Biology subjects

Iyer, N. R.

Publications and source records attributed to Iyer, N. R..

7 recordsLinked to original sources

bioRxiv2026

Complementary constraints in germ and immune cells shape evolution of gene regulation and phenotype

New mutations arise in germ cells and must preserve fertility while altering somatic phenotype to confer a fitness advantage. To understand how whole organisms balance these constraints, we generated mice with humanized noncoding promoter sequence at Traf6, an NF-{kappa}B pathway component with recently diverged chromatin state in mouse germ cells. The humanized allele (Traf6h) reinstated human-like expression in germ and immune cells, which we traced to a 13 base pair deletion that impairs CTCF binding. Traf6h had no effect on fertility but induced a sensitized innate immune response to endotoxin. Other NF-{kappa}B pathway genes followed a similar pattern, suggesting that their expression has evolved in tandem and consistent with known mouse-human differences in inflammatory response. Our results reveal distinct roles for germ and immune cells in shaping gene regulatory evolution, where immune cells quickly adjust phenotype, enabling selection, while germ cells robustly maintain fertility despite changing expression levels, enabling inheritance.

genetics↗

bioRxiv2025

Rapid, Growth Factor-Reduced Differentiation of Functional Neurons from hiPSCs

IntroductionHuman induced pluripotent stem cells (hiPSCs) can be rapidly converted into neurons via NGN2 overexpression, but many protocols require costly reagents during the initial induction phase that may limit adoption by labs without routine neuronal differenitation experience. We developed a simplified, low-cost protocol using a tetracyline-inducible (TET-on) NGN2 system in minimal media to generate cortical neurons in as little as 6 days. MethodsKOLF2.1J hiPSCs were stably transfected with a TET-on NGN2 cassette using the nonviral PiggyBac system and induced with doxycycline in Essential 6 media. The impact of adding the Notch inhibitor, DAPT, during doxycycline induction to enhance neurogenesis to was evaluated with immunocytochemistry (ICC) and RT-PCR. Following induction neurons were matured and characterized with ICC for mature neuronal markers and by multielectrode array recordings for functional network activity. ResultsDAPT markedly improved conversion efficiency, reducing non-neuronal cells and increasing pan-neuronal TUJ1 expression. Resulting neurons expressed cortical markers and matured into functional glutamatergic neurons. MEA recordings showed spontaneous activity by day 14 and synchronous network firing by day 35. Secondary PB transfection enabled Td-Tomato labelling of KOLF2.1J:pB-TO-NGN2 hiPSCs, allowing 24-hour live imaging of neurite outgrowth. ConclusionThis streamlined, growth-factor-free workflow provides an accessible route for generating functional neurons from patient-derived hiPSCs, including in labs with limited hiPSC or neuronal culture experience.

synthetic biology↗

bioRxiv2025

CD8 T cells mediate immunosurveillance for neoantigen+ epithelial stem cells in the colon

Epithelial cells in the colon accumulate substantial numbers of somatic mutations, some of which can be recognised as neoantigens. The ability of CD8 T cells to survey for neoantigen+ cells in the healthy colon would provide an early detection mechanism to prevent cancer, but it is unclear whether neoantigen-specific CD8 T cells can mediate this process of immunosurveillance without becoming tolerant. To address this question, we used a genetically engineered mouse model to express a neoantigen in the epithelial cells of the adult proximal colon. Induction of neoantigen expression led to rapid elimination of neoantigen+ epithelial cells from the colon in a CD8 T cell-dependent manner. Neoantigen-specific CD8 T cells acquired cytolytic function within the colon tissue under steady-state conditions, which was required for elimination of the neoantigen+ epithelial cells. Despite the elimination of [~]25% of their epithelial cells over a two-day period, the colons looked histologically normal. Immunofluorescence and single-cell transcriptomic analyses revealed that neoantigen-specific CD8+ T cells specifically target neoantigen+ stem cells at the crypt base, which was associated with Ki67 in the crypt wall and abundance of neoantigen-negative stem cells. Infiltrating neoantigen-specific CD8 T cells made IFNg and expressed PD-1, raising the question of why PD-1-dependent suppression did not prevent the acquisition of effector functions by these neoantigen-specific CD8 T cells. Despite an increased signature of interferon-stimulated genes in colonic epithelial cells, PD-L1 expression was surprisingly absent. Moreover, we found that colonic epithelial stem cells also did not express PD-L1 under conditions of chronic inflammation, such as ulcerative colitis, immune checkpoint-induced colitis, and ageing, or when directly stimulated with IFN-{gamma} in vitro. Analyses of the PD-L1 gene promoter across humans and mice showed hypermethylation at sites associated with PD-L1 repression in cancer. Thus, our data support a model in which the acquisition of neoantigens by colonic epithelial cells triggers CD8 T cell-mediated immunosurveillance. This results in the elimination of PD-L1-negative neoantigen+ stem cells by effector CD8 T cells and simultaneous repair of the colon by neoantigen-negative epithelial cells to prevent immunopathology.

immunology↗

bioRxiv2025

Human microphysiological model of dorsal root ganglion-spinal cord dorsal horn circuitry recapitulates opioid induced effects

Microphysiological systems (MPSs) are engineered, in vitro platforms which have been established as viable alternatives to animal models for pre-clinical research with unique advantages over conventional model systems. Many MPSs utilize 3-dimensional (3D) tissue constructs that enable biomimetic cell-cell interactions, allow for extended culture periods, and provide the time necessary for the emergence of physical and physiological characteristics of more mature tissues. Here, we present a novel MPS using human induced pluripotent stem cell (hiPSC)-derived spinal cord dorsal horn (SCDH) spheroids co-cultured with hiPSC-derived dorsal root ganglion (DRG) sensory spheroids in a microengineered hydrogel system to create a "connectoid" model of afferent pain circuitry. SCDH spheroids were functionally innervated by peripheral sensory neurons, and prolonged maturation of hiPSC-derived SCDH neurons within the connectoid system enabled derivation of crucial late-born cell types unattainable using 2D differentiations. Furthermore, hiPSC-derived SCDH spheroids spontaneously generate rhythmic, complex, synaptically-driven electrophysiological waveforms that are disinhibited by morphine exposure, consistent with spinal mechanisms of opioid-induced pruritus and hypersensitivity. One Sentence SummaryhiPSC-derived afferent sensory circuitry model, with NK1R+ spinal cord dorsal horn neurons, yields electrophysiologically mimetic response to opioids.

bioengineering↗

bioRxiv2023

A connection between the ribosome and two S. pombe tRNA modification mutants subject to rapid tRNA decay

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation in the ribosome. In both the yeast Saccharomyces cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two S. pombe mutants subject to RTD, mutations in ribosomal protein genes suppress the temperature sensitivity without altering tRNA levels. Prior work showed that S. pombe trm8{Delta} mutants, lacking 7-methylguanosine, were temperature sensitive due to RTD and that one class of suppressors had mutations in the general amino acid control (GAAC) pathway, which was activated concomitant with RTD, resulting in further tRNA loss. We now find that another class of S. pombe trm8{Delta} suppressors have mutations in rpl genes, encoding 60S subunit proteins, and that suppression occurs with minimal restoration of tRNA levels and reduced GAAC activation. Furthermore, trm8{Delta} suppression extends to other mutations in the large or small ribosomal subunit. We also find that S. pombe tan1{Delta} mutants, lacking 4-acetylcytidine, are temperature sensitive due to RTD, that one class of suppressors have rpl mutations, associated with minimal restoration of tRNA levels, and that suppression extends to other rpl and rps mutations. However, although S. pombe tan1{Delta} temperature sensitivity is associated with some GAAC activation, suppression by an rpl mutation does not significantly inhibit GAAC activation. These results suggest that ribosomal protein mutations suppress the temperature sensitivity of S. pombe trm8{Delta} and tan1{Delta} mutants due to reduced ribosome concentrations, leading to both a reduced requirement for tRNA, and reduced ribosome collisions and GAAC activation. Results with S. cerevisiae trm8{Delta} trm4{Delta} mutants are consistent with this model, and fuel speculation that similar results will apply across eukaryotes.

molecular biology↗

bioRxiv2021

Modular Derivation and Unbiased Single-cell Analysis of Regional Human Hindbrain And Spinal Neurons Enables Discovery of Nuanced Transcriptomic Patterns along Developmental Axes

Our inability to derive the vast neuronal diversity of the posterior central nervous system (pCNS) using human pluripotent stem cells (hPSCs) poses a major impediment to understanding human neurodevelopment and disease in the hindbrain and spinal cord. Here we establish a modular differentiation paradigm that recapitulates patterning along both the rostrocaudal (R/C) and dorsoventral (D/V) axes of the pCNS, enabling derivation of any neuronal phenotype with discrete regional specificity. First, neuromesodermal progenitors (NMPs) with discrete Hox profiles are efficiently converted to pCNS progenitors (pCNSPs). Then by tuning D/V signaling, pCNSPs are directed to ventral Shh-dependent MNs (MNs) and locomotor interneurons (INs) or dorsal TGF-{beta}-dependent proprioceptive INs and TGF-{beta}-independent sensory INs. We applied D/V protocols to NMPs spanning the R/C axis for expansive single-cell RNA-sequencing (scRNAseq) analysis. By implementing a novel computational pipeline comprising sparse non-negative matrix factorization, consensus clustering, and combinatorial gene expression pattern identification, we detect hundreds of transcriptional markers within region-specific neuronal phenotypes, enabling discovery of gene expression patterns along the developmental axes. These findings highlight the potential of these resources to advance a mechanistic understanding of pCNS development, expand the potential and accuracy of in vitro models, and inform novel regenerative therapeutic strategies.

neuroscience↗

bioRxiv2021

Passive clearing and 3D lightsheet imaging of the intact and injured spinal cord in mice

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.

neuroscience↗