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Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here,  Hintw  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  Hintw  in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&amp;E staining were used to detect the effects of  Hintw  on gonadal development of chicken embryos. Results showed that  Hintw  exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that  Foxl2 ,  Cyp19a1  in females were upregulated under the overexpression of  Hintw , while  Sox9  and  Dmrt1  were downregulated  Hintw . Overexpression of  Hintw  can promote the development of gonadal cortex, while interference with  Hintw  show the opposite result. Additionally, we found that overexpression of the  Hintw  in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with  Hintw  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.

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