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Brown C.Y.,University of Texas at Austin | Seok Eom D.,University of Texas at Austin | Seok Eom D.,University of Washington | Amarnath S.,Section of Molecular Cell and Developmental Biology | Agarwala S.,University of Texas at Austin
Developmental Dynamics | Year: 2012

Background: The amenability of the chick embryo to a variety of manipulations has made it an ideal experimental model organism for over 100 years. The ability to manipulate gene function via in ovo electroporations has further revolutionized its value as an experimental model in the last 15 years. Although in ovo electroporations are simple to conduct in embryos ≥ E2, in ovo electroporations at early E1 stages have proven to be technically challenging due to the tissue damage and embryonic lethality such electroporations produce. Results and Conclusions: Here we report our success with in vivo microelectroporations of E1 embryos as young as Hamburger-Hamilton Stage 4 (HH4). We provide evidence that such electroporations can be varied in size and can be spatially targeted. They cause minimal disruption of tissue-size, 3-dimensional morphology, cell survival, proliferation, and cell-fate specification. Our paradigm is easily adapted to a variety of experimental conditions since it does not depend upon the presence of a lumen to enclose the DNA solution during electroporation. It is thus compatible with the in vivo examination of E1 morphogenetic events (e.g., neural tube closure) where preservation of 3-dimensional morphology is critical. © 2012 Wiley Periodicals, Inc. Source


Brown C.Y.,University of Texas at Austin | Eom D.S.,University of Texas at Austin | Eom D.S.,University of Washington | Amarnath S.,Section of Molecular Cell and Developmental Biology | Agarwala S.,University of Texas at Austin
Cold Spring Harbor Protocols | Year: 2012

In ovo electroporation of chick embryos at ages ≥ E2 is simple to conduct and widely used to manipulate gene function. However, in ovo electroporation at early E1 stages has so far been unsuccessful because of unacceptable levels of tissue damage and embryonic lethality. Early E1 manipulations in the chick have therefore relied on in vitro electroporation, posing problems for morphogenetic studies in which the long-term preservation (>24 h) of three-dimensional tissue organization is critical. This article describes a simple technique for in vivo electroporation of E1 embryos as young as Hamburger- Hamilton stage 4 (HH4). It uses thin microelectrodes and low voltages, which permit precise localization of gene misexpression while causing minimal tissue damage and embryonic lethality. Critically, it does not depend on the presence of a lumen for DNA injections and can easily be adapted for a wide variety of tissues. © 2012 Cold Spring Harbor Laboratory Press. Source


Nah G.,Section of Molecular Cell and Developmental Biology | Jeffrey Chen Z.,Section of Molecular Cell and Developmental Biology | Jeffrey Chen Z.,Institute for Cellular and Molecular Biology | Jeffrey Chen Z.,University of Texas at Austin
New Phytologist | Year: 2010

•Flowering time is an important adaptive trait and varies among Arabidopsis thaliana and its related species, including allopolyploids that are formed between A. thaliana and Arabidopsis arenosa. FLOWERING LOCUS C (FLC) inhibits early flowering in A. thaliana. A previous study showed that late-flowering A. arenosa contained two or more FLC alleles that were differentially expressed in Arabidopsis allotetraploids, but the genomic organization and evolution of FLC locus were unknown.•Comparative sequence and evolutionary analyses were performed in FLC-containing genomic regions in A. thaliana, A. arenosa and Arabidopsis lyrata, and expression of FLC loci and alleles was examined in Arabidopsis allopolyploids.•The FLC locus was tandemly duplicated in A. lyrata and triplicated in A. arenosa, and the tandem duplication event occurred after divergence from A. thaliana. Although FLC duplicates were highly conserved, their upstream sequences rapidly diverged. The third FLC copy in A. arenosa acquired a new splicing site through a point mutation in the intron and generated the new exon followed by an early stop codon, resulting in a novel MADS box gene.•Flowering time variation in Arabidopsis allopolyploids is probably related to the expression diversity and/or copy number of multiple FLC loci. Moreover, exonization of intronic sequence is a mechanism for the origin of new genes. © The Authors (2010). Journal compilation © New Phytologist Trust (2010). Source


Lackey E.,Section of Molecular Cell and Developmental Biology | Ng D.W.-K.,Section of Molecular Cell and Developmental Biology | Chen Z.J.,Section of Molecular Cell and Developmental Biology | Chen Z.J.,Institute for Cellular and Molecular Biology | Chen Z.J.,University of Texas at Austin
New Phytologist | Year: 2010

•Both natural and newly synthesized allopolyploids display nonadditive gene expression changes through genetic and epigenetic mechanisms. The nonadditively expressed genes include many microRNA (miRNA) targets, suggesting a role for miRNAs and their targets in morphological variation in the allopolyploids and their progenitors.•We produced dominant-negative transgenic allotetraploid plants in Arabidopsis using RNA interference (RNAi) that downregulates the expression of miRNA biogenesis genes, including DCL1 and AGO1.•RNAi of DCL1 and AGO1 led to dominant negative phenotypes and decreased accumulation of several miRNAs and a tasiRNA tested in the transgenic resynthesized allotetraploids.•The results demonstrated that miRNA biogenesis genes are effectively downregulated in the resynthesized allotetraploids containing redundant homoeologous genes that are difficult to be manipulated by conventional mutation screens. These lines will be useful for studying the effects of miRNA biogenesis genes on growth and developmental variation in the allopolyploids. © The Authors (2010). Journal compilation © New Phytologist Trust (2010). Source


Clark G.,Section of Molecular Cell and Developmental Biology | Darwin C.,1Section of Molecular Cell and Developmental Biology | Mehta V.,1Section of Molecular Cell and Developmental Biology | Jackobs F.,1Section of Molecular Cell and Developmental Biology | And 3 more authors.
Plant signaling & behavior | Year: 2013

In Arabidopsis leaves there is a bi-phasic dose-response to applied nucleotides; i.e., lower concentrations induce stomatal opening, while higher concentrations induce closure. Two mammalian purinoceptor antagonists, PPADS and RB2, block both nucleotide-induced stomatal opening and closing. These antagonists also partially block ABA-induced stomatal closure and light-induced stomatal opening. There are two closely related Arabidopsis apyrases, AtAPY1 and AtAPY2, which are both expressed in guard cells. Here we report that low levels of apyrase chemical inhibitors can induce stomatal opening in the dark, while apyrase enzyme blocks ABA-induced stomatal closure. We also demonstrate that high concentrations of ATP induce stomatal closure in the light. Application of ATPγS and chemical apyrase inhibitors at concentrations that have no effect on stomatal closure can lower the threshold for ABA-induced closure. The closure induced by ATPγS was not observed in gpa1-3 loss-of-function mutants. These results further confirm the role of extracellular ATP in regulating stomatal apertures. Source

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