Biology subjects

Page, J.

Publications and source records attributed to Page, J..

13 recordsLinked to original sources

bioRxiv2025

The dynamics of ciliogenesis in prepubertal mouse meiosis reveal new clues about testicular maturation during puberty

The primary cilium, a solitary and non-motile extension of the plasma membrane, has recently been identified in adult male mouse spermatocytes. However, very little is known about when these cilia emerge during testicular maturation and what their function is. In the context of fertility establishment during puberty, this study investigates the dynamics of ciliogenesis in prepubertal mouse spermatocytes. Our findings reveal that primary cilia are not an intrinsic feature of spermatocytes during the first wave of meiosis, which initiates at 8 days post-partum (dpp). Instead, cilia begin polymerizing at 20 dpp, after first meiotic wave has been completed, and are present in spermatocytes across all stages of prophase I. Thus, no direct correlation between cilia polymerization and the initiation of synapsis or desynapsis was found, although chemical ablation of cilia may delay DNA repair during prophase I. Typical adult cilia features, which are shorter and restricted to zygotene spermatocytes, are settled upon acquisition of sexual maturity. This study also highlights that the emergence of ciliated spermatocytes in prepuberal mice coincides with the onset of flagellogenesis, hinting at a potential link between the regulation of the formation of both types of axonemes within the tissue developing environment. Proteomic analyses further identify temporal regulators of axoneme assembly, providing valuable targets for future research to unravel the molecular pathways underlying ciliogenesis, flagellogenesis, and their roles in spermatogenesis. We explored distinct regulatory mechanisms of ciliogenesis during the first meiotic wave and found that Aurora kinase A (AURKA) is a critical regulator of cilia disassembly during late diplotene, with evidence suggesting that centrosome migration and cilia depolymerization are mutually exclusive events during meiosis. In summary, this study provides the first detailed characterization of primary cilia dynamics during early testicular maturation in mice, revealing their spatiotemporal regulation, candidate molecular mediators, and potential roles during meiosis. These findings lay the groundwork for understanding the physiological relevance of meiotic cilia in spermatogenesis and testicular development.

cell biology↗

bioRxiv2025

Leveraging Dynamic Stability to Infer Regulation in Protein-Protein Interaction Networks: A Study of Infectious Vulnerability in COPD.

The fourth leading cause of death in the US, Chronic Obstructive Pulmonary Disease (COPD) is punctuated by frequent viral and bacterial infections causing severe acute exacerbations (AECOPD) and increased mortality. In previous work we have shown that altered immune cell signaling may confer increased and persistent susceptibility to infection. Here we continue this investigation by conducting broad-spectrum proteomic profiling of circulating white blood cells to assemble an empirical protein-protein interaction network associated with frequency of infectious exacerbation. In a novel extension of conventional cross-sectional data analyses, we translate these undirected protein-protein interactions into candidate regulatory relationships with both direction and mode of action. The latter are inferred by formulating and solving a constraint satisfaction problem (SAT) whereby predicted dynamic behaviors of any valid regulatory network must support the expected persistent nature of low and high vulnerability phenotypes. Solving this SAT problem produced a set of competing candidate protein regulatory network architectures and signalling rules that unanimously highlighted several novel candidate pathway elements involved in oxidative stress response. Analysis of the overall dynamics supported by these networks, again supported the hypothesis that progression beyond an immune tipping point may confer persistent susceptibility to infection and that this may constitute a stable phenotype or regulatory trap in COPD characterized by a reactive oxygen cascade. HIGHLIGHTSO_LIInfectious vulnerability phenotypes in COPD may represent immune regulatory traps. C_LIO_LIA 15-protein oxidative stress response interactome offers a phenotypic signature. C_LIO_LIHomeostasis in cross-sectional data can support inference of co-regulatory actions. C_LIO_LI8 of 11 documented regulatory actions recovered in candidate model majority vote C_LIO_LI17 new candidate protein co-regulatory actions proposed unanimously by all models. C_LI

systems biology↗

bioRxiv2023

Loss of Slc35a2 alters development of the mouse cerebral cortex

Brain somatic variants in SLC35A2 are associated with clinically drug-resistant epilepsy and developmental brain malformations, including mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). SLC35A2 encodes a uridine diphosphate galactose translocator that is essential for protein glycosylation; however, the neurodevelopmental mechanisms by which SLC35A2 disruption leads to clinical and histopathological features remain unspecified. We hypothesized that focal knockout (KO) or knockdown (KD) of Slc35a2 in the developing mouse cortex would disrupt cerebral cortical development through altered neuronal migration and cause changes in network excitability. We used in utero electroporation (IUE) to introduce CRISPR/Cas9 and targeted guide RNAs or short-hairpin RNAs to achieve Slc35a2 KO or KD, respectively, during early corticogenesis. Following Slc35a2 KO or KD, we observed disrupted radial migration of transfected neurons evidenced by heterotopic cells located in lower cortical layers and in the sub-cortical white matter. Slc35a2 KO in neurons did not induce changes in oligodendrocyte number, suggesting that the oligodendroglial hyperplasia observed in MOGHE originates from distinct cell autonomous effects. Spontaneous seizures were not observed, but intracranial EEG recordings after focal KO showed a reduced seizure threshold following pentylenetetrazol injection. These results demonstrate that Slc35a2 KO or KD in vivo disrupts corticogenesis through altered neuronal migration.

neuroscience↗

bioRxiv2023

Up-regulation of cholesterol synthesis pathways and limited neurodegeneration in a knock-in Sod1 mutant mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder affecting brain and spinal cord motor neurons. Mutations in the copper/zinc superoxide dismutase gene (SOD1) are associated with [~]20% of inherited and 1-2% of sporadic ALS cases. Much has been learned from mice expressing transgenic copies of mutant SOD1, which typically involve high-level transgene expression, thereby differing from ALS patients expressing one mutant gene copy. To generate a model that more closely represents patient gene expression, we created a knock-in point mutation (G85R, a human ALS-causing mutation) in the endogenous mouse Sod1 gene, leading to mutant SOD1G85R protein expression. Heterozygous Sod1G85R mutant mice resemble wild type, whereas homozygous mutants have reduced body weight and lifespan, a mild neurodegenerative phenotype, and express very low mutant SOD1 protein levels with no detectable SOD1 activity. Homozygous mutants exhibit partial neuromuscular junction denervation at 3-4 months of age. Spinal cord motor neuron transcriptome analyses of homozygous Sod1G85R mice revealed up-regulation of cholesterol synthesis pathway genes compared to wild type. Transcriptome and phenotypic features of these mice are similar to Sod1 knock-out mice, suggesting the Sod1G85R phenotype is largely driven by loss of SOD1 function. By contrast, cholesterol synthesis genes are down-regulated in severely affected human TgSOD1G93A transgenic mice at 4 months. Our analyses implicate dysregulation of cholesterol or related lipid pathway genes in ALS pathogenesis. The Sod1G85R knock-in mouse is a useful ALS model to examine the importance of SOD1 activity in control of cholesterol homeostasis and motor neuron survival. SIGNIFICANCE STATEMENTAmyotrophic lateral sclerosis is a devastating disease involving the progressive loss of motor neurons and motor function for which there is currently no cure. Understanding biological mechanisms leading to motor neuron death is critical for developing new treatments. Using a new knock-in mutant mouse model carrying a Sod1 mutation that causes ALS in patients, and in the mouse, causes a limited neurodegenerative phenotype similar to Sod1 loss-of-function, we show that cholesterol synthesis pathway genes are up-regulated in mutant motor neurons, whereas the same genes are down-regulated in transgenic SOD1 mice with a severe phenotype. Our data implicate dysregulation of cholesterol or other related lipid genes in ALS pathogenesis and provide new insights that could contribute to strategies for disease intervention.

neuroscience↗

bioRxiv2023

Divergent patterns of meiotic double strand breaks and synapsis initiation dynamics suggest an evolutionary shift in the meiosis program between American and Australian marsupials

In eutherian mammals, hundreds of programmed DNA double-strand breaks (DSBs) are generated at the onset of meiosis. The DNA damage response is then triggered. Although the dynamics of this response is well studied in eutherian mammals, recent findings have revealed different patterns of DNA damage signaling and repair in marsupial mammals. To better characterize these differences, here we analyzed synapsis and the chromosomal distribution of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Dromiciops gliorides, and Macropus eugenii) that represent South American and Australian Orders. Our results revealed inter-specific differences in the chromosomal distribution of DNA damage and repair proteins, which were associated with differing synapsis patterns. In the American species T. elegans and D. gliroides, synapsis progressed exclusively from the chromosomal ends towards interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends, which appeared conspicuously polarized in a bouquet configuration at early stages of prophase I. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophaseI in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, {gamma}H2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes.

genetics↗

bioRxiv2023

The organotypic culture of mouse seminiferous tubules as a reliable methodology for the study of meiosis in vitro

Male mouse meiosis has been traditionally studied using descriptive methods like histological sections, spreading or squashing techniques, which allow the observation of fixed meiocytes in either wildtype or genetically modified mice. For these studies, the sacrifice of the males and the extraction of the testicles are required to obtain the material of study. Other functional in vivo studies include the administration of intravenous or intraperitoneal drugs, or the exposure to mutagenic agents or generators of DNA damage, in order to study their impact on meiosis progression. However, in these studies, the exposure times or drug concentration are important limitations to consider when acknowledging animal welfare. Recently, several approaches have been proposed to offer alternative methodologies that allow the in vitro study of spermatocytes with a considerable reduction in the use of animals. Here we revisit and validate an optimal technique of organotypic culture of fragments of seminiferous tubules for meiotic studies. This technique is a trustable methodology to develop functional studies that preserve the histological configuration of the seminiferous tubule, aim homogeneity of the procedures (the use of the same animal for different study conditions), and allow procedures that would compromise the animal welfare. Therefore, this methodology is highly recommendable for the study of meiosis and spermatogenesis, while it supports the principle of 3R's for animal research.

cell biology↗

bioRxiv2022

The male mouse meiotic cilium emanates from the mother centriole at zygotene prior to centrosome duplication

Cilia are hair-like projections of the plasma membrane with an inner microtubule skeleton known as axoneme. Motile cilia and flagella beat to displace extracellular fluids, playing important roles in the airways and reproductive system, among others. Instead, primary cilia function as cell type-dependent sensory organelles, detecting chemical, mechanical or optical signals from the extracellular environment. Cilia dysfunction is associated with genetic diseases called ciliopathies, and with some types of cancer. Cilia have been recently identified in zebrafish gametogenesis as an important regulator of the bouquet conformation and recombination. However, there is very little information about the structure and functions of cilia in mammalian meiosis. Here we describe the presence of cilia in male mouse meiotic cells. These solitary cilia form transiently in 20% of zygotene spermatocytes and reach considerable lengths (up to 15 m). CEP164 and CETN3 localization studies indicate that these cilia emanate from the mother centriole, prior to centrosome duplication. In addition, the study of telomeric TFR2 suggests that these cilia are not directly related to the bouquet conformation during early male mouse meiosis. Instead, based on TEX14 labeling of intercellular bridges in spermatocyte cysts, we suggest that mouse meiotic cilia may have sensory roles affecting cyst function during prophase I.

cell biology↗

bioRxiv2022

The kleisin subunit controls the function of meiotic cohesins by determining the mode of DNA binding and differential regulation by SCC-2 and WAPL-1

The cohesin complex plays essential roles in chromosome segregation, 3D genome organisation, and DNA damage repair through its ability to modify DNA topology. In higher eukaryotes, meiotic chromosome function, and therefore fertility, requires cohesin complexes containing meiosis-specific kleisin subunits: REC8 and RAD21L in mammals and REC-8 and COH-3/4 in C. elegans. How these complexes perform the multiple functions of cohesin during meiosis and whether this involves different modes of DNA binding or dynamic association with chromosomes is poorly understood. Combining time-resolved methods of protein removal with live imaging and exploiting the temporospatial organisation of the C. elegans germline, we show that REC-8 complexes provide sister chromatid cohesion (SCC) and DNA repair, while COH-3/4 complexes control higher-order chromosome structure. High-abundance COH-3/4 complexes associate dynamically with individual chromatids in a manner dependent on cohesin loading (SCC-2) and removal (WAPL-1) factors. In contrast, low-abundance REC-8 complexes associate stably with chromosomes, tethering sister chromatids from S-phase until the meiotic divisions. Our results reveal that kleisin identity determines the function of meiotic cohesin by controlling the mode and regulation of cohesin-DNA association, and are consistent with a model in which SCC and DNA looping are performed by variant cohesin complexes that coexist on chromosomes.

cell biology↗

bioRxiv2022

The origin of a new chromosome in gerbils

Chromosome-scale genome assemblies based on ultra-long read sequencing technologies are able to illuminate previously intractable aspects of genome biology such as fine-scale centromere structure and large-scale variation in genome features such as heterochromatin, GC content, recombination rate, and gene content. We present here a new chromosome-scale genome of the Mongolian gerbil (Meriones unguiculatus) which includes the complete sequence of all centromeres. Gerbil centromeres are composed of four different repeats of length 6pb, 37bp, 127bp, or 1747bp which occur in simple alternating arrays and span 1-6Mb. Gerbil genomes have both an extensive set of GC-rich genes and chromosomes strikingly enriched for constitutive heterochromatin. We sought to determine if there was a link between these two phenomena and found that the two heterochromatic chromosomes of the Mongolian gerbil have distinct underpinnings: Chromosome 5 has a large block of intra-arm heterochromatin as the result of a massive expansion of centromeric repeats, while chromosome 13 is comprised of extremely large (>150kb) repeated sequences. In addition to characterizing centromeres, our results demonstrate the importance of including karyotypic features such as chromosome number and the locations of centromeres in the interpretation of genome sequence data, and highlight novel patterns involved in the evolution of chromosomes.

genomics↗

bioRxiv2021

Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This is an essential step towards the accuracy of cell division and therefore must be precisely regulated in both mitosis and meiosis. One of the main centromeric regulatory signaling pathways is the Haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. In mitosis, Haspin kinase phosphorylates H3 at threonine 3 (H3T3ph), the essential histone mark that recruits the CPC whose catalytic component is Aurora B kinase. To date, no data has yet been presented about the action of the centromeric Haspin-H3T3ph-CPC pathway in mammalian male meiosis. We have analyzed the consequences of Haspin chemical inhibition in cultured spermatocytes using LDN-192960. Our in vitro studies suggest that Haspin kinase activity is required for proper chromosome congression during both meiotic divisions and for the recruitment of phosphorylated Aurora B at meiotic centromeres. These results have been confirmed by the characterization of the meiotic phenotype of the genetic mouse model Haspin-/-, which displays similar defects. In addition, our work demonstrates that the absence of H3T3ph histone mark does not alter SGO2 localization to meiotic centromeres. These results add new and relevant information regarding the regulation of centromere function during meiosis.

cell biology↗

bioRxiv2020

Unpacking the eagle collision risk model: practical guidance for wind energy

Climate change is one of the greatest threats facing biodiversity, and solutions to reduce carbon emissions are needed to conserve species. Renewable energies are a prominent means to achieve this goal, but the potential for direct harm to wildlife has raised concerns as these technologies proliferate. To protect biodiversity, approaches that facilitate renewable energy development while protecting species are needed. In the United States wind energy developers must obtain a permit for any Bald or Golden eagles that might be killed at a facility. The U.S. Fish & Wildlife Service estimates fatalities using a Bayesian modeling framework, which combines pre-construction eagle surveys with prior information. The ways in which prior information is incorporated and how pre-construction monitoring affects model outcomes can be unclear to regulated entities and other stakeholders, creating uncertainty in the permitting process and retarding both the build-out of renewable energy and conservation measures for eagles. We conducted a simulation study quantifying the differences in predicted eagle fatalities obtained by incorporating prior information and using only site-specific survey data across a range of scenarios, evaluating the impact of survey effort on the magnitude of this effect. We identified predictable relationships between survey effort, eagle activity, facility size and discrepancies between estimates. We also translated these patterns into real-world financial costs, illustrating the interaction between pre-construction surveys, fatality estimates, and compensatory mitigation obligations in determining permit timing and expense.

ecology↗

bioRxiv2020

Meiosis reveals the early steps in the evolution of a neo-XY sex chromosome pair in the African pygmy mouse Mus minutoides

Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in most mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouse Mus minutoides, which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution. AUTHOR SUMMARYThe early steps in the evolution of sex chromosomes are particularly difficult to study. Cessation of recombination around the sex-determining locus is thought to initiate the differentiation of sex chromosomes. Several studies have investigated this process from a genetic point of view. However, the cellular context in which recombination arrest occurs has not been considered as an important factor. In this report, we show that meiosis, the cellular division in which pairing and recombination between chromosomes takes place, can affect the incipient differentiation of X and Y chromosomes. Combining cytogenetic and genomic approaches, we found that in the African pygmy mouse Mus minutoides, which has recently undergone a sex chromosome-autosome fusion, synapsis and DNA repair dynamics are altered along the newly added region of the sex chromosomes, likely interfering with recombination and thus contributing to the genetic isolation of a large segment of the Y chromosome. Therefore, the cellular events that occur during meiosis are crucial to understand the very early stages of sex chromosome differentiation. This can help to explain why sex chromosomes evolve very fast in some organisms while in others they have barely changed for million years.

genetics↗

bioRxiv2018

Transition from a meiotic to a somatic-like DNA damage response during the pachytene stage in mouse meiosis

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of {gamma}H2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid pachytene to diplotene, {gamma}H2AX appeared as large discrete foci. This late repair pattern was mediated first by NHEJ, involving Ku70/80 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early \"meiotic\" repair pathway that acts by default at the beginning of meiosis is replaced from mid pachytene onwards by a \"somatic-like\" repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, expression of a new gene profile and acquisition of competence to proceed to metaphase.

genetics↗