Imp1 acts as a dosage- and stage-dependent temporal rheostat orchestrating radial glial fate transitions and cortical morphogenesis
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eLife Assessment
This important study presents new insights into the post-transcriptional mechanisms that govern cortical development. Through state-of-the-art methodology to track neuronal birth order, the data provide compelling evidence that Imp1 (Igf2bp1/Zbp1) orchestrates radial glia fate transitions and cortical neurogenesis. The findings establish a new framework for understanding how post-transcriptional mechanisms integrate with transcriptional and epigenetic regulatory layers to control cortical temporal patterning.
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Abstract
Cortical neurogenesis proceeds through a precise temporal program in which radial glia sequentially generate distinct neuronal subtypes and later glia, yet how post-transcriptional regulators coordinate these transitions remain poorly understood. We previously identified that a decreasing temporal gradient of the RNA-binding protein Imp encodes neural stem cell age in Drosophila. In this work, we extend our investigation to Imp1, a mammalian homologue of Imp, and its role in murine neocortical development. Using TEMPO to track birth-order dynamics, we demonstrate that sustained Imp1 overexpression during early neurogenesis arrests temporal fate progression, shifting neuronal populations toward deeper cortical layers V-VI. Immunostaining with layer-specific transcription factors Cux1 and Ctip2 confirmed that laminar repositioning results from genuine changes in neuronal identity rather than migratory defects, with neurons adopting molecular identities matching their final positions. Temporal window-specific manipulations reveal distinct stage-specific effects where early-stage Imp1 induction produces cascading effects on fate specification and moderately delays the neuronal-to-gliogenic transition, while mid-stage induction induces neuronal accumulation in the subplate region. Live imaging of organotypic cultures reveals continuous neuronal recruitment within intermediate and ventricular zones, with mid-stage-born neurons accumulating at significantly faster rates than earlier cohorts. Strikingly, mid-stage Imp1 overexpression also induces ectopic glial-like foci distributed throughout the cortical plate, featuring dramatic cellular expansion and morphological heterogeneity. These findings establish Imp1 as a dosage- and stage-dependent temporal rheostat orchestrating developmental transitions in radial glial progenitors, controlling neuronal fate decisions and spatial organization. This work advances our understanding of molecular timing mechanisms governing neuronal diversity in the mammalian cortex.
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eLife Assessment
This important study presents new insights into the post-transcriptional mechanisms that govern cortical development. Through state-of-the-art methodology to track neuronal birth order, the data provide compelling evidence that Imp1 (Igf2bp1/Zbp1) orchestrates radial glia fate transitions and cortical neurogenesis. The findings establish a new framework for understanding how post-transcriptional mechanisms integrate with transcriptional and epigenetic regulatory layers to control cortical temporal patterning.
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Reviewer #1 (Public review):
Summary:
A hallmark of cortical development is the temporal progression of lineage programs in radial glia progenitors (RGs) that orderly generate a large set of glutamatergic projection neuron types, which are deployed to the cortex in a largely inside-out sequence. This process is thought to contribute to the formation of proper cortical circuitry, but the underlying cellular and molecular mechanisms remain poorly understood. To a large extent, this is due to technical limitations that can fate map RGs and their progeny with cell type resolution, and manipulate gene expression with proper cell and temporal resolution. Building on the TEMPO technique that Tsumin Lee group developed, here Azur et al show that the RNA binding protein Imp1 functions as a dosage- and stage-dependent post-transcriptional …
Reviewer #1 (Public review):
Summary:
A hallmark of cortical development is the temporal progression of lineage programs in radial glia progenitors (RGs) that orderly generate a large set of glutamatergic projection neuron types, which are deployed to the cortex in a largely inside-out sequence. This process is thought to contribute to the formation of proper cortical circuitry, but the underlying cellular and molecular mechanisms remain poorly understood. To a large extent, this is due to technical limitations that can fate map RGs and their progeny with cell type resolution, and manipulate gene expression with proper cell and temporal resolution. Building on the TEMPO technique that Tsumin Lee group developed, here Azur et al show that the RNA binding protein Imp1 functions as a dosage- and stage-dependent post-transcriptional mechanism that orchestrates developmental stage transitions in radial glial progenitors, and controls neuronal fate decisions and spatial organization of neuronal and glial cell progeny. Their results suggest that while transcriptional regulators define available cellular states and gate major transitions, post-transcriptional mechanisms like Imp1 provide an additional layer of control by modulating stage-specific transcript stability. Imp1 thus acts as a temporal coordinator whose dosage and timing determine whether developmental transitions are temporarily delayed or blocked. These findings establish a new framework for understanding how post-transcriptional mechanisms integrate with transcriptional and epigenetic regulatory layers to control cortical temporal patterning.
Strengths:
The authors apply a novel genetic fate mapping and gene manipulation technology (TEMPO) with cellular resolution. This reveals a dosage- and stage-dependent post-transcriptional mechanism that orchestrates developmental stage transitions in radial glial progenitors, and controls neuronal fate decisions and spatial organization of neuronal and glial cell/astrocyte progeny.
Weaknesses:
The endogenous developmental expression pattern of Imp1 and TEMPO-mediated overexpression are not well described or characterized with cellular resolution (whether only in radial glial cells or also in post-mitotic neurons). Thus, the interpretations of the overexpression phenotypes are not always clear.
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Reviewer #2 (Public review):
Summary:
In this manuscript, Azur et al seek to determine the role of Imp1/Igf2bp1 in regulating the temporal generation of cortical neuron types. The authors showed that overexpression of Imp1 changes the laminar distribution of cortical neurons and suggest that Imp1 plays a temporal role in specifying cell fates.
Strengths:
The study uniquely used TEMPO to investigate the temporal effects of Imp1/Igf2bp1 in cortical development. The disrupted laminar distribution and delayed fate transition are interesting. The results are presented with proper quantification, they are generally well interpreted, and suggest important roles for Imp1.
Weaknesses:
(1) While the results suggest Imp1 is important in regulating cortical neurogenesis, it remains unclear when and where it is expressed to execute such temporal …
Reviewer #2 (Public review):
Summary:
In this manuscript, Azur et al seek to determine the role of Imp1/Igf2bp1 in regulating the temporal generation of cortical neuron types. The authors showed that overexpression of Imp1 changes the laminar distribution of cortical neurons and suggest that Imp1 plays a temporal role in specifying cell fates.
Strengths:
The study uniquely used TEMPO to investigate the temporal effects of Imp1/Igf2bp1 in cortical development. The disrupted laminar distribution and delayed fate transition are interesting. The results are presented with proper quantification, they are generally well interpreted, and suggest important roles for Imp1.
Weaknesses:
(1) While the results suggest Imp1 is important in regulating cortical neurogenesis, it remains unclear when and where it is expressed to execute such temporal functions. For instance, where is Imp1 expressed in the developing brain? Is it specific to the radial glial cells or ubiquitous in progenitors and neurons? Does it show temporal expression in RGCs?
(2) The advantage and interpretation of TEMPO need further clarification. TEMPO is an interesting method and appears useful in simultaneously labelling cells and controlling gene expression. Since the reporter, Cas9, and gRNA triggers are all driven by ubiquitous promoters and integrated into the genome using piggyBac, it appears logical that the color transition should happen in all cells over time. The color code appears to track the time when the plasmids got integrated instead of the birthday of neurons. Is this logically true? If the TEMPO system is introduced into postmitotic neurons and the CAG promoter is not silenced, would the tri-color transition happen?
(3) The accumulation of neurons at the subplate region would benefit from showing larger views of the affected hemisphere. IUE is invasive. The glass pipette may consistently introduce focal damages and truncate RGCs. It is important to examine slices covering the whole IUE region.
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Reviewer #3 (Public review):
Summary:
The work by Azur and colleagues makes use of the TEMPO (Temporal Encoding and Manipulation in a Predefined Order) methodology to trace cortical neurogenesis in combination with overexpression of Imp1. Imp1 is a mammalian homologue of the Drosophila Imp, which has been shown to control temporal identity in a stem cell context. In their work, they show that overexpression of Imp1 in radial glia, which generate neurons and macroglia in a sequential manner during cortical development, leads to a disruption of faithful neuron/glia generation. They show that continuous overexpression leads to a distinct phenotypic outcome when compared to paradigms where Imp1 was specifically overexpressed in defined temporal windows, enabled by the unique TEMPO approach. Interestingly, the observed phenotype with …
Reviewer #3 (Public review):
Summary:
The work by Azur and colleagues makes use of the TEMPO (Temporal Encoding and Manipulation in a Predefined Order) methodology to trace cortical neurogenesis in combination with overexpression of Imp1. Imp1 is a mammalian homologue of the Drosophila Imp, which has been shown to control temporal identity in a stem cell context. In their work, they show that overexpression of Imp1 in radial glia, which generate neurons and macroglia in a sequential manner during cortical development, leads to a disruption of faithful neuron/glia generation. They show that continuous overexpression leads to a distinct phenotypic outcome when compared to paradigms where Imp1 was specifically overexpressed in defined temporal windows, enabled by the unique TEMPO approach. Interestingly, the observed phenotype with 'ectopic' generation of mainly lower cortical layer neurons appears not to be due to migration deficits. Strikingly, the overexpression of Imp1 specifically at later stages also leads to ectopic glia-like foci throughout the developing cortical plate. Altogether, the new data provide new insights regarding the role of the post-transcriptional Imp1 regulator in controlling temporal fate in radial glia for the faithful generation of neurons and glia during cortical development.
Strengths:
The TEMPO approach provides excellent experimental access to probe Imp1 gene function at defined temporal windows. The data is very robust and convincing. The overexpression paradigm and its associated phenotypes match very well the expected outcome based on Imp1 loss-of-function. Overall, the study contributes significantly to our understanding of the molecular cues that are associated with the temporal progression of radial glia fate potential during cortical development.
Weaknesses:
The authors provide some experimental evidence, including live imaging, that deficits related to Imp1 overexpression and subsequent overabundance of lower-layer neurons, or accumulation at the subplate, appear to evolve independently of neuronal migration deficits. However, the analysis at the population level might not suffice to make the claim robust. To analyze neuronal migration in more depth, the authors could trace individual neurons and establish speed and directional parameters for comparison.
In their analysis, the authors mainly rely on temporal parameters/criteria to associate the generation of certain neuron fates. While two markers were used to identify the neuronal fate, the variance seems quite high. The authors could consider utilizing an antibody against Satb2, which would provide additional data points that could help to establish statistical significance in some of the analyses.
The analysis of glia was done at postnatal day 10, although gliogenesis and, in particular, astrocyte maturation last at least until postnatal day 28. The authors could consider extending their analysis to capture the full spectrum of their astrocyte phenotype.
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