Multifunctional requirements for ERK1/2 signaling in the development of ganglionic eminence derived glia and cortical inhibitory neurons
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This is an important study on the role of the Ras/MEK/Erk signaling cascade in brain development, with both theoretical and practical implications in multiple fields. The authors reveal functional redundancy and shared function for Erk1 and Erk2 in determining medial ganglionic eminence derived glial number, and exclude a role for Erk1 and Erk2 in the regulation of GABAergic neuron number or initial specification of GABAergic identity. They combine multiple methods including Ribotags, electrophysiology, and chemogenetic stimulation using DREADDs towards comprehensive conclusions. Reviewers expressed concerns about the coherence of the findings, and about the selection of controls mouse lines. Heterozygous ERK1/2 mice are used as 'het controls' throughout the manuscript, however, reviewers felt that there was not sufficient evidence that ERK levels in heterozygous mice are similar to wildtype mice. Nevertheless, these compelling findings will be of interest to a broad readership, and especially readers interested in kinase function and neurodevelopmental syndromes.
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Abstract
Abstract
The RAS/RAF/MEK/ERK1/2 intracellular signaling pathway is activated by numerous cues during brain development and dysregulated in neurodevelopmental syndromes, particularly the RASopathies and certain forms of autism. Cortical excitatory/inhibitory imbalance is thought to be critical in the neuropathogenesis of these conditions. However, the developmental functions of ERK1/2 signaling in cortical inhibitory neurons (CINs) and other medial ganglionic eminence (MGE)-derived non-neuronal cells are poorly understood. Here, we genetically modulated ERK1/2 signaling in mouse MGE neural progenitors or GABAergic neurons in vivo. We find that MEK-ERK1/2 signaling is essential for regulating MGE-derived oligodendrocyte number in the anterior commissure. While Erk1/2 inactivation does not alter CIN number, we discovered a significant and persistent reduction in somatostatin, but not parvalbumin, expression in a subset of CINs. ERK1/2 signaling is also necessary for chemogenetic activity-dependent FOSB expression in CINs in vivo. Interestingly, one week of chronic chemogenetic stimulation in juvenile or adult animals partially rescues the decrease in somatostatin expression in Erk1/2 mutant CINs. Our data demonstrate ERK1/2 signaling is required for the establishment of MGE-derived glia, whereas in CINs, ERK1/2 drives activity dependent-responses and the expression of somatostatin in a subset of neurons.
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eLife assessment
This is an important study on the role of the Ras/MEK/Erk signaling cascade in brain development, with both theoretical and practical implications in multiple fields. The authors reveal functional redundancy and shared function for Erk1 and Erk2 in determining medial ganglionic eminence derived glial number, and exclude a role for Erk1 and Erk2 in the regulation of GABAergic neuron number or initial specification of GABAergic identity. They combine multiple methods including Ribotags, electrophysiology, and chemogenetic stimulation using DREADDs towards comprehensive conclusions. Reviewers expressed concerns about the coherence of the findings, and about the selection of controls mouse lines. Heterozygous ERK1/2 mice are used as 'het controls' throughout the manuscript, however, reviewers felt that there was not …
eLife assessment
This is an important study on the role of the Ras/MEK/Erk signaling cascade in brain development, with both theoretical and practical implications in multiple fields. The authors reveal functional redundancy and shared function for Erk1 and Erk2 in determining medial ganglionic eminence derived glial number, and exclude a role for Erk1 and Erk2 in the regulation of GABAergic neuron number or initial specification of GABAergic identity. They combine multiple methods including Ribotags, electrophysiology, and chemogenetic stimulation using DREADDs towards comprehensive conclusions. Reviewers expressed concerns about the coherence of the findings, and about the selection of controls mouse lines. Heterozygous ERK1/2 mice are used as 'het controls' throughout the manuscript, however, reviewers felt that there was not sufficient evidence that ERK levels in heterozygous mice are similar to wildtype mice. Nevertheless, these compelling findings will be of interest to a broad readership, and especially readers interested in kinase function and neurodevelopmental syndromes.
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Reviewer #1 (Public Review):
The Ras/MEK/Erk signaling cascade is a ubiquitous pathway activated by many extracellular signals and is critical for a wide variety of cell function. In this manuscript, the authors generate Erk1/2 double knockouts specifically in Nkx2.1-derived cells (basically MGE/POA-derived cells in the forebrain) and explore changes in oligodendrocyte number and cortical interneuron function. They observe a striking loss of Nkx2.1-lineage oligos (and astrocytes) in the anterior commissure, although the mechanism for this specific loss is unclear. While there is no significant change in the number of cortical interneurons, the authors do note a decrease in SST+/Calb- INs in the mutant. The authors then use DREADDs to manipulate activity in Nkx2.1-lineage cells. Surprisingly, chemogenetic activation of Nkx2.1-lineage KO …
Reviewer #1 (Public Review):
The Ras/MEK/Erk signaling cascade is a ubiquitous pathway activated by many extracellular signals and is critical for a wide variety of cell function. In this manuscript, the authors generate Erk1/2 double knockouts specifically in Nkx2.1-derived cells (basically MGE/POA-derived cells in the forebrain) and explore changes in oligodendrocyte number and cortical interneuron function. They observe a striking loss of Nkx2.1-lineage oligos (and astrocytes) in the anterior commissure, although the mechanism for this specific loss is unclear. While there is no significant change in the number of cortical interneurons, the authors do note a decrease in SST+/Calb- INs in the mutant. The authors then use DREADDs to manipulate activity in Nkx2.1-lineage cells. Surprisingly, chemogenetic activation of Nkx2.1-lineage KO cells led to an upregulation of SST protein in SST+ INs, while other characteristics in KO mice (cFos expression, open field locomotion) were not changed (or altered at much lower levels) in KOs compared to similar stimulation in control mice. Overall, the paper contains numerous insightful observations, but a coherent, overall theme for what Erk1/2 is doing in Nkx2.1-lineage cells at different development timepoints is somewhat lacking. For example, the authors focus on changes in SST levels in the KO mice, justifiably because that is where they see the biggest difference, yet they perform e-phys experiments only on PV+, fast spiking cells in Figure 5. While it may be more challenging to find SST+ cells in the KO, the logic of recording from PV cells was not clear. Sometimes this paper reads as a series of data points where the overall theme of the story is not always evident.
More importantly, the authors use heterozygous ERK1/2 mice as 'het controls' throughout the manuscript. However, they have not sufficiently demonstrated that the ERK levels in hets are similar to WT. Figure 1B-J purports to show that ERK1/2 levels in a handful of cells from heterozygous mice are equivalent to WT, but there is no quantification of this observation. It is unconventional to use heterozygous mice as controls without clearly demonstrating that they are similar/identical to controls. Especially in a scenario such as this, where one would expect to see 50% of protein levels in hets compared to WT mice. As such, readers are cautioned for how to interpret some of these findings. For example, there may be instances where there is no significant difference between KO and 'het controls', but if they had compared to true WT controls, then it's possible some differences could emerge.
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Reviewer #2 (Public Review):
Knowles et al. investigated the developmental roles of Erk1/2 expression in cells from the Nkx2.1-lineage, which includes the PV and SST classes of cortical inhibitory interneurons (CINs) and glial subtypes. They find that embryonic expression of Erk1/2 regulates the number of Nkx2.1-derived oligodendrocytes and astrocytes, but not CINs, observed in postnatal mice. However, Erk1/2 is necessary for the expression of SST in subset of Nkx2.1-derived CINs, which can be partially rescued by postnatal depolarization via chemogenetic stimulation with DREADDs. Finally, loss of Erk1/2 from these cells impairs activity-dependent expression of FOSB. Collectively, this revised paper demonstrates differential roles of Erk1/2 for the development of glia and neurons. Furthermore, it suggests SST CINs may be particularly …
Reviewer #2 (Public Review):
Knowles et al. investigated the developmental roles of Erk1/2 expression in cells from the Nkx2.1-lineage, which includes the PV and SST classes of cortical inhibitory interneurons (CINs) and glial subtypes. They find that embryonic expression of Erk1/2 regulates the number of Nkx2.1-derived oligodendrocytes and astrocytes, but not CINs, observed in postnatal mice. However, Erk1/2 is necessary for the expression of SST in subset of Nkx2.1-derived CINs, which can be partially rescued by postnatal depolarization via chemogenetic stimulation with DREADDs. Finally, loss of Erk1/2 from these cells impairs activity-dependent expression of FOSB. Collectively, this revised paper demonstrates differential roles of Erk1/2 for the development of glia and neurons. Furthermore, it suggests SST CINs may be particularly vulnerable to loss of Erk1/2 signaling during both early embryonic and later postnatal developmental stages.
Strengths:
This paper uses multiple transgenic mouse lines to investigate the contributions of Erk1/2 loss and over-expression and MEK overexpression for interneuron and glial development. Furthermore, they consider how Erk1/2 signaling may evolve over the course of development from embryonic to postnatal juvenile and adult stages. Thus, they investigate Erk1/2's early role in cell differentiation and its later role in activity dependent signaling. This approach to studying gene function throughout development is important but not often attempted within a single study.The authors investigate Erk1/2 using several techniques, including immunohistochemistry, sequencing of translated genes using the Ribotag method, electrophysiology, and chemogenetic stimulation using DREADDs. Thus, they aim to apply a comprehensive battery of approaches to assay Erk1/2 signaling in Nkx2.1-derived cells throughout development.
Weaknesses:
This paper describes a series of mostly separate observations that are not directly linked. The mechanisms underlying their observations and the significance of the findings are often unclear.The authors use Erk1-/-; Erk2fl/wt; Nkx2.1Cre as "het" controls throughout the manuscript. However, there is no explanation for why this is a valid control except for a statement that they are "grossly intact", without elaboration. It is unclear why the authors did not use Nkx2.1Cre mice for their control. Figure 1 - Supplemental Figure 1 provides the only comparison between Erk1-/-; Erk2fl/wt; Nkx2.1Cre and Erk1-/-; Erk2wt/wt; Nkx2.1Cre mice. This figure shows a single example of immune staining for Erk2, but it is not obvious that Nkx2.1 control or "het control" cells even express Erk2 in this image. There is no quantification. Thus, their choice of control condition is not obviously appropriate.
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