton, suggesting that the genes are involved in the specification of these elements. Another large group of mutations affects differentiation and morphogenesis of cartilage, and may provide insight into the genetic control of chondrogenesis. The last group of mutations leads to the abnormal arrangement of skeletal elements and may uncover important tissue-tissue interactions underlying jaw development.
Development, 1996
The notochord is critical for the normal development of vertebrate embryos. It serves both as the... more The notochord is critical for the normal development of vertebrate embryos. It serves both as the major skeletal element of the embryo and as a signaling source for the establishment of pattern within the neurectoderm, the paraxial mesoderm and other tissues. In a large-scale systematic screen of mutations affecting embryogenesis in zebrafish we identified 65 mutations that fall into 29 complementation groups, each leading to a defect in the formation and/or maintenance of the notochord. These mutations produce phenotypic abnormalities at numerous stages of notochord development, thereby establishing a phenotypic pathway, which in turn suggests a genetic pathway for the development of the notochord. Perturbations within adjacent tissues in mutant embryos further indicate the importance of notochord-derived signals for patterning within the embryo and suggest that these mutations will yield additional insight into the cues that regulate these patterning processes.
Development, 1996
One of the major challenges of developmental biology is understanding the inductive and morphogen... more One of the major challenges of developmental biology is understanding the inductive and morphogenetic processes that shape the vertebrate embryo. In a large-scale genetic screen for zygotic effect, embryonic lethal mutations in zebrafish we have identified 25 mutations that affect specification of cell fates and/or cellular rearrangements during gastrulation. These mutations define at least 14 complementation groups, four of which correspond to previously identified genes. Phenotypic analysis of the ten novel loci revealed three groups of mutations causing distinct effects on cell fates in the gastrula. One group comprises mutations that lead to deficiencies in dorsal mesodermal fates and affect central nervous system patterning. Mutations from the second group affect formation of ventroposterior embryonic structures. We suggest that mutations in these two groups identify genes necessary for the formation, maintenance or function of the dorsal organizer and the ventral signaling pat...
Development, 1996
Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the... more Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development. Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for developmental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according ...
Development, 1994
In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover t... more In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500-1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer. At the onset of epiboly, the ex...
In a large scale screen for genetic defects in zebrafish embryogenesis we identified 49 mutations... more In a large scale screen for genetic defects in zebrafish embryogenesis we identified 49 mutations affecting development of the retina. Based on analysis of living embryos as well as histological sections, we grouped the isolated mutations into six phenotypic categories. (1) Mutations in three loci result in a loss of wild-type laminar pattern of the neural retina. (2) Defects in four loci lead to an abnormal specification of the eye anlagen. Only one eye frequently forms in this class of mutants. (3) Seven loci predominantly affect development of the outer retinal layers. Mutants in this category display cell loss mainly in the photoreceptor cell layer. (4) Nine mutations cause retardation of eye growth without any other obvious abnormalities in the retina. (5) A group of twelve mutations is characterized by nonspecific retinal degeneration. (6) Four mutations display retinal degeneration associated with a pigmentation defect. Finally, two mutations, one with absence of the ventral ...
The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence ... more The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence with the constitutively expressed beta 1B-tubulin and the myxamoeba-specific beta 1A-tubulin isotypes. A polyclonal antibody specific for beta 2-tubulin was used to monitor the subcellular distribution of the beta 2-tubulin antigen in the mitotic spindle of the mature plasmodium - the sole microtubular array in that stage of Physarum. By immunofluorescence, the beta 2-tubulin antigen was detected throughout this anastral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No beta 2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the beta 2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial beta 2-tubulin isotype in a mitotic spindle does not determine a closed, anastra...
Encyclopedia of Molecular Biology, 2002
Proceedings of the …, 2000
During zebrafish development, zygotic gene expression initiated at the midblastula transition con... more During zebrafish development, zygotic gene expression initiated at the midblastula transition converts maternal information on embryo polarity into a transcriptional read-out. Expression of a homeobox gene, vega1, is activated at midblastula transition in all blastomeres, but is down-regulated dorsally before gastrulation. Ubiquitous expression of vega1 is maintained in bozozok mutants, in which the dorsal-specific homeobox gene bozozok͞dharma (boz͞dha) is disrupted and organizer formation is impaired. Vega1 inhibits expression of boz͞dha and organizer-specific genes, and causes ventralization resulting in a headless phenotype. In contrast, VP16-vega1, a fusion including the Vega1 homeodomain and VP16 activation domain, elicits ectopic expression of organizer genes and suppresses several aspects of the boz mutant phenotype. We propose that boz͞dha-dependent down-regulation of vega1 in the dorsal region is an early essential step in organizer formation in zebrafish.
…, 2007
Human ARHGEF11, a PDZ-domain-containing Rho guanine nucleotide exchange factor (RhoGEF), has been... more Human ARHGEF11, a PDZ-domain-containing Rho guanine nucleotide exchange factor (RhoGEF), has been studied primarily in tissue culture, where it exhibits transforming ability, associates with and modulates the actin cytoskeleton, regulates neurite outgrowth, and mediates activation of Rho in response to stimulation by activated G␣12/13 or Plexin B1. The fruit fly homolog, RhoGEF2, interacts with heterotrimeric G protein subunits to activate Rho, associates with microtubules, and is required during gastrulation for cell shape changes that mediate epithelial folding. Here, we report functional characterization of a zebrafish homolog of ARHGEF11 that is expressed ubiquitously at blastula and gastrula stages and is enriched in neural tissues and the pronephros during later embryogenesis. Similar to its human homolog, zebrafish Arhgef11 stimulated actin stress fiber formation in cultured cells, whereas overexpression in the embryo of either the zebrafish or human protein impaired gastrulation movements. Loss-of-function experiments utilizing a chromosomal deletion that encompasses the arhgef11 locus, and antisense morpholino oligonucleotides designed to block either translation or splicing, produced embryos with ventrally-curved axes and a number of other phenotypes associated with ciliated epithelia. Arhgef11-deficient embryos often exhibited altered expression of laterality markers, enlarged brain ventricles, kidney cysts, and an excess number of otoliths in the otic vesicles. Although cilia formed and were motile in these embryos, polarized distribution of F-actin and Na + /K +-ATPase in the pronephric ducts was disturbed. Our studies in zebrafish embryos have identified new, essential roles for this RhoGEF in ciliated epithelia during vertebrate development.
Developmental …, 1999
The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone mo... more The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone morphogenetic protein (BMP) activity, which, in part, arises through the interaction of dorsally expressed antagonists Chordin and Noggin with the ventralizing BMPs. The zebrafish mercedes tm305 , ogon m60 , and short tail b180 mutations produce ventralized phenotypes, including expanded bmp2b/4 expression domains. We find that the three mutations are allelic and that the locus they define, renamed ogon (ogo), maps to linkage group 25. The ogo m60 and ogo b180 mutations are deficiencies and thus represent null alleles, whereas the ENU-induced allele ogo tm305 retains partial function. Aspects of the ogo m60 and ogo tm305 mutant phenotypes are fully suppressed by overexpression of BMP antagonists. Moreover, swirl tc300 , a null mutation in bmp2b, is epistatic to ogo m60 mutation, providing further evidence that ogo normally functions in a BMP-dependent manner. Embryonic patterning is highly sensitive to maternal and zygotic ogo gene dosage, especially when the level of zygotic chordin activity is also reduced. However, elimination of the zygotic activity of both genes does not result in a completely ventralized embryo. Thus, while ogo and chordin are required to limit activity of BMPs, additional mechanisms must exist to block these ventralizing signals. We have ruled out zebrafish noggin homologues as candidates for the ogo gene, including a newly identified gene, nog1, which is specifically expressed in the gastrula organizer. The results suggest that ogo encodes an as yet unidentified dorsalizing factor that mediates dorsoventral patterning by directly or indirectly antagonizing BMP activity.