Biomedical subjects

Daniel L Weeks

Publications and source records attributed to Daniel L Weeks.

13 recordsLinked to original sources

PubMed2007

Chordin affects pronephros development in Xenopus embryos by anteriorizing presomitic mesoderm.

Spemann's organizer emits signals that pattern the mesodermal germ layer during Xenopus embryogenesis. In a previous study, we demonstrated that FGFR1 activity within the organizer is required for the production of both the somitic muscle- and pronephros-patterning signals by the organizer and the expression of chordin, an organizer-specific secreted protein (Mitchell and Sheets [2001] Dev. Biol. 237:295-305). Studies from others in both chicken and Xenopus embryos provide compelling evidence that pronephros forms by means of secondary induction signals emitted from anterior somites (Seufert et al. [1999] Dev. Biol. 215:233-242; Mauch et al. [2000] Dev. Biol. 220:62-75). Here we provide several lines of evidence in support of the hypothesis that chordin influences pronephros development by directing the formation of anterior somites. Chordin mRNA was absent in ultraviolet (UV) -irradiated embryos lacking pronepheros (average DAI<2) but was always found in UV-irradiated embryos that retain pronepheros (average DAI>2). Furthermore, ectopic expression of chordin in embryos and in tissue explants leads to the formation of anterior somites and pronephros. In these experiments, pronephros was only observed in association with muscle. Chordin diverted somatic muscle cells to more anterior positions within the somite file in chordin-induced secondary trunks and induced the expression of the anterior myogenic gene myf5. Finally, depletion of chordin mRNA with DEED antisense oligonucleotides substantially reduced somitic muscle and pronephric tubule and duct formation in whole embryos. These data and previous studies on ectoderm and endoderm (Sasai et al. [1995] Nature 377:757) support the idea that chordin functions as an anteriorizing signal in patterning the germ layers during vertebrate embryogenesis. Our data support the hypothesis that chordin directs the formation of anterior somites that in turn are necessary for pronephros development.

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PubMed2006

Reduction of XNkx2-10 expression leads to anterior defects and malformation of the embryonic heart.

Normal vertebrate heart development depends upon the expression of homeodomain containing proteins related to the Drosophila gene, tinman. In Xenopus laevis, three such genes have been identified in regions that will eventually give rise to the heart, XNkx2-3, XNkx2-5 and XNkx2-10. Although the expression domains of all three overlap in early development, distinctive differences have been noted. By the time the heart tube forms, there is little XNkx2-10 mRNA detected by in situ analysis in the embryonic heart while both XNkx2-3 and XNkx2-5 are clearly present. In addition, unlike XNkx2-3 and XNkx2-5, injection of XNkx2-10 mRNA does not increase the size of the embryonic heart. We have reexamined the expression and potential role of XNkx2-10 in development via oligonucleotide-mediated reduction of XNkx2-10 protein expression. We find that a decrease in XNkx2-10 leads to a broad spectrum of developmental abnormalities including a reduction in heart size. We conclude that XNkx2-10, like XNkx2-3 and XNkx2-5, is necessary for normal Xenopus heart development.

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PubMed2006

Retinoic acid signaling is essential for formation of the heart tube in Xenopus.

Retinoic acid is clearly important for the development of the heart. In this paper, we provide evidence that retinoic acid is essential for multiple aspects of cardiogenesis in Xenopus by examining embryos that have been exposed to retinoic acid receptor antagonists. Early in cardiogenesis, retinoic acid alters the expression of key genes in the lateral plate mesoderm including Nkx2.5 and HAND1, indicating that early patterning of the lateral plate mesoderm is, in part, controlled by retinoic acid. We found that, in Xenopus, the transition of the heart from a sheet of cells to a tube required retinoic acid signaling. The requirement for retinoic acid signaling was determined to take place during a narrow window of time between embryonic stages 14 and 18, well before heart tube closure. At the highest doses used, the lateral fields of myocardium fail to fuse, intermediate doses lead to a fusion of the two sides but failure to form a tube, and embryos exposed to lower concentrations of antagonist form a heart tube that failed to complete all the landmark changes that characterize looping. The myocardial phenotypes observed when exposed to the retinoic acid antagonist resemble the myocardium from earlier stages of cardiogenesis, although precocious expression of cardiac differentiation markers was not seen. The morphology of individual cells within the myocardium appeared immature, closely resembling the shape and size of cells at earlier stages of development. However, the failures in morphogenesis are not merely a slowing of development because, even when allowed to develop through stage 40, the heart tubes did not close when embryos were exposed to high levels of antagonist. Indeed, some aspects of left-right asymmetry also remained even in hearts that never formed a tube. These results demonstrate that components of the retinoic acid signaling pathway are necessary for the progression of cardiac morphogenesis in Xenopus.

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PubMed2005

Antisense knockdown of cyclin E does not affect the midblastula transition in Xenopus laevis embryos.

In Xenopus laevis embryos, cyclin E protein remains constitutively high throughout the first 12 cell cycles following fertilization until the onset of the midblastula transition (MBT) (after the 12(th) cell cycle) when it undergoes a dramatic reduction. The disappearance of cyclin E at the MBT occurs independently of active cell cycle progression, zygotic transcription, protein synthesis and the nuclear to cytoplasmic ratio. This has suggested that cyclin E is part of an autonomous maternal timer that regulates the onset of the MBT. To determine how constitutively high levels of cyclin E are maintained prior to the MBT and to investigate if the reduction in cyclin E protein affects the timing of the MBT, we have knocked down endogenous cyclin E mRNA using an N,N-diethyl-ethylene-diamine modified antisense oligonucleotide targeted to its open reading frame. We report that maintenance of high levels of cyclin E protein before the MBT is due to a balance between ongoing translation and proteolytic degradation. In support of our antisense experiments, polysome analysis demonstrates that cyclin E mRNA is associated with the translated fraction prior to the MBT. Furthermore, knockdown of cyclin E was not associated with defects in the timing of developmental events. Our data suggests that cyclin E is not required for the later cell cycles of embryonic development and that the pathway effecting downregulation of cyclin E rather then cyclin E degradation itself may be part of a maternal timer that affects the onset of the MBT.

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PubMed2005

Triplex-induced recombination and repair in the pyrimidine motif.

Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Both purine and pyrimidine TFOs, when linked to psoralen, have been shown to direct psoralen adduct formation in cells, leading to mutagenesis or recombination. However, only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct. In this work, we report the ability of a series of pyrimidine TFOs, with selected chemical modifications, to induce repair and recombination in two distinct episomal targets in mammalian cells in the absence of any DNA-reactive conjugate. We find that TFOs containing N3'-->P5' phosphoramidate (amidate), 5-(1-propynyl)-2'-deoxyuridine (pdU), 2'-O-methyl-ribose (2'-O-Me), 2'-O-(2-aminoethyl)-ribose, or 2'-O, 4'-C-methylene bridged or locked nucleic acid (LNA)-modified nucleotides show substantially increased formation of non-covalent triplexes under physiological conditions compared with unmodified DNA TFOs. However, of these modified TFOs, only the amidate and pdU-modified TFOs mediate induced recombination in cells and stimulate repair in cell extracts, at levels comparable to those seen with purine TFOs in similar assays. These results show that amidate and pdU-modified TFOs can be used as reagents to stimulate site-specific gene targeting without the need for conjugation to DNA-reactive molecules. By demonstrating the potential for induced repair and recombination with appropriately modified pyrimidine TFOs, this work expands the options available for triplex-mediated gene targeting.

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PubMed2005

Distance and affinity dependence of triplex-induced recombination.

Triplex-forming oligonucleotides (TFOs) have the potential to serve as gene therapeutic agents on the basis of their ability to mediate site-specific genome modification via induced recombination. However, high-affinity triplex formation is limited to polypurine/polypyrimidine sites in duplex DNA. Because of this sequence restriction, careful analysis is needed to identify suitable TFO target sites within or near genes of interest. We report here an examination of two key parameters which influence the efficiency of TFO-induced recombination: (1) binding affinity of the TFO for the target site and (2) the distance between the target site and the mutation to be corrected. To test the influence of binding affinity, we compared induced recombination in human cell-free extracts by a series of G-rich oligonucleotides with an identical base composition and an increasing number of mismatches in the third strand binding code. As the number of mismatches increased and, therefore, binding affinity decreased, induced recombination frequency also dropped. There was an apparent threshold at an equilibrium dissociation constant (K(d)) of 1 x 10(-)(7) M. In addition, TFO chemical modification with N,N-diethylethylenediamine (DEED) internucleoside linkages to confer improved binding was found to yield increased levels of induced recombination. To test the ability of triplex formation to induce recombination at a distance, episomal targets with informative reporter genes were constructed to contain polypurine TFO target sites at varying distances from the mutations to be corrected. TFO-induced recombination in mammalian cells between a plasmid vector and a donor oligonucleotide was detected at distances ranging from 24 to 750 bp. Together, these results indicate that TFO-induced recombination requires high-affinity binding but can affect sites hundreds of base pairs away from the position of triplex formation.

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PubMed2005

Transgenic Xenopus laevis embryos can be generated using phiC31 integrase.

Bacteriophage phiC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA. Here we show that the coinjection of mRNA encoding phiC31 integrase with plasmid DNA encoding the green fluorescent protein (GFP) can be used to generate transgenic X. laevis embryos. Despite integration into the genome, appropriate promoter expression required modification of the reporter plasmid by bracketing the GFP reporter gene with tandem copies of the chicken beta-globin 5' HS4 insulator to relieve silencing owing to chromatin position effects. These experiments demonstrate that the integration of insulated gene sequences using phiC31 integrase can be used to efficiently create transgenic embryos in X. laevis and may increase the practical use of phiC31 integrase in other systems as well.

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PubMed2005

EYA1 expression in the developing inner ear.

OBJECTIVES: We sought to determine the developmental anatomy and EYA1 protein distribution in the inner ear of Xenopus laevis. METHODS: Xenopus laevis embryos were stained with monoclonal antibodies and imaged with confocal microscopy. RESULTS: At stage 27, the otocyst fully forms, with strong tubulin staining of early sensory cells at its ventromedial aspect. Neuronal ingrowth follows at stage 33/34. At stage 50, the semicircular canals are complete. EYA1 localizes to the anterior aspect of the otocyst from stages 37 to 44. By stage 50, EYA1 distribution is localized primarily to the sensory maculae and the endolymphatic duct of the developing inner ear. CONCLUSIONS: Whole mount confocal imaging of the developing Xenopus inner ear delineates the exact timing of otic development, sensory cell differentiation, and innervation. EYA1 protein expression has a distinct distribution pattern at the anterior aspect of the developing otocyst in stages 41 and 44. Later stages have a more localized pattern, in which EYA1 is detected only in the sensory epithelium and endolymphatic duct. This specific pattern of expression indicates a possible role in the determination of the anterior-posterior orientation of the inner ear, as well as a later role in sensory cell differentiation.

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PubMed2004

Specialized and redundant roles of TBP and a vertebrate-specific TBP paralog in embryonic gene regulation in Xenopus.

The general transcription factor TATA-binding protein (TBP) is a key initiation factor involved in transcription by all three eukaryotic RNA polymerases. In addition, the related metazoan-specific TBP-like factor (TLF/TRF2) is essential for transcription of a distinct subset of genes. Here we characterize the vertebrate-specific TBP-like factor TBP2, using in vitro assays, in vivo antisense knockdown, and mRNA rescue experiments, as well as chromatin immunoprecipitation. We show that TBP2 is recruited to promoters in Xenopus oocytes in the absence of detectable TBP recruitment. Furthermore, TBP2 is essential for gastrulation and for the transcription of a subset of genes during Xenopus embryogenesis. In embryos, TBP2 protein is much less abundant than TBP, and moderate overexpression of TBP2 partially rescues an antisense knockdown of TBP levels and restores transcription of many TBP-dependent genes. TBP2 may be a TBP replacement factor in oocytes, whereas in embryos both TBP and TBP2 are required even though they exhibit partial redundancy and gene selectivity.

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PubMed2004

Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using noninvasive imaging.

Congenital heart defects often include altered conduction as well as morphological changes. Model organisms, like the frog Xenopus laevis, offer practical advantages for the study of congenital heart disease. X. laevis embryos are easily obtained free living, and the developing heart is readily visualized. Functional and morphological evidence for a conduction system is available for adult frog hearts, but information on the normal properties of embryonic heart contraction is lacking, especially in intact animals. With the use of fine glass microelectrodes, we were able to obtain cardiac recordings and make standard electrophysiological measurements in 1-wk-old embryos (stage 46). In addition, a system using digital analysis of video images was adapted for measurement of the standard cardiac intervals and compared with invasive measurements. Video images were obtained of the heart in live, pharmacologically paralyzed, stage 46 X. laevis embryos. Normal values for the timing of the cardiac cycle were established. Intervals determined by video analysis (n = 53), including the atrial and ventricular cycle lengths (473 +/- 10 ms and 464 +/- 19 ms, respectively) and the atrioventricular interval (169 +/- 5 ms) were not statistically different from those determined by intrathoracic cardiac recordings. We also present the data obtained from embryos treated with standard medications that affect the human conduction system. We conclude that the physiology of embryonic X. laevis cardiac conduction can be noninvasively studied by using digital video imaging. Additionally, we show the response of X. laevis embryonic hearts to chronotropic agents is similar but not identical to the response of the human heart.

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PubMed2003

Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis.

The experimental manipulation of early embryologic events, resulting in the misexpression of the homeobox transcription factor pitx2, is associated with subsequent defects of laterality in a number of vertebrate systems. To clarify the role of one pitx2 isoform, pitx2c, in determining the left-right axis of amphibian embryos, we examined the heart and gut morphology of Xenopus laevis embryos after attenuating pitx2c mRNA levels using chemically modified antisense oligonucleotides. We demonstrate that the partial depletion of pitx2c mRNA in these embryos results in alteration of both cardiac morphology and intestinal coiling. The most common cardiac abnormality seen was a failure of rightward migration of the outflow tract, while the most common intestinal laterality phenotype seen was a full reversal in the direction of coiling, each present in 23% of embryos injected with the pitx2c antisense oligonucleotide. An abnormality in either the heart or gut further predisposed to a malformation in the other. In addition, a number of other cardiac anomalies were observed after pitx2c mRNA attenuation, including abnormalities of atrial septation, extracellular matrix restriction, relative atrial-ventricular chamber positioning, and restriction of ventricular development. Many of these findings correlate with cardiac defects previously reported in pitx2 null and hypomorphic mice, but can now be assigned specifically to attenuation of the pitx2c isoform in Xenopus.

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PubMed2003

Conserved requirement of Lim1 function for cell movements during gastrulation.

To investigate Lim1 function during gastrulation, we used transcript depletion through DEED antisense oligonucleotides in Xenopus and cell transplantation in mice. Xenopus embryos depleted of Lim1 lack anterior head structures and fail to form a proper axis as a result of a failure of gastrulation movements, even though mesodermal cell identities are specified. Similar disruption of cell movements in the mesoderm is also observed in Lim1(-/-) mice. Paraxial protocadherin (PAPC) expression is lost in the nascent mesoderm of Lim1(-/-) mouse embryos and in the organizer of Lim1-depleted Xenopus embryos; the latter can be rescued to a considerable extent by supplying PAPC exogenously. We conclude that a primary function of Lim1 in the early embryo is to enable proper cell movements during gastrulation.

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PubMed2002

Transcription factor AP-2 is an essential and direct regulator of epidermal development in Xenopus.

Expression of the Xenopus homolog of the mammalian transcription factor AP-2alpha (XAP-2) is activated throughout the animal hemisphere shortly after the midblastula transition, and becomes restricted to prospective epidermis by the end of gastrulation, under the control of BMP signal modulation. Elevated expression in the future neural crest region begins at this time. Ectopic expression of XAP-2 can restore transcription of epidermal genes in neuralized ectoderm, both in ectodermal explants and in the intact embryo. Likewise, loss of XAP-2 function, accomplished by injection of antisense oligonucleotides or by overexpression of antimorphic XAP-2 derivatives, leads to loss of epidermal and gain of neural gene expression. These treatments also result in gastrulation failure. Thus, AP-2 is a critical regulator of ectodermal determination that is required for normal epidermal development and morphogenesis in the frog embryo.

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