Suppressive regulatory T cells (Treg cells) play a vital role in preventing autoimmunity and restraining excessive immune response to both self- and non-self-antigens. Studies on humans and mice show that the Forkhead box p3 (Foxp3) is a key regulatory gene for the development and function of Treg cells. In zebrafish, Treg cells have been identified by using foxp3a as a reliable marker. However, little is known about the function of foxp3a and Treg cells in gonadal development and sex differentiation. Here, we show that foxp3a is essential for maintaining immune homeostasis in zebrafish testis development. We found that foxp3a was specifically expressed in a subset of T cells in zebrafish testis, while knockout of foxp3a led to deficiency of foxp3a-positive Treg cells in the testis. More than 80% of foxp3a^–/– mutants developed as subfertile males, and the rest of the mutants developed as fertile females with decreased ovulation. Further study revealed that foxp3a^–/– mutants had a delayed juvenile ovary-to-testis transition in definite males and sex reversal in about half of the definite females, which led to a dominance of later male development. Owing to the absence of foxp3a-positive Treg cells in the differentiating testis of foxp3a^–/– mutants, abundant T cells and macrophages expand to disrupt an immunosuppressive milieu, resulting in defective development of germ cells and gonadal somatic cells and leading to development of infertile males. Therefore, our study reveals that foxp3a-positive Treg cells play an essential role in the orchestration of gonadal development and sex differentiation in zebrafish.

抑制性调节性T细胞（Treg细胞）在预防自身免疫和抑制对自身和非自身抗原的过度免疫反应中起着至关重要的作用。对人类和小鼠的研究表明，叉头盒p3（Foxp3）是Treg细胞发育和功能的关键调控基因。在斑马鱼中，已经通过使用foxp3a作为可靠的标记物鉴定了Treg细胞。然而，关于foxp3a和Treg细胞在性腺发育和性别分化中的功能知之甚少。在这里，我们显示foxp3a对于维持斑马鱼睾丸发育中的免疫稳态是必不可少的。我们发现foxp3a在斑马鱼睾丸的T细胞亚群中特异性表达，而敲除foxp3a导致睾丸中foxp3a阳性Treg细胞缺乏。超过80％的foxp3a ^{– / –}突变体发育为不育雄性，其余突变体发育为可育雌性，排卵减少。进一步的研究表明，foxp3a ^{– / –}突变体在确定的男性中延迟了从卵巢到睾丸的转变，并且在大约一半的确定的女性中发生了性逆转，从而导致后来的男性发育占主导地位。由于在分化的foxp3a睾丸中没有foxp3a阳性Treg细胞^{– / –}突变体，丰富的T细胞和巨噬细胞会扩展以破坏免疫抑制环境，从而导致生殖细胞和性腺体细胞发育不良，并导致不育男性发育。因此，我们的研究表明，foxp3a阳性Treg细胞在斑马鱼性腺发育和性别分化的编排中起着至关重要的作用。