Functional Analysis of Hoxd13 in Limb Development and Regeneration in Axolotl
YAN Xiwen1#, LIU Ning2#, QIU Yuanhui2,3, LU Binbin4, FEI Jifeng1,2,3*
Hoxd13, a member of the Hox13 paralog group, is an important functional gene involved in the regulation of distal appendage patterning in vertebrates. This study aimed to investigate the expression characteristics of Hoxd13 during forelimb development and regeneration in the axolotl (Ambystoma mexicanum) and to further evaluate its role in these processes. Sequence alignment and section in situ hybridization showed that the HOXD13 homeodomain is highly conserved among vertebrates. Both Hoxd13 and Hoxa13 were expressed in the limb buds of axolotl larvae, with Hoxd13 exhibiting a broader distribution; in addition, distinct Hoxd13-positive signals were detected near the spinal cord and dorsal root ganglia in tail tissues. Moreover, in situ hybridization showed differential expression patterns of Hoxd13 and Hoxa13 during limb regeneration. To further assess the function of Hoxd13, three sgRNAs targeting Hoxd13 were designed for CRISPR/Cas9-mediated targeted editing. Screening results showed that sgRNA1 and sgRNA3 achieved relatively high editing efficiencies and generated relatively high proportions of high-KO-score F0 mosaic animals, accounting for 12.3% (9/73, KO score 86.0±4.8) and 14.3% (6/42, KO score 86.2±5.1), respectively. Forelimb developmental phenotyping and alcian blue-alizarinred S skeletal staining of high-knockdown F0 mosaics revealed no obvious abnormalities in overall forelimb morphology, digit number, or skeletal structure. Further tracking of post-amputation regeneration, together with endpoint skeletal staining, showed no evident defects in regeneration progression or skeletal patterning of regenerated forelimbs. KO scores in regenerated digit-tip tissues remained between 84 and 90, indicating that limb regeneration after Hoxd13 editing was unlikely to result from the competitive contribution of unedited or low-edited cells.
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