Supplementary Material for: Characterization of DMRT1 variants for testis determination and differentiation in emu

Introduction: DMRT1 on the Z chromosome is a conserved male sex-determining gene in birds. In chickens, a representative model species of Neognathae, the function of DMRT1 has been well characterized. In contrast, Palaeognathae species such as the emu possess less differentiated sex chromosomes and thus provide a valuable system for investigating avian sex determination, yet molecular studies remain limited. We investigated the timing of sex determination and the expression of key genes involved in gonadal differentiation in emu, and further characterized DMRT1 variants. Methods: Sex determination stage was identified by anatomical comparison of male and female embryonic gonads. Expression of seven genes (DMRT1, AMH, SOX9, NR5A1, FOXL2, CYP19A1, and RSPO1) was examined by mRNA-seq and RT-PCR. DMRT1 splicing variants were predicted by in silico analysis and 3’ RACE was used to identify alternative polyadenylation (APA) variants. Results: The gonadal differentiation occurred at HH25–28 based on gonadal morphology. Gene expression analysis revealed emu-specific patterns not observed in chickens. Notably, RSPO1 was highly expressed in females at HH24–25, preceding DMRT1 expression in males at HH28–29, suggesting ovarian differentiation begins earlier. We identified three splicing variants and four APA variants of DMRT1, with variant 1 predominant during gonadal development. Conclusion: These findings suggest that while molecular sex differentiation mechanisms are largely conserved between Palaeognathae and Neognathae, they differ in parts. In particular, early RSPO1 expression may initiate ovarian differentiation prior to testis determination by DMRT1. The presence of emu-specific DMRT1 variants further indicates possible species-specific mechanisms in testis development.

引言：Z染色体上的DMRT1是鸟类中保守的雄性性别决定基因。作为新颚类（Neognathae）的代表性模式物种，家鸡的DMRT1功能已得到充分解析。与之相对，鸸鹋等古颚类（Palaeognathae）物种的性染色体分化程度较低，因此为鸟类性别决定研究提供了极具价值的实验体系，但相关分子层面的研究仍较为有限。本研究以鸸鹋为研究对象，探究了其性别决定的时序以及性腺分化相关关键基因的表达情况，并进一步解析了DMRT1的可变剪接变体。
方法：通过对雌雄胚胎性腺进行解剖学比较，确定了性别决定的关键阶段。采用mRNA测序（mRNA-seq）和逆转录聚合酶链式反应（RT-PCR）检测了7个基因（DMRT1、AMH、SOX9、NR5A1、FOXL2、CYP19A1及RSPO1）的表达水平。通过计算机模拟分析预测了DMRT1的剪接变体，并利用3'末端快速扩增（3' RACE）技术鉴定了可变多聚腺苷酸化（APA）变体。
结果：根据性腺形态学特征，性腺分化发生在HH25~28阶段。基因表达分析揭示了鸸鹋特有的表达模式，该模式在家鸡中并未被观察到。值得注意的是，RSPO1在HH24~25阶段的雌性个体中呈高表达，早于雄性DMRT1在HH28~29阶段的表达，这表明卵巢分化的启动时间更早。本研究共鉴定出DMRT1的3种剪接变体和4种APA变体，其中变体1在性腺发育过程中占主导地位。
结论：本研究结果表明，尽管古颚类与新颚类的分子性别分化机制整体上较为保守，但仍存在部分差异。具体而言，早期RSPO1的表达可能在DMRT1介导的睾丸决定之前启动卵巢分化。鸸鹋特异性DMRT1变体的存在进一步提示，睾丸发育过程中可能存在物种特异性的调控机制。