Biology subjects

Wills, A. E.

Publications and source records attributed to Wills, A. E..

7 recordsLinked to original sources

bioRxiv2025

Spinal cord regeneration deploys cell-type specific developmental and non-developmental strategies to restore neuron diversity

A major goal of spinal cord injury research is to develop a path to endogenous regeneration. This approach has been heavily informed by animal models of natural regeneration. An unresolved question is whether these models rebuild the spinal cord by exclusively accessing developmental mechanisms of neuron differentiation. To address this question, we contrasted single-cell gene expression during regeneration with stage-matched controls in the conditionally regenerative frog Xenopus tropicalis. We generated an expanded atlas of neuronal diversity, annotating several neurons in Xenopus for the first time. From this atlas, we found that the neuron composition of the developing and regenerating spinal cord differ. So do the strategies employed, which favor waves of cell-type specific neuron morphogenesis, proliferation, and proliferative neurogenesis during regeneration. Low levels of early neurogenesis are then compensated by movement of post-mitotic neurons. Our work highlights the use of distinct developmental versus regenerative paths to heal post-injury.

developmental biology↗

bioRxiv2025

An IMPDH2 variant associated with neurodevelopmental disorder disrupts purine biosynthesis and somitogenesis

IMP dehydrogenase (IMPDH) controls a key regulatory node in purine biosynthesis. Gain-of-function mutations in human IMPDH2 are associated with neurodevelopmental disorders and neuromuscular symptoms including dystonia, but the developmental mechanisms underlying these defects are unknown. We previously showed that these mutants are insensitive to GTP inhibition and hypothesized that their hyperactivity would affect nucleotide metabolism in vivo. Here, we characterize the metabolic and developmental consequences of the neurodevelopmental disorder-associated IMPDH2 mutant, S160del, in Xenopus tropicalis. We show that expressing S160del but not WT human IMPDH2 disrupts purine pools and somitogenesis in the developing tadpole. We also show that S160del disrupts in vivo IMPDH filament assembly, a well-described IMPDH regulatory mechanism. Cryo-EM structures show that S160del disrupts filament assembly by destabilizing the dimerization of regulatory Bateman domains. Dimerization of Bateman domains and subsequent filament formation can be restored with a high affinity ligand, but this does not restore sensitivity to GTP inhibition, suggesting S160del also disrupts allostery of IMPDH2 filaments. This work demonstrates that the structural effects of patient IMPDH2 variants can cause disruptions both to nucleotide levels and to the normal development of sensorimotor structures, helping us better understand the physiological basis of disease in these patients. SIGNIFICANCE STATEMENTIMPDH2 is a critical enzyme for de novo purine biosynthesis, regulating the balance between adenine and guanine nucleotides. Under purine stress, it forms filaments that resist feedback inhibition by GTP. Patients with gain-of-function variants of this enzyme suffer from early-onset neuromotor symptoms including dystonia. Here, we express one gain-of-function variant of IMPDH2, S160del, in Xenopus tropicalis. S160del is particularly powerful for structural and developmental studies, as it impedes filament formation and also is insensitive to feedback inhibition by GTP. Here, we show S160del can perturb vertebrate development, metabolism, and filament formation in a dominant fashion. Insights from this work will open the door to a new suite of studies defining the function of purine metabolism in development and disease.

biochemistry↗

bioRxiv2024

Shh signaling directs dorsal ventral patterning in the regenerating X. tropicalis spinal cord

Tissue development and regeneration rely on the deployment of embryonic signals to drive progenitor activity and thus generate complex cell diversity and organization. One such signal is Sonic Hedgehog (Shh), which establishes the dorsal-ventral (D/V) axis of the spinal cord during embryogenesis. However, the existence of this D/V axis and its dependence on Shh signaling during regeneration varies by species. Here we investigate the function of Shh signaling in patterning the D/V axis during spinal cord regeneration in Xenopus tropicalis tadpoles. We find that neural progenitor markers Msx1/2, Nkx6.1, and Nkx2.2 are confined to dorsal, intermediate and ventral spatial domains, respectively, in both the uninjured and regenerating spinal cord. These domains are altered by perturbation of Shh signaling. Additionally, we find that these D/V domains are more sensitive to Shh perturbation during regeneration than uninjured tissue. The renewed sensitivity of these neural progenitor cells to Shh signals represents a regeneration specific response and raises questions about how responsiveness to developmental patterning cues is regulated in mature and regenerating tissues.

developmental biology↗

bioRxiv2024

Appendage regeneration requires IMPDH2 and creates a sensitized environment for enzyme filament formation

Regeneration of lost tissue requires biosynthesis of metabolites needed for cell proliferation and growth. Among these are the critical purine nucleotides ATP and GTP. The abundance and balance of these purines is regulated by inosine monophosphate dehydrogenase 2 (IMPDH2), which catalyzes the committing step of GTP synthesis. IMPDH2 assembles into filaments that resist allosteric inhibition under conditions of high GTP demand. Here we asked whether IMPDH2 is required in the highly proliferative context of regeneration, and whether its assembly into filaments takes place in regenerating tissue. We find that inhibition of IMPDH2 leads to impaired tail regeneration and reduced cell proliferation in the tadpole Xenopus tropicalis. We find that both endogenous and fluorescent fusions of IMPDH2 robustly assemble into filaments throughout the tadpole tail, and that the regenerating tail creates a sensitized condition for filament formation. These findings clarify the role of purine biosynthesis in regeneration and reveal that IMPDH2 enzyme filament formation is a biologically relevant mechanism of regulation in vertebrate regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/605679v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1006d64org.highwire.dtl.DTLVardef@10ddf85org.highwire.dtl.DTLVardef@1cdc99eorg.highwire.dtl.DTLVardef@16fc4cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2021

Hif1α is required for Wnt regulated gene expression during Xenopus tropicalis tail regeneration

Regeneration of complex tissues is initiated by an injury-induced stress response, eventually leading to activation of developmental signaling pathways such as Wnt signaling. How early injury cues are interpreted and coupled to activation of these developmental signals and their targets is not well understood. Here, we show that Hif1, a stress induced transcription factor, is required for tail regeneration in Xenopus tropicalis. We find that Hif1 is required for regeneration of differentiated axial tissues, including axons and muscle. Using RNA-sequencing, we find that Hif1 and Wnt converge on a broad set of genes required for posterior specification and differentiation, including the posterior hox genes. We further show that Hif1 is required for transcription via a Wnt-responsive element, a function that is conserved in both regeneration and early neural patterning. Our findings indicate a regulatory role for Hif1 in Wnt mediated gene expression across multiple tissue contexts.

developmental biology↗

bioRxiv2020

A temporally resolved transcriptome for developing Keller explants of the Xenopus laevis dorsal marginal zone

Explanted tissues from vertebrate embryos reliably develop in culture and have provided essential paradigms for understanding embryogenesis, from early embryological investigations of induction, to the extensive study of Xenopus animal caps, to the current studies of mammalian gastruloids. Cultured explants of the Xenopus dorsal marginal zone ("Keller" explants) serve as a central paradigm for studies of convergent extension cell movements, yet we know little about the global patterns of gene expression in these explants. In an effort to more thoroughly develop this important model system, we provide here a time-resolved bulk transcriptome for developing Keller explants.

developmental biology↗

bioRxiv2018

Extreme nuclear branching in healthy epidermal cells of the Xenopus tail fin

Changes in nuclear morphology contribute to regulation of complex cell properties, including differentiation and tissue elasticity. Perturbations of nuclear morphology are associated with pathologies that include, progeria, cancer, and muscular dystrophy. The mechanisms governing nuclear shape changes in healthy cells remain poorly understood, partially because there are few healthy models of nuclear shape variation. Here, we introduce nuclear branching in epidermal fin cells of Xenopus tropicalis as a model for extreme variation of nuclear morphology in a diverse population of healthy cells. We find that nuclear branching arises and elaborates during embryonic development. They contain broadly distributed marks of transcriptionally active chromatin and heterochromatin and have active cell cycles. We find that nuclear branches are disrupted by loss of filamentous actin and depend on epidermal expression of the nuclear lamina protein Lamin B1. Inhibition of nuclear branching disrupts fin morphology, suggesting that nuclear branching may be involved in fin development. This study introduces the nuclei of the fin as a powerful new model for extreme nuclear morphology in healthy cells to complement studies of nuclear shape variation in pathological contexts.\n\nList of abbreviations and symbolsLINC-Linker of Nucleoskeleton and Cytoskeleton, HGPS - Hutchinson-Gilford Progeria Syndrome, TEM - Transmission electron microscopy, PH3 - Phosphorylated Histone 3, Lat B - Latrunculin B, WT-Wild type, Cyto D-Cytochalasin D, Lmnb1 CRISPR - lmnb1 mutants generated by CRISPR/Cas9, E-Lmnb1 CRISPR - epidermal specific lmnb1 mutants generated by CRISPR/Cas9, Scrmbl-tadpoles injected with a scrambled version of the lmnb1 targeted sgRNA, Lmnb1-rod - dominant-negative Lamin B1 containing only the rod domain\n\nSummary StatementNuclei are highly branched throughout the heterogeneous population of healthy epidermal cells that comprise the Xenopus tail fin periphery, and disruption of nuclear branching mechanisms results in improper fin morphology.

cell biology↗