Biology subjects

Brown, H. E.

Publications and source records attributed to Brown, H. E..

11 recordsLinked to original sources

bioRxiv2025

The pos-1 3' untranslated region governs germline specification and proliferation to ensure reproductive robustness

During fertilization, haploid gametes combine to form a zygote. The male (sperm) and female (oocyte) gametes contribute a similar amount of DNA, but the oocyte contributes nearly all the cytoplasm. Oocytes are loaded with maternal mRNAs thought to be essential for embryonic patterning after fertilization. A conserved suite of RNA-binding proteins (RBPs) regulates the spatiotemporal translation and stability of maternal mRNAs. POS-1 is a CCCH-type tandem zinc finger RBP expressed in fertilized Caenorhabditis elegans zygotes from maternally supplied mRNA. POS-1 accumulates in the posterior of the embryo where it promotes posterior cell fate. Here, we show that the pos-1 3' untranslated region (UTR) is essential for POS-1 patterning and contributes to maximal reproductive fecundity. We engineered a pos-1 mutant where most of the endogenous pos-1 3'UTR was removed using CRISPR genome editing. Our results show that the 3'UTR represses POS-1 expression in the maternal germline but increases POS-1 protein levels in embryos after fertilization. In a wild-type background, POS-1 repression via the 3'UTR has little impact on fertility. In a sensitized background, the deletion mutant has a complex pleiotropic phenotype where most adult homozygous progeny lack either one or both gonad arms. Most phenotypes become more penetrant at elevated temperature. Together, our results support an emerging model where the 3'UTRs of maternal transcripts, rather than being essential, contribute to reproductive robustness during stress.

developmental biology↗

bioRxiv2025

Integrating molecular and physiological approaches to quantify genetic controls for wheat development and improve phenotyping

Disentangling genotype x environment (GxE) effects is critical to understand the performance of wheat across different environments. A framework for doing this was previously presented in a model that integrated knowledge of crop physiology and the Vrn gene feedback loop to explain and predict the time of anthesis. The aims of this study were: 1) provide an updated description of the Cereal Anthesis Molecular Phenology (CAMP) model; 2) to verify the models assumptions regarding the relationship between Vrn gene expression and the timing of phenological stages in a set of diverse genotypes and environments; 3) to use the CAMP model to establish a phenotyping strategy for use in genetic studies and model parameterisation. Six wheat genotypes with a range of cool temperature and photoperiod sensitivities were evaluated. Apical development, final leaf number (FLN) and temporal expression of Vrn1, Vrn2 and Vrn3 were compared with model predictions. There was a clear relationship between FLN responses to cool temperature and photoperiod, the timing of phenological events and the patterns of Vrn gene expression for all genotypes. There was general agreement between the temporal patterns of foliar gene expression observed with those assumed by CAMP, but some obvious discrepancies. These may be related to differences between gene expression in foliar (observed) and apical (assumed by the model) parts of the plant, or differences in the way observed and modelled gene expression are scaled. Overall, the model described all the observed development responses to environment and provides a basis for building quantitative predictions of field-based development from genotypic and environmental data. A protocol is presented for phenotyping wheat using FLN measured in specific combinations of temperature and photoperiod. It allows easy and unconfounded measure of key developmental phenotypes that clearly relate to the genetic make-up of the plants and underlying gene expression profiles.

plant biology↗

bioRxiv2024

Differential regulation of eye specification in Drosophila by Polycomb Group (PcG) epigenetic repressors

During metazoan development, chromatin plasticity and genetic regulation are tightly connected to establish proper tissue fate and patterning. Drosophila imaginal discs are excellent models to study these processes as both genetic and mechanical injury can redirect their fate during regeneration. Reducing expression of Polycomb (Pc) results in the ectopic activation of the wing selector gene vestigial (vg) which in turn interacts with its DNA-binding partner Scalloped (Sd) - this forces the eye to transform into a wing. Reductions of other PcG members alone does not phenocopy this transformation. However, knocking down Sex combs on midleg (Scm) or Scm-related gene containing four mbt domains (Sfmbt) alongside the Pax6 gene twin of eyeless (toy) enables the eye-to-wing transformation. Using high throughput sequencing we show that toy-Sfmbt and toy-Scm knockdowns alter expression of wing selector and Hox genes, respectively. These findings provide new insights into how the fate of the eye is specified. Author SummaryDistinct gene sets are differentially expressed in response to the knockdown of Polycomb Group (PcG) members suggesting multiple avenues may exist for the eye to be transformed into a wing.

developmental biology↗

bioRxiv2024

The mex-3 3' untranslated region is essential for reproduction during temperature stress

Organisms must sense temperature and modify their physiology to survive environmental stress. Elevated temperature reduces fertility in most sexually reproducing organisms. Maternally supplied mRNAs are required for embryogenesis. They encode proteins that govern early embryonic patterning. RNA-binding proteins (RBPs) are major effectors of maternal mRNA regulation. MEX-3 is a conserved RBP essential for anterior patterning of Caenorhabditis elegans embryos. We previously demonstrated that the mex-3 3 untranslated region (3UTR) represses MEX-3 abundance in the germline yet is mostly dispensable for fertility. Here, we show that the 3UTR is essential during thermal stress. Deletion of the 3UTR causes a highly penetrant temperature sensitive embryonic lethality phenotype distinct from a mex-3 null. Loss of the 3UTR decreases MEX-3 abundance specifically in maturing oocytes and early embryos during temperature stress. Dysregulation of mex-3 reprograms the thermal stress response by reducing the expression of hundreds of heat shock genes. We propose that the primary role of the mex-3 3UTR is to buffer MEX-3 expression during fluctuating temperature, ensuring the robustness of oocyte maturation and embryogenesis.

developmental biology↗

bioRxiv2023

Slit-independent guidance of longitudinal axons by Drosophila Robo3

Drosophila Robo3 is a member of the evolutionarily conserved Roundabout (Robo) receptor family and one of three Drosophila Robo paralogs. During embryonic ventral nerve cord development, Robo3 does not participate in canonical Slit-dependent midline repulsion, but instead regulates the formation of longitudinal axon pathways at specific positions along the medial-lateral axis. Longitudinal axon guidance by Robo3 is hypothesized to be Slit dependent, but this has not been directly tested. Here we create a series of Robo3 variants in which the N-terminal Ig1 domain is deleted or modified, in order to characterize the functional importance of Ig1 and Slit binding for Robo3s axon guidance activity. We show that Robo3 requires its Ig1 domain for interaction with Slit and for proper axonal localization in embryonic neurons, but deleting Ig1 from Robo3 only partially disrupts longitudinal pathway formation. Robo3 variants with modified Ig1 domains that cannot bind Slit retain proper localization and fully rescue longitudinal axon guidance. Our results indicate that Robo3 guides longitudinal axons independently of Slit, and that sequences both within and outside of Ig1 contribute to this Slit-independent activity.

developmental biology↗

bioRxiv2023

Polycomb safeguards imaginal disc specification through control of the Vestigial-Scalloped complex

A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are mis-regulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate. Summary StatementHere we describe a novel mechanism by which Pc promotes an eye fate during normal development and how the eye is reprogrammed into a wing in its absence.

developmental biology↗

bioRxiv2022

The C-type Lectin Receptor Dectin-2 is a receptor for Aspergillus fumigatus galactomannan

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly amongst immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient sera and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2 dependent signaling including activation of spleen tyrosine kinase and potent TNF production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan which leads to a pro-inflammatory immune response in the lung. IMPORTANCEAspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptos can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

immunology↗

bioRxiv2020

Internalization of the host alkaline pH signal in a fungal pathogen

The ability for cells to internalize extracellular cues allows them to adapt to novel and stressful environments. This adaptability is especially important for microbial pathogens that must sense and respond to drastic changes when encountering the human host. Cryptococcus neoformans is an environmental fungus and opportunistic pathogen that naturally lives in slightly acidic reservoirs, but must adapt to the relative increase in alkalinity in the human host in order to effectively cause disease. The fungal-specific Rim alkaline response signaling pathway effectively converts this extracellular signal into an adaptive cellular response allowing the pathogen to survive in its new environment. The newly identified Rra1 protein, the most upstream component of the C. neoformans Rim pathway, is an essential component of this alkaline response. Previous work connected Rra1-mediated signaling to the dynamics of the plasma membrane. Here we identify the specific mechanisms of Rim pathway signaling through detailed studies of the activation of the Rra1 protein. Specifically, we observe that the Rra1 protein is internalized and recycled in a pH-dependent manner, and that this dynamic pattern of localization further depends on specific residues in its C-terminal tail, clathrin-mediated endocytosis, and the integrity of the plasma membrane. The data presented here continue to unravel the complex and intricate processes of pH-sensing in a relevant human fungal pathogen. These studies will further elucidate general mechanisms by which cells respond to and internalize extracellular stress signals. Author SummaryThe work described here explores the genetics and mechanics of a cellular signaling pathway in a relevant human fungal pathogen, Cryptococcus neoformans. The findings presented in this manuscript untangle the complex interactions involved in the activation of a fungal-specific alkaline response pathway, the Rim pathway. Specifically, we find that C. neoformans is able to sense an increase in pH within the human host, internalize a membrane-bound pH-sensor, and activate a downstream signaling pathway enabling this pathogen to adapt to a novel host environment and effectively cause disease. Revealing the mechanisms of Rim pathway activation within the larger context of the fungal cell allows us to understand how and when this microorganism interprets relevant host signals. Furthermore, understanding how this pathogenic organism converts extracellular stress signals into an adaptive cellular response will elucidate more general mechanisms of microbial environmental sensing and stress response.

cell biology↗

bioRxiv2020

Minimal structural elements required for midline repulsive signaling and regulation of Drosophila Robo1

The Roundabout (Robo) family of axon guidance receptors has a conserved ectodomain arrangement of five immunoglobulin-like (Ig) domains plus three fibronectin (Fn) repeats. Based on the strong evolutionary conservation of this domain structure among Robo receptors, as well as in vitro structural and domain-domain interaction studies of Robo family members, this ectodomain arrangement is predicted to be important for Robo receptor signaling in response to Slit ligands. Here, we define the minimal ectodomain structure required for Slit binding and midline repulsive signaling in vivo by Drosophila Robo1. We find that the majority of the Robo1 ectodomain is dispensable for both Slit binding and repulsive signaling. We show that a significant level of midline repulsive signaling activity is retained when all Robo1 ectodomain elements apart from Ig1 are deleted, and that the combination of Ig1 plus one additional ectodomain element (Ig2, Ig5, or Fn3) is sufficient to restore midline repulsion to wild type levels. Further, we find that deleting four out of five Robo1 Ig domains ({Delta}Ig2-5) does not affect negative regulation of Robo1 by Commissureless (Comm) or Robo2, while variants lacking all three fibronectin repeats ({Delta}Fn1-3 and {Delta}Ig2-Fn3) are insensitive to regulation by both Comm and Robo2, signifying a novel regulatory role for Robo1s Fn repeats. Our results provide an in vivo perspective on the importance of the conserved 5+3 ectodomain structure of Robo receptors, and suggest that specific biochemical properties and/or ectodomain structural conformations observed in vitro for domains other than Ig1 may have limited significance for in vivo signaling in the context of midline repulsion.

developmental biology↗

bioRxiv2020

Sterol-response pathways mediate alkaline survival in diverse fungi

The ability for cells to maintain homeostasis in the presence of extracellular stress is essential for their survival. Stress adaptations are especially important for microbial pathogens to respond to rapidly changing conditions, such as those encountered during the transition from the environment to the infected host. Many fungal pathogens have acquired the ability to quickly adapt to changes in extracellular pH to promote their survival in the various micro-environments encountered during a host infection. For example, the fungal-specific Rim/Pal alkaline response pathway has been well characterized in many fungal pathogens, including Cryptococcus neoformans. However, alternative mechanisms for sensing and responding to host pH have yet to be extensively studied. Recent observations from a genetic screen suggest that the C. neoformans sterol homeostasis pathway is required for growth at elevated pH. This work explores interactions among mechanisms of membrane homeostasis, alkaline pH tolerance, and Rim pathway activation. We find that the sterol homeostasis pathway is necessary for growth in an alkaline environment, and that an elevated pH is sufficient to induce Sre1 activation. This pH-mediated activation of the Sre1 transcription factor is linked to the biosynthesis of ergosterol, but is not dependent on Rim pathway signaling, suggesting that these two pathways are responding to alkaline pH independently. Furthermore, we discover that C. neoformans is more susceptible to membrane-targeting antifungals in alkaline conditions highlighting the impact of micro-environmental pH on the treatment of invasive fungal infections. Together, these findings further connect membrane integrity and composition with the fungal pH response and pathogenesis.

microbiology↗

bioRxiv2017

Increased microglial synapse elimination in patient-specific models of schizophrenia

Schizophrenia patients display decreased synaptic density in postmortem studies, suggesting aberrant microglial synapse elimination during neurodevelopment. Here, we use cellular reprogramming to create patient-specific in vitro models of microglia-mediated synapse engulfment that demonstrate increased synapse elimination in schizophrenia-derived models compared to healthy controls. We show that excessive synaptic pruning in schizophrenia reflects abnormalities in microglia-like cells as well as synaptic structures. Further, we find that schizophrenia risk-associated variants within the complement component 4 locus contribute to the increased uptake in schizophrenia models. Finally, we demonstrate that the antibiotic minocycline reduces microglia-mediated synapse uptake and show that minocycline treatment for acne is associated with a reduction in incident schizophrenia risk compared to other treatments in a cohort of more than 9,000 young adults drawn from health records. Specific pharmacological interventions targeting excessive pruning merit further study for their capacity to delay or prevent the onset of schizophrenia in high-risk individuals.

neuroscience↗