Identification of Autophagy-Related Gene 7 and Autophagic Cell Death in the Planarian Dugesia japonica

Abstract

Planarians undergo continuous body size remodeling under starvation or during regeneration. This process likely involves autophagy and autophagic cell death, but this hypothesis is supported by few studies. To test this hypothesis, we cloned and characterized autophagy-related gene 7 (Atg7) from the planarian Dugesia japonica (DjAtg7). The full-length cDNA of DjAtg7 measures 2272 bp and includes a 2082-bp open reading frame encoding 693 amino acids with a molecular weight of 79.06 kDa. The deduced amino acid sequence of DjAtg7 contains a conserved ATP-binding site and a catalytic active site of an E1-like enzyme belonging to the ATG7 superfamily. DjAtg7 transcripts are mainly expressed in intestinal tissues of the intact animals. After amputation, DjAtg7 was highly expressed at the newly regenerated intestinal branch on days 3-7 of regeneration and in the old tissue of the distal intestinal branch on day 10 of regeneration. However, knockdown of DjAtg7 by RNAi did not affect planarian regeneration and did not block autophagosome formation, which indicates that autophagy is more complex than previously expected. Interestingly, TEM clearly confirmed that autophagy and autophagic cell death occurred in the old tissues of the newly regenerated planarians and clearly revealed that the dying cell released vesicles containing cellular cytoplasmic contents into the extracellular space. Therefore, the autophagy and autophagic cell death that occurred in the old tissue not only met the demand for body remodeling but also met the demand for energy supply during planarian regeneration. Collectively, our work contributes to the understanding of autophagy and autophagic cell death in planarian regeneration and body remodeling.

Keywords: DjAtg7; RNAi; autophagic cell death; autophagy; planarian; regeneration.

FIGURE 1

Multi-alignment of ATG7 homologs in invertebrate and vertebrate species. The highly conserved ATP-binding sites (GxGxxG) and catalytic active sites (CTxxxP) are boxed.

FIGURE 2

The phylogenetic tree of the ATG7 protein family, constructed with the neighbor-joining method. The number on each branch indicates the confidence percentage from a bootstrap test of 1000 pseudoreplicates. GenBank accession numbers are in brackets.

FIGURE 3

Expression pattern of DjAtg7 during planarian regeneration. (A) Colorimetric WISH in intact and regenerating animals. Intense staining signals are indicated by black arrows. For each time point, n = 10 animals. Scale bars: 1 mm. Colorimetric WISH was repeated three times. (B) Double FISH of DjAtg7 (red) and DjPk-1/DjWi-1 (green) with DAPI (blue). n = 6 animals. Scale bars: 200 μm.

FIGURE 4

Morphological features of autophagy and autophagic cell death during planarian body remodeling. (A) Capture of an intact, normal cell. (B–G) The morphological features of each stage of autophagy and autophagic cell death. (H) A vesicle being released to the outside of the cell through the plasma membrane. (I) A large vesicle containing cellular cytoplasmic contents being released to the outside of the cell. (J) Lower magnification showing many cells with the emptied cytoplasms. The white arrow indicates the autophagic vesicles. The double white arrow indicates autophagosomes with multiple membranes. The black arrow indicates the released vesicles containing cellular cytoplasmic contents. The star indicates the emptied cytoplasm. n, nucleus; Scale bar: 2 μm.

FIGURE 5

Effects of RNAi-DjAtg7 on planarian regeneration. The dashed brown line represents the border between blastema and old tissue. RNAi experiments were repeated three times. Scale bar: 0.5 mm.

FIGURE 6

Detection of DjAtg7 expression in RNAi-animals by WISH and qPCR. (A) the control animals, n = 10. (B) RNAi animals, n = 10. (C) qPCR shown are averages of three independent experiments; error bars = SEM, ^∗∗P < 0.01 (Student’s t-test). Asterisks indicate significant differences compared to the control. Samples were collected at day 10 of regeneration. Scale bar: 0.5 mm.

FIGURE 7

Autophagosome formation in RNAi-DjAtg7 animals. Six samples were collected at 7 and 10 days of regeneration for TEM. (A) The process of autophagosome formation: (1) single-vesicle structure → (2) double-vesicle structure → (3) multiple-vesicle structure → (4) early autophagosomes with multiple membranes → (5) late autophagosome with multiple membranes. The black box indicates the double-membrane vesicle. (B) Mitochondria sequestered into double-membrane vesicles. The double black arrow indicates mitochondria. The white arrow indicates the double-membrane. n: nucleus; scale bar: 2 μm.

FIGURE 8

qPCR showing the relative expression level of Atgs in RNAi-DjAtg7 animals. Shown are averages of three independent experiments; error bars = SEM, ^∗P < 0.05, ^∗∗P < 0.01 (Student’s t-test). Asterisks indicate significant differences compared to the control. Samples were collected at day 10 of regeneration.