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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.


Failure of subtype specification of the cells positioned ectopically in L2/3 by knockdown of Dcx.(A–J) Images shown in Figure 6A–M are presented at low magnification. (K, L) Images shown in Figure 6O and P are presented at low magnification. (M–O) Expression of PC20sh and Dcx_sh resulted in both phenotypes. Dcx_sh vector together with PC20sh and a GFP vector was electroporated into E14.0 brains, then, P7 brains were fixed and analyzed. The sections were counterstained with PI (magenta). The boxed region in N is shown at a higher magnification in O. The neurons were positioned in a random manner throughout the neocortex. In addition, some of them showed well-developed apical dendrites, a feature of Pcdh20-knockdown cells, suggesting that both RNAi vectors were effective. Scale bars, 100 µm (A, K); 200 µm (M); 50 µm (O).DOI:http://dx.doi.org/10.7554/eLife.10907.015
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fig6s1: Failure of subtype specification of the cells positioned ectopically in L2/3 by knockdown of Dcx.(A–J) Images shown in Figure 6A–M are presented at low magnification. (K, L) Images shown in Figure 6O and P are presented at low magnification. (M–O) Expression of PC20sh and Dcx_sh resulted in both phenotypes. Dcx_sh vector together with PC20sh and a GFP vector was electroporated into E14.0 brains, then, P7 brains were fixed and analyzed. The sections were counterstained with PI (magenta). The boxed region in N is shown at a higher magnification in O. The neurons were positioned in a random manner throughout the neocortex. In addition, some of them showed well-developed apical dendrites, a feature of Pcdh20-knockdown cells, suggesting that both RNAi vectors were effective. Scale bars, 100 µm (A, K); 200 µm (M); 50 µm (O).DOI:http://dx.doi.org/10.7554/eLife.10907.015

Mentions: The phenotypic consequences of Pcdh20 knockdown, disrupted neuronal positioning and change of layer identity, raise at least three possibilities. One is that the two phenotypes might arise independently, and the other two are that the disrupted neuronal positioning may affect the neuronal specification, or vice versa. To distinguish among these possibilities, we sought to disrupt the neuronal positioning of “future L4 neurons” using another method. We took advantage of knockdown of Doublecortin (Dcx), since knockdown of this gene in the mouse neocortex has been reported to cause random disruption of neuronal positioning (Baek et al., 2014; Ramos et al., 2006). We focused on the cells that came to reside in the superficial layers (L2–4) for the purpose of this study. The proportions of Rorb- as well as KCNH5-positive cells among the GFP-positive cells located in L2–4 were dramatically decreased by Dcx knockdown (Figure 6A–E; Figure 6—figure supplement 1A–D). Correspondingly, the proportions of Lhx2- as well as Satb2-positive cells were markedly increased by Dcx knockdown (Figure 6F–K; Figure 6—figure supplement 1E–H), implying the importance of cell positioning. On the other hand, no change in the proportion of Tbr1-positive cells was noted in association with Dcx knockdown (Figure 6L–N; Figure 6—figure supplement 1I,J), suggesting that Tbr1 expression may not be regulated simply by cell positioning. To investigate the relationship between subtype specification and the ultimate laminar positioning, we further examined the proportions of Rorb-positive cells among Dcx-knockdown neurons residing in L2/3 or L4. The majority of the neurons positioned in L4 expressed Rorb (72%), whereas none of the neurons in L2/3 expressed this marker (Figure 6B). These results suggest that the ultimate cell positioning determines most of the neuronal characteristics of the neurons labeled on E14.0, that is, into L2/3 or L4 neurons.10.7554/eLife.10907.014Figure 6.Failure of subtype specification of the cells positioned ectopically in L2/3 by knockdown of Dcx.


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)

Failure of subtype specification of the cells positioned ectopically in L2/3 by knockdown of Dcx.(A–J) Images shown in Figure 6A–M are presented at low magnification. (K, L) Images shown in Figure 6O and P are presented at low magnification. (M–O) Expression of PC20sh and Dcx_sh resulted in both phenotypes. Dcx_sh vector together with PC20sh and a GFP vector was electroporated into E14.0 brains, then, P7 brains were fixed and analyzed. The sections were counterstained with PI (magenta). The boxed region in N is shown at a higher magnification in O. The neurons were positioned in a random manner throughout the neocortex. In addition, some of them showed well-developed apical dendrites, a feature of Pcdh20-knockdown cells, suggesting that both RNAi vectors were effective. Scale bars, 100 µm (A, K); 200 µm (M); 50 µm (O).DOI:http://dx.doi.org/10.7554/eLife.10907.015
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fig6s1: Failure of subtype specification of the cells positioned ectopically in L2/3 by knockdown of Dcx.(A–J) Images shown in Figure 6A–M are presented at low magnification. (K, L) Images shown in Figure 6O and P are presented at low magnification. (M–O) Expression of PC20sh and Dcx_sh resulted in both phenotypes. Dcx_sh vector together with PC20sh and a GFP vector was electroporated into E14.0 brains, then, P7 brains were fixed and analyzed. The sections were counterstained with PI (magenta). The boxed region in N is shown at a higher magnification in O. The neurons were positioned in a random manner throughout the neocortex. In addition, some of them showed well-developed apical dendrites, a feature of Pcdh20-knockdown cells, suggesting that both RNAi vectors were effective. Scale bars, 100 µm (A, K); 200 µm (M); 50 µm (O).DOI:http://dx.doi.org/10.7554/eLife.10907.015
Mentions: The phenotypic consequences of Pcdh20 knockdown, disrupted neuronal positioning and change of layer identity, raise at least three possibilities. One is that the two phenotypes might arise independently, and the other two are that the disrupted neuronal positioning may affect the neuronal specification, or vice versa. To distinguish among these possibilities, we sought to disrupt the neuronal positioning of “future L4 neurons” using another method. We took advantage of knockdown of Doublecortin (Dcx), since knockdown of this gene in the mouse neocortex has been reported to cause random disruption of neuronal positioning (Baek et al., 2014; Ramos et al., 2006). We focused on the cells that came to reside in the superficial layers (L2–4) for the purpose of this study. The proportions of Rorb- as well as KCNH5-positive cells among the GFP-positive cells located in L2–4 were dramatically decreased by Dcx knockdown (Figure 6A–E; Figure 6—figure supplement 1A–D). Correspondingly, the proportions of Lhx2- as well as Satb2-positive cells were markedly increased by Dcx knockdown (Figure 6F–K; Figure 6—figure supplement 1E–H), implying the importance of cell positioning. On the other hand, no change in the proportion of Tbr1-positive cells was noted in association with Dcx knockdown (Figure 6L–N; Figure 6—figure supplement 1I,J), suggesting that Tbr1 expression may not be regulated simply by cell positioning. To investigate the relationship between subtype specification and the ultimate laminar positioning, we further examined the proportions of Rorb-positive cells among Dcx-knockdown neurons residing in L2/3 or L4. The majority of the neurons positioned in L4 expressed Rorb (72%), whereas none of the neurons in L2/3 expressed this marker (Figure 6B). These results suggest that the ultimate cell positioning determines most of the neuronal characteristics of the neurons labeled on E14.0, that is, into L2/3 or L4 neurons.10.7554/eLife.10907.014Figure 6.Failure of subtype specification of the cells positioned ectopically in L2/3 by knockdown of Dcx.

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.