Publications

Latest Publications

Spatial identification of transcripts and biological processes in laser micro-dissected sub-regions of waterlogged corn roots with altered expression of phytoglobin

1 June, 2019

Youssef M, Mira M, Millar J, Becker M, Belmonte M, Hill R, Stasolla C

Over-expression of the corn phytoglobin ZmPgb1.2 increases tolerance to waterlogging, while 3 suppression of ZmPgb1.2 compromises plant growth. To unravel compartment-specific transcriptional 4 changes evoked by ZmPgb1.2 during hypoxia, laser micro-dissected sub-regions from waterlogged roots 5 of WT and ZmPgb1.2 overexpressing [ZmPgb1.2(S)] plants were probed for global transcriptional 6 analysis using next generation RNA sequencing. These sub-regions included compartments within the 7 meristematic, elongation, and maturation zone. Of the 149 genes differentially expressed by the up8 regulation of ZmPgb1.2, 78 occurred within the meristematic region and included genes involved in 9 jasmonic acid synthesis and response, ascorbic acid metabolism, and ethylene signalling. The 10 ZmPgb1.2 regulation of these genes, discussed in the context of known functions of Pgbs, was further 11 validated by monitoring their expression in meristematic cells of waterlogged roots suppressing 12 ZmPgb1.2. Of the 27 genes differentially expressed by the over-expression of ZmPgb1.2 in the 13 elongation zone, pyruvate kinase and alcohol dehydrogenase showed an expression pattern correlated to 14 the level of ZmPgb1.2 in the tissue. The transcriptional induction of these two enzymes in hypoxic 15 domains of the elongation zone over-expressing ZmPgb1.2 suggests the activation of the fermentation 16 pathway which might be required to sustain metabolic flux and production of ATP in support of cell 17 elongation.

Pages: 350-365 doi: 10.1016/j.plaphy.2019.03.036

Molecularly defined cortical astroglia subpopulation modulates neurons via secretion of Norrin

1 May, 2019

Miller SJ, Philips T, Kim N, Dastgheyb R, Chen Z, Hsieh YC, Daigle JG, Datta M, Chew J, Vidensky S, Pham JT, Hughes EG, Robinson MB, Sattler R, Tomer R, Suk JS, Bergles DE, Haughey N, Pletnikov M, Hanes J, Rothstein JD

Despite expanding knowledge regarding the role of astroglia in regulating neuronal function, little is known about regional or functional subgroups of brain astroglia and how they may interact with neurons. We use an astroglia-specific promoter fragment in transgenic mice to identify an anatomically defined subset of adult gray matter astroglia. Using transcriptomic and histological analyses, we generate a combinatorial profile for the in vivo identification and characterization of this astroglia subpopulation. These astroglia are enriched in mouse cortical layer V; express distinct molecular markers, including Norrin and leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6), with corresponding layer-specific neuronal ligands; are found in the human cortex; and modulate neuronal activity. Astrocytic Norrin appears to regulate dendrites and spines; its loss, as occurring in Norrie disease, contributes to cortical dendritic spine loss. These studies provide evidence that human and rodent astroglia subtypes are regionally and functionally distinct, can regulate local neuronal dendrite and synaptic spine development, and contribute to disease.

Pages: 741-752 doi: 10.1038/s41593-019-0366-7

Global donor and acceptor splicing site kinetics in human cells

26 April, 2019

Wachutka L, Caizzi L, Gagneur J, Cramer P

RNA splicing is an essential part of eukaryotic gene expression. Although the mechanism of splicing has been extensively studied in vitro, in vivo kinetics for the two-step splicing reaction remain poorly understood. Here we combine transient transcriptome sequencing (TT-seq) and mathematical modeling to quantify RNA metabolic rates at donor and acceptor splice sites across the human genome. Splicing occurs in the range of minutes and is limited by the speed of RNA polymerase elongation. Splicing kinetics strongly depends on the position and nature of nucleotides flanking splice sites, and on structural interactions between unspliced RNA and small nuclear RNAs in spliceosomal intermediates. Finally, we introduce the 'yield' of splicing as the efficiency of converting unspliced to spliced RNA and show that it is highest for mRNAs and independent of splicing kinetics. These results lead to quantitative models describing how splicing rates are encoded in the human genome.

doi: 10.7554/eLife.45056

Transcriptome analysis of mouse and human sinoatrial node cells reveals a conserved genetic program

25 April, 2019

van Eif VWW, Stefanovic S, van Duijvenboden K, Bakker M, Wakker V, de Gier-de Vries C, Zaffran S, Verkerk AO, Boukens BJ, Christoffels VM

The rate of contraction of the heart relies on proper development and function of the sinoatrial node, which consists of a small heterogeneous cell population, including Tbx3+ pacemaker cells. Here, we have isolated and characterized the Tbx3+ cells from Tbx3+/Venus knock-in mice. We studied electrophysiological parameters during development and found that Venus-labeled cells are genuine Tbx3+ pacemaker cells. We analyzed the transcriptomes of late fetal FACS-purified Tbx3+ sinoatrial nodal cells and Nppb-Katushka+ atrial and ventricular chamber cardiomyocytes, and identified a sinoatrial node-enriched gene program, including key nodal transcription factors, BMP signaling and Smoc2, the disruption of which in mice did not affect heart rhythm. We also obtained the transcriptomes of the sinoatrial node region, including pacemaker and other cell types, and right atrium of human fetuses, and found a gene program including TBX3, SHOX2, ISL1 and HOX family members, and BMP and NOTCH signaling components conserved between human and mouse. We conclude that a conserved gene program characterizes the sinoatrial node region and that the Tbx3+/Venus allele provides a reliable tool for visualizing the sinoatrial node, and studying its development and function.

doi: 10.1242/dev.173161

Chemosynthetic symbiont with a drastically reduced genome serves as primary energy storage in the marine flatworm Paracatenula

23 April, 2019

Jäckle O, Seah BKB, Tietjen M, Leisch N, Liebeke M, Kleiner M, Berg JS, Gruber-Vodicka HR

Hosts of chemoautotrophic bacteria typically have much higher biomass than their symbionts and consume symbiont cells for nutrition. In contrast to this, chemoautotrophic Candidatus Riegeria symbionts in mouthless Paracatenula flatworms comprise up to half of the biomass of the consortium. Each species of Paracatenula harbors a specific Ca Riegeria, and the endosymbionts have been vertically transmitted for at least 500 million years. Such prolonged strict vertical transmission leads to streamlining of symbiont genomes, and the retained physiological capacities reveal the functions the symbionts provide to their hosts. Here, we studied a species of Paracatenula from Sant'Andrea, Elba, Italy, using genomics, gene expression, imaging analyses, as well as targeted and untargeted MS. We show that its symbiont, Ca R. santandreae has a drastically smaller genome (1.34 Mb) than the symbiont´s free-living relatives (4.29-4.97 Mb) but retains a versatile and energy-efficient metabolism. It encodes and expresses a complete intermediary carbon metabolism and enhanced carbon fixation through anaplerosis and accumulates massive intracellular inclusions such as sulfur, polyhydroxyalkanoates, and carbohydrates. Compared with symbiotic and free-living chemoautotrophs, Ca R. santandreae's versatility in energy storage is unparalleled in chemoautotrophs with such compact genomes. Transmission EM as well as host and symbiont expression data suggest that Ca R. santandreae largely provisions its host via outer-membrane vesicle secretion. With its high share of biomass in the symbiosis and large standing stocks of carbon and energy reserves, it has a unique role for bacterial symbionts-serving as the primary energy storage for its animal host.

Pages: 8505-8514 doi: 10.1073/pnas.1818995116