Nicolas Renier

iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging

The visualization of molecularly labeled structures within large intact tissues in three dimensions is an area of intense focus. We describe a simple, rapid, and inexpensive method, iDISCO, that permits whole-mount immunolabeling with volume imaging of large cleared samples ranging from perinatal mouse embryos to adult organs, such as brains or kidneys. iDISCO is modeled on classical histology techniques, facilitating translation of section staining assays to intact tissues, as evidenced by compatibility with 28 antibodies to both endogenous antigens and transgenic reporters like GFP. When applied to degenerating neurons, iDISCO revealed unexpected variability in number of apoptotic neurons within individual sensory ganglia despite tight control of total number in all ganglia. It also permitted imaging of single degenerating axons in adult brain and the first visualization of cleaved Caspase-3 in degenerating embryonic sensory axons in vivo, even single axons. iDISCO enables facile volume imaging of immunolabeled structures in complex tissues. PAPERCLIP:

Genetic Dissection of the Function of Hindbrain Axonal Commissures

The Robo3 receptor controls midline crossing by axons. Deleting Robo3 in specific commissural neurons with a conditional knockout reveals their contribution to sensory and motor integration, and models human neurological conditions.

Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism

Dissecting how signaling directs axon growth During development of the nervous system, nerve cells send out projections called axons that must be guided to their proper targets. Netrins are secreted proteins that bind to receptors to either attract or repel the growing axons. Xu et al. present x-ray structures that show that complexes of netrin with two different receptors, neogenin and DCC, have different architectures. How netrin signals remains to be understood in detail, but netrins ability to create different assemblies probably plays a role in the diverse signaling outcomes it mediates. Science, this issue p. 1275 The axon guidance protein netrin binds to two receptors with different architectures, providing a basis for diverse signaling outcomes. Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. We provide evidence that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes.

Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

A neuronal PI(3,4,5)P 3 -dependent program of oligodendrocyte precursor recruitment and myelination

The molecular trigger of CNS myelination is unknown. By targeting Pten in cerebellar granule cells and activating the AKT1–mTOR pathway, we increased the caliber of normally unmyelinated axons and the expression of numerous genes encoding regulatory proteins. This led to the expansion of genetically wild-type oligodendrocyte progenitor cells, oligodendrocyte differentiation and de novo myelination of parallel fibers. Thus, a neuronal program dependent on the phosphoinositide PI(3,4,5)P3 is sufficient to trigger all steps of myelination.

Robo3-Driven Axon Midline Crossing Conditions Functional Maturation of a Large Commissural Synapse

During the formation of neuronal circuits, axon pathfinding decisions specify the location of synapses on the correct brain side and in correct target areas. We investigated a possible link between axon midline crossing and the subsequent development of output synapses formed by these axons. Conditional knockout of Robo3 in the auditory system forced a large commissural synapse, the calyx of Held, to be exclusively formed on the wrong, ipsilateral side. Ipsilateral calyx of Held synapses showed strong transmission defects, with reduced and desynchronized transmitter release, fewer fast-releasable vesicles, and smaller and more variable presynaptic Ca(2+) currents. Transmission defects were not observed in a downstream inhibitory synapse, and some defects persisted into adulthood. These results suggest that axon midline crossing conditions functional maturation of commissural synapses, thereby minimizing the impact of mislocalized synapses on information processing. This mechanism might be relevant to human disease caused by mutations in the ROBO3 gene.