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Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo

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Abstract

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7+ neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7+ and HNK-1+ cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1+ neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU+ neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

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Acknowledgements

We thank Maurice van den Hoff for the BMP4 plasmid. This study was supported by NSFC grant (81571436, 31401230), Science and Technology Planning Project of Guangdong Province (2016B030229002), Science and Technology Program of Guangzhou (201710010054, 201510010073), Guangdong Natural Science Foundation (2016A030311044), Research Grant of Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University (Nos. ZSYX-M-00001 and ZSYX-T-00001), Fund for Science and Technology Innovation of Guangdong College Student (pdjh 2017b060) and Students Research Training Program Fund (CX16014, 16112011).

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Authors and Affiliations

  1. Division of Histology & Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, Medical College, Jinan University, Guangzhou, 510632, China

    Guang Wang, En-ni Chen, Chang Liang, Jianxin Liang, Lin-rui Gao & Xuesong Yang

  2. Chinese Medicine College, Jinan University, Guangzhou, 510632, China

    Guang Wang & Jianxin Liang

  3. Division of Cell and Developmental Biology, University of Dundee, Dundee, DD1 5EH, UK

    Manli Chuai

  4. School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

    Andrea Münsterberg

  5. Norwich Medical School, University of East Anglia, Norwich, Norfolk, NR4 7UQ, UK

    Yongping Bao

  6. Key Laboratory of Medical Cell Biology, China Medical University, Shenyang, 110001, China

    Liu Cao

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  1. Guang Wang
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  2. En-ni Chen
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  3. Chang Liang
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Contributions

G.W., E.C., S.C., C.L., J.L., and L.G. performed the experiments and collected the data; G.W., M.C., A.M., L.C., and X. Y. designed the study and analyzed the data; and Y.B. and X.Y. wrote the manuscript.

Corresponding authors

Correspondence to Liu Cao or Xuesong Yang.

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The authors declare no competing interests.

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Guang Wang, En-ni Chen, and Chang Liang contribute to the work equally.

Electronic supplementary material

Fig. S1

Atg7 expression following the over-expression of Atg7 on neural tubes. Half side of HH10 chick neural tubes was transfected with FL-Atg7, and then the transfected embryos were further incubated for 10 h before Atg7 immunofluorescent staining was implemented and sectioned. A-D: The representative transverse sections of DAPI staining (A), autofluorescence (B), Atg7 staining (C) and the merge (D) of A and C. Scale bars = 50 μm in A-D. (GIF 131 kb)

High resolution image (TIFF 1764 kb)

Fig. S2

Assessment of HNK-1 + neural crest production following the over-expression of Atg7 on neural tubes. Half side of HH10 chick neural tubes was transfected with FL-Atg7 + GFP, and then the transfected embryos were further incubated for 10 h before HNK-1 immunofluorescent staining was implemented and sectioned. A-C: The representative transverse sections of FL-Atg7 + GFP transfection (A), HNK-1 staining (B) and the merge (C) of A and B at cranial level. A1-C1: The high magnification images from the sites indicated by dotted squares in A-C respectively. D-F: The representative transverse sections of FL-Atg7 + GFP transfection (D), HNK-1 staining (E) and the merge (F) of D and E at cardiac level. D1-F1: The high magnification images from the sites indicated by dotted squares in D-F respectively. G-H: The bar charts show the comparisons of the HNK-1+ areas between the transfected side and opposite control side. Abbreviations: NT, neural tube; HT, heart tube. Scale bars = 100 μm in A-F and 50 μm in A1-F1. (GIF 158 kb)

High resolution image (TIFF 2537 kb)

Fig. S3

Polarization and protrusion formation were changed following the up-regulation of Atg7 on NCCs. After transfection of neural tubes with either FL-Atg7 plasmids, the neural tubes were isolated and cultured to allow the NCCs to migrate out of the tubes. A-D: The representative images of cells, in which were stained with DAPI (A), Atg7 (B), F-actin (C), and (D) was merged image. E-F: Bar charts showing the ratio of long axis to short axis (E) and the numbers of the antennas (F). Scale bars = 20 μm in A-D. (GIF 78 kb)

High resolution image (TIFF 1262 kb)

Fig. S4

Assessment of HNK-1 + and Pax7 + neural crest production following the down-regulation of Atg7 on neural tubes. Half side of HH10 chick neural tubes was transfected with Mo-Atg7 + GFP, and then the transfected embryos were further incubated for 10 h before HNK-1 or Pax7 immunofluorescent staining was implemented and sectioned. A-C: The representative transverse sections of Mo-Atg7 + GFP transfection (A), Pax7 staining (B) and the merge (C) of A and B. DAPI stains in C. D-F: The representative transverse sections of Mo-Atg7 + GFP transfection (D), HNK-1 staining (E) and the merge (F) of D and E. DAPI stains in F. G-H: The bar charts show the comparisons of the Pax7+ cell numbers (G) and HNK-1+ areas (H) between the transfected side and opposite control side. Abbreviations: Mo-Atg7, morpholino-Atg7. Scale bars = 50 μm in A-F. (GIF 86 kb)

High resolution image (TIFF 1122 kb)

Fig. S5

Over-expressing Atg7 and Atg8 increased the numbers of BrdU positive cells at neural tubes. Half side of HH10 chick neural tubes was transfected with either Control GFP, FL-Atg7 + GFP or FL-Atg8-GFP, and then the transfected embryos were further incubated for 10 h before BrdU immunofluorescent staining was implemented. A-C: The representative transverse sections of GFP transfection (A), BrdU staining (B) and merge (C) of A and B from Control-GFP transfected chick embryo group. D-F: The representative transverse sections of FL-Atg7 + GFP transfection (D), BrdU staining (E) and merge (F) of D and E from FL-Atg7 + GFP transfected chick embryo group. G-I: The representative transverse sections of FL-Atg8-GFP transfection (G), BrdU staining (H) and merge (I) of G and H from FL-Atg8-GFP transfected chick embryo group. J-K: The bar chart shows the ratios of BrdU+ neural crest cell number in transfected side and BrdU+ neural crest cell numbers in opposite control side between control and FL-Atg7 + GFP or FL-Atg8-GFP transfection groups. Scale bars = 20 μm in A-I. (GIF 123 kb)

High resolution image (TIFF 1269 kb)

Fig. S6

Assessment of Atg8 expression following the over-regulation of Atg8 on neural tubes. Half side of HH10 chick neural tubes was transfected with either Control GFP or FL-Atg8-GFP, and then the transfected embryos were further incubated for 10 h before Atg8 immunofluorescent staining was implemented and sectioned. A-C: The representative transverse sections of Control-GFP transfection (A), Atg8 staining (B) and the merge (C) of A and B from Control-GFP transfected embryos. DAPI stains in C. D-F: The representative transverse sections of FL-Atg8-GFP transfection (D), Atg8 staining (E) and the merge (F) of D and E from FL-Atg8-GFP transfected embryos. DAPI stains in F. Scale bars = 50 μm in A-F. (GIF 192 kb)

High resolution image (TIFF 2927 kb)

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Wang, G., Chen, En., Liang, C. et al. Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo. Mol Neurobiol 55, 3523–3536 (2018). https://doi.org/10.1007/s12035-017-0583-6

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  • DOI: https://doi.org/10.1007/s12035-017-0583-6

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