Abstract
SoxB subfamily is an important branch of Sox family and plays a key role in animal physiological process, but little is known about their function in planarian regeneration. This study aims to evaluate the function of DjSoxB family genes in intact and regenerating planarians Dugesia japonica. Here, we amplify the full-length cDNA of DjSoxB1 and DjSoxB2 in D. japonica by rapid amplification of the cDNA ends (RACE), detect the expression of DjSoxB family genes in planarian. The results show that DjSoxBs are expressed in parenchymal tissue and the hybridization signals partially disappear after irradiation indicates DjSoxB family genes are expressed in neoblasts. After the RNA interference (RNAi) of DjSoxB1, DjSoxB2 and DjSoxB3 separately, the numbers of proliferative cells are all reduced that causes planarians show slower growth of blastema in the early stage of regeneration, and nerves of planarians are affected that the movement speed of planarians decreases in varying degrees. Specially, planarians in the DjSoxB3 RNAi group show shrinkage and twisting. Overall, this study reveals that DjSoxB family genes play a role in cell proliferation during regeneration. They also play an important role in the maintenance of normal nerve function and nerve regeneration. These results provide directions for the functional study of SoxB family genes and provide an important foundation for planarian regeneration.
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Availability of data and materials
The data sets generated for this study are available from the GenBank of NCBI. Accession no. MK945596.2 (https://www.ncbi.nlm.nih.gov/nuccore/MK945596.2), MK945597.1 (https://www.ncbi.nlm.nih.gov/nuccore/MK945597.1).
Abbreviations
- AGAT-2:
-
L-arginine:glycine amidinotransferase -2
- ANOVA:
-
One-way analysis of variance
- AP:
-
Alkaline phosphatase
- Chat:
-
Choline O-acetyltransferase
- DEPC:
-
Diethyl pyrocarbonate
- dpa:
-
Day post amputation
- dsRNA:
-
Double-stranded RNA
- ef2:
-
Elongation factor-2
- H3P:
-
Anti-phospho-Histone H3 (Ser10)
- HMG-box:
-
High-mobility group box
- IPTG:
-
Isopropyl β-D-Thiogalactoside
- LSD:
-
Least significant difference
- NBT/BCIP:
-
Nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate
- NJ:
-
Neighbor-joining
- OD:
-
Optical density
- otx:
-
Homeobox protein OTX
- PC2:
-
Prohormone convertase 2
- pcna:
-
Proliferating cell nuclear antigen
- piwiA:
-
Piwi-like protein A
- qPCR:
-
Real-time quantitative PCR
- RACE:
-
Rapid amplification of the cDNA ends
- RNAi:
-
RNA interference
- tbh:
-
Tyramine-beta hydroxylase
- th:
-
Tyrosine hydroxylase
- TUNEL:
-
TdT-mediated dUTP nick end labeling
- WISH:
-
Whole-mount in situ hybridization
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant numbers: 32070427, 32270501, 32200376), the Major Public Welfare project of Henan Province (grant number: 201300311700), and by the Puyang Field Scientific Observation and Research Station for Yellow River Wetland Ecosystem, Henan Province.
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Yibo Yang: Formal analysis, Writing—Original Draft, Visualization. Ziyi Lin: Validation. Nannan Li: Investigation. Ning Li: Writing—Review & Editing, Funding acquisition. Dezeng Liu: Writing—Review & Editing. Zimei Dong: Conceptualization, Methodology, Resources, Writing—Review & Editing, Supervision, Funding acquisition. Guangwen Chen: Conceptualization, Methodology, Resources, Writing—Review & Editing, Project administration, Funding acquisition.
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The authors declare no conflicts of interest.
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All handling procedures were strictly compliant with the current Animal Protection Law of China. This study did not involve endangered or protected species. No approvals were required for collections of specimens from the locations in this study. Ethical approvals are not required at Henan Normal University for research conducted on planarians in this study.
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438_2024_2142_MOESM3_ESM.tif
Supplementary file3 Figure S1 a Flowchart of feeding and amputation time. b Amputation scheme. c Detection of DjSoxBs RNAi effect. d Morphology of intact worms after DjSoxB1 and DjSoxB2 RNAi separately. e Diagram of motion test device. f Brain size was quantified by measuring the brain area (mm2) and normalized to the body area (mm2) of the whole planarian. g Effects of DjSoxBs RNAi on nerve during regeneration of head fragments. * (p < 0.05) and ** (p < 0.01) respectively indicated significant difference and highly significant difference comparing with control group. Scale bar: 200 μm. dpa, day post amputation (TIF 13321 KB)
438_2024_2142_MOESM4_ESM.tif
Supplementary file4 Figure S2 Expression pattern prediction of DjSoxBs homologous genes in S. mediterranea. NBs, neoblasts. Ne, nerve. Mus, muscle (TIF 23863 KB)
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Yang, Y., Lin, Z., Li, N. et al. SoxB family genes delay regeneration and cause abnormal movement in Dugesia japonica. Mol Genet Genomics 299, 53 (2024). https://doi.org/10.1007/s00438-024-02142-5
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DOI: https://doi.org/10.1007/s00438-024-02142-5