Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
The question addressed in this manuscript is of potential interest for neurodevelopmental biologists studying the role of molecular factors released from thalamocortical axons in the establishment of cortical layers. The work clarifies the contribution of distinct thalamic secretory proteins in cortical development. Overall, although the data are properly controlled and analyzed, there are several aspects of the manuscript that need to be addressed and improved to support the conclusions raised by the authors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (eLife)
Abstract
Neuronal abundance and thickness of each cortical layer are specific to each area, but how this fundamental feature arises during development remains poorly understood. While some of area-specific features are controlled by intrinsic cues such as morphogens and transcription factors, the exact influence and mechanisms of action by cues extrinsic to the cortex, in particular the thalamic axons, have not been fully established. Here, we identify a thalamus-derived factor, VGF, which is indispensable for thalamocortical axons to maintain the proper amount of layer 4 neurons in the mouse sensory cortices. This process is prerequisite for further maturation of the primary somatosensory area, such as barrel field formation instructed by a neuronal activity-dependent mechanism. Our results provide an actual case in which highly site-specific axon projection confers further regional complexity upon the target field through locally secreting signaling molecules from axon terminals.
Article activity feed
-
Evaluation Summary:
The question addressed in this manuscript is of potential interest for neurodevelopmental biologists studying the role of molecular factors released from thalamocortical axons in the establishment of cortical layers. The work clarifies the contribution of distinct thalamic secretory proteins in cortical development. Overall, although the data are properly controlled and analyzed, there are several aspects of the manuscript that need to be addressed and improved to support the conclusions raised by the authors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
Sato et al. investigated the role of the thalamus-derived factor, VGF, as extrinsic cue that controls layer 4 development in the cortex. They show that this process is necessary for further maturation of the primary somatosensory cortex (S1) and the barrel field formation. To explore the role of thalamocortical axon (TCA) projections in cortical layer formation, the authors developed a mouse model with TCA ablation from the ventrobasal (VB) thalamic nucleus by the administration of diphtheria toxin (DT) from P0. They induced a decrease in the VB nucleus size and the TCAs terminals were also diminished in layer 4 of S1. Sato et al. demonstrated that the number of layer 4 cells in S1 is reduced in TCA-ablated model and to verify that these TCA ablation from the VB was indeed responsible for this laminar …
Reviewer #1 (Public Review):
Sato et al. investigated the role of the thalamus-derived factor, VGF, as extrinsic cue that controls layer 4 development in the cortex. They show that this process is necessary for further maturation of the primary somatosensory cortex (S1) and the barrel field formation. To explore the role of thalamocortical axon (TCA) projections in cortical layer formation, the authors developed a mouse model with TCA ablation from the ventrobasal (VB) thalamic nucleus by the administration of diphtheria toxin (DT) from P0. They induced a decrease in the VB nucleus size and the TCAs terminals were also diminished in layer 4 of S1. Sato et al. demonstrated that the number of layer 4 cells in S1 is reduced in TCA-ablated model and to verify that these TCA ablation from the VB was indeed responsible for this laminar phenotype, they used a different approach to specifically ablate only the VB neurons. By performing a technically impressive in utero electroporation at e11.5 in the thalamus with a diphtheria toxin receptor (DTR) expression plasmid and then administered DT at P0, they mimicked the laminar phenotype in the cortex and the results were similar to the TCA-ablated mice. Moreover, they showed that, apparently, the rest of the cortical layers remain intact. Interestingly, the authors demonstrated that VGF and NRN1 as TCA-derived factors play an important role to maintain the layer 4 neuronal number during cortical development by restoring these cell number after the overexpression of these factors in TCA-ablated model in vivo. To further address this question, they induced a genetic inactivation of Vgf by using CRISPR/Cas9-mediated gene editing, and they proved that VGF is necessary for the maintenance of layer 4 neurons number.
The manuscript shows potentially interesting findings but there are some open questions and experiments that should be done to better support the conclusions of the paper. Moreover, some aspects of image acquisition and data analysis need to be clarified.
-
Reviewer #2 (Public Review):
Sato H. and colleagues here investigate the role of extrinsic factors in the development of the murine neocortex. It was previously shown by these authors that thalamocortical neurons, through the expression of Vgf and Nrn1 among others, contribute specifically to layer 4 neurons development in vitro. In the current study, by postnatally ablating thalamocortical projections and studying a Vgf knockout, they further investigate the role of these projections in layer 4 establishment and bring some light to the in vivo role of VGF.
Although this is an interesting study, the novelty is relatively limited as it incrementally builds on previous work from this laboratory as well as previous work from several laboratories directly addressing the effects of ablation of specific thalamic nuclei on cortical neuron …
Reviewer #2 (Public Review):
Sato H. and colleagues here investigate the role of extrinsic factors in the development of the murine neocortex. It was previously shown by these authors that thalamocortical neurons, through the expression of Vgf and Nrn1 among others, contribute specifically to layer 4 neurons development in vitro. In the current study, by postnatally ablating thalamocortical projections and studying a Vgf knockout, they further investigate the role of these projections in layer 4 establishment and bring some light to the in vivo role of VGF.
Although this is an interesting study, the novelty is relatively limited as it incrementally builds on previous work from this laboratory as well as previous work from several laboratories directly addressing the effects of ablation of specific thalamic nuclei on cortical neuron identity. In order to increase interest and relevance, several key experiments should be performed.
-
Reviewer #3 (Public Review):
The first extrinsic influences that shape the cortical neuroepithelium are secretory factors, emanating from signaling centers adjacent to the telencephalic vesicles. These centers set up the gross areal pattern of the neocortex without any extrinsic signals. While it is clear that there are intrinsic gradients from the beginning of cortical neurogenesis, there are also extrinsic cues that contribute to the differences. The best candidate to deliver the area-specific cues to the cortex is via area-specific thalamocortical projections. These arrive to the cortex very early, at the peak of the cortical neurogenesis and neuronal migration. The impact of thalamic lesions on cortical lamination was demonstrated by Windrem and Finlay, 1991. Moreover the influence of ephrin A5 on cortical progenitor cells and the …
Reviewer #3 (Public Review):
The first extrinsic influences that shape the cortical neuroepithelium are secretory factors, emanating from signaling centers adjacent to the telencephalic vesicles. These centers set up the gross areal pattern of the neocortex without any extrinsic signals. While it is clear that there are intrinsic gradients from the beginning of cortical neurogenesis, there are also extrinsic cues that contribute to the differences. The best candidate to deliver the area-specific cues to the cortex is via area-specific thalamocortical projections. These arrive to the cortex very early, at the peak of the cortical neurogenesis and neuronal migration. The impact of thalamic lesions on cortical lamination was demonstrated by Windrem and Finlay, 1991. Moreover the influence of ephrin A5 on cortical progenitor cells and the effect of secreted Wnt3 on neuronal differentiation has been previously described.
The Sato et al., paper studies two thalamus-derived factors that might mediate some of the area-specific differences to the cortex. The authors used the results of their previous screens to identify secreted molecules that are not produced in the cortex, but delivered to the cortex through the thalamocortical projections. The authors screened for thalamus-specific genes by comparing expression profiles of the thalamus and the cortex (Sato et al., 2012). These screens identified neuritin 1 (NRN1) and VGF nerve growth factor inducible (Vgf) transported to the cortex through TCAs. This study demonstrates that VGF maintains the proper amount of layer 4 neurons in S1.
The study is reported in a logical sequence. First the authors established that birthdates of all cortical layers in various cortical areas are all prenatal. Since all neurons were generated before birth, no NeuN positive birthdated cells were found after postnatal EdU injection and therefore it is unlikely that layer 4 neurons are additively generated in S1. The study also analysed the emergence of RORβ (RAR-related orphan receptor beta) expression in the postnatal primary somatosensory cortex.
Then, they performed toxin-mediated ventrobasal complex ablation in vivo. Cre expression was the highest in VB among the thalamic nuclei in 5HTT-Cre mice, therefore the lesion was the greatest there. This reduced the thalamocortical axons that project to primary somatosensory cortex. The ablation triggered accumulation of Iba1-immunoreactive microglial cells in VB. This indicated that these are the regions with dead cells, in the thalamus. After this ablation VB was reduced at P5, and RORα-expressing thalamic neurons were decreased.
The authors examined the possibility that other thalamic neurons project to the S1, but detailed tracing from S1 did not produce backlabeling pattern in the thalamus that would indicate TCA ingrowth from the dLGN or MG to S1. No such re-wiring was observed in the PO VB TCA-ablated mice.
The number of RORβ-expressing cortical cells was decreased to 67% as compared with control cortex. Moreover the absolute number of layer 4 neurons also decreased in the TCA-ablated S1. This argued against the possibilities of altered RORβ expression or fate change of the layer 4-destined cells to those of other layer. The authors suggested cell death as a possible mechanisms for getting these differences. However, all the conventional cell death detection methods (ssDNA, cleaved caspase 3, Iba1, mRNA of Bax, Bad, and Bak, and DAPI), they could not obtain convincing evidence for significant cell death induction in layer 4 upon TCA ablation.
The toxin-mediated ventrobasal complex ablation in vivo is based on cre expression. Since Cre expression was the highest in VB among the thalamic nuclei in 5HTT-Cre mice, this was the region for the greatest damage. Nevertheless there was additional cre expression in layer 6 and also in the raphe nucleus the authors designed experiments to exclude that the layer 4 reduction in 5HTT-Cre; R26-DTR mice is due to ablation of these brain parts rather than the VB in the thalamus. The authors used DTR expression plasmid that was electroporated into the embryonic dorsal thalamus in utero at E11.5, when VB neurons are generated. The authors demonstrated that the effect was S1 specific. The cortical areas with dLGN and MGN innervation were not affected. Layers 2/3 (revealed with Brn2), layer 5 (revealed with Ctip2) and layer 6 (revealed with Tbr1) appear intact upon TCA elimination suggesting that the effect was specific for layer 4. The number of layer 4 neurons is restored by forced expression of NRN1 and VGF in the cortex of TCA-ablated mice NRN1 and VGF was lost in the thalamic nuclei and their axon terminals in layer 4 of S1 in TCA-ablated mice
The next group of experiments demonstrated that genetic inactivation of Vgf in thalamocortical projection neurons results in a reduction in layer 4 neurons in S1 cortex and that NRN1 is dispensable in this process. The authors used three single-guide RNAs (sgRNAs) cutting exons of Nrn1 and Vgf to induce frame shifts resulting in failure of protein translation of both NRN1 and VGF. They electroporated the sgRNAs and Cas9 protein into fertilized eggs, mutations were induced in the genomic sequences of Nrn1 and Vgf allele near designed sgRNAs. The loss of TCA-derived VGF from the cortex resulted in the significant reduction of RORβ immunoreactive layer 4 cells in S1 and in V1. These observations suggest that the regulation of the neuronal number of layer 4 by TCAs via VGF is a common mechanism operating widely in sensory areas.
VGF released from TCA terminals sets the exact numbers of cortical layer 4 neurons. Then, the activity dependent sorting of thalamocortical afferents will impose the cytoarchitectonic changes that will form the cytoarchitectonic barrels. This interaction between thalamic and layer 4 neurons to form the cytoarchitectonic barrel formation in S1 was significantly impaired in Vgf-KO mice despite the presence of TCAs. The paper convincingly demonstrates that thalamocortical axons play instructive roles in the regulation of layer 4 cell numbers and the specification of area properties of somatosensory and visual cortices.
I consider this study a very important and significant step in our field. This is a direct demonstration of the instructive role of the thalamocortical projections. The major conclusions of the study will have to be supported by additional experiments (cell death - fate change distinction).
-
-
