HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway

Yingjie Fu; Xueyan Zhang; Haibin Wu; Pingping Zhang; Shoupei Liu; Tingting Guo; Huanhuan Shan; Yan Liang; Honglin Chen; Jinghe Xie; Yuyou Duan

doi:10.3389/fimmu.2023.1258074

HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway

Front Immunol. 2024 Jan 8:14:1258074. doi: 10.3389/fimmu.2023.1258074. eCollection 2023.

Authors

Yingjie Fu^#^{1

2}, Xueyan Zhang^#^{1

2}, Haibin Wu^{1

2}, Pingping Zhang³, Shoupei Liu^{1

2}, Tingting Guo^{1

2}, Huanhuan Shan^{1

2}, Yan Liang^{1

2}, Honglin Chen^{2

4}, Jinghe Xie^{2

5}, Yuyou Duan^{1

2

6

7}

Affiliations

¹ Laboratory of Stem Cells and Translational Medicine, Institute for Clinical Medicine, the Second Affiliation Hospital, School of Medicine, South China University of Technology, Guangzhou, China.
² Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou, China.
³ Department of Laboratory Medicine, the Second Affiliation Hospital, School of Medicine, South China University of Technology, Guangzhou, China.
⁴ Medical Research Institute, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, China.
⁵ School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou, China.
⁶ National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, China.
⁷ The Innovation Centre of Ministry of Education for Development and Diseases, the Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou, China.

^# Contributed equally.

Abstract

Objectives: Normal commitment of the endoderm of the third pharyngeal pouch (3PP) is essential for the development and differentiation of the thymus. The aim of this study was to investigate the role of transcription factor HOXA3 in the development and differentiation of 3PP endoderm (3PPE) from human embryonic stem cells (hESCs).

Methods: The 3PPE was differentiated from hESC-derived definitive endoderm (DE) by mimicking developmental queues with Activin A, WNT3A, retinoic acid and BMP4. The function of 3PPE was assessed by further differentiating into functional thymic epithelial cells (TECs). The effect of HOXA3 inhibition on cells of 3PPE was subsequently investigated.

Results: A highly efficient approach for differentiating 3PPE cells was developed and these cells expressed 3PPE related genes HOXA3, SIX1, PAX9 as well as EpCAM. 3PPE cells had a strong potential to develop into TECs which expressed both cortical TEC markers K8 and CD205, and medullary TEC markers K5 and AIRE, and also promoted the development and maturation of T cells. More importantly, transcription factor HOXA3 not only regulated the differentiation of 3PPE, but also had a crucial role for the proliferation and migration of 3PPE cells. Our further investigation revealed that HOXA3 controlled the commitment and function of 3PPE through the regulation of Wnt signaling pathway by activating EPHB2.

Conclusion: Our results demonstrated that HOXA3 functioned as the on-off switch to regulate the development of hESC-derived 3PPE through EPHB2-mediated Wnt pathway, and our findings will provide new insights into studying the development of 3PP and thymic organ in vitro and in vivo.

Keywords: EPHB2; HOXA3; Wnt; human embryonic stem cell differentiation; third pharyngeal pouch; thymus.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Endoderm
Genes, Homeobox
Homeodomain Proteins* / genetics
Human Embryonic Stem Cells*
Humans
Transcription Factors
Wnt Signaling Pathway*

Substances

Homeodomain Proteins
HOXA3 protein, human
SIX1 protein, human
Transcription Factors

Associated data

GEO/GSE237751

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported in part by the National Key Research and Development Program of China (2018YFA0108200, 2018YFA0108201), by Research Starting Funding of South China University of Technology (D6201880, K5180910, K5204120, and D6212440), by Research Starting Funding of the Second Affiliation Hospital of South China University of Technology (KY09060026), by Research Agreement between South China University of Technology and Guangzhou First People’s Hospital (D9194290), by Guangzhou Basic and Applied Basic Research Foundation (202201010538), and by Guangzhou Postdoctoral Research Support (L2230330).