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Identity of neocortical layer 4 neurons is specified through correct positioning into the cortex.

Oishi K, Nakagawa N, Tachikawa K, Sasaki S, Aramaki M, Hirano S, Yamamoto N, Yoshimura Y, Nakajima K - Elife (2016)

Bottom Line: Moreover, restoration of positioning of the Pcdh20-knockdown neurons into L4 rescued the specification failure.We further suggest that the thalamocortical axons provide a positional cue to specify L4 identity.These results suggest that the L4 identity is not completely determined at the time of birth but ensured by the surrounding environment after appropriate positioning.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Keio University School of Medicine, Tokyo, Japan.

ABSTRACT
Many cell-intrinsic mechanisms have been shown to regulate neuronal subtype specification in the mammalian neocortex. However, how much cell environment is crucial for subtype determination still remained unclear. Here, we show that knockdown of Protocadherin20 (Pcdh20), which is expressed in post-migratory neurons of layer 4 (L4) lineage, caused the cells to localize in L2/3. The ectopically positioned "future L4 neurons" lost their L4 characteristics but acquired L2/3 characteristics. Knockdown of a cytoskeletal protein in the future L4 neurons, which caused random disruption of positioning, also showed that those accidentally located in L4 acquired the L4 characteristics. Moreover, restoration of positioning of the Pcdh20-knockdown neurons into L4 rescued the specification failure. We further suggest that the thalamocortical axons provide a positional cue to specify L4 identity. These results suggest that the L4 identity is not completely determined at the time of birth but ensured by the surrounding environment after appropriate positioning.

No MeSH data available.


Pcdh20 knockdown resulted in “future L4 neurons” acquiring L2/3 characteristics.(A–J) Images shown in Figure 3A–M are presented at low magnification. (K, L) CONsh or PC20sh vector together with a GFP vector was electroporated into E14.0 brains, then, P7 brains were fixed and analyzed. The sections were counterstained with DAPI (blue) and immunostained for NetrinG1 (red). (M–T) CONsh or PC20sh vector together with a GFP vector was electroporated into E14.0 brains, then, E18.0 (M–P) and P2 (Q–T) brains were analyzed. The sections were immunostained for Rorb and Lhx2. Scale bars, 100 µm (A, K); 50 µm (M, Q).DOI:http://dx.doi.org/10.7554/eLife.10907.007
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fig3s1: Pcdh20 knockdown resulted in “future L4 neurons” acquiring L2/3 characteristics.(A–J) Images shown in Figure 3A–M are presented at low magnification. (K, L) CONsh or PC20sh vector together with a GFP vector was electroporated into E14.0 brains, then, P7 brains were fixed and analyzed. The sections were counterstained with DAPI (blue) and immunostained for NetrinG1 (red). (M–T) CONsh or PC20sh vector together with a GFP vector was electroporated into E14.0 brains, then, E18.0 (M–P) and P2 (Q–T) brains were analyzed. The sections were immunostained for Rorb and Lhx2. Scale bars, 100 µm (A, K); 50 µm (M, Q).DOI:http://dx.doi.org/10.7554/eLife.10907.007

Mentions: We next characterized these ectopic “future L4 neurons” malpositioned in L2/3 using several subtype-specific molecular markers. First, we examined the expression of Rorb, a well-known L4 marker in the mature neocortex (Nakagawa and O'Leary, 2003). Immunohistochemical analysis of the P7 brains showed that while most of the GFP-positive cells in the control samples expressed Rorb, the percentage of Rorb-positive cells among the GFP-positive cells was markedly decreased by knockdown of Pcdh20 (Figure 3A–C; Figure 3—figure supplement 1A,B). Expression of KCNH5 (a potassium voltage-gated channel, subfamily H), another marker of L4 neurons, was also not observed in the ectopically located GFP-positive cells by Pcdh20 knockdown (Figure 3D,E; Figure 3—figure supplement 1C,D). Moreover, immunostaining for NetrinG1, a marker of thalamocortical axons (TCAs), showed that the malpositioned neurons by Pcdh20 knockdown did not grossly change the growth of TCAs (Figure 3—figure supplement 1K,L). These results suggest that the malpositioned Pcdh20-knockdown neurons lost the characteristics of L4 neurons.


Identity of neocortical layer 4 neurons is specified through correct positioning into the cortex.

Oishi K, Nakagawa N, Tachikawa K, Sasaki S, Aramaki M, Hirano S, Yamamoto N, Yoshimura Y, Nakajima K - Elife (2016)

Pcdh20 knockdown resulted in “future L4 neurons” acquiring L2/3 characteristics.(A–J) Images shown in Figure 3A–M are presented at low magnification. (K, L) CONsh or PC20sh vector together with a GFP vector was electroporated into E14.0 brains, then, P7 brains were fixed and analyzed. The sections were counterstained with DAPI (blue) and immunostained for NetrinG1 (red). (M–T) CONsh or PC20sh vector together with a GFP vector was electroporated into E14.0 brains, then, E18.0 (M–P) and P2 (Q–T) brains were analyzed. The sections were immunostained for Rorb and Lhx2. Scale bars, 100 µm (A, K); 50 µm (M, Q).DOI:http://dx.doi.org/10.7554/eLife.10907.007
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764574&req=5

fig3s1: Pcdh20 knockdown resulted in “future L4 neurons” acquiring L2/3 characteristics.(A–J) Images shown in Figure 3A–M are presented at low magnification. (K, L) CONsh or PC20sh vector together with a GFP vector was electroporated into E14.0 brains, then, P7 brains were fixed and analyzed. The sections were counterstained with DAPI (blue) and immunostained for NetrinG1 (red). (M–T) CONsh or PC20sh vector together with a GFP vector was electroporated into E14.0 brains, then, E18.0 (M–P) and P2 (Q–T) brains were analyzed. The sections were immunostained for Rorb and Lhx2. Scale bars, 100 µm (A, K); 50 µm (M, Q).DOI:http://dx.doi.org/10.7554/eLife.10907.007
Mentions: We next characterized these ectopic “future L4 neurons” malpositioned in L2/3 using several subtype-specific molecular markers. First, we examined the expression of Rorb, a well-known L4 marker in the mature neocortex (Nakagawa and O'Leary, 2003). Immunohistochemical analysis of the P7 brains showed that while most of the GFP-positive cells in the control samples expressed Rorb, the percentage of Rorb-positive cells among the GFP-positive cells was markedly decreased by knockdown of Pcdh20 (Figure 3A–C; Figure 3—figure supplement 1A,B). Expression of KCNH5 (a potassium voltage-gated channel, subfamily H), another marker of L4 neurons, was also not observed in the ectopically located GFP-positive cells by Pcdh20 knockdown (Figure 3D,E; Figure 3—figure supplement 1C,D). Moreover, immunostaining for NetrinG1, a marker of thalamocortical axons (TCAs), showed that the malpositioned neurons by Pcdh20 knockdown did not grossly change the growth of TCAs (Figure 3—figure supplement 1K,L). These results suggest that the malpositioned Pcdh20-knockdown neurons lost the characteristics of L4 neurons.

Bottom Line: Moreover, restoration of positioning of the Pcdh20-knockdown neurons into L4 rescued the specification failure.We further suggest that the thalamocortical axons provide a positional cue to specify L4 identity.These results suggest that the L4 identity is not completely determined at the time of birth but ensured by the surrounding environment after appropriate positioning.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Keio University School of Medicine, Tokyo, Japan.

ABSTRACT
Many cell-intrinsic mechanisms have been shown to regulate neuronal subtype specification in the mammalian neocortex. However, how much cell environment is crucial for subtype determination still remained unclear. Here, we show that knockdown of Protocadherin20 (Pcdh20), which is expressed in post-migratory neurons of layer 4 (L4) lineage, caused the cells to localize in L2/3. The ectopically positioned "future L4 neurons" lost their L4 characteristics but acquired L2/3 characteristics. Knockdown of a cytoskeletal protein in the future L4 neurons, which caused random disruption of positioning, also showed that those accidentally located in L4 acquired the L4 characteristics. Moreover, restoration of positioning of the Pcdh20-knockdown neurons into L4 rescued the specification failure. We further suggest that the thalamocortical axons provide a positional cue to specify L4 identity. These results suggest that the L4 identity is not completely determined at the time of birth but ensured by the surrounding environment after appropriate positioning.

No MeSH data available.