Characterizing novel markers for Kranocytes, the "forgotten" NMJ-capping cells

N Pelaez-Poblet(1) S Fuertes-Alvarez(1) A Izeta(1)

1:Biodonostia Institute

Neuromuscular junctions (NMJs) enable nerve transmission to muscular fibres, triggering muscle contraction. NMJs are covered by terminal Schwann cells (tSCs) and mesenchymal cells known as Kranocytes. tSC functions are well characterised: they are essential in the maintenance of NMJ structure and in the reinnervation process after nerve injury. In contrast, little is known about the function of Kranocytes in homeostasis. In response to denervation, Kranocytes migrate earlier than tSCs and generate mesenchymal bridges that tSCs and axons will later colonize for reinnervation. Known kranocyte markers (CD34 and Tenascin C; the latter only in response to nerve injury) are not specific. To be able to isolate Kranocytes and make a deeper transcriptomic and functional characterization of this ill-known cell type, we first aimed to describe new membrane markers as a tool to isolate them. Skeletal muscles from the hindlimbs of C57BL/6 adult mice were analysed by confocal microscopy and flow cytometry. Immunofluorescence staining was performed on iDisco-clarified whole mount muscles. A screen of 50 antibodies yielded a new specific transmembrane glycoprotein marker for Kranocytes, Podoplanin. In muscle whole mounts, Podoplanin+ cells were limited to Kranocytes covering NMJs and lymphatic vessels. We are currently isolating Podoplanin+CD34+ cells by FACS, and will further characterise Kranocytes by single cell RNA sequencing. The identification of Podoplanin as a new and almost specific marker for Kranocytes will pave the way to shed light on the function of these cells in homeostasis and injury of skeletal muscle.

Key words: Kranocytes, neuromuscular junction (NMJ), podoplanin.