Biology subjects

McNerney, C.

Publications and source records attributed to McNerney, C..

3 recordsLinked to original sources

bioRxiv2025

DIO3 coordinates photoreceptor development timing and fate stability in human retinal organoids

The mechanisms governing the generation of neuronal subtypes at distinct times and proportions during human retinal development are poorly understood. While thyroid hormone (TH) signaling specifies cone photoreceptor subtypes, how this regulation changes over time remains unclear. To address this question, we studied the expression and function of type 3 iodothyronine deiodinase (DIO3), an enzyme that degrades TH, in human retinal organoids. We show that DIO3 is a master regulator of human photoreceptor developmental timing and cell fate stability. DIO3 is highly expressed in retinal progenitor cells (RPCs) and decreases as these cells asynchronously differentiate into neurons, progressively reducing TH degradation and increasing TH signaling. DIO3 mutant organoids display precocious development of S cones, L/M cones, and rods, increased photoreceptor (PR) density, and adoption of L/M cone fate characteristics by S cones and rods. Our multiomics and chimeric organoid experiments show that cell autonomous and non-autonomous mechanisms locally coordinate and maintain DIO3 expression and TH signaling levels among cells. Computational modeling reveals a mechanism that couples TH levels and fate specification, providing robustness to photoreceptor development as compared to a probabilistic, cell-intrinsic mechanism. Based on our findings, we propose an hourglass hypothesis, in which the proportion of progenitors to neurons decreases over time to relieve TH degradation, which triggers development of PR subtypes at specific times. Our study identifies how local regulation of thyroid hormone signaling influences neural cell fate specification, which may be a consideration for designing regenerative therapies.

developmental biology↗

bioRxiv2025

Application of machine learning to discriminate photoreceptor cell species in xenotransplanted chimeric retinas

Photoreceptor transplantation is being studied to improve visual function in retinal diseases causing blindness, such as age-related macular degeneration, hereditary eye diseases, and traumatic retinopathy, among others. Preclinical studies often involve the delivery of exogenous human photoreceptor cells into the retinas of animal models. In such experiments, a key readout is the differential frequency of donor cell somatic integration versus artificial labeling secondary to material transfer of cytosolic or nuclear labels from donor to recipient cells. For this analysis, the ability to recognize photoreceptor nuclei as being of donor (human) versus animal is key, but purely immunohistology discrimination can be challenging because of antigenic species overlap or intercellular antigen transfer. To address this challenge, we sought to develop and validate a computational technique to discriminate between photoreceptor cells of different animal species based on machine learning of nuclear morphology. Here, we aimed to evaluate the feasibility of using computer-assisted detection of separate nuclei and employing random forest classification to automate the species differentiation, among DAPI-stained photoreceptors after xeno-transplantation of human photoreceptors into the retinas of mice and pigs. Our models were trained on single-species samples and validated with mixed-species samples. We then transplanted human embryonic stem cell-derived retinal organoid cells into rodent and pig retinal degeneration models. The random forest model accurately determined cell identity post-xenotransplantation, validated by histological assessment using an anti-human nuclear antibody. Our results support the potential efficacy of employing machine learning image analysis and classification techniques that may promote experimental rigor, minimize observer bias, and enable high throughput semi-automated workflows for transplantation outcomes analysis. The methodological framework reported here may enable a more nuanced and precise analysis of the behavior of transplanted photoreceptors for the purposes of human retinal regeneration.

neuroscience↗

bioRxiv2020

Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by E. coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

FRET (fluorescence energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes showed extensive bending/wrapping of {lambda}PR promoter DNA on E. coli RNA polymerase (RNAP) in closed and open complexes (CC, OC). Here we determine the kinetics and mechanism of DNA bending/wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye fluorescence enhancements (PIFE). FRET/PIFE kinetics exhibit two phases: rapidly-reversible steps forming a CC ensemble ({CC}c of four intermediates (initial (RPC), early (I1E), mid-(I1M), late (I1L)), followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET/PIFE together reveal large-scale bending/wrapping of upstream and downstream DNA as RPC advances to I1E, reducing -100/+14 distance to [~]75[A] and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper {beta}-clamp while downstream DNA contacts the top of the {beta}-pincer in I1E. Converting I1E to I1M (~1s time-scale) reduces FRET efficiency with little change in -100/+14PIFE, interpreted as clamp-opening that moves far-upstream DNA (on {beta}) away from downstream DNA (on {beta}) to increase the -100/+14 distance by ~14[A]. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp-closing to reduce the -100/+14 distance by ~21[A]. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L converts to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream-binding, DNA-bending transcription activators are discussed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/932780v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1068460org.highwire.dtl.DTLVardef@dcd6d9org.highwire.dtl.DTLVardef@9d4727org.highwire.dtl.DTLVardef@1aa8e47_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗