CLONAL MAPPING OF THE ONTOGENY OF THE MOUSE SUPRACHIASMATIC NUCLEUS

The circadian system aligns organisms’ physiology to 24-h rhythms, anticipating recurring environmental changes (light/dark cycles). In mammals, the hypothalamic suprachiasmatic nucleus (SCN), acting as the master pacemaker, orchestrates these rhythms. The ~20,000 cells composing the mouse SCN are organised into a ventral core responding directly to light and a dorsal shell organising rhythmic output. Almost all SCN neurons produce and receive GABA, together with ≥100 neuropeptides. SCN astrocytes also play essential roles as timekeepers. Although the adult mouse SCN circuit is well characterized, its ontogeny during development remains obscure.
To investigate the mouse SCN ontogeny, we used StarTrack, a state-of-the-art cell lineage-tracing tool. By in utero electroporation at gestational day 12, we introduced 12 plasmids encoding six fluorescent proteins with or without a nuclear localization signal. The plasmids randomly integrated into SCN progenitors, labelling clones—clusters of sibling cells derived from a common progenitor—with a unique color code. A detailed clonal analysis was performed on brain slices at postnatal day 30. Confocal imaging was followed by image analysis using an ad-hoc ImageJ macro. To determine cell types, we performed immunohistochemical mapping of SCN neurons, astrocytes, and oligodendrocytes.
Remarkably, we detected SCN clones comprising different cell types, suggesting that SCN progenitors are multipotent and that SCN neurons, astrocytes, and oligodendrocytes can share a common progenitor. Using three-dimensional clonal dispersion analysis, we provide a high-resolution anatomical reconstruction of SCN clones distribution. These findings first describe the mouse SCN clonal ontogeny, revealing key developmental signatures of the master circadian clock.