Probes for INS

Primary cilia are ubiquitous microtubule-based organelles that serve as signaling hubs for numerous developmental pathways, most notably the Hedgehog (Hh) pathway. Defects in the structure or function of primary cilia result in a class of diseases called ciliopathies. It is well known that primary cilia participate in transducing a Hh signal, and as such ciliopathies frequently present with phenotypes indicative of aberrant Hh function. Interestingly, the exact mechanisms of cilia-dependent Hh signaling transduction are unclear as some ciliopathic animal models simultaneously present with gain-of-Hh phenotypes in one organ system and loss-of-Hh phenotypes in another. To better understand how Hh signaling is perturbed across different tissues in ciliopathic conditions, we examined four distinct Hh-dependent signaling centers in the naturally occurring avian ciliopathic mutant talpid2 (ta2). In addition to the well-known and previously reported limb and craniofacial malformations, we observed dorsal-ventral patterning defects in the neural tube, and a shortened gastrointestinal tract. Molecular analyses for elements of the Hh pathway revealed that the loss of cilia impact transduction of an Hh signal in a tissue-specific manner at variable levels of the pathway. These studies will provide increased knowledge into how impaired ciliogenesis differentially regulates Hh signaling across tissues and will provide potential avenues for future targeted therapeutic treatments.

Delayed oligodendrocyte (OL) maturation caused by hypoxia (Hx)-induced neonatal brain injury results in hypomyelination and leads to neurological disabilities. Previously, we characterized Sirt1 as a crucial regulator of OL progenitor cell (OPC) proliferation in response to Hx. We now identify Sirt2 as a critical promoter of OL differentiation during both normal white matter development and in a mouse model of Hx. Importantly, we find that Hx reduces Sirt2 expression in mature OLs and that Sirt2 overexpression in OPCs restores mature OL populations. Reduced numbers of Sirt2+ OLs were also observed in the white matter of preterm human infants. We show that Sirt2 interacts with p27Kip1/FoxO1, p21Cip1/Cdk4, and Cdk5 pathways, and that these interactions are altered by Hx. Furthermore, Hx induces nuclear translocation of Sirt2 in OPCs where it binds several genomic targets. Overall, these results indicate that a balance of Sirt1 and Sirt2 activity is required for developmental oligodendrogenesis, and that these proteins represent potential targets for promoting repair following white matter injury.

Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8-10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.

Neural and oligodendrocyte precursor cells (NPCs and OPCs) in the subventricular zone (SVZ) of the brain contribute to oligodendrogenesis throughout life, in part due to direct regulation by chemokines. The role of the chemokine fractalkine is well established in microglia; however, the effect of fractalkine on SVZ precursor cells is unknown. We show that murine SVZ NPCs and OPCs express the fractalkine receptor (CX3CR1) and bind fractalkine. Exogenous fractalkine directly enhances OPC and oligodendrocyte genesis from SVZ NPCs in vitro. Infusion of fractalkine into the lateral ventricle of adult NPC lineage-tracing mice leads to increased newborn OPC and oligodendrocyte formation in vivo. We also show that OPCs secrete fractalkine and that inhibition of endogenous fractalkine signaling reduces oligodendrocyte formation in vitro. Finally, we show that fractalkine signaling regulates oligodendrogenesis in cerebellar slices ex vivo. In summary, we demonstrate a novel role for fractalkine signaling in regulating oligodendrocyte genesis from postnatal CNS precursor cells.

For precise motor control, distinct subpopulations of corticospinal neurons (CSN) must extend axons to distinct spinal segments, from proximal targets in the brainstem and cervical cord to distal targets in thoracic and lumbar spinal segments. We find that developing CSN subpopulations exhibit striking axon targeting specificity in spinal white matter, which establishes the foundation for durable specificity of adult corticospinal circuitry. Employing developmental retrograde and anterograde labeling, and their distinct neocortical locations, we purified developing CSN subpopulations using fluorescence-activated cell sorting to identify genes differentially expressed between bulbar-cervical and thoracolumbar-projecting CSN subpopulations at critical developmental times. These segmentally distinct CSN subpopulations are molecularly distinct from the earliest stages of axon extension, enabling prospective identification even before eventual axon targeting decisions are evident in the spinal cord. This molecular delineation extends beyond simple spatial separation of these subpopulations in the cortex. Together, these results identify candidate molecular controls over segmentally specific corticospinal axon projection targeting.