Biology subjects

Delgado, R. N.

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

4 recordsLinked to original sources

bioRxiv2025

Programmable orthogonal and rapid DNA strand displacement for fluidic-exchange-free highly multiplexed fluorescent imaging

Multiplexed fluorescent imaging methods are essential for studying cellular function by visualization of biomolecules in cells and tissues with high-resolution spatial information, but most suffer from limitations such as low multiplexity because of spectral overlap between the used fluorophores. Fluidic exchange based sequential multiplexed imaging overcomes this limitation but requires time-consuming incubation and washing steps. We introduce a novel multiplexed fluorescent imaging method that uses rapid, orthogonal DNA strand displacement reactions to enable unlimited multiplexed fluorescent imaging without fluidic exchange. The signal switching between targets is achieved by strand displacement with added non-fluorescent DNA displacer strands, no fluidic washing step is required for sequential multiplexed imaging. We experimentally screened a set of rapid and orthogonal DNA displacement sequences for probe design and applied it for RNA imaging, which takes less than 30 seconds per round to complete in fixed cells. Because of the vast sequence design space of DNA probes, theoretically unlimited multiplexity can be achieved. Using 25 developed rapid orthogonal probes, we achieved 25-plex RNA imaging in a single fluorophore channel in fixed cells within 20 minutes. To further demonstrate robustness and practical usage of DIRSE-based imaging, we showed 24-plex RNA imaging with the method in retinal tissues and resolved different cell types. This DIRSE mechanism significantly simplifies the high-plex fluorescent imaging process with pre-programmed DNA probes and has broad biotechnical applications for future medicine and diagnostics.

bioengineering↗

bioRxiv2025

Massively parallel reporter assay for mapping gene-specific regulatory regions at single nucleotide resolution

Precise gene regulation is essential for the development and function of complex tissues, yet comprehensive mapping of cis-regulatory modules (CRMs) remains challenging due to limitations in throughput, resolution, and the ability to assay within specific cell types. Here, we introduce two complementary approaches--a locus-specific massively parallel reporter assay (LS-MPRA) and a degenerate MPRA (d-MPRA)--specifically designed to address some of these shortcomings. LS-MPRA leverages bacterial artificial chromosomes (BACs) to generate high-complexity libraries spanning large genomic regions, enabling unbiased interrogation of millions of DNA fragments potentially relevant for the regulation of a specific gene or set of genes. The d-MPRA employs systematic mutagenesis to resolve the functional architecture of CRMs at nucleotide resolution, thereby nominating critical nucleotides as potential TF binding sites, or for other regulatory roles. We applied these methods to retinal genes that are stably expressed in differentiated cells of the retina, in rod photoreceptors and in subsets of bipolar interneurons, using both in vivo and ex vivo preparations of mouse tissue. LS-MPRA recapitulated some of the known CRMs for these genes--such as the proximal promoter region of Rho--and identified potentially novel CRMs, including those located within neighboring genes. The method was then applied to a gene that is dynamically expressed in subsets of retinal progenitor cells, Olig2, where it identified three distinct CRM regions (Olig2-NR1, NR2, and NR3). D-MPRA and subsequent motif analyses nominated critical TF binding sites within these regions. CUT&RUN experiments confirmed direct binding of these candidates. Moreover, extending LS-MPRA to chick retina and spinal cord demonstrated the applicability of these methods across species and tissues. Together, the integrated LS-MPRA and d-MPRA strategies provide a robust, high-resolution platform for discovery of the cis-regulatory code underlying tissue-specific gene expression. It does not require prior knowledge of potential CRMs, and is quite rapid and straightforward to deploy, using typical molecular biology methods. The fragment size can be scaled to create short CRMs, e.g. for cell type-specific expression within viral vectors. It should enable CRM discovery at a scale and affordability for laboratories wishing to focus on a particular locus or set of loci.

developmental biology↗

bioRxiv2025

Autism-Associated Genes and Neighboring lncRNAs Converge on Key Gene Regulatory Networks

Autism spectrum disorder (ASD) is highly heritable, and mutations in hundreds of genes have been implicated as individually rare causes of ASD1-3. Understanding how disruptions to these functionally diverse genes lead to the core features of ASD remains a major challenge4. Moreover, ASD is three- to four-fold more common in males than females5, and autistic females tend to carry more autosomal risk alleles for ASD compared to autistic males6,7, but the biological basis of this "female protective effect" (FPE) is unknown8,9. Here we show that individual perturbations of 18 ASD genes in human neural progenitor cells converge on shared effects on gene expression, including widespread downregulation of other ASD genes. De novo reconstruction of a gene regulatory network (GRN) enabled the identification of central transcriptional regulators, including the prominent ASD gene CHD8 as well as novel candidates such as REST, that drive this transcriptomic convergence. Furthermore, the X-linked transcription factor ZFX, which is expressed from both the active and the inactive X chromosomes in females10, emerged as a key activator of many ASD genes: we propose that the higher ZFX expression level observed in female brain can buffer damaging mutations in diverse ASD genes, contributing to the FPE. Together, these results reveal how key GRNs can become broadly and similarly dysregulated upon disruption of individual ASD genes and provide molecular insight into the female protective effect in ASD.

genetics↗

bioRxiv2024

Chromatin regulator Kdm6b is required for the establishment and maintenance of neural stem cells in mouse hippocampus

Neural stem cells (NSCs) in the mouse hippocampal dentate gyrus (DG) - a structure important to learning and memory - generate new neurons postnatally and throughout adult life. However, the regulators that enable this lifelong neurogenesis remain incompletely understood. Here we show that the chromatin regulator KDM6B is required for both the establishment and maintenance of NSCs in the mouse DG. Conditional deletion of Kdm6b in embryonic DG precursors results in an adult hippocampus that is essentially devoid of NSCs, and hippocampal-dependent behaviors are defective. Kdm6b-deletion causes precocious neuronal differentiation, and the NSC population fails to become established in the postnatal DG. Using single cell RNA sequencing (scRNA-seq), we observed that Kdm6b-deletion disrupts the transcriptomic signature of NSC maintenance. Furthermore, deleting Kdm6b in adult DG NSCs induces early neuronal differentiation, and the NSC population is not properly maintained. These data illustrate the critical role that Kdm6b plays in adult DG neurogenesis, which may help understand how mutations in this chromatin regulator result in cognitive disorders in human patients.

neuroscience↗