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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.


Postmitotic function of Pcdh20 regulates the development of L4 neurons.(A) The experimental procedure to identify postmitotic cells from VZ cells. (B) The brains treated as in A were immunostained for BrdU, GFP and Rorb. Typical images of this experiment are shown. (C) The percentages of Rorb-positive cells among the GFP-positive and BrdU-negative cells were analyzed. The Pcdh20-knockdown cells showed loss of expression of Rorb even in a postmitotic population. (D) The percentages of BrdU-negative cells among GFP-positive cells are presented. The percentages of the BrdU-negative cells were not affected by the knockdown of Pcdh20. (E, F) PC20sh vector together with the empty vector (pTα1) or Tα1 promoter-driven resPcdh20 expression vector (pTα1-resPcdh20) was electroporated into E14.0 brains, and P7 brains were analyzed. Results of quantitative analyses of E are presented in F. *p = 0.0056; NS, no significance (p = 0.62). Scale bars, 20 µm (B); 200 µm (E).DOI:http://dx.doi.org/10.7554/eLife.10907.008
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fig3s2: Postmitotic function of Pcdh20 regulates the development of L4 neurons.(A) The experimental procedure to identify postmitotic cells from VZ cells. (B) The brains treated as in A were immunostained for BrdU, GFP and Rorb. Typical images of this experiment are shown. (C) The percentages of Rorb-positive cells among the GFP-positive and BrdU-negative cells were analyzed. The Pcdh20-knockdown cells showed loss of expression of Rorb even in a postmitotic population. (D) The percentages of BrdU-negative cells among GFP-positive cells are presented. The percentages of the BrdU-negative cells were not affected by the knockdown of Pcdh20. (E, F) PC20sh vector together with the empty vector (pTα1) or Tα1 promoter-driven resPcdh20 expression vector (pTα1-resPcdh20) was electroporated into E14.0 brains, and P7 brains were analyzed. Results of quantitative analyses of E are presented in F. *p = 0.0056; NS, no significance (p = 0.62). Scale bars, 20 µm (B); 200 µm (E).DOI:http://dx.doi.org/10.7554/eLife.10907.008

Mentions: The timing of terminal mitosis has been supposed to be critical for determination of the ultimate laminar fates (Dehay and Kennedy, 2007; Molyneaux et al., 2007). It is thus possible that misspecification of the neuronal laminar identity after Pcdh20 knockdown was caused as a result of additional or delayed cell divisions. To address this question and determine whether the laminar specification was indeed changed by Pcdh20 knockdown in the postmitotic cells, we performed electroporation of the shRNA and GFP vectors on E14.0 and subsequently labeled the entire population of mitotic cells by serial injections of BrdU every 5 hr for 20 hr immediately after the electroporation (Figure 3—figure supplement 2A). In this experiment, the population of GFP-positive and BrdU-negative cells (referred to as GFP+/BrdU– cells) was thought to represent postmitotic neurons or cells in the very late stages of the cell cycle in terminal mitosis in the VZ at the time of the electroporation, based on the following reasons. First, plasmid vectors are incorporated primarily into VZ cells by in utero electroporation. Second, since the duration of the S phase at this stage is about 4 hr, and since the nuclei in the S phase can be labeled for at least 2 hr after a single injection of BrdU (Takahashi et al., 1992), injections of BrdU at intervals of up to 6 hr are sufficient for labeling all the mitotic VZ cells. Finally, the total length of the cell cycle is about 15 hr, which is shorter than the total period of BrdU application (see ref. (Tabata et al., 2009) for more details). These GFP+/BrdU– cells are included in the “slowly exiting population”, a population that becomes postmitotic in the VZ and migrates out slowly into the multipolar cell accumulation zone above the VZ (Tabata et al., 2009).


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)

Postmitotic function of Pcdh20 regulates the development of L4 neurons.(A) The experimental procedure to identify postmitotic cells from VZ cells. (B) The brains treated as in A were immunostained for BrdU, GFP and Rorb. Typical images of this experiment are shown. (C) The percentages of Rorb-positive cells among the GFP-positive and BrdU-negative cells were analyzed. The Pcdh20-knockdown cells showed loss of expression of Rorb even in a postmitotic population. (D) The percentages of BrdU-negative cells among GFP-positive cells are presented. The percentages of the BrdU-negative cells were not affected by the knockdown of Pcdh20. (E, F) PC20sh vector together with the empty vector (pTα1) or Tα1 promoter-driven resPcdh20 expression vector (pTα1-resPcdh20) was electroporated into E14.0 brains, and P7 brains were analyzed. Results of quantitative analyses of E are presented in F. *p = 0.0056; NS, no significance (p = 0.62). Scale bars, 20 µm (B); 200 µm (E).DOI:http://dx.doi.org/10.7554/eLife.10907.008
© Copyright Policy
Related In: Results  -  Collection

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

fig3s2: Postmitotic function of Pcdh20 regulates the development of L4 neurons.(A) The experimental procedure to identify postmitotic cells from VZ cells. (B) The brains treated as in A were immunostained for BrdU, GFP and Rorb. Typical images of this experiment are shown. (C) The percentages of Rorb-positive cells among the GFP-positive and BrdU-negative cells were analyzed. The Pcdh20-knockdown cells showed loss of expression of Rorb even in a postmitotic population. (D) The percentages of BrdU-negative cells among GFP-positive cells are presented. The percentages of the BrdU-negative cells were not affected by the knockdown of Pcdh20. (E, F) PC20sh vector together with the empty vector (pTα1) or Tα1 promoter-driven resPcdh20 expression vector (pTα1-resPcdh20) was electroporated into E14.0 brains, and P7 brains were analyzed. Results of quantitative analyses of E are presented in F. *p = 0.0056; NS, no significance (p = 0.62). Scale bars, 20 µm (B); 200 µm (E).DOI:http://dx.doi.org/10.7554/eLife.10907.008
Mentions: The timing of terminal mitosis has been supposed to be critical for determination of the ultimate laminar fates (Dehay and Kennedy, 2007; Molyneaux et al., 2007). It is thus possible that misspecification of the neuronal laminar identity after Pcdh20 knockdown was caused as a result of additional or delayed cell divisions. To address this question and determine whether the laminar specification was indeed changed by Pcdh20 knockdown in the postmitotic cells, we performed electroporation of the shRNA and GFP vectors on E14.0 and subsequently labeled the entire population of mitotic cells by serial injections of BrdU every 5 hr for 20 hr immediately after the electroporation (Figure 3—figure supplement 2A). In this experiment, the population of GFP-positive and BrdU-negative cells (referred to as GFP+/BrdU– cells) was thought to represent postmitotic neurons or cells in the very late stages of the cell cycle in terminal mitosis in the VZ at the time of the electroporation, based on the following reasons. First, plasmid vectors are incorporated primarily into VZ cells by in utero electroporation. Second, since the duration of the S phase at this stage is about 4 hr, and since the nuclei in the S phase can be labeled for at least 2 hr after a single injection of BrdU (Takahashi et al., 1992), injections of BrdU at intervals of up to 6 hr are sufficient for labeling all the mitotic VZ cells. Finally, the total length of the cell cycle is about 15 hr, which is shorter than the total period of BrdU application (see ref. (Tabata et al., 2009) for more details). These GFP+/BrdU– cells are included in the “slowly exiting population”, a population that becomes postmitotic in the VZ and migrates out slowly into the multipolar cell accumulation zone above the VZ (Tabata et al., 2009).

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.