A longitudinal single-cell and spatial multiomic atlas of pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is an incurable central nervous system malignancy that is a leading cause of pediatric cancer death. While pHGG shares many similarities to adult glioma, it is increasingly recognized as a molecularly distinct, yet highly heterogeneous disease. In this study, we longitudinally profiled a molecularly diverse cohort of 16 pHGG patients before and after standard therapy through single-nucleus RNA and ATAC sequencing, whole-genome sequencing, and CODEX spatial proteomics to capture the evolution of the tumor microenvironment during progression following treatment. We found that the canonical neoplastic cell phenotypes of adult glioblastoma are insufficient to capture the range of tumor cell states in a pediatric cohort and observed differential tumor-myeloid interactions between malignant cell states. We identified key transcriptional regulators of pHGG cell states and did not observe the marked proneural to mesenchymal shift characteristic of adult glioblastoma. We showed that essential neuromodulators and the interferon response are upregulated post-therapy along with an increase in non-neoplastic oligodendrocytes. Through in vitro pharmacological perturbation, we demonstrated novel malignant cell-intrinsic targets. This multiomic atlas of longitudinal pHGG captures the key features of therapy response that support distinction from its adult counterpart and suggests therapeutic strategies which are targeted to pediatric gliomas.

Pioneer Axons Utilize a Dcc Signaling-Mediated Invasion Brake to Precisely Complete Their Pathfinding Odyssey.

Axons navigate through the embryo to construct a functional nervous system. A missing part of the axon navigation puzzle is how a single axon traverses distinct anatomic choice points through its navigation. The dorsal root ganglia (DRG) neurons experience such choice points. First, they navigate to the dorsal root entry zone (DREZ), then halt navigation in the peripheral nervous system to invade the spinal cord, and then reinitiate navigation inside the CNS. Here, we used time-lapse super-resolution imaging in zebrafish DRG pioneer neurons to investigate how embryonic axons control their cytoskeleton to navigate to and invade at the correct anatomic position. We found that invadopodia components form in the growth cone even during filopodia-based navigation, but only stabilize when the axon is at the spinal cord entry location. Further, we show that intermediate levels of DCC and cAMP, as well as Rac1 activation, subsequently engage an axon invasion brake. Our results indicate that actin-based invadopodia components form in the growth cone and disruption of the invasion brake causes axon entry defects and results in failed behavioral responses, thereby demonstrating the importance of regulating distinct actin populations during navigational challenges.SIGNIFICANCE STATEMENT Correct spatiotemporal navigation of neuronal growth cones is dependent on extracellular navigational cues and growth cone dynamics. Here, we link dcc-mediated signaling to actin-based invadopodia and filopodia dynamics during pathfinding and entry into the spinal cord using an in vivo model of dorsal root ganglia (DRG) sensory axons. We reveal a molecularly-controlled brake on invadopodia stabilization until the sensory neuron growth cone is present at the dorsal root entry zone (DREZ), which is ultimately essential for growth cone entry into the spinal cord and behavioral response.

Generating intravital super-resolution movies with conventional microscopy reveals actin dynamics that construct pioneer axons.

Super-resolution microscopy is broadening our in-depth understanding of cellular structure. However, super-resolution approaches are limited, for numerous reasons, from utilization in longer-term intravital imaging. We devised a combinatorial imaging technique that combines deconvolution with stepwise optical saturation microscopy (DeSOS) to circumvent this issue and image cells in their native physiological environment. Other than a traditional confocal or two-photon microscope, this approach requires no additional hardware. Here, we provide an open-access application to obtain DeSOS images from conventional microscope images obtained at low excitation powers. We show that DeSOS can be used in time-lapse imaging to generate super-resolution movies in zebrafish. DeSOS was also validated in live mice. These movies uncover that actin structures dynamically remodel to produce a single pioneer axon in a 'top-down' scaffolding event. Further, we identify an F-actin population - stable base clusters - that orchestrate that scaffolding event. We then identify that activation of Rac1 in pioneer axons destabilizes stable base clusters and disrupts pioneer axon formation. The ease of acquisition and processing with this approach provides a universal technique for biologists to answer questions in living animals.