Biology subjects

Murawala, P.

Publications and source records attributed to Murawala, P..

9 recordsLinked to original sources

bioRxiv2026

A uniform tissue-clearing framework and mesoSPIM-ultra enable cm-scale single-neuron tracing

Tissue-clearing and light-sheet microscopy have transformed volumetric imaging of intact organs, yet limited mechanistic understanding of dehydration-based clearing continues to constrain rational protocol design and broader applicability. Here, we define the cardinal chemical and physical principles underlying dehydration-based tissue-clearing and establish a new pipeline for large-volume imaging. To maximize imaging performance, we developed the "mesoSPIM-ultra", an upgraded mesoSPIM platform with a temperature-controlled sample chamber, a large field-of-view (FoV) camera and specialized optics to achieve long-working-distance, high-resolution imaging of cleared samples. We applied this approach to investigate the projectome of Chx10+ neurons, a cell population with complex axonal morphologies along the entire mouse spinal-cord and brain, and implicated in ipsilateral orienting behaviors. By combining behavioral analysis with post-hoc single-neuron reconstructions, we revealed previously inaccessible branching architectures and long-range projections extending from the brainstem to the spinal cord. Together, our work establishes a mechanistic foundation for tissue-clearing and scalable imaging.

neuroscience↗

bioRxiv2026

Adult regenerative defects arise from discordant scaling of signal dependent growth and patterning

Orders of magnitude distinguish an organs size in adulthood from its size when patterning was established during embryonic development. The prospect of engineering adult organ regeneration raises the fundamental question of whether regenerative stem cell patterning should be elicited at embryonic or adult scale. Axolotls regenerate their limbs at all stages of post-embryonic life, encompassing an order of magnitude increase in animal size, but the mechanisms allowing this robust capacity remain unclear. Limb regeneration occurs by the formation of an embryonic-like progenitor zone, the blastema, whose dimensions increase with animal size, suggesting that at least some aspects of limb regeneration must scale. Here, by combining spatial transcriptomics, biophysical modelling, quantitative imaging, and functional perturbations, we found distinct scaling signatures among key signaling pathways, arguing against a body-size-dependent hormonal scaling mechanism. SHH signaling showed partial scaling that saturated in largest adult sizes, with a correspondingly early termination of blastema growth while Wnt9a-modulated digit patterning scaled at all animal sizes. As a result of this differential scaling, digit patterning wavelength was mismatched with respect to domain growth in the largest blastemas, preventing the addition of the last digit. In agreement with this model, regenerative failure can be rescued by timed supplementation of SHH. Our results show that coordinated scaling of morphogen signaling is a key requirement for adult organ regeneration.

systems biology↗

bioRxiv2025

The essential role of connective-tissue cells during axolotl limb regeneration

Axolotls (Ambystoma mexicanum) are known for their remarkable limb-regeneration abilities, which involve the formation of the blastema, a specialized structure consisting of progenitor cells contributed by all major tissues of the limb. Lateral plate mesoderm (LPM)-derived connective tissue (CT) cells dedifferentiate and play a critical role in blastema formation and subsequent limb regeneration. However, the complexity of the blastemas cellular composition and the extent of CT participation and necessity have not been rigorously explored. To address this gap, we conducted spatial transcriptomics using a select array of probes, revealing that CT cells constitute up to 75% of the blastema cells at their peak. Genetic ablation of CT cells significantly delays or truncates limb regeneration, underscoring their necessity during this process. Finally, we analyzed the molecular profile of CT cells throughout the stages of blastema formation and made it accessible through an interactive web platform. Our work reaffirms the central role of CT cells in axolotl limb regeneration and lays the foundation for identifying molecular mechanisms that govern blastema formation during the initial phases of limb regeneration.

developmental biology↗

bioRxiv2025

Zebrafish genetic model of neuromuscular degeneration associated with Atrogin-1 expression

The degenerative loss of muscle associated with aging leading to muscular atrophy is called sarcopenia. Currently, practicing regular physical exercise is the only efficient way to delay sarcopenia onset. Identification of therapeutic targets to alleviate the symptoms of aging requires in vivo model organisms of accelerated muscle degeneration and atrophy. The zebrafish undergoes aging, with hallmarks including mitochondrial dysfunction, telomere shortening, and accumulation of senescent cells. However, zebrafish age slowly, and no specific zebrafish models of accelerated muscle atrophy associated with molecular events of aging are currently available. We have developed a new genetic tool to efficiently accelerate muscle-fiber degeneration and muscle-tissue atrophy in zebrafish larvae and adults. We used a gain-of-function strategy with a molecule that has been shown to be necessary and sufficient to induce muscle atrophy and a sarcopenia phenotype in mammals: Atrogin-1 (also named Fbxo32). We report the generation, validation, and characterization of a zebrafish genetic model of accelerated neuromuscular atrophy, the atrofish. We demonstrated that Atrogin-1 expression specifically in skeletal muscle tissue induces a muscle atrophic phenotype associated with locomotion dysfunction in both larvae and adult fish. We identified degradation of the myosin light chain as an event occurring prior to muscle-fiber degeneration. Biological processes associated with muscle aging such as proteolysis, inflammation, stress response, extracellular matrix (ECM) remodeling, and apoptosis are upregulated in the atrofish. Surprisingly, we observed a strong correlation between muscle-fiber degeneration and reduced numbers of neuromuscular junctions in the peripheral nervous system, as well as neuronal cell bodies in the spinal cord, suggesting that muscle atrophy could underly a neurodegenerative phenotype in the central nervous system. Finally, while atrofish larvae can recover locomotive functions, adult atrofish have impaired regenerative capacities, as is observed in mammals during muscle aging. In the future, the atrofish could serve as a platform for testing molecules aimed at treating or alleviating the symptoms of muscle aging, thereby opening new therapeutic avenues in the fight against sarcopenia.

developmental biology↗

bioRxiv2024

Somite-independent regeneration of the axolotl primary body axis

Primary body-axis development is a highly conserved process that proceeds through somitogenesis and subsequent subdivision into dermatome, myotome, and sclerotome. Defects in somitic-clock genes such as Hes7 lead to vertebral-segmentation defects in mice and fish. Here we show that in the axolotl, although Hes7 is necessary for proper embryonic vertebral segmentation, it is-- surprisingly--dispensable during tail regeneration. We investigated the mechanism of vertebral segmentation during regeneration which initially occurs through extension of a cartilage rod ventral to the spinal cord. We find that the regenerating cartilage rod undergoes a periodic wrinkling that provides a template for vertebral segmentation. Via direct mechanical measurements and biophysical perturbations, we show that a model of compression-induced buckling instability can predict vertebral segmentation. The cartilage rod and other somitic derivatives (muscle, cartilage, tendon, fibroblasts) arise from tendon-like, Lfng+ multi-potent mesenchymal progenitors, which display a gene regulatory state distinct from somitic progenitors. In summary, we uncover a mechanism of vertebral segmentation during axolotl tail regeneration that is distinct from the somite-based developmental mechanism.

developmental biology↗

bioRxiv2023

Collagen IV of basement membranes: I. Origin and diversification of COL4 genes enabling animal evolution

Collagen IV is a major component of basement membranes, a specialized form of extracellular matrix that enabled the assembly of multicellular epithelial tissues. In mammals, collagen IV assembles from a family of six -chains (1 to 6), forming three supramolecular scaffolds: Col-IV121, Col-IV345 and Col-IV121-556. The -chains are encoded by six genes (COL4A1-6) that occur in pairs in a head-to-head arrangement. In Alport syndrome, variants in COL4A3, 4 or 5 genes, encoding Col-IV345 scaffold in glomerular basement membrane (GBM), the kidney ultrafilter, cause progressive renal failure in millions of people worldwide. How variants cause dysfunction remains obscure. Here, we gained insights into Col-IV345 function by determining its evolutionary lineage, as revealed from phylogenetic analyses and tissue expression of COL4 gene-pairs. We found that the COL4A[<]1|2[>] gene-pair emerged in basal Ctenophores and Cnidaria phyla and is highly conserved across metazoans. The COL4A[<]1|2[>] duplicated and arose as the progenitor to the COL4A[<]3|4[>] gene-pair in cyclostomes, coinciding with emergence of kidney GBM, and expressed and conserved in jawed-vertebrates, except for amphibians, and a second duplication as the progenitor to the COL4A[<]5|6[>] gene-pair and conserved in jawed-vertebrates. These findings revealed that Col-IV121 is the progenitor scaffold, expressed ubiquitously in metazoan basement membranes, and which evolved into vertebrate Col-IV345 and expressed in GBM. The Col-IV345 scaffold, in comparison, has an increased number of cysteine residues, varying in number with osmolarity of the environment. Cysteines mediate disulfide crosslinks between protomers, an adaptation enabling a compact GBM that withstands the high hydrostatic pressure associated with glomerular ultrafiltration.

genomics↗

bioRxiv2022

Skeletal muscle lineage is dispensable for appendage regeneration in axolotl

Regeneration of a complex appendage structure such as limb requires hierarchical coordination of multiple types of tissues. Muscles, as one of the major cell masses in limbs, have been reported recently to be critical to guide other tissue regeneration in planaria, but its function and relationship to other cells in vertebrate complex regeneration have been unclear. Here, we use Pax7 mutant axolotls, in which the limb muscle is developmentally lost, to investigate limb regeneration in the absence of skeletal muscle. We find that the pattern of regenerated limbs is normal in Pax7 mutants compared to the controls. Lack of muscles do not affect the proliferation of fibroblasts, another major population in limbs. Furthermore, using single cell RNA-sequencing, we demonstrate that the cell type composition in completely regenerated limbs in Pax7 mutants is similar to that in the controls, except the lack of cell types in muscle lineage. Our study reveals skeletal muscle is not required for the guidance of complex tissue regeneration in axolotls, and provides new views of the tissue hierarchy in vertebrate appendage regeneration.

cell biology↗

bioRxiv2019

The Prrx1 limb enhancer marks an adult population of injury-responsive, multipotent dermal fibroblasts

The heterogeneity of adult tissues has been posited to contribute toward the loss of regenerative potential in mammals. Here we characterize an adult population of dermal fibroblasts that maintain expression of a Prrx1 enhancer which originally marked mesenchymal limb progenitors. Prrx1 enhancer-positive cells (Prrx1enh+) make up a small subset of adult dermal cells (~0.2%) and reside mainly within specific dermal perivascular and hair follicle niches. Upon injury, however, Prrx1enh+ cells readily migrate into the wound bed and amplify on average 16-fold beyond their uninjured numbers. Additionally, Prrx1enh+ cells emigrate out of their dermal niches following wounding and contribute to subcutaneous tissue. Prrx1enh+ cells are uniquely injury-responsive and do not contribute to tissue homeostasis or enriched by neonatal-like Wnt signaling. Prrx1enh+ cells represent a potent regenerative cell population that, despite being a meager minority in adult skin, demonstrate the potential to tip the balance of mammalian wound healing toward scar-free healing.

developmental biology↗

bioRxiv2018

Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure

Turbidity and opaqueness are inherent properties of tissues which limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here we describe 2Eci (2nd generation Ethyl cinnamate based clearing method) which can be used to clear a wide range of tissues, including cerebral organoids, Drosophila melanogaster, zebrafish, axolotl, and Xenopus laevis in as little as 1-5 days while preserving a broad range of fluorescence proteins including GFP, mCherry, Brainbow, and alexa-fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method will open up clearing to a much broader group of researchers, due to its broad applicability, ease of use, and non-toxic nature of Ethyl cinnamate.\n\nSummary statementThe non-toxic, broadly applicable, and simplified protocol of 2Eci tissue clearing makes it possible for non-specialist labs to use clearing approaches on conventional inverted microscopes.

developmental biology↗