After amputation, axolotl salamanders can regenerate their limbs, but the degree to which limb regeneration recapitulates limb development remains unclear. One limitation in answering this question is our lack of knowledge about salamander limb development. Here, we address this question by studying expression patterns of genes important for limb patterning during axolotl salamander limb development and regeneration. We focus on the Wnt signaling pathway because it regulates multiple functions during tetrapod limb development, including limb bud initiation, outgrowth, patterning, and skeletal differentiation. We use fluorescence in situ hybridization to show the expression of Wnt ligands, Wnt receptors, and limb patterning genes in developing and regenerating limbs. Inhibition of Wnt ligand secretion permanently blocks limb bud outgrowth when treated early in limb development. Inhibiting Wnt signaling during limb outgrowth decreases the expression of critical signaling genes, including Fgf10, Fgf8, and Shh, leading to the reduced outgrowth of the limb. Patterns of gene expression are similar between developing and regenerating limbs. Inhibition of Wnt signaling during regeneration impacted patterning gene expression similarly. Overall, our findings suggest that limb development and regeneration utilize Wnt signaling similarly. It also provides new insights into the interaction of Wnt signaling with other signaling pathways during salamander limb development and regeneration.
Amputation of a salamander tail leadsto functional spinal cord regeneration through activation of endogenous stem cells. Identifying the signaling pathways that control cell proliferation in these neural stem cells will help elucidate the mechanisms underlying the salamander's regenerative ability. Here, we show that neuregulin 1 (Nrg1)/ErbB2 signaling is an important pathway in the regulation of neural stem cell proliferation in the spinal cord of the axolotl salamander (Ambystoma mexicanum ). Simultaneous localization of nrg1 mRNA and Nrg1 protein was performed by utilizing a hybridization chain reaction fluorescence in situ hybridization (FISH) methodology in tissue sections. Multiplexed FISH also permitted the phenotyping of multiple cell types on a single fixed section allowing the characterization of mRNA expression, protein expression, and tissue architecture. Pharmacological inhibition of ErbB2 showed that intact Nrg1/ErbB2 signaling is critical for adult homeostatic regeneration as well as for injury-induced spinal cord regeneration. Overall, our results highlight the importance of the NRG1/ErbB2 signaling pathway in neural stem cell proliferation in the axolotl.
The study of neurotransmitter signaling helps to understand fundamental neuronal circuits and develop strategies against neurodegenerative diseases. Acetylcholine (ACh) plays a pivotal role in modulating neuron functions. Monitoring its physiological level in real‐time and probing its biological distribution is of crucial importance. However, the current techniques are subject to temporal and spatial limitations which impede their further in vivo applications. Herein we report the development of a ACh nanosensor by using DNA as a scaffold, acetylcholinesterase as a recognition component, pH‐sensitive fluorophores as signal generator, and α‐bungarotoxin as a targeting ligand. The nanosensor was delivered to the submandibular ganglion in living mice by microinjection for in vivo imaging of endogenous ACh release. The ACh nanosensor selectively binds to acetylcholine receptors on the post‐synaptic neurons, and display a sensitive response to physiological ACh range from 0.228 μM to 358 μM in ex vivo calibration. The ACh nanosensors also respond to endogenous ACh release triggered by electrical stimulation in a reversible and dose‐dependent manner. We also found that the treatment of cholinergic inhibitor, Vesamicol, significant suppresses the response of the ACh nanosensor. This work provides a tool for in vivo imaging of ACh dynamics with unprecedented capabilities to enhance the spatial and temporal resolution of detection. We envision such a sensor platform could be applied to imaging other neurotransmitters in mammalian system as well as extended to other organ systems in the peripheral nervous system Support or Funding Information NIH common fund_SPARC_Project number: 1OT2OD024909‐01 DNA‐based acetylcholine (ACh) nanosensors detect the ACh in the submandibular ganglion (SMG) of living mice. (A) ACh sensors were microinjected into the SMG of a living mouse. With the targeting ability of bungarotoxin, the ACh nanosensor can immobilize in the synaptic cleft of the mouse. (B) The mechanism of ACh nanosensors. (D) ΔF/F from pHAb channel in response to electrical stimulation (indicated by red arrow).
The patterning of limbs in development utilizes spatial and temporal signaling dynamics which guide the formation of a complete limb from undefined tissue. Although they may play different roles, a group of key factors guide the development of most species. This process is recapitulated in the limb regeneration of the Mexican axolotl, and the possibility of regeneration in humans will require a precise understanding of how these factors guide this patterning. Previously, observation of limb patterning was restricted by in situ hybridization imaging techniques which had limited resolution and could not reliably compare the collection of essential genes on a single tissue. Now, we have applied V3 Fluorescent in situ Hybridization by Hybridization Chain Reaction (HCR‐FISH) to the model to generate clear, bright, multiplexed images of the expression of these factors. These multiplexed images are the first of their kind in the field, and allow for the overlapping of key factors in both axolotl salamander and murine models to draw essential conclusions regarding each gene’s role in limb patterning within and between species. Development of an efficient probe‐design pipeline and oligonucleotide amplification parameters has improved signal strength, selectivity, and cost effectiveness. Furthermore, we have designed novel V3 HCR‐FISH probes targeting the introns of actively transcribed sequences allows for the identification of actively transcribed RNA via nuclear‐specific signal. Our techniques have not only been applied to confirm predictions derived from single cell RNA‐seq data regarding the roles of Sonic Hedghog (Shh), Fibroblast Growth Factor 8 (Fgf8), and Gremlin 1 (Grem1), in axolotl limb patterning, but also showcased significant differences between the axolotl and murine limb models. These results indicate that application of V3 HCR‐FISH allows for the robust visualization of multi‐gene transcriptional dynamics with a clarity previously impossible in both regenerative and developmental models. Support or Funding Information This project was supported by NSF grants 1558017 and 1656429
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