Elsevier

Differentiation

Volume 112, March–April 2020, Pages 1-6
Differentiation

A novel long non-coding RNA, Leat1, causes reduced anogenital distance and fertility in female mice

https://doi.org/10.1016/j.diff.2019.10.007Get rights and content

Abstract

Defective anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. Only a few molecular cues have been identified in early formation of the female urogenital system. Here we describe a novel long non-coding RNA molecule known as Leat1 (long non-coding RNA, EphrinB2 associated transcript 1). This lncRNA is syntenic with EfnB2 (which encodes EphrinB2) and expressed during embryonic development of the genital tubercle. While lncRNAs have varied functions, many are known to regulate their neighbouring genes. Eph/Ephrin bidirectional signaling molecules mediate many patterning pathways in early embryonic development, including cloacal septation and urethral development. Here we investigate the role of Leat1 and its possible regulation of EphrinB2 during development of the female reproductive tract. We show that a loss of Leat1 leads to reduced EfnB2 expression in the developing female genital tubercle, reduced anogenital distance and decreased fertility.

Introduction

Development of female genitalia occurs through the differentiation of the genital tubercle (GT) in conjunction with cloacal septation (Perriton et al., 2002; Runck et al., 2014). Defective cloacal development and resulting anorectal and urogenital malformations are some of the most severe congenital anomalies in children (Gupta et al., 2014). However, the mechanisms that govern anogenital development are poorly understood, particularly in females (He et al., 2016).

In mammals, the anorectal and urogenital systems arise from a common transient embryonic structure called the cloaca, present between E10.5–12.5 in the mouse (Seifert et al., 2008). In females, the embryonic cloaca is subsequently divided (septated), first into the ventral urogenital sinus and separate dorsal hindgut, and then into three distinct openings – the anal canal, vaginal and urethral openings (Warne et al., 2011). The molecular and morphogenetic mechanisms that pattern and subdivide the embryonic cloaca are still unclear. Several mechanisms have been proposed to explain the processes of cloacal division, involving the formation and subsequent migration of the urorectal septum (URS) to divide the cloaca (Matsumaru et al., 2015). Several signaling molecules that regulate epithelial-mesenchymal interactions, including Hedgehog (Mo et al., 2001), ephrin-Eph (Dravis et al., 2004) and Wnt signaling related genes (Li et al., 2011; Tai et al., 2009), have been identified as potential candidates driving cloacal septation.

In the mouse, the external genitalia first arise as lateral genital swellings on either side of the cloaca at around E10.5 that merge to form a single genital tubercle at E11.5 (Perriton et al., 2002). By E13.5, paired preputial swellings are observed at the lateral margins at the base of the genital tubercle. In the male, these preputial swellings fuse medially and form the external prepuce (Cohn, 2011). In male and female mice, the preputial swellings grow over the genital tubercle to form the perineal appendage that is present in the adult (Liu et al., 2018a) (Cunha et al., this issue). The mouse clitoris forms deep to the perineal appendage, which is formed by growth of the preputial swellings along the sides of the genital tubercle to form the circumferential preputial lamina (Cunha et al., this issue).

Long non-coding RNAs (lncRNAs) are essential regulators of many developmental processes and can regulate expression of genes at transcriptional, post-transcriptional and epigenetic levels. The role of lncRNAs in reproductive processes is not well understood (Liu et al., 2018b). Our lab has identified a novel long non-coding RNA molecule known as Leat1 (long non-coding RNA, EphrinB2 associated transcript 1). This lncRNA is located downstream of EfnB2 in the genome. EfnB2 is a Eph/Ephrin bidirectional signaling molecule that mediates many patterning pathways in early embryonic development, including cloacal septation and urethral development. Here we investigate the role of Leat1 in female development and hypothesise that this long non-coding RNA is essential for the patterning of the female urogenital region and development of the perineal region.

Section snippets

Methods

The OVE442 mouse line was obtained from P.A. Overbeek laboratory (Baylor College of Medicine, Houston, USA) and was maintained on a FVB/NJ genetic background. OVE442 mice have a deleted region 300 kb downstream of the EphrinB2 (EfnB2) gene that contains a long non-coding RNA (UCSC accession number AK042353). This lncRNA was therefore named long non-coding RNA, EphrinB2 associated transcript 1 (Leat1) due to its proximity to EfnB2. Wild type FVB/NJ mice were obtained from Monash University

Leat1 is expressed in the female genital tubercle during embryonic development

Expression of Leat1 was examined in whole GTs during embryonic development to determine its temporal profile using quantitative RT-PCR (qRT-PCR). Leat1 levels peaked between E13.5 and E14.5 (Fig. 1A), coinciding with sexual differentiation of the GT. At E15.5, Leat1 expression decreased before a small peak again at E16.5.

The proximity of Leat1 to the EfnB2 gene, and the known role of EfnB2 in urorectal development, led us to investigate the expression of EfnB2 in mice that are carrying a

Discussion

Malformations of the anorectal and urogenital systems are common congenital conditions. In humans, they can present as a spectrum of disorders, from simple malformations such as hypospadias, to complex malformations, such as cloacal malformation in females with a single channel into the perineum (Gupta et al., 2014; Runck et al., 2014). Disorders of anorectal and genitourinary systems frequently occur together, highlighting a mechanistic link during early development. However the mechanisms

Declaration of competing interest

None.

Acknowledgements

Research reported in this publication was supported by The National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award number R01DK096263 to AJP.

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