Biology subjects

Phelps, W. A.

Publications and source records attributed to Phelps, W. A..

4 recordsLinked to original sources

bioRxiv2022

Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis

After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis, an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization.

developmental biology↗

bioRxiv2020

Optimized design of antisense oligomers for targeted rRNA depletion

RNA sequencing (RNA-seq) has become a standard method for quantifying gene expression transcriptome-wide. Although RNA-seq is often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, selective rRNA digestion, using RNaseH and antisense DNA oligomers that tile the entire length of target RNAs, has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion method with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA, in a digestion reaction of only 5 minutes. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by designing oligo sets to deplete rRNA in Xenopus laevis and in zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/169102v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@c05b57org.highwire.dtl.DTLVardef@ace8a3org.highwire.dtl.DTLVardef@1dd25adorg.highwire.dtl.DTLVardef@1d4adeb_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Zinc protection of fertilized eggs is an ancient feature of sexual reproduction in animals

One of the earliest and most prevalent barriers to successful reproduction is polyspermy, or fertilization of an egg by multiple sperm. To prevent these supernumerary fertilizations, eggs have evolved multiple mechanisms. It has recently been proposed that zinc released by mammalian eggs at fertilization may block additional sperm from entering. Here, we demonstrate that eggs from amphibia and teleost fish also release zinc. Using Xenopus laevis as a model, we document that zinc reversibly blocks fertilization. Finally, we demonstrate that extracellular zinc similarly disrupts early embryonic development in eggs from diverse phyla, including: Cnidaria, Echinodermata, and Chordata. Our study reveals that a fundamental strategy protecting human eggs from fertilization by multiple sperm may have evolved more than 650 million years ago.Competing Interest StatementThe authors have declared no competing interest.View Full Text

physiology↗

bioRxiv2018

The TMEM16A Channel Mediates the fast polyspermy block in Xenopus Laevis

In externally fertilizing animals, such as sea urchins and frogs, prolonged depolarization of the egg immediately after fertilization inhibits the entry of additional sperm - a phenomenon known as the fast block to polyspermy. In the African clawed frog, Xenopus laevis, this depolarization is driven by a Ca2+-activated Cl- efflux. Although the prominent Ca2+-activated Cl- currents generated by immature X. laevis oocytes are conducted by xTMEM16A channels, little is known about which channels contribute to fertilization-competency in mature eggs. Moreover, the gamete undergoes a gross transformation as it matures from an immature oocyte into a fertilization-competent egg. Here we report the results of our approach to identify the Ca2+-activated Cl- channel that triggers the fast block. Querying published proteomics and RNA-seq data, we identified two Ca2+-activated Cl- channels expressed in fertilization-competent X. laevis eggs: xTMEM16A and xBEST2A. Furthermore, transcripts for these channels increase in abundance during gamete maturation. To determine if either of these mediates the fast block, we characterized exogenously expressed xTMEM16A and xBEST2A using pharmacologic inhibitors. None of the inhibitors tested blocked xBEST2A currents specifically. However, Ani9 and MONNA each reduced xTMEM16A currents by more than 70%, while only nominally inhibiting those generated by xBEST2A. Using whole-cell recordings during fertilization, we found that Ani9 and MONNA effectively diminished fertilization-evoked depolarizations. These results indicate that fertilization activates TMEM16A channels in X. laevis eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy.\n\nHIGHLIGHTSO_LIProtein for the channels xBEST2A and xTMEM16A is present in X. laevis eggs.\nC_LI\n\nO_LIThe inhibitors MONNA and Ani9 effectively block xTMEM16A compared to xBEST2A.\nC_LI\n\nO_LIXenopus laevis fertilization opens TMEM16A to trigger egg depolarization.\nC_LI\n\nO_LIThe TMEM16A-mediated depolarization is critical for the fast block to polyspermy.\nC_LI

developmental biology↗