Role and function of the <i>Hintw</i> in early sex differentiation in chicken (<i>Gallus gallus</i>) embryo

Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here, <i>Hintw</i> was identified as a potential key gene in sex-determination process in chickens via RNA-seq. Then we developed an effective method to interfere or overexpress <i>Hintw</i> in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&amp;E staining were used to detect the effects of <i>Hintw</i> on gonadal development of chicken embryos. Results showed that <i>Hintw</i> exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that <i>Foxl2</i>, <i>Cyp19a1</i> in females were upregulated under the overexpression of <i>Hintw</i>, while <i>Sox9</i> and <i>Dmrt1</i> were downregulated <i>Hintw</i>. Overexpression of <i>Hintw</i> can promote the development of gonadal cortex, while interference with <i>Hintw</i> show the opposite result. Additionally, we found that overexpression of the <i>Hintw</i> in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with <i>Hintw</i> in female chicken embryos decreased estrogen levels and increased androgen levels. In conclusion, this work sets the basis for the understanding of the molecular regulatory network for the sex-determination process in chicken embryos as well as providing the theoretical basis for mono-sex culturing of poultry.