Suppression of limb regeneration by RNA interference of WNT4 in the swimming crab Portunus trituberculatus

May 11, 2019Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology

Stopping limb regrowth by blocking WNT4 gene in the swimming crab

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Abstract

The PtWNT4 gene, encoding a 323 amino acid polypeptide, is significantly involved in limb regeneration stages of Portunus trituberculatus.

PtWNT4 was expressed during all regenerative limb stages, peaking in stage III before declining in stages IV and V.
Strong localization of PtWNT4 was observed in loose connective tissue during limb regeneration, with decreased protein levels as muscle fibers formed.
Injection of WNT4 dsRNA led to significant decreases in PtWNT4 mRNA levels, confirming effective gene silencing.
Knockdown of PtWNT4 expression resulted in notably slower limb regeneration rates compared to control groups.
Limb regeneration rates were measured as 0%, 31.5%, and 40.5% for the dsWNT4 group at 36 hours, 3 days, and 5 days, respectively, versus 29.95%, 83.0%, and 92.5% for controls.

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The functions of Wnt signalling in crab limb regeneration are poorly understood. Herein, we isolated and characterised the full-length cDNA of WNT4 from Portunus trituberculatus, designated PtWNT4. The 4831 bp cDNA encodes 323 amino acid polypeptide. Protein structure prediction showed that PtWNT4 has a conserved WNT domain. The PtWNT4 gene was expressed in all regenerative limb stages, and was upregulated from stage I, with highest expression in stage III, and expression then declined in stages IV and V. Immunohistochemistry revealed strong localisation at loose connective tissue during limb regeneration, but PtWNT4 protein levels decreased upon formation of muscle fibers. Injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels from 36 h to 5 days after injection, indicating successful gene silencing. RNA interference knockdown of PtWNT4 expression greatly retarded limb regeneration compared with the control group. Blastema emergence phenotype analysis revealed limb regeneration rates of 0, 31.5% and 40.5% for the dsWNT4 group at 36 h, 3 days and 5 days, respectively, compared with 29.95%, 83.0% and 92.5% for the saline-injected control groups (p <0.05). Expression analysis on the WNT4 using RNAi provide important information for understanding its functional mechanism during limb regeneration in P. trituberculatus. The results also contribute to our understanding of the role of Wnt signalling during limb regeneration in crustaceans.

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Suppression of limb regeneration by RNA interference of WNT4 in the swimming crab Portunus trituberculatus

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