Summary: A new study challenges the classical “inside-out” model of cortical development. Using Mosaic Analysis with Double Markers (MADM) single-cell lineage tracing, the team demonstrated that radial glial progenitor cells split early in brain development into distinct, parallel developmental branches.
Rather than a single progenitor pool sequentially producing deeper-layer extra-telencephalic projection neurons (ET-PNs) followed by superficial-layer intra-telencephalic projection neurons (IT-PNs), cortical neurogenesis involves early lineage branching. One progenitor branch exclusively generates IT-PNs across all layers, while a parallel branch yields both ET-PNs and IT-PNs.
These findings reshape fundamental assumptions about neural stem cell fate determination, neurodevelopmental timing, and cortical layer construction.
Key Facts
- Early Lineage Divergence: Cortical neural stem cells (radial glial cells) split into two distinct lineage branches much earlier in development than previously recognized.
- Refutation of Strict Sequential Switching: Cortical projection neurons do not arise from a single homogeneous stem cell population that strictly switches cell-type production over time.
- Lineage-Specific Subtype Output: One progenitor branch gives rise exclusively to intra-telencephalic projection neurons (IT-PNs), whereas a parallel branch generates both extra-telencephalic projection neurons (ET-PNs) and IT-PNs.
- Differential Neurogenic Dynamics: ET-PN-producing lineages generate smaller neuron clusters that exhaust their neurogenic potential early, while IT-PN lineages produce larger, layer-spanning neuronal clones.
- Resolution of Temporal Paradox: The early exhaustion of ET-PN progenitor clones explains why ET-PNs settle predominantly in early-forming deep layers, while ongoing IT-PN production continues into upper cortical layers.
Source: ISTA
Science thrives on exchange. Whether at conferences, symposia, Zoom calls, or visits to other research institutes and universities—Irene Varela-Martínez can tell you a thing or two about it.
In her PhD project at the Centro Nacional de Biotecnología (CNB-CSIC) in Madrid, in the lab of Marta Nieto, the neuroscientist investigated how precursor cells—stem cells—in the cerebral cortex develop and how they form different groups of nerve cells. During the data collection, she also spent time (supported by an EMBO short-term fellowship) at the Institute of Science and Technology Austria (ISTA) in Simon Hippenmeyer’s group. A time she looks back on fondly—and one she is now building on as a postdoctoral researcher at ISTA.
The results of this work have now been published in Science Advances and show that precursor cells, different than previously thought, split very early into two lineages and thereby give rise to distinct types of neurons.
Part of the gray matter
“It’s time to get the gray matter going” or “a bit of input for the gray matter”: there are many sayings about putting the brain to work. A large part of this gray matter is formed by the cerebral cortex—the outermost layer of our brain. There, neurons and glial cells sit densely packed, playing an enormous role in attention, perception, consciousness, thinking, memory, and language.
In the cerebral cortex, projection neurons—nerve cells that “project,” or send, signals over longer distances into brain regions or the nervous system—can be broadly divided into two groups. These include the intra-telencephalic projection neurons—IT-PNs for short—which establish connections to other areas of the cerebral cortex, including the opposite brain hemisphere. Another group is the extra-telencephalic projection neurons, ET-PNs for short. They send their processes out of the cerebral cortex, for example toward the spinal cord.
All of these neurons are born during neurogenesis—the developmental phase of nerve cells. Until now, however, it remained unclear how neural stem cells ‘decide’ which type of projection neuron to produce.
“The cerebral cortex consists of six layers,” explains Varela-Martínez. “During brain development, these layers are gradually populated with neurons. According to the ‘inside-out’ model, cortical nerve cells for the deeper layers arise first, followed by neurons that settle in progressively more superficial layers.” This led researchers to assume that neural stem cells first generated ET-PNs, which only populate the deep layers, and only later switched to producing IT-PNs, which are enriched in the upper layers.
However, it does not seem to be that simple. The first results from Varela-Martínez and her colleagues showed that, in mice, rather than following a strict temporal switch from one neuronal subtype to another, the production of ET-PNs and IT-PNs partially overlaps. Perhaps more importantly, each subtype follows its own distinct neurogenic dynamics.
Neuronal branches
With these results in hand, the then-PhD student came to the Hippenmeyer group as a visiting scientist.
“The group has great expertise in the gold-standard MADM technique. This method allows you to precisely follow cell division during the development of neurons,” explains Varela-Martínez. “With it, daughter cells can be made visible—and beyond that, the entire cell lineages and cell clones can be reconstructed.”
Using this technique, Varela-Martínez set out to search for how the two cell types, ET-PNs and IT-PNs, differ in their lineages. So-called radial glial cells were the starting point—neuronal precursor cells from which the projection neurons of the cerebral cortex arise.
This lineage analysis showed that there are at least two developmental branches that originate in parallel from this precursor cell and separate from each other already early on. One branch gives rise exclusively to IT-PNs, while the other produces both ET-PNs and IT-PNs. This finding refines the long-standing view that radial glial progenitors follow a single developmental program, producing ET-PNs first and IT-PNs later.
Importantly, the production of the two neuronal groups does not follow the same developmental dynamics. While ET-PNs are generated in small clusters that become exhausted early, IT-PN lineages consist of larger groups of neurons, distributed across all cortical layers. This explains why ET-PNs predominate early in development, whereas later neurogenesis produces almost exclusively IT-PNs.
Overall, the study suggests that the cerebral cortex is built through an early branching process, in which different neuronal lineages arise in parallel from the very beginning.
New project at ISTA
For almost a year now, Varela-Martínez has been a postdoc at ISTA. Although the researcher is still dedicated to the cerebral cortex, the question is now a different one. The neuroscientist is trying to understand how the size and complexity of the cerebral cortex have changed over the course of evolution.
To do so, she investigates how the developmental programs of neural stem cells have evolved to generate larger numbers of neurons.
“Across evolution, brains have become larger and more complex,” explains Varela-Martínez. “How have neural stem cells adapted to generate more and even more diverse neurons? How is this achieved at the lineage level?”
Key Questions Answered:
A: The classical model held that a single pool of radial glial stem cells sequentially produced deep-layer neurons first (like ET-PNs) and later switched entirely to producing upper-layer neurons (like IT-PNs). This study shows that neural stem cells branch early into parallel lineages with distinct neurogenic fates and clone sizes, meaning subtype production is dictated by lineage commitment rather than a simple clock-like switch.
A: Mosaic Analysis with Double Markers (MADM) is a genetic single-cell labeling technique that allows scientists to visualize individual cell divisions and track the precise lineage trees (clones) derived from a single progenitor cell. Without single-cell resolution, population-level assays mask individual stem cell fates, making parallel progenitor branches appear as a single homogenous population.
A: Intra-telencephalic projection neurons (IT-PNs) extend axons locally or across the corpus callosum to connect regions within the cerebral cortex. Extra-telencephalic projection neurons (ET-PNs) send long-range projections out of the cerebral cortex entirely to subcortical targets such as the brainstem, thalamus, and spinal cord to coordinate motor control and autonomic signaling.
Editorial Notes:
- This article was edited by a Neuroscience News editor.
- Journal paper reviewed in full.
- Additional context added by our staff.
About this genetics and neurodevelopment research news
Author: Andreas Rothe
Source: ISTA
Contact: Andreas Rothe – ISTA
Image: The image is credited to Irene Varela-Martínez/CNB-CSIC
Original Research: Open access.
“Early Fate Diversification of Radial Glial Progenitors During Corticogenesis” by Irene Varela-Martínez, Ana Villalba, Jorge García-Marqués, Alfonso Aguilera, Diogo S. Castro, Simon Hippenmeyer, Marta Nieto. Science Advances
DOI:10.1126/sciadv.adw5487
Abstract
Early Fate Diversification of Radial Glial Progenitors During Corticogenesis
Radial glial progenitors (RGPs) generate all projection neurons (PNs) in the cerebral cortex through incompletely understood processes.
We combined Mosaic Analysis with Double Markers at embryonic stages (E)12.5 and E13.5 with early postnatal callosal tracing to dissect RGP lineage progression. We find that multipotent RGPs generate all extra-telencephalic (ET) and intra-telencephalic (IT) PNs via parallel sublineages that emerge simultaneously at neurogenesis onset. ET-PN production progresses exclusively via small, self-consuming lineages; IT-PN lineages feature RGPs generating large translaminar outputs.
The early emergence of IT-PN–fated RGPs, coinciding with a switch to direct neurogenesis, contributes to the stereotyped population-level progression of the multipotent lineage. We also identify POU3F transcription factors as candidate regulators of IT-PN fate via noncanonical mitotic chromatin binding.
The results support a model whereby IT- and ET-PNs arise from an early bifurcation and parallel specification within the multipotent RGP lineage.
