A novel method to follow meiotic progression in Arabidopsis using confocal microscopy and 5-ethynyl-2′-deoxyuridine labeling

Stronghill PE, Azimi W, Hasenkampf CA

Plant Methods 2014;10(1):33

PMID: 25337148

Abstract

BACKGROUND: Meiosis progression in the more recent past has been investigated using 5-bromo-2′-deoxyuridine (BrdU) uptake by S-phase meiocytes undergoing DNA replication. BrdU uptake is detected by reaction with BrdU antibody followed by epifluorescent microscopy examination of chromosome spreads and/or squashes. We here report using confocal microscopic examination of intact meiocytes in conjunction with the new thymidine analog 5-ethynyl-2′-deoxyuridine (EdU). The simplicity of the EdU detection coupled with confocal examination of anthers provides a more exact temporal description of meiotic prophase I progression in Arabidopsis and opens up the possibility of examining the coordination of microsporocyte development with the other tissues of the anther.

RESULTS: Using our time course protocol, we have determined the duration of wild type Arabidopsis leptotene to be 5 h, zygotene -6 h, pachytene -10 h and a diplotene duration of approximately 1 h. We estimate G2 duration to be approximately 7 h based on the timing of the initial appearance of EdU signal in early leptotene meiocytes. In addition we have found that DNA replication in meiocytes is not done synchronously with the associated tapetal layer of cells. The EdU labeling suggests that S-phase replication of meiocyte DNA precedes the duplication of tapetal cell DNA.

CONCLUSIONS: The increased number of meiotic staging criteria that can be assessed in our confocal analysis, as compared to chromosome spreading or squashing, makes the identification of even the early and late portions of the prophase I substages attainable. This enhanced staging coupled with the ability to easily generate large data sets at hourly time points makes it possible to more exactly determine substage duration and to detect modest temporal abnormalities involving meiocyte entrance into and/or exit from leptotene, zygotene and pachytene. Confocal analysis also makes it possible to study the relationships between different cell types within the flower bud as meiosis proceeds.

Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity

Meyer M, Reimand J, Lan X, Head R, Zhu X, Kushida M, Bayani J, Pressey JC, Lionel AC, Clarke ID, Cusimano M, Squire JA, Scherer SW, Bernstein M, Woodin MA, Bader GD, Dirks PB

Proc. Natl. Acad. Sci. U.S.A. 2015 Jan;112(3):851-6

PMID: 25561528

Abstract

Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.

Parvalbumin and GAD65 interneuron inhibition in the ventral hippocampus induces distinct behavioral deficits relevant to schizophrenia

Nguyen R, Morrissey MD, Mahadevan V, Cajanding JD, Woodin MA, Yeomans JS, Takehara-Nishiuchi K, Kim JC

J. Neurosci. 2014 Nov;34(45):14948-60

PMID: 25378161

Abstract

Hyperactivity within the ventral hippocampus (vHPC) has been linked to both psychosis in humans and behavioral deficits in animal models of schizophrenia. A local decrease in GABA-mediated inhibition, particularly involving parvalbumin (PV)-expressing GABA neurons, has been proposed as a key mechanism underlying this hyperactive state. However, direct evidence is lacking for a causal role of vHPC GABA neurons in behaviors associated with schizophrenia. Here, we probed the behavioral function of two different but overlapping populations of vHPC GABA neurons that express either PV or GAD65 by selectively inhibiting these neurons with the pharmacogenetic neuromodulator hM4D. We show that acute inhibition of vHPC GABA neurons in adult mice results in behavioral changes relevant to schizophrenia. Inhibiting either PV or GAD65 neurons produced distinct behavioral deficits. Inhibition of PV neurons, affecting ∼80% of the PV neuron population, robustly impaired prepulse inhibition of the acoustic startle reflex (PPI), startle reactivity, and spontaneous alternation, but did not affect locomotor activity. In contrast, inhibiting a heterogeneous population of GAD65 neurons, affecting ∼40% of PV neurons and 65% of cholecystokinin neurons, increased spontaneous and amphetamine-induced locomotor activity and reduced spontaneous alternation, but did not alter PPI. Inhibition of PV or GAD65 neurons also produced distinct changes in network oscillatory activity in the vHPC in vivo. Together, these findings establish a causal role for vHPC GABA neurons in controlling behaviors relevant to schizophrenia and suggest a functional dissociation between the GABAergic mechanisms involved in hippocampal modulation of sensorimotor processes.