Biology subjects

Becker, J. D.

Publications and source records attributed to Becker, J. D..

6 recordsLinked to original sources

bioRxiv2025

High throughput isolation of male gametophyte cells of Solanum lycopersicum var. Micro-Tom by fluorescence-activated cell sorting.

Efficient isolation of male gametes has enabled unprecedented advances in omics research, crucial for elucidating the molecular mechanisms governing male gametogenesis and fertilization. In this study, we developed a method for isolating generative and sperm cells from the economically important crop Solanum lycopersicum. A double fluorescent marker line was generated in the tomato variety Micro-Tom, employing mTurquoise and mScarlet-I fluorescent proteins under the control of promoters exhibiting preferential activity in generative and sperm cells, respectively. Then we developed a protocol that combines male gamete release from pollen of this line and SYTOX Red live/dead cell stain to obtain viable cells by Fluorescence-activated cell sorting. This allows the isolation of generative cells from mature pollen grains, and of sperm cells from pollen tubes after semi-in vivo growth, both in high quantity and purity. Additionally, an unexpected mScarlet-I signal in the vegetative nucleus, that persists until the sperm cells are formed, allows the sorting of vegetative nuclei. We anticipate that our novel double-marker line will accelerate research into tomato male gametogenesis, thereby enhancing efforts to improve the resilience of fertilization processes to climate change.

plant biology↗

bioRxiv2024

OsbHLH089 and OsbHLH094 Modulate OsSLR1 Levels to Maintain Male Reproductive Fitness in Rice

DELLA proteins are a unique class of transcriptional regulators in plants, playing critical roles in diverse biological processes. Far from being only negative regulators of the Gibberellin (GA) signalling pathway, DELLAs act as central signalling hubs due to their versatile binding capacity and responsiveness to GA fluctuations. This adaptability allows DELLAs to interact with a wide array of proteins but also makes their functional study challenging: disruptions in DELLA function lead to pleiotropic effects across multiple pathways. To address this complexity, understanding DELLA interactors provides valuable insights into DELLAs nuanced functions and regulation. Building on this approach, we investigated novel OsSLR1 interactors, OsbHLH089 and OsbHLH094, members of the basic-helix-loop-helix transcription factor family. Our findings reveal that OsbHLH089 and OsbHLH094 are expressed in mature pollen grains, with the Osbhlh089/94 double mutant displaying smaller, indehiscent anthers and non-viable pollen, indicating their redundant role in pollen development during its late stages. Through ChIP-Seq and RNA-Seq analyses, we identified five target genes repressed by OsbHLH089 and OsbHLH094 (OsTDL1A, OsSPS1, OsDGD2{beta}, OspPGM, and OsDPE2) that are essential for pollen and anther development. Interestingly, we also observed the binding of these TFs to the OsSLR1 promoter. Notably, while other target genes were repressed, OsSLR1 was induced, with a significant protein accumulation in the double mutant compared to the Kitaake background in the late stages of pollen development. This suggests that OsSLR1 accumulation may compromise pollen viability, further highlighting the critical regulatory role of OsbHLH089 and OsbHLH094 in repressing OsSLR1 levels for proper pollen and anther development.

plant biology↗

bioRxiv2024

The LOTUS-domain containing protein PpLDCP3 controls germline and dispersal unit formation in the moss Physcomitrium patens

O_LIDispersal is crucial for the survival and thriving of plant populations, yet the mechanisms of dispersal unit formation are poorly understood. LOTUS domain-containing proteins are essential for animal reproduction, promoting cell cycle control, transposon silencing, and transgenerational inheritance in the germline. In this study, we demonstrate that the formation of dispersal units, sperm and spores, in the model moss Physcomitrium patens relies on the novel LOTUS domain-containing protein 3 (PpLDCP3). C_LIO_LIPpldcp3 knock-out mutants revealed pleiotropic phenotypes in both gametogenesis and sporogenesis. Ppldcp3 sperm were rarely released and appeared to be partially non-viable, leading to fewer sporophytes produced. Sporophyte development in Ppldcp3 was impaired, resulting in abnormal dehiscence of spores, which were incapable of germinating. C_LIO_LIFluorescent reporter lines revealed the presence of PpLDCP3 in developing gametangia. Within the premature egg cell and ventral canal cell of archegonia, PpLDCP3 was identified as a component of cytoplasmic fluorescent foci, which we interpret as P-bodies. This suggests a role for PpLDCP3 in RNA metabolism, analogous to animal LDCPs. Moreover, bioinformatic predictions indicate PpLDCP3 as a downstream player of WOX13 and bHLH transcription factors. C_LIO_LIWe conclude that PpLDCP3 is integrated into evolutionary conserved mechanisms of dispersal unit formation in land plants. C_LI

plant biology↗

bioRxiv2022

The conserved transcriptional program of metazoan male germ cells uncovers ancient origins of human infertility

Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein-coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross-species analytical pipeline that can be repurposed to other cell types and pathologies.

systems biology↗

bioRxiv2020

The 3D architecture and molecular foundations of de novo centriole assembly via bicentrioles

Abstract/SummaryCentrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes form centrioles de novo through uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, combining electron microscopy and tomography, we show that during spermatogenesis of the moss Physcomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. We observe that microtubules emanate from those bicentrioles, which localize to the spindle poles during cell division. Thereafter, each bicentriole breaks apart, and the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two cilia are assembled which are capable of beating asynchronously. We further show that conserved cartwheel and centriole wall components, SAS6, BLD10 and POC1 are expressed during spermatogenesis and are required for this de novo biogenesis pathway. Our work supports a scenario where centriole biogenesis is more diverse than previously thought and that conserved molecular modules underlie diversification of this essential pathway.

cell biology↗

bioRxiv2018

The Trithorax group protein dMLL3/4 instructs the assembly of the zygotic genome at fertilization

The transition from fertilized oocyte to totipotent embryo relies on maternally-provided factors that are synthetized and accumulated in developing oocytes. Yet, it is still unclear how oocytes regulate the expression of these embryo fate-promoting genes within the general transcriptional program of oogenesis. Here we report that the Drosophila Trithorax group protein MLL3/4 (dMLL3/4, also known as Trr) is essential for the transition to embryo fate at fertilization. In the absence of dMLL3/4, oocytes develop normally but fail to initiate the embryo mitotic divisions after fertilization. This incapability results from defects in both paternal genome reprogramming and maternal meiotic completion. We show that, during oogenesis, dMLL3/4 promotes the expression of a functionally coherent gene subset that is later required for the correct assembly of the zygotic genome. Accordingly, we identify the evolutionarily-conserved IDGF4 glycoprotein (known as oviductin in mammals) as a new oocyte-to-embryo transition gene under dMLL3/4 transcriptional control. Based on these observations, we propose that dMLL3/4 plays an instructive role in the oocyte-to-embryo transition that is functionally uncoupled from the requirements of normal oocyte differentiation.

developmental biology↗