Abstract
Numb (Nb) and Numb-like (Nbl) are functionally redundant adaptor proteins that critically regulate cell fate and morphogenesis in a variety of organs. We selectively deleted Nb and Nbl in testicular germ cells by breeding Nb/Nbl floxed mice with a transgenic mouse line Tex101-Cre. The mutant mice developed unilateral or bilateral cystic dilation in the rete testis (RT). Dye trace indicated partial blockages in the testicular hilum. Morphological and immunohistochemical evaluations revealed that the lining epithelium of the cysts possessed similar characteristics of RT epithelium, suggesting that the cyst originated from dilation of the RT lumen. Spermatogenesis and the efferent ducts were unaffected. In comparisons of isolated germ cells from mutants to control mice, the Notch activity considerably increased and the expression of Notch target gene Hey1 significantly elevated. Further studies identified that germ cell Fgf4 expression negatively correlated the Notch activity and demonstrated that blockade of FGF receptors mediated FGF4 signaling induced enlargement of the RT lumen in vitro. The crucial role of the FGF4 signaling in modulation of RT development was verified by the selective germ cell Fgf4 ablation, which displayed a phenotype similar to that of germ cell Nb/Nbl null mutant males. These findings indicate that aberrant over-activation of the Notch signaling in germ cells due to Nb/Nbl abrogation impairs the RT development, which is through the suppressing germ cell Fgf4 expression. The present study uncovers the presence of a lumicrine signal pathway in which secreted/diffusible protein FGF4 produced by germ cells is essential for normal RT development.
Highlights
Numb (Nb) and its homolog Numb-like (Nbl) in mammals are functionally redundant intracellular adaptor proteins that critically regulate cell fate choice, cell–cell adhesion, and morphogenesis in a variety of tissues and organs
Notch intracellular domain (NICD) is transported into the nucleus, where NICD acts as a coactivator with other transcription factors to regulate the expression of downstream target genes, including hairy and enhancer of split (Hes) and Hes related with YRPW motif (Hey), two families of transcription repressors that modulate a number of genes to influence multiple cellular processes in response to ligand-induced activation of all Notch proteins (Jarriault et al 1995; Kopan and Ilagan 2009; Schroeter et al 1998; Wahi et al 2016)
To circumvent the early embryonic lethal phenotype of Nb/ Nbl null mutation, we explored the role of Nb/Nbl in testicular germ cells by deleting the exon 1 of Nb and the first 3 exons of Nbl within the seminiferous tubules, and subsequently breeding Tex-Cre transgenic mice and Nb/ Nbl–floxed mice (Nbf/f/Nblf/f)
Summary
Numb (Nb) and its homolog Numb-like (Nbl) in mammals are functionally redundant intracellular adaptor proteins that critically regulate cell fate choice, cell–cell adhesion, and morphogenesis in a variety of tissues and organs. Antagonizing Notch signaling is one of well characterized Nb/Nbl mediated actions in regulation of these cellular and developmental processes (Gulino et al 2010; Katoh and Katoh 2006; Shao et al 2017; Verdi et al 1996; Wahi et al 2016; Zhang et al 2016). NICD is transported into the nucleus, where NICD acts as a coactivator with other transcription factors to regulate the expression of downstream target genes, including hairy and enhancer of split (Hes) and Hes related with YRPW motif (Hey), two families of transcription repressors that modulate a number of genes to influence multiple cellular processes in response to ligand-induced activation of all Notch proteins (Jarriault et al 1995; Kopan and Ilagan 2009; Schroeter et al 1998; Wahi et al 2016). Nb/Nbl-dependent inhibition of Notch signaling is mediated by targeting activated Notch NICD to polyubiquitination and degradation as well as stimulating endocytosis and sequestration of pre-cleaved Notch or membrane tethered NICD (Berdnik et al 2002; McGill and McGlade 2003; Shao et al 2017)
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