Genetic evidence against a role for W-linked histidine triad nucleotide binding protein (HINTW) in avian sex determination

Abstract

Birds have a ZZ/ZW sex chromosome system, but the mechanism of sex determination remains unknown. The heterogametic sex is female (ZW) and one hypothesis holds that the W chromosome carries a dominant-acting ovary-determining gene. The strongest candidate ovary-determinant on the W chromosome is HINTW, which encodes an aberrant nucleotide hydrolase enzyme. HINTW is conserved amongst all carinate (flying) birds and it is strongly expressed in the gonads and other tissues of female chicken embryos. This and other lines of circumstantial evidence support the proposal that HINTW is the female-determining gene in birds. However, in vivo gain-of-function or loss-of-function studies have not hitherto been reported to test this hypothesis. We tested the potential role of HINTW by mis-expressing it in genetically male (ZZ) embryos, using the RCASBP avian retroviral vector. Strong, widespread expression was delivered throughout the embryo, including the urogenital system, as assessed by whole mount in situ hybridisation. This expression pattern mimicked that seen in normal ZW females, in which HINTW is widely expressed. Strong mis-expression was observed throughout the gonads of genetic male (ZZ) embryos. However, despite strong HINTW expression, ZZ gonads developed normally as bilateral testes. In tissue sections of ZZ urogenital systems transgenic for HINTW, normal testicular histology was observed. Female (ZW) gonads over-expressing HINTW also developed normally, with normal ovarian structure and left/right asymmetry. These results provide genetic evidence against a dominant role for HINTW in avian sex determination.

Rowley Review

“…In vitro enzyme data support a dominant negative influence of HINTW on HINTZ enzymatic activity (Parks et al 2004). However, the HINTW gene is apparently absent in ratites, and overexpression in early Chicken embryos does not induce ovarian development, undermining this gene as a candidate female determinant (Smith et al 2009b). At present, therefore, there is little evidence in support of the dominant W hypothesis for avian sex determination.…”

Left-Right Asymmetry in Chicken Embryonic Gonads

“…At an early stage, DMRT1 (Raymond et al, 1999;Oreal et al, 2002;Smith et al, 2003;Yamamoto et al, 2003;Koba et al, 2008;Smith et al, 2009b;Yang et al, 2013) and Sox9 (Kent et al, 1996;Morais da Silva et al, 1996;Yamamoto et al, 2003;Takada et al, 2006) are expressed in male (ZZ) embryos, whereas HINTW (Smith, 2007;Smith et al, 2009a), FET1 (Reed and Sinclair, 2002), FOXL2 (Hudson et al, 2005b) and aromatase Nakabayashi et al, 1998;Yamamoto et al, 2003) are expressed in female (ZW) embryos.…”

<b><i>SMARCE1</i></b> Promotes Chicken Embryonic<b> </b>Gonad Development by Regulating<b><i> ER</i></b>α and <b><i>AR </i></b>Expression

“…Viral titer was assessed by adding serial dilutions (10 -3 -10 -8 ) of concentrated virus to the fresh DF-1 cells as described previously [Logan and Tabin, 1998;Smith et al, 2009b]. DF-1 cells were infected with RCASBP.B-SMARCE1 virus and harvested several days later.…”

“…Among them, DMRT1 was found to be required for testis determination in chicken [Smith et al, 2009a], while PKCIW did not play a dominant role in avian sex determination [Smith et al, 2009b]. The last candidate FET1 was once considered to be a unique ovary-determining gene because no counterpart on the Z chromosome was reported, although our experiments (unpublished result) found several homologs on the Z chromosome which had an identity of 91% with FET1 , which indicated that FET1 might not be so unique in avian sex determination.…”