Biology subjects

Leigh, N. D.

Publications and source records attributed to Leigh, N. D..

7 recordsLinked to original sources

bioRxiv2026

Adaptive immunity is dispensable for salamander appendage regeneration

Complex multi-tissue regeneration capacity varies across vertebrates. Mammals are amongst the least regenerative species, while salamanders can regenerate complex tissues such as limbs and tails throughout life. Previous studies have shown that innate and adaptive immune cells are present during salamander limb regeneration. While innate immune cells have been shown to promote limb regeneration, it is unknown whether adaptive immunity is responsive to amputation or plays a role in appendage regeneration. Here we show that during limb regeneration in axolotls, the immune response is characterized by a coordinated immunoregulatory signature including the downregulation of antigen presentation, cytokine secretion, and T cell activation. We corroborate this transcriptomic data in vivo using skin allografts in newts and define the blastema as an immunosuppressed niche. To test the role of adaptive immune cells in regeneration, we generated Recombination activating gene 1 deficient (Rag1-/-) newts. Rag1-/- newts lack antigen receptor recombination and show a marked reduction of adaptive immune cells. We find that Rag1-/- newts do not reject allografts, confirming their functional immunodeficiency. Finally, we demonstrate that both larval and adult newts regenerate appendages in the absence of adaptive immunity. Our work demonstrates that the adaptive arm of the immune system is not required for appendage regeneration and establishes an important model for novel experimental approaches in comparative immunology and regenerative biology.

immunology↗

bioRxiv2026

Novabrowse: A Tool for High-Resolution Synteny Analysis, Ortholog Detection, and Gene Signal Discovery

Accurate genome annotation remains challenging as assembly quality often exceeds annotation reliability. Resolving ambiguities of gene presence, absence, and orthology typically requires integrating two complementary lines of evidence: sequence homology between species and the conservation of gene order (i.e., synteny). BLAST remains the standard for homology detection, yet its raw output can be difficult to interpret. Existing tools address this challenge but operate at opposing scales. Alignment viewers provide detailed pairwise statistics without genomic context, while synteny tools offer chromosome-scale perspectives without sequence-level resolution. To fill this intermediate gap, we developed Novabrowse, an interactive BLAST results interpretation framework featuring high-resolution multi-species synteny analysis, chromosomal re-arrangement investigation, ortholog detection, and gene signal discovery. Users define a genomic region of interest in a query species and/or use custom sequences, then select one or more subject species for comparison. The pipeline retrieves query gene sequences via NCBI API integration and performs BLAST searches against each subject transcriptome or genome. Results are presented via an interactive HTML file featuring alignment statistics, chromosomal maps, coverage visualizations, ribbon plots, and distance-based clustering of high-scoring segment pairs into putative gene units. We demonstrate these capabilities by investigating Foxp3, Aire, and Rbl1, three highly conserved vertebrate genes, in the recently assembled genome of the newt Pleurodeles waltl. Foxp3 and Aire have not been described in any salamander species to date, despite availability of multiple assemblies and extensive transcriptomic datasets. Using Novabrowse, we discovered conserved loci and gene signals for both genes in P. waltl, the presence of which was subsequently confirmed via Nanopore long-read RNA sequencing. In contrast, Rbl1 analysis uncovered a chromosomal rearrangement at its expected locus with no gene signal detected, indicating a gene loss specific to P. waltl despite the genes retention in the closely related axolotl (Ambystoma mexicanum). Our findings demonstrate Novabrowses capacity for evidence-based evaluation of annotation artifacts, an essential capability as high-quality assemblies become more available for phylogenetically diverse species. Novabrowse is open source (MIT license) and freely available at: https://github.com/RegenImm-Lab/Novabrowse.

genomics↗

bioRxiv2023

Label-free high-resolution infrared spectroscopy for spatiotemporal analysis of complex living systems

Label-free chemical and structural imaging of complex living tissue and biological systems is the holy grail of biomedical research and clinical diagnostics. The current analysis techniques are time-consuming and/or require extensive sample preparation, often due to the presence of interfering molecules such as water, making them unsuitable for the analysis of such systems. Here, we demonstrate a proof-of-principle study using label-free optical photothermal mid-infrared microspectroscopy (O-PTIR) for fast, direct spatiotemporal chemical analysis of complex living biological systems at submicron resolution. While other analytical methods can provide only static snapshots of molecular structures, our O-PTIR approach enables time-resolved and in situ investigation of chemical and structural changes of diverse biomolecules in their native conditions. This comprises a technological breakthrough in infrared spectroscopy to analyze biomolecules under native conditions over time: in fresh unprocessed biopsies, living brain tissue, and vertebrates without compromising their viability. One-Sentence SummaryProof-of-principle application of non-destructive O-PTIR for high-resolution spatiotemporal chemical and structural analysis of unprocessed biopsies, living brain tissue, and vertebrates.

molecular biology↗

bioRxiv2022

Accurate genotype-based demultiplexing of single cell RNA sequencing samples from non-human animals

Single cell sequencing technologies (scRNA-seq, scATAC-seq, etc.) have revolutionized the study of complex tissues and unique organisms, providing researchers with a much needed species agnostic tool to study biological processes at the cellular level. To date, scRNA-seq technologies are expensive, require sufficient cell quantities, and need biological replicates to avoid batch effects or artifactual results. Pooling cells from multiple individuals into a single scRNA-seq library can address these problems. However, sample labeling protocols for facilitating the computational separation of pooled scRNA-seq samples, termed demultiplexing, have undesirable limitations, particularly in resource-limited organisms. One promising solution developed for use in humans exploits the genetic diversity between individuals (i.e., single nucleotide polymorphisms (SNP)) to demultiplex pooled scRNA-seq samples. The use of SNP-based demultiplexing methods has not been validated for use in non-human species, but the widespread use of SNP-based demuxers would greatly facilitate research in commonly used, emerging, and more obscure species. In this study we applied SNP-based demultiplexing algorithms to pooled scRNA-seq datasets from numerous species and applied diverse ground truth confirmation assays to validate genetic demultiplexing results. SNP-based demultiplexers were found to accurately demultiplex pooled scRNA-seq data from species including zebrafish, African green monkey, Xenopus laevis, axolotl, Pleurodeles waltl, and Notophthalmus viridescens. Our results demonstrate that SNP-based demultiplexing of unlabeled, pooled scRNA-seq samples can be used with confidence in all of the species studied in this work. Further, we show that the only genomic resource required for this approach is the single-cell sequencing data and a de novo transcriptome. The incorporation of pooling and SNP-demultiplexing into scRNA-seq study designs will greatly increase the reproducibility and experimental options for studying species previously limited by technical uncertainties, computational hurdles, or limited cell quantities.

bioinformatics↗

bioRxiv2022

Cell type profiling in salamanders identifies innovations in vertebrate forebrain evolution

The evolution of advanced cognition in vertebrates is associated with two independent innovations in the forebrain: the six-layered neocortex in mammals and the dorsal ventricular ridge (DVR) in sauropsids (reptiles and birds). How these novelties arose in vertebrate ancestors remains unclear. To reconstruct forebrain evolution in tetrapods, we built a cell type atlas of the telencephalon of the salamander Pleurodeles waltl. Our molecular, developmental, and connectivity data indicate that parts of the sauropsid DVR trace back to tetrapod ancestors. In contrast, the salamander dorsal pallium is devoid of cellular and molecular characteristics of the mammalian neocortex, yet shares similarities with entorhinal cortex and subiculum. Our findings chart the series of innovations that resulted in the emergence of the sauropsid DVR, and the mammalian six-layered neocortex.

neuroscience↗

bioRxiv2020

Isolation of high yield and quality RNA from human precision-cut lung slices for RNA-sequencing and computational integration with larger patient cohorts

Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology and disease, as well as for screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology and disease as compared to PCLS derived from animals (e.g. clinical heterogeneity), but access to human tissue is limited. A number of different experimental readouts have been established for use with PCLS, but obtaining high yield and quality RNA for downstream gene expression analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for non-biased and high through-put analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis (IPF). We show that the RNA isolated using this method is of sufficient quality for both RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS was comparable to data generated from native tissue and could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data sets. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS derived from distal lung tissue, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease, such as IPF.

molecular biology↗

bioRxiv2019

von Willebrand Factor D and EGF Domains is an evolutionarily conserved and required feature of blastemas capable of multi-tissue appendage regeneration

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema--a transient structure that houses a pool of progenitor cells that regenerate the missing tissue. We have identified the expression of von Willebrand Factor D and EGF Domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species. Further, vwde expression is tightly linked to the ability to regenerate appendages. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent mitogen in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily-informed approach for identifying conserved genetic components of regeneration.

developmental biology↗