HPM live µ for a full CLEM workflow.

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information-known as multimodal imaging or correlative microscopy (CM)-has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artifacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To minimize the delay separating live imaging from cryo-arrest, we developed a high-pressure freezing (HPF) system directly coupled to a light microscope. We address the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. To further explore the potential of cryo-fixation related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification, the HPF core technology has been revisited to allow easy modification of the environmental parameters during vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

PlexinA2 and Sema6A are required for retinal progenitor cell migration.

In the vertebrate retina six types of neurons and one glial cell type are generated from multipotent retinal progenitor cells (RPCs) whose proliferation and differentiation are regulated by intrinsic and extrinsic factors. RPCs proliferate undergoing interkinetic nuclear migration within the neuroblastic layer, with their nuclei moving up and down along the apico-basal axis. Moreover, they only differentiate and therefore exit the cell cycle at the apical side of the neuroblastic layer. Sema6A and its receptors PlexinA4 and PlexinA2 control lamina stratification of the inner plexiform layer in the mouse retina. Nevertheless, their function in earlier developmental stages is still unknown. Here, we analyzed the embryonic retina of PlexinA2 and Sema6A knockout mice. Using time-lapse videomicroscopy we provide evidence that Sema6A/PlexinA2 signaling participates to interkinetic nuclear migration of RPCs around birth. When disrupted, RPCs migration is blocked at the apical side of the neuroblastic layer. This is the first evidence supporting a role for transmembrane molecules in the regulation of interkinetic nuclear migration in the mouse retina.

Lithium enhances remyelination of peripheral nerves.

Glycogen synthase kinase 3ß (GSK3ß) inhibitors, especially the mood stabilizer lithium chloride, are also used as neuroprotective or anti-inflammatory agents. We studied the influence of LiCl on the remyelination of peripheral nerves. We showed that the treatment of adult mice with LiCl after facial nerve crush injury stimulated the expression of myelin genes, restored the myelin structure, and accelerated the recovery of whisker movements. LiCl treatment also promoted remyelination of the sciatic nerve after crush. We also demonstrated that peripheral myelin gene MPZ and PMP22 promoter activities, transcripts, and protein levels are stimulated by GSK3ß inhibitors (LiCl and SB216763) in Schwann cells as well as in sciatic and facial nerves. LiCl exerts its action in Schwann cells by increasing the amount of ß-catenin and provoking its nuclear localization. We showed by ChIP experiments that LiCl treatment drives ß-catenin to bind to T-cell factor/lymphoid-enhancer factor response elements identified in myelin genes. Taken together, our findings open perspectives in the treatment of nerve demyelination by administering GSK3ß inhibitors such as lithium.

Wnt/beta-catenin signaling is an essential and direct driver of myelin gene expression and myelinogenesis.

Wnt/ß-catenin signaling plays a major role in the development of the nervous system and contributes to neuronal plasticity. However, its role in myelination remains unclear. Here, we identify the Wnt/ß-catenin pathway as an essential driver of myelin gene expression. The selective inhibition of Wnt components by small interfering RNA or dominant-negative forms blocks the expression of myelin protein zero (MPZ) and peripheral myelin protein 22 (PMP22) in mouse Schwann cells and proteolipid protein in mouse oligodendrocytes. Moreover, the activation of Wnt signaling by recombinant Wnt1 ligand increases by threefold the transcription of myelin genes and enhances the binding of ß-catenin to T-cell factor/lymphoid-enhancer factor transcription factors present in the vicinity of the MPZ and PMP22 promoters. Most important, loss-of-function analyses in zebrafish embryos show, in vivo, a key role for Wnt/ß-catenin signaling in the expression of myelin genes and in myelin sheath compaction, both in the peripheral and central nervous systems. Inhibition of Wnt/ß-catenin signaling resulted in hypomyelination, without affecting Schwann cell and oligodendrocyte generation or axonal integrity. The present findings attribute to Wnt/ß-catenin pathway components an essential role in myelin gene expression and myelinogenesis.