Biology subjects

Monaghan, J. R.

Publications and source records attributed to Monaghan, J. R..

15 recordsLinked to original sources

bioRxiv2026

Anatomical mapping of dopamine D1 and muscarinic M4 receptor

Dopamine D1 receptors (D1R) and muscarinic M4 receptors (M4R) regulate overlapping functions in the cortex and the striatum, but the cellular relationship between D1R- and M4R-associated populations across brain regions remains incompletely defined. Using D1R-tdTomato/M4R-EGFP double-reporter mice, we quantified tdTomato-only, EGFP-only, and coexpressing cellular populations across the dorsal striatum, nucleus accumbens, and medial prefrontal cortex in 18 animals. Reporter composition differed strongly by anatomical region. Coexpression was prominent throughout the dorsal striatum and greatest in the dorsomedial striatum, whereas the nucleus accumbens shifted toward greater tdTomato-only and reduced EGFP-only representation, particularly in the core. In contrast, the medial prefrontal cortex was dominated by EGFP-only cells. These regional patterns were broadly retained across sex and age. HCR-FISH further supported spatial correspondence between endogenous Drd1 and Chrm4 transcript signal and the respective fluorescent reporters. Together, these findings demonstrate region-dependent organization of D1R- and M4R-associated cellular populations across the cortex and striatum and define distinct anatomical contexts in which M4R-directed signaling may influence D1R-associated neurons.

neuroscience↗

bioRxiv2026

Model recapitulates regenerative limb blastema formation through local softening of the wounded epithelium

Studies of appendage regeneration in vertebrates have shown that the fundamental building block of any regenerative tissue is a blastema. The blastema is a cone-shaped accumulation that forms at the site of amputation post wound-healing and is the result of a highly coordinated process involving a cluster of cells capable of growth, migration, and differentiation. Although several key signaling pathways involved in regeneration have been identified, which cellular processes they control and how these processes are coordinated across space and time are not yet fully understood. This study introduces a computational tool to examine how the outgrowth results from the interaction of two tissue layers: the bulk (mesenchyme) and the overlying epithelium. We developed a novel hybrid agent-based modeling framework and an accompanying parameter inference pipeline to uncover the cellular properties in the epithelium and the mesenchyme driving the formation of a normal regenerative blastema with a morphology similar to that observed in experiments. Using our model, we report two conditions for blastema formation: retained local softening of the epithelial layer at the site of injury, which was confirmed experimentally with atomic force microscopy (AFM) measurements, and the involvement of the Wnt signaling pathway in the directed migration of mesenchyme cells towards the distal tip. Taken together, this combined experimental-theoretical approach provides a framework for understanding how the Wnt signaling pathway influences the formation of the early blastema at multiple levels of organization and how key cellular behaviors contribute to its formation. Author SummaryA small number of tetrapods have retained the ancestral ability to regenerate tissues and even limbs. Indifferent of species or tissue, the decisive initial stage of limb regeneration is the formation of a specialized structure called the blastema, a heterogeneous mass of mesenchymal cells, in a relatively short timescale of 2-14 days post injury or amputation. To study the mechanical and cellular conditions for limb blastema formation in the axolotl model organism, we developed a novel hybrid agent-based modeling framework and accompanying kinetic parameter inference pipeline. By recapitulating blastema morphometrics of healthy and stalled regenerative states, our model finds two conditions for blastema formation: retained local softening of the epithelial layer at the injury site post wound-healing, which we confirmed with atomic force microscopy measurements, and that the Wnt signaling pathway plays a role in the migration of mesenchyme cells to the distal tip in order to produce the blastema.

developmental biology↗

bioRxiv2025

TRPV4 Inhibition Reduces Cartilage Growth During Axolotl Limb Regeneration

BackgroundAxolotls can regenerate entire limbs, recapitulating mammalian developmental processes. During development, mechanosensitive ion channels such as TRPV4, PIEZO1, and PIEZO2 regulate tissue morphogenesis by transducing mechanical signals. Their roles in regeneration, however, have yet to be thoroughly explored. To investigate this, we assessed the expression of these channels during limb regeneration using single-cell RNA sequencing dataset, hybridization chain reaction fluorescence in-situ hybridization, and immunofluorescence. Additionally, functional relevance was tested by pharmacological inhibition of TRPV4 and PIEZO1/2 mechanosensitive ion channels during limb regeneration. ResultsWhile PIEZO1 expression was undetected, we observed TRPV4 and PIEZO2 expression in uninjured cartilage and at both the mid-bud and palette blastemal stages of limb regeneration. TRPV4 and PIEZO2 were highly expressed in chondrocytes, with PIEZO2 enriched in fibroblasts. Inhibition with GSK205, a TRPV4 antagonist, significantly reduced calcium influx and humeral length without inhibiting cartilage differentiation. Treatment with gadolinium chloride, a broad spectrum mechanosensitive ion channel inhibitor, had no significant morphological impact. ConclusionsTRPV4 and PIEZO2 are dynamically regulated during axolotl limb regeneration. Selective TRPV4 inhibition altered final limb morphology, but chondrogenesis was unaffected. This suggests a role for these genes in shaping tissue architecture during limb regeneration. These findings underscore the importance of ion channel-mediated mechanotransduction in regenerative patterning. Key findingsO_LITRPV4 and PIEZO2 show distinct, stage- and cell type-specific expression patterns during axolotl limb regeneration C_LIO_LISelective pharmacological inhibition of TRPV4 significantly reduces calcium signaling in chondrocytes and leads to shorter limb and humeral lengths in vivo C_LI

bioengineering↗

bioRxiv2025

A Characterization of Axolotl Digit Regeneration: Conserved Mechanisms, Divergent Patterning, and a Critical Role for Hedgehog Signaling

Axolotl digits offer an experimentally versatile model for studying complex tissue regeneration. Here, we provide a comprehensive morphological and molecular characterization of digit regeneration, revealing both conserved features and notable divergences from classical limb regeneration. Digit blastemas progress through similar morphological stages, are nerve-dependent, contain key regenerative cell populations, and express many canonical morphogens and mitogens. However, they exhibit minimal expression of the A-P patterning genes Shh, Fgf8, and Grem1; suggesting distal outgrowth and patterning occur independently of these signals. Joint regenerative fidelity varies significantly across digits and cannot be explained by differences in nerve supply, cell proliferation, or differential expression of any patterning genes assessed in this study. Furthermore, functional experiments reveal Hedgehog signaling is essential for interphalangeal joint regeneration, but activation alone is insufficient to improve fidelity in less robust digits. This system combines experimental accessibility with intrinsic variation in regenerative outcomes, making it an ideal platform to identify critical determinants of successful tissue regeneration and refine models of appendage patterning.

developmental biology↗

bioRxiv2025

Growth and patterning in vertebrate limb developmentA timescale perspective on skeletal specification

1The vertebrate limb provides a powerful system to study how growth and molecular signaling interact to shape complex skeletal patterns. However, how these processes are coordinated across space and time is not fully understood. This study introduces a computational tool to examine how growth interacts with positional cues and self-organizing patterning mechanisms to shape skeletal structures in both mice and axolotl limbs. We developed the Growth-Processing-Propagation (GPP) framework, a reaction-diffusion system within a growing domain, in which the relative contribution of growth, processing (reaction) and propagation (diffusion) is modulated through two non-dimensional parameters, informed by experimental morphogen maps. This formulation normalizes the reaction-diffusion equation relative to growth, enabling investigation of how different spatiotemporal regimes of growth, processing and propagation interact to produce whole limb patterning. The GPP framework captures the progressive formation of limb segments and digit patterns by varying the relative contributions of reaction, diffusion, and growth to the pattern. Our models indicate that in the proximal region (humerus, radius/ulna) the contributions of growth, reaction and diffusion are equally important to patterning, but in the distal elements (hands) the reaction and diffusion contributions are much greater than the contribution of growth to formation of the digits. A single framework predicts whole-limb skeletal patterns in both mice and axolotls, despite their morphological differences, highlighting its potential to explore conserved and divergent features of limb development from an evolutionary perspective through a unified mechanism across species. O_TEXTBOXSignificanceUnderstanding limb skeletal patterning is a central question in developmental biology. Numerical models provide a means to explore this process. We introduce the Growth-Processing-Propagation (GPP) framework, which integrates tissue expansion (growth), gene regulation and cellular specification (processing), and signaling spread (propagation). By normalizing patterning dynamics relative to growth, the framework reveals how the relative contributions of growth, processing, and propagation vary across the limb, influencing the timing and positioning of skeletal elements. The model successfully predicts whole-limb skeletal patterning in both mice and axolotls, supporting the idea of a shared underlying mechanism. This cross-species framework offers a versatile computational tool for studying fundamental processes in morphogenesis, with potential applications in developmental biology, and evolutionary studies. C_TEXTBOX

developmental biology↗

bioRxiv2024

3D spatial distribution of Sost mRNA and Sclerostin expression in response to in vivo mechanical loading

Bones adapt to external mechanical loads through a process known as mechanoadaptation. Osteocytes are the bone cells that sense the mechanical environment and initiate a biological response. Investigating the changes in osteocyte molecular expression following mechanical loading has been instrumental in characterizing the regulatory pathways involved in bone adaptation. However, current methods for examining osteocyte molecular expression do not preserve the three-dimensional structure of the bone, which plays a critical role in the mechanical stimuli sensed by the osteocytes and their spatially controlled biological responses. In this study, we used WISH-BONE to investigate the spatial distribution of Sost-mRNA transcripts and its encoded protein, sclerostin, in 3D mouse tibia midshaft following in vivo tibia loading. Our findings showed a decrease in the percentage of Sost-positive osteocytes predominantly at 25% and 37% of the bone length, and in the posterior-lateral side of the tibia after loading. Sclerostin-positive osteocytes in the loaded legs were found to be similar to the contralateral legs after 2 weeks of loading. This work is the first to provide a 3D analysis of Sost and sclerostin distribution in loaded versus contralateral mouse tibia midshafts. It also highlights the importance of the bone region analyzed and the method utilized when interpreting mechanoadaptation results. WISH-BONE represents a powerful tool for further characterization of mechanosensitive genes regulation in bone and holds potential for advancing the development of new treatments targeting mechanosensitivity-related bone disorders.

bioengineering↗

bioRxiv2024

Retinoic acid breakdown is required for proximodistal positional identity during amphibian limb regeneration

Regenerating limbs retain their proximodistal (PD) positional identity following amputation. This positional identity is genetically encoded by PD patterning genes that instruct blastema cells to regenerate the appropriate PD limb segment. Retinoic acid (RA) is known to specify proximal limb identity, but how RA signaling levels are established in the blastema is unknown. Here, we show that RA breakdown via CYP26B1 is essential for determining RA signaling levels within blastemas. CYP26B1 inhibition molecularly reprograms distal blastemas into a more proximal identity, phenocopying the effects of administering excess RA. We identify Shox as an RA-responsive gene that is differentially expressed between proximally and distally amputated limbs. Ablation of Shox results in shortened limbs with proximal skeletal elements that fail to initiate endochondral ossification. These results suggest that PD positional identity is determined by RA degradation and RA-responsive genes that regulate PD skeletal element formation during limb regeneration.

developmental biology↗

bioRxiv2024

WISH-BONE: Whole-mount In Situ Histology, to label osteocyte mRNA and protein in 3D adult mouse bones

Bone is a three dimensional and highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or only provide two-dimensional spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH-BONE, to label osteocyte mRNA and protein in whole-mount mouse bone. For mRNA labeling, we used Hybridization Chain Reaction - Fluorescence In Situ Hybridization to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy-based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone. However, the relatively dim signal observed from nanobodies staining limited detection. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole-mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH-BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone-related fields of research.

bioengineering↗

bioRxiv2022

Regenerating axolotl retinas regrow diverse cell types with modulation by Notch signaling and reconnect to the brain

Some species successfully repair retinal injuries in contrast to non-regenerative mammalian retina. We show here that the Mexican axolotl salamander regrows its excised retina even in adulthood. During early regeneration, cell proliferation occurred in the retinal pigment epithelium (RPE). All dividing cells expressed Vimentin, and some also expressed Muller glia and neural progenitor cell marker Glast (Slc1a3), suggesting that regeneration is driven by RPE-derived retinal progenitor cells. Bulk RNA sequencing showed that genes associated with the extracellular matrix and angiogenesis were upregulated in early-to-mid retinal regeneration. The fully regenerated retina re-established nerve projections to the brain and contained all the original retinal cell types, including Muller glia. Regeneration of cellular diversity may be modulated by Notch signaling, as inhibiting Notch signaling in early regeneration promoted production of rod photoreceptors. Our study highlights the axolotl salamander as an advantageous model of adult tetrapod retinal regeneration and provides insights into its mechanisms. SummaryWe demonstrate that adult Mexican axolotl salamanders regenerate retinas after a retinectomy. We also show some cellular and molecular mechanisms that drive axolotl retinal regeneration.

neuroscience↗

bioRxiv2021

Local mechanical stimuli shape tissue growth in vertebrate joint morphogenesis

The correct formation of synovial joints is essential for proper motion throughout life. Movement-induced forces are critical to creating correctly shaped joints, but it is unclear how cells sense and respond to these mechanical cues. To determine how mechanical stimuli drive joint morphogenesis, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a TRPV4 agonist to impair chondrocyte mechanosensitivity during joint morphogenesis. We quantified growth and shape in regrown humeri from whole mount light sheet fluorescence images of the regenerated limbs. Results revealed statistically significant differences in morphology and cell proliferation between groups, indicating that mechanical stimuli play a role in the shaping of joints. We simulated local tissue growth in a finite element model with a biological contribution to growth proportional to chondrocyte density, and a mechanical contribution to growth proportional to fluid pressure. Computational predictions agreed with experimental outcomes, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.

developmental biology↗

bioRxiv2021

A constitutively expressed fluorescence ubiquitin cell cycle indicator (FUCCI) in axolotls for studying tissue regeneration

Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI). We demonstrate near-ubiquitous expression of FUCCI expression in developing and adult tissues and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in-situ hybridization, EdU click-chemistry, and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in-situ endpoint assays and in-vivo imaging in developing and regenerating animals. Summary statementWe generated a ubiquitous transgenic fluorescence ubiquitin cell cycle indicator (FUCCI) axolotl line for examination of cell cycle dynamics during tissue regeneration.

developmental biology↗

bioRxiv2020

A DNA-based optical nanosensor for in vivo imaging of acetylcholine in the peripheral nervous system

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological disease. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and -bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 M to 358 M. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose-dependent, reversible, and we observe a reduction of ~76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.

bioengineering↗

bioRxiv2020

Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBACKGROUND & AIMSC_ST_ABSThe enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (EN). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODSHuman myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA-Seq. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA-Seq (snRNA-Seq), totaling over four billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse snRNA-Seq data. Comparative informatics between human and mouse datasets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction (HCR). RESULTSSeveral EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of one or more marker genes. CONCLUSIONSOur combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human GI studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/187211v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1b41277org.highwire.dtl.DTLVardef@12856feorg.highwire.dtl.DTLVardef@1674dadorg.highwire.dtl.DTLVardef@11ca1c8_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

neuroscience↗

bioRxiv2020

3D Visualization of Macromolecule Synthesis

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here, we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

developmental biology↗

bioRxiv2018

Neuregulin-1 exerts molecular control over axolotl lung regeneration through ErbB family receptors

The induction of new lung tissue after disease or trauma has the potential to save lives and transform patient outcomes. Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration, primarily after limb amputation. While it is hypothesized that axolotls regenerate all of their tissues, exploration of lung regeneration has not been performed until now. Proliferation after lung injury was observed to be a global response, suggesting that regeneration utilizes a compensatory mechanism, in contrast to limb regenerations epimorphic response. ErbB signaling is crucial for the proliferative response during lung regeneration, likely through the ErbB2:ErbB4 receptor heterodimer. ErbB4 mRNA was found to be highly upregulated at both one and three weeks post amputation. Neuregulin-1p (NRG1) can induce proliferation in the lung and likely exerts molecular control over lung regeneration. Inhibition of ErbB2 was sufficient to both block regeneration and the proliferative response observed after NRG1 treatment.

developmental biology↗