Potential role of TGFΒ and autophagy in early crebellum development

Abstract

During development, the interconnected generation of various neural cell types within the cerebellar primordium is essential. Over embryonic (E) days E9-E13, Purkinje cells (PCs), and cerebellar nuclei (CN) neurons are among the created primordial neurons. The molecular and cellular mechanisms fundamental for the early cerebellar neurogenesis, migration/differentiation, and connectivity are not clear yet. Autophagy has a vital role in controlling cellular phenotypes, such as epithelial-to-mesenchymal transition (EMT) and endothelial to mesenchymal transition (EndMT). Transforming growth factor-beta 1 (TGF-β1) is the main player in pre-and postnatal development and controlling cellular morphological type via various mechanisms, such as autophagy. Thus, we hypothesized that TGF-β1 may regulate early cerebellar development by modifying the levels of cell adhesion molecules (CAMs) and consequently autophagy pathway in the mouse cerebellar primordium. We demonstrated the stimulation of the canonical TGF-β1 signaling pathway at the point that concurs with the generation of the nuclear transitory zone and PC plate in mice. Furthermore, our data show that the stimulated TGF-β1 signaling pathway progressively and chronologically could upregulate the expression of β-catenin (CTNNB1) and N-cadherin (CDH2) with the most expression at E11 and E12, leading to upregulation of chromodomain helicase DNA binding protein 8 (CDH8) and neural cell adhesion molecule 1 (NCAM1) expression, at E12 and E13. Finally, we demonstrated that the stimulated TGF-β signaling pathway may impede the autophagic flux at E11/E12. Nevertheless, basal autophagy flux happens at earlier developmental phases from E9-E10. Our study determined potential role of the TGF-β signaling and its regulatory impacts on autophagic flux during cerebellar development and cadherin expression, which can facilitate the proliferation, migration/differentiation, and placement of PCs and the CN neurons in their designated areas.

Keywords: Autophagy; Autophagy flux; Cerebellum early development; Transforming growth factor-beta.

Fig. 1

Workflow from embryonic cerebellar primordium sample collection, processing and analysis.

Fig. 2

Active Tgfbs and Smad signaling pathway elements changes in cerebellar primordium during early developmental stages at E9-E13 (n = 3). A, B) Tgfb1 highly expressed during earliest embryonic days at E9 and E10. Its expression levels decreased significantly with considerable differences from E11 to E13 (A). Tgfb2 values experienced almost the same expression pattern as Tgfb1, with higher and lower amounts at E9-E10 and E11-E13, respectively. However, the changing expression pattern of Tgfb2 is not statistically significant (B). C, D) mRNA expression of Tgfbr1 and Tgfbr2 were measured by RT-qPCR. Tgfbr1 transcription at E9 is higher with a significant reduction at E12 (C). mRNA values of Tgfbr2 maintained at the same level with no significant changes from E9- E13 (D) (The significance of the receptor expression was compared to E9 and the relative expression was compared to β-actin (universal gene control). E-G) Downregulation of total Smad2 & 3 and confirmation of Smad2 and Smad3 protein phosphorylation in E12 and E13. Anti-total Smad2 & 3 reactive bands in Western blot analyses (E) and quantification of total Smad2 &3; Total Smad2 and 3 values upregulated at E9 and E10, compared to their correspondent controls, with a non-significant reduction from E11 to E13 (F). Anti-phosphorylated Smad2 and Smad 3 showed its phosphorylation in E12 and E13 (G). The blots were quantified compared to the TGF-β1 treated cells using densitometry software Alpha Ease FC. The protein loading was confirmed using B-actin. The data in the bar graphs are presented as the mean ± SEM, and statistical analysis was performed using one-way ANOVA (P-Value > 0.05, considered as non significant, P-value ≤ 0.05 and lower were considered statistically significant. ** shows P < 0.01, **** shows P < 0.0001). H-M) Distribution of the Tgfb1 & 2, Tgfbr1 & 2, and Smad2 & 3 in the cerebellar primordium at E11.5, and E13.5. All images show cerebellar primordia and image credit: Allen Institute. © 2008 Allen Institute for Brain Science, Allen Developing Mouse Brain Atlas. Available online at: https://developingmouse.brain-map.org/. H) Tgfb1 was only detected at E11.5 in pia mater of the cerebellar primordium and very low level in the neuroepithelium and tela choroidea. I) ISH patterns of Tgfb2 on sections at E11.5, and E13.5 were not detectable. J) Sagittal sections show dynamic expression of Tgfbr1 express at both E11.5 and E13.5 which is detected in nuclear transitory zone (NTZ) at E11.5 with higher levels of expression in the NTZ and Purkinje cell palate at E13.5. K) Tgfbr2 expression is not detected at any given time points. L) Smad2 expression is strongly high throughout the whole cerebellar primordium at E11.5. No expression is detected at E13.5. M) Smad3 has moderate expression in the cerebellar primordium mainly in the Purkinje cell plate and rhombic lip of the cerebellar primordium at E11.5, but not at E13.5.

Fig. 2

Active Tgfbs and Smad signaling pathway elements changes in cerebellar primordium during early developmental stages at E9-E13 (n = 3). A, B) Tgfb1 highly expressed during earliest embryonic days at E9 and E10. Its expression levels decreased significantly with considerable differences from E11 to E13 (A). Tgfb2 values experienced almost the same expression pattern as Tgfb1, with higher and lower amounts at E9-E10 and E11-E13, respectively. However, the changing expression pattern of Tgfb2 is not statistically significant (B). C, D) mRNA expression of Tgfbr1 and Tgfbr2 were measured by RT-qPCR. Tgfbr1 transcription at E9 is higher with a significant reduction at E12 (C). mRNA values of Tgfbr2 maintained at the same level with no significant changes from E9- E13 (D) (The significance of the receptor expression was compared to E9 and the relative expression was compared to β-actin (universal gene control). E-G) Downregulation of total Smad2 & 3 and confirmation of Smad2 and Smad3 protein phosphorylation in E12 and E13. Anti-total Smad2 & 3 reactive bands in Western blot analyses (E) and quantification of total Smad2 &3; Total Smad2 and 3 values upregulated at E9 and E10, compared to their correspondent controls, with a non-significant reduction from E11 to E13 (F). Anti-phosphorylated Smad2 and Smad 3 showed its phosphorylation in E12 and E13 (G). The blots were quantified compared to the TGF-β1 treated cells using densitometry software Alpha Ease FC. The protein loading was confirmed using B-actin. The data in the bar graphs are presented as the mean ± SEM, and statistical analysis was performed using one-way ANOVA (P-Value > 0.05, considered as non significant, P-value ≤ 0.05 and lower were considered statistically significant. ** shows P < 0.01, **** shows P < 0.0001). H-M) Distribution of the Tgfb1 & 2, Tgfbr1 & 2, and Smad2 & 3 in the cerebellar primordium at E11.5, and E13.5. All images show cerebellar primordia and image credit: Allen Institute. © 2008 Allen Institute for Brain Science, Allen Developing Mouse Brain Atlas. Available online at: https://developingmouse.brain-map.org/. H) Tgfb1 was only detected at E11.5 in pia mater of the cerebellar primordium and very low level in the neuroepithelium and tela choroidea. I) ISH patterns of Tgfb2 on sections at E11.5, and E13.5 were not detectable. J) Sagittal sections show dynamic expression of Tgfbr1 express at both E11.5 and E13.5 which is detected in nuclear transitory zone (NTZ) at E11.5 with higher levels of expression in the NTZ and Purkinje cell palate at E13.5. K) Tgfbr2 expression is not detected at any given time points. L) Smad2 expression is strongly high throughout the whole cerebellar primordium at E11.5. No expression is detected at E13.5. M) Smad3 has moderate expression in the cerebellar primordium mainly in the Purkinje cell plate and rhombic lip of the cerebellar primordium at E11.5, but not at E13.5.

Fig. 3

Cadherins expression are upregulated in cerebellar primordium during early developmental stages at E9-E13. A-H) Expression levels of Cdh2, Ctnnb1, Cdh8 and Ncam1 proteins measured by Western blotting in mice cerebellar primordium at E9-E13 (n = 3). The protein loading was confirmed using β-actin. A, B) Anti-Cdh2 reactive bands in Western blot analyses and quantification of Cdh2; Protein expression is upregulated from E9 to E11 and reached its higher amount at E11, which is statistically significant. C, D) Western blot analyses and quantification of Ctnnb1; protein expression remained unchanged with no significant differences between embryonic days. E, F) Cdh8; protein blots show no expression during the earliest embryonic days at E9-E11. Cdh8 protein expression is upregulated from E12 to E13 and reached its higher amount at E13, which is statistically significant. G, H) Data show no Ncam1 expression during the earliest embryonic days at E9-E11. Ncam1 protein expression is upregulated from E12 to E13 and reached its higher amount at E13 which is statistically significant. I-L) mRNA expression levels of Cdh2, Ctnnb1, Cdh8 and Ncam1 measured by RT-qPCR in cerebellar primordium during early developmental stages at E9-E13 (n = 3). I) Cdh2 mRNA expression remained unchanged with constant level of expression during the earliest embryonic days at E9-E13. J) The trend for Ctnnb1 mRNA values is negative; transcription at E9 is higher with significant reduction at E11 and E12. K) Cdh8 mRNA expression level experienced an upward trend from E9 to E13, with lower expression levels at E9/E10, which is significantly increased at E13. L) Ncam1 mRNA levels increased from E9 to E13 with lower and higher levels of expression at E9 and E13, respectively. The data in the bar graphs are presented as the mean ± SEM, and statistical analysis was performed using one-way ANOVA (P-value ≤ 0.05 and lower were considered statistically significant. * shows P < 0.05, ** shows P < 0.01, *** shows P < 0.001, **** shows P < 0.0001). M-P) Distribution of the Cdh2, Ctnnb1, Cdh8 and Ncam1in the cerebellar primordium at E11.5, and E13.5. All images show cerebellar primordia and image credit: Allen Institute. © 2008 Allen Institute for Brain Science, Allen Developing Mouse Brain Atlas. Available online at: https://developingmouse.brain-map.org/. M) Cdh2 ISH data shows constant expression of Cdh2 mRNA at E11.5, and E13.5, with condense pattern in the nucmear transitory zone and Purkinje cell plate (mostly in Foxp2⁺ cells) at E13.5. N) ISH data shows enrich expression of Ctnnb1 in the entire cerebellar primordium at E11.5 which is localized in the Purkinje cell plate at E13.5 (less in nuclear transitory zone). O) Cdh8 mRNA is not detected at E11.5 but is detectable in caudal part of the Purkinje cell plate at E13.5. P) Ncam1 ISH data shows strong expression pattern in the nuclear transitory zone of the cerebellar primordium at E11.5, and both nuclear transitory zone and Purkinje cell plate at E13.5.

Fig. 3

Cadherins expression are upregulated in cerebellar primordium during early developmental stages at E9-E13. A-H) Expression levels of Cdh2, Ctnnb1, Cdh8 and Ncam1 proteins measured by Western blotting in mice cerebellar primordium at E9-E13 (n = 3). The protein loading was confirmed using β-actin. A, B) Anti-Cdh2 reactive bands in Western blot analyses and quantification of Cdh2; Protein expression is upregulated from E9 to E11 and reached its higher amount at E11, which is statistically significant. C, D) Western blot analyses and quantification of Ctnnb1; protein expression remained unchanged with no significant differences between embryonic days. E, F) Cdh8; protein blots show no expression during the earliest embryonic days at E9-E11. Cdh8 protein expression is upregulated from E12 to E13 and reached its higher amount at E13, which is statistically significant. G, H) Data show no Ncam1 expression during the earliest embryonic days at E9-E11. Ncam1 protein expression is upregulated from E12 to E13 and reached its higher amount at E13 which is statistically significant. I-L) mRNA expression levels of Cdh2, Ctnnb1, Cdh8 and Ncam1 measured by RT-qPCR in cerebellar primordium during early developmental stages at E9-E13 (n = 3). I) Cdh2 mRNA expression remained unchanged with constant level of expression during the earliest embryonic days at E9-E13. J) The trend for Ctnnb1 mRNA values is negative; transcription at E9 is higher with significant reduction at E11 and E12. K) Cdh8 mRNA expression level experienced an upward trend from E9 to E13, with lower expression levels at E9/E10, which is significantly increased at E13. L) Ncam1 mRNA levels increased from E9 to E13 with lower and higher levels of expression at E9 and E13, respectively. The data in the bar graphs are presented as the mean ± SEM, and statistical analysis was performed using one-way ANOVA (P-value ≤ 0.05 and lower were considered statistically significant. * shows P < 0.05, ** shows P < 0.01, *** shows P < 0.001, **** shows P < 0.0001). M-P) Distribution of the Cdh2, Ctnnb1, Cdh8 and Ncam1in the cerebellar primordium at E11.5, and E13.5. All images show cerebellar primordia and image credit: Allen Institute. © 2008 Allen Institute for Brain Science, Allen Developing Mouse Brain Atlas. Available online at: https://developingmouse.brain-map.org/. M) Cdh2 ISH data shows constant expression of Cdh2 mRNA at E11.5, and E13.5, with condense pattern in the nucmear transitory zone and Purkinje cell plate (mostly in Foxp2⁺ cells) at E13.5. N) ISH data shows enrich expression of Ctnnb1 in the entire cerebellar primordium at E11.5 which is localized in the Purkinje cell plate at E13.5 (less in nuclear transitory zone). O) Cdh8 mRNA is not detected at E11.5 but is detectable in caudal part of the Purkinje cell plate at E13.5. P) Ncam1 ISH data shows strong expression pattern in the nuclear transitory zone of the cerebellar primordium at E11.5, and both nuclear transitory zone and Purkinje cell plate at E13.5.

Fig. 4

Autophagic-flux is potentially inhibited in cerebellar primordium during early developmental stages at E9-E13. A-E) LC3β I & II and p62 protein expression levels measured by Western blotting in cerebellar primordium at E9-E13. A) Anti-LC3β-I & II reactive bands in Western blot analyses and B) quantification of LC3β-I & II; protein expression levels increase by time; however, LC3β-II value is lower than LC3β-I at each embryonic day. C) Quantification of LC3β-II/LC3β-I ratio increase gradually from E9 to E13, and the values reached the highest levels at E12 and E13, which is statistically significant compared to E9. D) Anti-P62 reactive bands in Western blot analyses and E) quantification of P62; protein expression levels experienced an upward trend from E9 to E12, followed by a rapid reduction at E13. The P62 value is statistically significant at E11 and E12 compared to E9. The blots were quantified using densitometry software Alpha Ease FC. The protein loading was confirmed using β-actin. Data in the bar graphs are presented as the mean ± SEM, and statistical analysis was performed using one-way ANOVA (P-value ≤ 0.05 and lower were considered statistically significant. * shows P < 0.05, ** shows P < 0.01).

Fig. 5

The regulatory roles of Tgfb1 in formation of the cerebellar primordium during early developmental stages at E9-E13. Based on the neurogenesis timing of the cerebellum, the only postmitotic neural populations that exist in the cerebellar primordium between E9 to E13 are the Purkinje cells and cerebellar nuclei neurons located at the Purkinje cell plate and nuclear transitory zone, respectively. Expression of active Tgfb1 is intensely high at earliest embryonic days at both E9 and E10, followed by a substantial reduction in its value from E11 to E13. Our study shows that Tgfb1 through the canonical TGF-β signaling pathway could upregulate the expression of CAMs. On the other hand, activation of the Tgf-β signaling pathway and its consequent reduction in available Tgfb1 amounts, autophagic-flux, which the Tgf-β1 regularly induces, is inhibited from E9 to E13. A) Activation of the Smad-dependent Tgf-β1 signaling pathway in the mouse cerebellar primordium at E9-E13; Our finding showed the presence of main components of the canonical Tgf-β signaling pathway, including Tgfbr1, Tgfbr2and cytoplasmic total and phosphorylated Smad signaling molecules during the given embryonic period. B) Tgfb1 upregulates the expression of Cadherins in the mouse cerebellar primordium at E9-E13; Tgfb1 by upregulating the expression of cell adhesion proteins, including cdh2, Cdh8, Ncam, and cadherin binding protein β-catenin (Ctnnb1), contribute to the physical interactions and connectivity between postmitotic Purkinje cells and cerebellar nuclei neurons, locating in the Purkinje cell plate and nuclear transitory zone during earliest developmental stages from E9 to E13 with a peak at E11. These data show the direct association between upregulation of both Cdh2 and Ctnnb1, Cdh8 and Ncam upon activation of the Tgf-β signaling pathway, correlated to time points during which Purkinje cells and cerebellar nuclei neurons are born, migrating and positioning in cerebellar primordium. C) Tgfb1 inhibits autophagic-flux in the mouse cerebellar primordium at E9-E13; Tgfb1, by regulating the autophagic-flux, responds to the extracellular signals such as stress (starvation, hypoxia, aggregation of unwanted materials) to maintain cellular homeostasis and provide neural protection during cerebellar development. The results from our study pinpoint a potential regulatory role of autophagy inhibition by Smad-dependent Tgf-β signaling pathway in the formation of the Purkinje cell plate and accumulation of cerebellar nuclei neurons in nuclear transitory zone during early cerebellar development.