Biology subjects

Spencer, A. P.

Publications and source records attributed to Spencer, A. P..

4 recordsLinked to original sources

bioRxiv2025

Fusome morphogenesis is sufficient to promote female germline stem cell self-renewal in Drosophila

Germline cysts, or interconnected groups of germ cells, promote the synchronization of gamete development in vertebrates and invertebrates. In the Drosophila ovary, cyst formation is coordinated by the fusome, a unique endoplasmic reticulum-like organelle. Although many structural components of the fusome have been characterized, little is known about the genetic factors that control fusome growth and distribution between germ cells. Here, we identify the {beta}-importin, Tnpo-SR, as an important regulator of fusome morphogenesis in germline stem cells (GSCs) and their dividing daughters. Although Tnpo-SR does not aggregate at fusomes or centrosomes, Tnpo-SR null mutants fail to form proper fusomes and knock-down of Tnpo-SR reduces fusome accumulation, ultimately leading to cyst fragmentation and improper numbers of germ cells within an egg chamber. Tnpo-SR depletion disrupts microtubule organization during interphase and sub-cellular localization of the microtubule associated protein Asp. However, overexpression of asp is not sufficient to restore fusome size or cyst architecture when Tnpo-SR is depleted, suggesting that Tnpo-SR-dependent regulation of asp does not solely control fusome morphogenesis. Instead, we find that restoring fusome size by overexpressing the core fusome component hu-li tai shao or the polarity factor Par3/bazooka is sufficient to restore fusome area in Tnpo-SR-depleted GSCs. Moreover, restoration of fusome area in the absence of Tnpo-SR also rescues cyst organization and oocyte specification, suggesting that Tnpo-SR functions upstream of fusome structural component production. Taken together, these data functionally link nuclear import/export machinery to fusome morphogenesis during Drosophila germline cyst development. ARTICLE SUMMARYThe fusome plays a key role in cyst formation in the Drosophila ovarian germline, yet regulation of fusome growth and distribution between germ cells after mitosis remains understudied. Through loss-of-function analyses, the authors identify Tnpo-SR as a novel regulator of fusome morphogenesis. Tnpo-SR depletion reduces the core fusome structural component Hts in germline stem cells (GSCs) and disrupts microtubule organization and nucleation. Overexpression of Hts or Par3/bazooka in Tnpo-SR-depleted GSCs restores fusome size, which in turn rescues cyst organization and oocyte specification. These findings establish Tnpo-SR as an upstream regulator of Hts during fusome morphogenesis.

developmental biology↗

bioRxiv2024

Analysis of a hypomorphic mei-P26 mutation reveals developmental control of CO patterning mechanisms

Female gametogenesis in Drosophila requires differentiation and mitotic division of germ cells, acquisition of oocyte fate, and entry into meiosis. Each of these processes is well understood individually; however, little is known about the mechanisms that ensure proper temporal integration of germ cell differentiation and meiotic chromosome dynamics. Here, we take advantage of a hypomorphic mutation in mei-P26, a well-characterized gene with multiple diverse functions in germ cell development, to determine the consequences of disrupting the coordination between development and meiosis. While null mutations in mei-P26 lead to tumorous ovaries, the hypomorphic allele mei-P261 allows sufficient germ cell differentiation and fertility to support analysis of meiotic chromosome dynamics. Unlike wildtype germaria, 60% of cysts in mei-P261 germaria co-express the differentiation factor Bag of marbles (Bam) and the oocyte specification factor Orb, suggesting that mitotic division is delayed. In this context, the synaptonemal complex rarely assembles into full length continuous tracks and instead is missing or present only as foci. Despite these phenotypes, meiotic double-strand breaks still form and are repaired as crossovers, but the crossovers are mis-patterned and form in centromere proximal regions rather than chromosome arms. The strength of crossover interference is significantly reduced and the centromere effect is lost, but crossover assurance is intact and the meiosis-specific machinery is used to form crossovers. We suggest a model where the failure to exit mitosis in a timely fashion causes cells to enter meiosis while still receiving mitotic signals, resulting in abnormal meiotic chromosome dynamics and impaired crossover formation. Article summaryFemale gametogenesis in Drosophila requires the precise temporal integration of germ cell differentiation and meiotic entry. Using a hypomorphic mutation in mei-P26, we investigated how disrupting this coordination affects meiotic chromosome dynamics. In mei-P261 mutants, delayed mitotic exit causes cells to enter meiosis while still receiving mitotic signals. This results in fragmented synaptonemal complex and the loss of some but not all CO patterning mechanisms. These results suggest that timely mitotic exit is critical for establishing the proper regulatory landscape required for meiotic recombination.

genetics↗

bioRxiv2024

Engineered chitosan-derived nanocarrier for efficient siRNA delivery to peripheral and central neurons

Gene therapy using small interfering RNA (siRNA) holds promise for treating neurological disorders by silencing specific genes, such as the phosphatase and tensin homolog (PTEN) gene, which restricts axonal growth. Yet, delivering siRNA to neurons efficiently is challenging due to premature degradation and unspecific delivery. Chitosan-based delivery systems have shown great potential due to their well-established biocompatibility. However, their limited transfection efficiency and lack of neuronal tropism require further modification. Building on our previous successes with neuron-targeted DNA delivery using chitosan, a novel approach for siRNA delivery aimed at PTEN downregulation is proposed. This involves using thiolated trimethyl chitosan (TMCSH)-based siRNA nanoparticles functionalized with the neurotropic C-terminal fragment of the tetanus neurotoxin heavy chain (HC) for efficient delivery to both peripheral and central neurons. These polyplexes demonstrated suitable physicochemical properties, biocompatibility, and no adverse effects on neuronal electrophysiology. Diverse neuronal models, including 3D ex vivo cultures and microfluidics, confirmed polyplexes efficiency and neurospecificity. HC targeting significantly enhanced nanoparticle neuronal binding, and live cell imaging revealed five times faster retrograde transport along axons. Furthermore, siRNA delivery targeting PTEN promoted axonal outgrowth in embryonic cortical neurons. Thus, these polyplexes represent a promising platform for siRNA delivery, offering potential for clinical translation and therapeutic applications.

bioengineering↗

bioRxiv2024

Unveiling the potential of neuron-targeted dendriplexes for siRNA delivery using a PNS-CNS-on-Chip

Neurological disorders, a leading global cause of death, encompass conditions affecting the peripheral and central nervous systems (PNS and CNS, respectively). Limited axon regeneration is a significant challenge in these disorders, and it is linked to proteins like PTEN. RNA-based therapeutics, particularly siRNAs, hold potential for silencing these inhibitory pathways, but their clinical application is hindered by poor stability and cellular uptake. Our study addressed this challenge with the development of novel, fully biodegradable dendritic nanoparticles designed specifically for neuron targeting. These nanoparticles were functionalized with the neurotropic binding domain of tetanus toxin, enhancing selective neuronal targeting and cellular internalization. We demonstrated that these dendriplexes not only maintain biocompatibility and efficient siRNA delivery in neuronal cultures but also significantly enhance axonal growth, as shown in microfluidic models. In a groundbreaking PNS-CNS-on-Chip, dendriplexes exhibited effective migration from PNS to CNS neurons, highlighting their potential for targeted therapeutic delivery. This study pioneers the application of microfluidics to demonstrate the CNS targeting of dendriplexes, paving the way for innovative treatments in the field of nanomedicine.

bioengineering↗