Evolutionary Turnover in Wnt Gene Expression but Conservation of Wnt Signaling during Ovary Determination in a TSD Reptile

Abstract

Temperature-dependent sex determination (TSD) is a well-known characteristic of many reptilian species. However, the molecular processes linking ambient temperature to determination of gonad fate remain hazy. Here, we test the hypothesis that Wnt expression and signaling differ between female- and male-producing temperatures in the snapping turtle <i>Chelydra serpentina</i>. Canonical Wnt signaling involves secretion of glycoproteins called WNTs, which bind to and activate membrane bound receptors that trigger β-catenin stabilization and translocation to the nucleus where β-catenin interacts with TCF/LEF transcription factors to regulate expression of Wnt targets. Non-canonical Wnt signaling occurs via 2 pathways that are independent of β-catenin: one involves intracellular calcium release (the Wnt/Ca<sup>2+</sup> pathway), while the other involves activation of RAC1, JNK, and RHOA (the Wnt/planar cell polarity pathway). We screened 20 Wnt genes for differential expression between female- and male-producing temperatures during sex determination in the snapping turtle. Exposure of embryos to the female-producing temperature decreased expression of 7 Wnt genes but increased expression of 2 Wnt genes and <i>Rspo1</i> relative to embryos at the male-producing temperature. Temperature also regulated expression of putative Wnt target genes in vivo and a canonical Wnt reporter (6x TCF/LEF sites drive H2B-GFP expression) in embryonic gonadal cells in vitro. Results indicate that Wnt signaling was higher at the female- than at the male-producing temperature. Evolutionary analyses of all 20 Wnt genes revealed that thermosensitive Wnts, as opposed to insensitive Wnts, were less likely to show evidence of positive selection and experienced stronger purifying selection within TSD species.

Characterization and expression pattern of Wnt5b gene in Pelodiscus sinensis

“…An increasing number of researchers recognize that Wnt signalling is essential for early embryonic development. Recently, some studies have found that the expression of two Wnt target genes, Axin2 and Osr1, were upregulated in snapping turtle embryos at a female-producing temperature (Rhen et al, 2021) Cooper et al, 2007). The research found that overexpression of the Wnt5 gene has been reported to inactivate canonical Wnt signalling, it also inhibited the downstream target genes of this pathway in granulosa cells (Wu et al, 2017).…”

“…An increasing number of researchers recognize that Wnt signalling is essential for early embryonic development. Recently, some studies have found that the expression of two Wnt target genes, Axin2 and Osr1, were upregulated in snapping turtle embryos at a female‐producing temperature (Rhen et al, 2021), indicating that Wnt signalling seem essential for turtle embryogenesis. Wnt agonists, as potent modulators of the Wnt/β‐catenin signalling pathway, have been employed in embryos of P .…”

“…For example, in red‐eared slider turtles, Wnt signalling activates β‐catenin, which has been shown to repress the transcription of Sox9, potentially influencing sexual determination (Mork & Capel, 2013). Besides, the canonical Wnt2 ligands are expressed in embryonic gonads and display sexually dimorphic expression in snapping turtle and red‐eared slider turtle (Rhen et al, 2021). Similarly, Rspo1, a canonical Wnt signalling ligand, is also sex‐determining genes in mammals, which is necessary for primary female sexual differentiation in P .…”

“…. Besides, the canonical Wnt2 ligands are expressed in embryonic gonads and display sexually dimorphic expression in snapping turtle and red-eared slider turtle (Rhen et al, 2021). Similarly, Rspo1, a canonical Wnt signalling ligand, is also sex-determining genes in mammals, which is necessary for primary female sexual differentiation in P. sinensis (zhang et al, 2021).…”

Becoming female: Ovarian differentiation from an evolutionary perspective

“…OSR1 is expressed in differentiated chicken pre-granulosa cells, colocalizing with FOXL2 ( Estermann et al, 2020 ). OSR1 is also significantly enriched in Muscovy duck embryonic ovaries ( Bai et al, 2020 ), and in snapping turtle ( Chelydra serpentina ) embryonic gonads at the female-producing temperature ( Rhen et al, 2021 ). In Xenopus and zebrafish, Osr1 was shown to control kidney development, but its contribution to gonadal development remains unexplored ( Tena et al, 2007 ).…”