<i>DMXL2</i> is mutated in affected patients.

<p>(A) Pedigree of the affected family. Closed symbols indicate affected individuals. (B) Linkage analysis delineated two candidate regions on chromosomes 13 and 15 with a LOD score of 2.5. (C) Next-generation sequencing characterized a deletion of 15 nucleotides (c.5824_5838del) in exon 24 of <i>DMXL2</i>. This deletion removes five amino acids (p.1942_1946del). Rbcn-3α is a protein with 17 WD domains (green box) and one Rav1p_C domain, which is involved in regulating the glucose-dependent assembly and disassembly of the V1 and V0 subunits of the vacuolar ATPase (purple box) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952-Seol1" target="_blank">[57]</a>. (D) Quantification, by RT-qPCR, of <i>DMXL2</i> mRNA levels relative to RNA 18S levels in blood lymphocytes. Error bars, SEM. * <i>p</i><0.05. Numerical data used to generate graph 1D may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a>.</p

Summary of the clinical phenotype of the three affected patients with <i>DMXL2</i> mutations (see Figure 1 for patient numbering).

<p>SDS, standard deviation score.</p><p>Summary of the clinical phenotype of the three affected patients with <i>DMXL2</i> mutations (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio-1001952-g001" target="_blank">Figure 1</a> for patient numbering).</p

Rbcn-3α is expressed in exocytosis vesicles in the external layer of the median eminence.

<p>(A) ISH with a mouse <i>Dmxl2</i> antisense probe (AS) and a sense probe (S). (B) Immunolabeling with an antibody against Rbcn-3α revealed high levels of <i>Dmxl2</i>/Rbcn-3α expression in the dentate gyrus, the CA1 and CA3 regions of the hippocampus, and the cortex (black arrowheads). Scale bars, 200 µm. (C and D) Rbcn-3α was found to be strongly expressed in the external layer of the ME (C) and the OVLT (D). Scale bars, 100 µm. (E) Confocal analysis with an antibody against Rbcn-3α showed punctate staining in the median eminence and staining of the long processes extending from the cell bodies lining the third ventricle. (F and G) Rbcn-3α immunoreactivity was observed in small clear vesicles and LDCVs at the extremities of the axons in the ME (white arrow). Scale bar, 0.2 µm.</p

Hypothalamic GnRH mRNA and GnRH-IR neuron levels are lower in the hypothalamus of <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice.

<p>(A) Rbcn-3α is expressed in GnRH neurons in the median eminence. (B) Rbcn-3α is located in small clear vesicles and in LDCVs in GnRH neurons. White arrows indicate Rbcn-3α DAB staining; white arrow heads indicate GnRH nanogold staining. (C) <i>GnRH1</i> mRNA levels relative to RNA18S were lower in the hypothalamus of <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt male mice than in WT mice. (D) The total number of GnRH-ir neurons in the brain was lower in <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt male mice than in WT mice. (E) GnRH immunostaining in the OVLTs in <i>Dmxl2</i>^lox/wt and <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt male mice. (F) An analysis of the rostral–caudal distribution of GnRH-ir neurons in the hypothalamus revealed that <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt male mice had fewer GnRH-ir cell bodies in the OVLT (see inset) than their WT littermates. * <i>p</i><0.05, *** <i>p</i><0.0001. White bars, <i>Dmxl2^lox/wt</i>; black bars, <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt. Numerical data used to generate graphs 6C, 6D, 6F, and 6F inset may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a>.</p

Female <i>Nes-cre;Dmxl2</i>^–/wt mice displayed delayed puberty and were infertile.

<p>(A) Postnatal growth curve of female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice (black line, <i>Dmxl2^lox/wt</i>; hatched gray line, <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt). (B and C) VO and first estrus occurred significantly later in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice than in their WT littermates. (D) The interval from VO to first estrus was significantly longer in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice than in WT mice, suggesting a defect in maturation of the HPG axis. (E) Very few complete estrous cycles were observed in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (F) Female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt spent less time in high estradiol concentration (M, miestrus; E, estrus; D, diestrus; P, proestrus). (G) A significant difference in AGD was observed between male <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice and their WT littermates. Black line, <i>Dmxl2^lox/wt</i>; hatched gray line, <i>nes-Cre</i>;<i>Dmxl2</i>^-/wt. White bars, <i>Dmxl2^lox/wt</i>. Black bars, <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt. Numerical data used to generate graphs 4B, 4C, 4D, 4E, 4F, or graphs 4A, 4G may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s013" target="_blank">Table S6</a>, respectively. Error bars are SEM. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

<i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice displayed a partial gonadotropin deficiency.

<p>(A and B) Weights of testes and ovaries were low in <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (C) Histological analysis of ovaries showed a normal number of antral follicles but very few corpora lutea in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (D) Estradiol concentrations were normal in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (E) Plasma testosterone concentration was low in male <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (F) Plasma LH concentrations were moderately high in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (G) Despite their lower testosterone concentrations, male <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice had normal plasma LH concentrations. (H) The GnRH-induced increase in LH concentration was normal in <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (I) The administration of PMSG to young mice induced a normal increase in estradiol concentration in <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice, similar to that observed in their WT littermates (asterisks indicate significant differences: * <i>p</i><0.05, ** <i>p</i><0.001; ***<i>p</i><0.0001). Error bars: SEM. P, postnatal day. White bars, <i>Dmxl2^lox/wt</i>; black bars, <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt. Numerical data used to generate these graphs may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a>.</p

<i>Dmxl2</i> knockdown in INS-1E cells decreases glucose-induced insulin release.

<p>(A) Rbcn-3α is expressed in insulin-secreting cells in the islets of Langerhans in the mouse pancreas. (B) Seventy-two hours after the transfection of INS-1E cells with <i>Dmxl2-</i>siRNA, <i>Dmxl2</i> mRNA levels had decreased by 75% (error bars: SEM; from three independent experiments). (C) Seventy-two hours after siRNA transfection, the release of insulin was evaluated by quantifying insulin concentrations in cell supernatants. In the absence of glucose, insulin concentrations in <i>Dmxl2</i>-siRNA–transfected cells were double those in cells transfected with NT siRNA (NT-siRNA). In the presence of various concentrations of glucose (5 and 20 mM), <i>Dmxl2</i>-siRNA–transfected cells displayed only a small increase in insulin release, at a glucose concentration of 20 mM only, whereas a 2- to 3-fold increase was observed with NT siRNA-transfected cells. Error bars: SEM from one representative experiment performed twice, in hextuplicate. * <i>p</i><0.05, ** <i>p</i><0.001, *** <i>p</i><0.0001. Numerical data used to generate graphs 7B and 7C may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a>.</p

Hormonal status of the three affected patients.

<p>Testosterone, 3–12 ng/ml; GnRH test (100 µg i.v.): normal values for FSH, 1–8.5 IU/l; LH, 1.5–8 IU/l. 60 minutes after GnRH administration, LH and FSH basal concentrations should have increased by factors of 4.5 and 2, respectively. TRH test (200 µg i.v.): normal values for TSH, 0.5–6, 14±7 and <8 mU/l for basal, peak, and 120-min post-TRH. Inhibin B, normal values in young adults 135–350 pg/ml; Free T4 (FT4), normal values 11–21 pmol/l; Prolactin, normal values <20 ng/ml; HbA1c, normal values<5.8%. Insulin concentrations in an intravenous glucose tolerance test (IGTT) at T1+T3 min after glucose administration; normal values >40 mU/l. C-peptide, normal basal values: 0.3–1.3 nmol/l.</p><p>Hormonal status of the three affected patients.</p

Rbcn-3α is specifically expressed in gonadotropes in the pituitary.

<p>(A) Double-immunostaining for Rbcn-3α and LH or FSH in the rat pituitary gland showed that Rbcn-3α was expressed in gonadotropes. Scale bars, 10 µm. (B) Double-immunostaining for Rab3GAP or Rab3GEP revealed that these two proteins were present in both LH- and FSH-expressing cells. (C) Immunostaining with antibodies against Rbcn-3α, Rab3GAP, or Rab3GEP and ACTH, TSH, and GH showed that Rbcn-3α, Rab3GAP, and Rab3GEP were not expressed in corticotropes, thyreotropes, and somatotropes. Scale bars, 40 µm. Red staining, antibodies against Rbcn-3α, Rab3GAP, and Rab3GEP. Green staining, antibodies against pituitary hormones.</p

Growth data 5-18y

Growth data 5-18