Distinct embryonic precursors generate gray and white matter astrocytes in the mouse spinal cord

Astrocytes display two canonical morphologies—protoplasmic in the gray matter (GM) and fibrous in the white matter (WM)—yet recent transcriptomic studies indicate that their heterogeneity is far more extensive. In the developing mouse spinal cord, discrete dorsoventral progenitor domains generate astrocytes that populate both GM and WM. We hypothesized that heterogeneous, fate-restricted ventricular precursors generate either GM or WM astrocytes, explaining their distinct identities despite a shared origin. To test that, we performed in vivo lineage tracing using two complementary approaches: tamoxifen-inducible expression of fluorescent reporters in Glast CreER mice, and the Mosaic Analysis with Double Markers (MADM) system, a Cre-loxP–based strategy inducing intrachromosomal recombination to reconstitute chimeric fluorescent proteins (Tomato and GFP). Sparce fate mapping revealed that astrocyte clones are frequently composed of two daughter cells of the same subtype (either GM or WM), sharing morphology, molecular marker expression, and spatial position. These pairs appear to originate from symmetric divisions occurring after migration near-final settling location. Our study reveals that, in addition to dorsoventral patterning, astrocyte diversity is established by distinct embryonic precursors within each progenitor domain that selectively generate GM or WM astrocytes. These results shed light on the differentiation programs underlying astrocyte heterogeneity.