The vertebrate limb serves as an experimental paradigm to study mechanisms that regulate development of the stereotypical skeletal elements. In this study, we simultaneously inactivated Sall4 using Hoxb6Cre and Plzf in mouse embryos, and found that their combined function regulates development of the proximal-anterior skeletal elements in hindlimbs. The Sall4; Plzf double knockout exhibits severe defects in the femur, tibia, and anterior digits, distinct defects compared to other allelic series of Sall4; Plzf. We found that Sall4 regulates Plzf expression prior to hindlimb outgrowth. Further expression analysis indicated that Hox10 genes and GLI3 are severely downregulated in the Sall4; Plzf double knockout hindlimb bud. In contrast, PLZF expression is reduced but detectable in Sall4; Gli3 double knockout limb buds, and SALL4 is expressed in the Plzf; Gli3 double knockout limb buds. These results indicate that Plzf, Gli3, and Hox10 genes downstream of Sall4, regulate femur and tibia development. In the autopod, we show that Sall4 negatively regulates Hedgehog signaling, which allows for development of the most anterior digit. Collectively, our study illustrates genetic systems that regulate development of the proximal-anterior skeletal elements in hindlimbs.
Bibliographical noteFunding Information:
We are grateful to Eric Feil, Jennifer Kim, Cailin McMahon, Aditi Soni, Justin Wang, and Julia Wong for their excellent technical assistance. We are also grateful to Drs. Maria Barna, Mario Capecchi, C-c Hui, Juan Carlos Izpisua Bel-monte, and Licia Selleri for providing in situ probes; Drs. Mark Lewandoski and Yasushi Nakagawa for mouse lines; Dr. Michael O’Connor for the use of the Zeiss LSM710; and Dr. Naoyuki Wada for critical reading of the manuscript. K.Q.C. and A.A. were partially supported by the University of Minnesota’s Undergraduate Research Opportunity Program. This study was supported by a grant from the National Institutes of Health to Y.K. (R01AR064195). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing or financial interests.
Copyright © 2020 by the Genetics Society of America
- Proximal-anterior skeleton
- Sonic Hedgehog-Gli3