preprint submitted I Varela-Martínez author Ana Villalba Requena author 68cb85a0-39f7-11eb-9559-9aaab4f6a2470000-0002-5615-5277 J. Garcia-Marqués author Simon Hippenmeyer author 37B36620-F248-11E8-B48F-1D18A9856A870000-0003-2279-1061 M. Nieto author SiHi department Radial glial progenitors (RGPs) generate all projection neurons (PNs) in the cerebral cortex through incompletely understood processes. Herein, we combine Mosaic Analysis with Double Markers (MADM)-based clonal analysis at embryonic days 12.5 and 13.5 with early postnatal callosal tracing to reveal a lineage progression that challenges the inside-outside model of cortical development and the conventional view of an invariable sequence of asymmetric neurogenic divisions. Our data demonstrate that early multipotent RGPs generate all extra-telencephalic (ET) and intra-telencephalic (IT) PNs across all layers through parallel sublineages and the random specification, during the earliest neurogenic divisions, of fate-restricted daughter RGPs. While the neuronal production of the parental multipotent RGPs consists of small ET-PN or IT-PN outputs, fate-restricted RGPs produce larger translaminar outputs spanning deep and upper layers of only IT-PNs, the predominant mammalian PN subtype. We further show that the emergence of IT-PN fate-restricted RGPs also leads to quantitatively and temporally stereotyped neurogenesis population-wise. 2025 eng 10.1101/2025.05.07.652665 Varela-Martínez I, Villalba Requena A, Garcia-Marqués J, Hippenmeyer S, Nieto M. Early emergence of projection-subtype fate-restricted radial glial progenitors orchestrates neocortical neurogenesis. bioRxiv, <a href="https://doi.org/10.1101/2025.05.07.652665">10.1101/2025.05.07.652665</a>. Varela-Martínez I, Villalba Requena A, Garcia-Marqués J, Hippenmeyer S, Nieto M. Early emergence of projection-subtype fate-restricted radial glial progenitors orchestrates neocortical neurogenesis. <i>bioRxiv</i>. doi:<a href="https://doi.org/10.1101/2025.05.07.652665">10.1101/2025.05.07.652665</a> I. Varela-Martínez, A. Villalba Requena, J. Garcia-Marqués, S. Hippenmeyer, and M. Nieto, “Early emergence of projection-subtype fate-restricted radial glial progenitors orchestrates neocortical neurogenesis,” <i>bioRxiv</i>. . Varela-Martínez, I, Ana Villalba Requena, J. Garcia-Marqués, Simon Hippenmeyer, and M. Nieto. “Early Emergence of Projection-Subtype Fate-Restricted Radial Glial Progenitors Orchestrates Neocortical Neurogenesis.” <i>BioRxiv</i>, n.d. <a href="https://doi.org/10.1101/2025.05.07.652665">https://doi.org/10.1101/2025.05.07.652665</a>. I. Varela-Martínez, A. Villalba Requena, J. Garcia-Marqués, S. Hippenmeyer, M. Nieto, BioRxiv (n.d.). Varela-Martínez, I., Villalba Requena, A., Garcia-Marqués, J., Hippenmeyer, S., &#38; Nieto, M. (n.d.). Early emergence of projection-subtype fate-restricted radial glial progenitors orchestrates neocortical neurogenesis. <i>bioRxiv</i>. <a href="https://doi.org/10.1101/2025.05.07.652665">https://doi.org/10.1101/2025.05.07.652665</a> Varela-Martínez, I., et al. “Early Emergence of Projection-Subtype Fate-Restricted Radial Glial Progenitors Orchestrates Neocortical Neurogenesis.” <i>BioRxiv</i>, doi:<a href="https://doi.org/10.1101/2025.05.07.652665">10.1101/2025.05.07.652665</a>. 197172025-05-20T10:19:29Z2026-08-13T07:16:48Z