2
Maëva Elzaiat1¶, Clara Gobé1¶, Bruno Passet2, Nicolas Meunier3,4,5, Marjolaine André1, Ikrame 3
Naciri6 , Luc Jouneau1, Patrice Congar3,4, Aurélie Allais-Bonnet1,7, Jean-Luc Vilotte2, Eric Pailhoux1, 4
Maëlle Pannetier1* 5
6
1INRA, UMR 1198, Biologie du Développement et Reproduction, F-78350 Jouy-en-Josas, France. 7
2INRA, UMR1313 Génétique Animale et Biologie Intégrative, F-78350 Jouy-en-Josas, France. 8
3INRA, UR1197 Neurobiologie de l’Olfaction, F-78350 Jouy-en-Josas, France. 9
4NeuroSud Paris, IFR144, Gif-sur-Yvette, France 10
5Université de Versailles Saint-Quentin en Yvelines, Versailles, France 11
6Epigenetics and Cell Fate, University Paris Diderot, Sorbonne Paris Cité, UMR 7216 CNRS, 12
75013 Paris, France 13
7
ALLICE , 149 rue de Bercy, 75012 Paris, France. 14 15 16 * Corresponding author 17 E-mail: maelle.pannetier@jouy.inra.fr (MP) 18 19
¶These authors contributed equally to this work 20
2
Abstract
21
DMXL2 encodes a scaffold protein containing WD40 domains, which was initially characterized for
22
its function in vesicle exocytosis in the brain. DMXL2 has also been involved in the Notch signaling 23
pathway via the V-ATPase pump regulation, which was described in both mammalian cell lines and 24
drosophila ovary. Interestingly, high-throughput sequencing data we obtained in the goat species
25
pinpointed DMXL2 as a female-associated gene when sex-determination takes place. DMXL2 was 26
found highly expressed in the differentiating goat ovary, decreasing slightly after germ cells meiosis 27
onset and primordial follicle formation. To apprehend the role of DMXL2 during ovarian 28
development in mammals, we studied the phenotype of Dmxl2 knocked-out mice. 29
Dmxl2 invalidation was found to be lethal a few hours after birth. Dmxl2-/- newborn pups showed an
30
abnormal feeding behavior that was linked to the alteration of olfaction; odorant transmission from 31
the olfactory mucosa to the olfactory bulb was markedly impaired. In addition, Dmxl2 was found to 32
be expressed in both female and male gonads in mice, showing higher level in female around 33
folliculogenesis, and higher level in male during spermatogenesis. Both germ cells and supporting 34
cells expressed DMXL2. Microarray data analyses at birth showed some molecular deregulations 35
in the female Dmxl2-/- gonad, despite normal morphology. The development of these knocked-out 36
ovaries was achieved few extra days through transplant experiments, revealing folliculogenesis 37
alterations. In conclusion, as it was suggested previously, DMXL2 plays an important role in 38
synaptic transmission, in particular in the olfactory system. In addition to its role in olfaction, 39
DMXL2 protein appeared also to take important part in female fertility in the mouse species. 40
3
Introduction
41
DMX-Like 2 (DMXL2) gene encodes a protein containing multiple WD40-domains which
42
mediate protein-protein and protein-DNA interactions [Nagano et al., 2002 ; Stirmimann et al., 43
2010 ; Xu and Min 2011]. These WD40-domains correspond to a series of sequence repeats of 44 44
to 60 amino acids with characteristic sequence terminating in a tryptophan – aspartic acid (WD) 45
dipeptide [Neer et al., 1994]. The WD40 proteins function as hubs, capable of recruiting multiple 46
partners, involving these proteins in a wide range of cellular functions such as signal transduction, 47
vesicular trafficking, cell cycle control, chromatin dynamics and transcriptional regulation 48
[Stirnimann et al., 2010, Xu and Min, 2011]. One of DMXL2 (also known as Rabconnectin-3 or 49
Rbcn-3 ) well characterized partners is WDR7 (Rabconnectin-3 or Rbcn-3β) and together they 50
form the stoechiometric complex of Rabconnectin-3 in the brain [Kawabe et al., 2003]. In this 51
tissue, where DMXL2/Rbcn-3α has been firstly described, Rabconnectin-3 complex might be 52
involved in modulating Ca2+-exocytosis of neurotransmitters, interacting with the small G-proteins 53
Rab3-GEP and Rab3-GAP [Nagano et al., 2002; Geppert et al., 1997]. In addition DMXL2 interacts 54
with some subunits of the vacuolar-ATPase proton pump (V-ATPase) [Yan et al., 2009, Li et al., 55
2012], promoting its assembly and activity in different species such as yeast [Seol et al. 2001 ; 56
Smardon and Kane 2007], drosophila [Yan et al., 2009], zebrafish [Einhorn et al., 2012] and in 57
mammalian cell lines [Sethi et al., 2010]. V-ATPases are responsible for the acidification of 58
intracellular compartments including endosomes, lysosomes, secretory vesicles and the Golgi 59
apparatus, and have well established role in protein sorting, trafficking and turnover [Forgac 2007; 60
Jefferies et al., 2008]. In particular, V-ATPase pumps were shown to be a key player in the Notch 61
signaling pathway in both drosophila [Yan et al., 2009, Vaccari et al., 2010] and mice [Lange et al., 62
2011]. Indeed γ-secretase, whose activity is essential for Notch processing (S3 cleavage 63
generating the NICD), has been shown to have optimal activity in acidic endocytic vesicles where 64
pH is controlled by proton pumps [Pasternak et al, 2003; Vaccari et al., 2010 ; Lange et al., 2011]. 65
The Notch pathway is required for early follicle cell differentiation during oogenesis in drosophila. 66
Loss of this signal transduction in follicle cell epithelium disrupts the normal transition of the follicle 67
cells from mitotic cycle to endocycle [Deng et al., 2001]. Mutant flies presenting an absence of 68
4 DMXL2/Rbcn-3α or WDR7/Rbcn-3 in follicular cells were shown to recapitulate the Notch mutant 69
phenotype [Yan et al., 2009]. Demonstration that Rabconnectin-3 complex affects Notch signaling 70
and trafficking through regulating V-ATPase function was also confirmed in mammals, by in vitro 71
experiments realized on several human and mouse cell lines [Sethi et al., 2010; Faronato et al., 72
2015]. 73
In mammalian female gonad, the Notch signaling pathway was also shown to be involved in 74
ovarian follicle formation [Trombly et al., 2009]. Because follicles are required to support oocyte 75
development, the establishment of these structures is essential for fertility. In mice, primordial germ 76
cells (PGCs) originating from epiblast cells, migrate from outside embryo and populate the gonads 77
around the embryonic day 10.5 (E10.5). At E13.5, germ cells enter meiosis and are afterwards 78
called oocytes (arrested in the diplotene stage of meiotic prophase I) [Borum et al., 1961]. At birth 79
oocytes exist as cyst surrounded by somatic cells [Pepling and Spradling 1998]. Then, 80
fragmentation of these syncytia starts, to undergo follicle formation in which individual oocytes are 81
encapsulated by a single layer of squamous pregranulosa cells to form primordial follicles (around 82
post natal day 0, P0 to post natal day 2, P2, depending on the mouse strain) [Tingen et al., 2009]. 83
The Notch signaling pathway in the mouse ovary occurs in somatic pregranulosa cells between 84
E17.5 and P0 [Vanorny et al., 2014]. Inhibition of -secretase activity in ex-vivo experiments or 85
invalidation of Notch2 receptor in pregranulosa cells or of its Jagged1 ligand in germ cells reduces 86
oocyte cyst breakdown and primary follicle assembly [Trombly et al., 2009; Chen et al., 2014; 87
Vanorny et al., 2014]. 88
In this context, DMXL2 implication in Notch signaling regulation or to a more general point 89
of view in protein trafficking and cell-to-cell cross-talking is unknown. Communication between the 90
oocyte and surrounding granulosa cells has been shown to be crucial for folliculogenesis [Eppig, 91
1991], but signaling molecules and pathways inside mammalian follicles that control formation and 92
early development of ovarian follicles have not been described extensively. On the contrary, 93
several transcription factors, whose function is required at different level of folliculogenesis 94
processing, have been well established, like FIGL [Soyal et al., 2000], LHX8 [Choi et al., 2008], 95
NOBOX [Choi et al., 2007], or FOXL2 [Schmidt et al., 2004; Uda et al., 2004]. In addition to its role 96