Respiratory pattern and apnea in <i>Ndn</i>−/− young adult versus <i>Ndn</i>+m/−p mice.

<p>While the mean apnea duration was similar between genotypes (∼900 msec), evaluation of the total apnea duration expressed as the total recording time (i.e. apnea scores) revealed a two-fold increase in the percentage of total apnea duration in <i>Ndn−/−</i> compared to <i>Ndn+m/−p</i> individuals.</p><p>Values are represented as Mean±SD; n = 20 for <i>Ndn+m/−p</i> and 18 for <i>Ndn</i> −/− mice.</p><p>Mann Whitney t-test, two-tailed. P value = 0.02 (a) and 0.03 (b).</p

<i>NDN</i> expression in PWS patients.

<p>Detection of <i>NDN</i> transcripts revealed by ISH, and using a <i>NDN</i> anti-sense probe, on PVN brain sections from control individuals (A,C,E) and PWS patients (B,D,F). A <i>NDN</i>-sense probe was used as a negative control. IHC on SON brain sections, using a NECDIN specific antibody, was performed on the same control and PWS patients. The expression was studied in the 94-118 adult control male (A) and the 95104 adult PWS patient with a maternal uniparental disomy (B), in the 88-017 adult control male (C) and the 00-028 PWS adult patient with a deletion (D), in the 97-153 control infant (E) and the 99-079 PWS infant with a deletion (F). Scale bar: 20 µm.</p

<i>Ndn</i> expression in <i>Ndn+m/−p</i> E12.5 embryos.

<p>Expression of <i>Ndn</i> in the nervous system of WT and <i>Ndn+m/−p</i> embryos at E12.5 revealed by IHC or ISH on frozen sections using a Necdin specific antibody (Ndn,red) or <i>Ndn</i> RNA probe (green). Tissue sections are visualized using a Hoechst labeling (blue). Expression is detected in both genotypes, at the protein and transcript levels, in the preoptic area (A), supraoptic area (B), thalamus (C), pons (D) and in the dorsal root ganglia (E). Note that the level of expression is weaker and more restricted in <i>Ndn+m/−p</i> embryos. Other structures, like the tegmentum, the subthalamus and the spinal cord also express the <i>Ndn</i> maternal allele. Scale bar: 50 µm.</p

<i>Ndn</i> expression analyzed by RT-qPCR in <i>Ndn+m/−p</i> mice.

<p>RT-qPCR analysis of <i>Ndn</i> transcripts. A) <i>Ndn</i> transcripts in E12 WT embryos and selected brain tissues from P1 (whole brain, hypothalamus, pons) or adult (hypothalamus) WT mice. B) <i>Ndn</i> transcripts in <i>Ndn+m/−p</i> E12 embryos and brain tissues from P1 (whole brain, hypothalamus, pons) or adult (hypothalamus) <i>Ndn+m/−p</i> mice. C–F) Quantification of <i>Ndn</i> transcripts in <i>Ndn</i>+m/−p E12 embryos (C,D) and whole brain (C,E) and pons (C,F) from P1 mice with respect to parental genotype (C) and to the maternal or paternal genotype contribution indicating a grandparental influence (D–F). G) Quantification of <i>Ndn</i> transcripts in <i>Ndn+m/−p</i> E12 embryos, P1 and adult <i>Ndn+m/−p</i> brain tissues in male (M) and female (F) mice. H) Quantification of <i>Ndn</i> transcripts in <i>Ndn+m/−p</i> E12 embryos and <i>Ndn+m/−p</i> P1 mice in C57Bl/6J and S129Sv/Pas mouse strains. The <i>Ndn</i> transcript copy number in <i>Ndn+m/−p</i> offspring (E12, P1 brain, P1 pons; n = 71) issued from a cross between a WT female and a <i>Ndn</i>+/− male is significantly more than 2 fold higher than the <i>Ndn</i> copy number in <i>Ndn+m/−p</i> offspring (n = 51) issued from a <i>Ndn+/−</i> female crossed with a <i>Ndn−/−</i> male (C). Considering separately the effect of the maternal or paternal genotype, we showed that when the mother is WT and the father is (+/−), with a maternal <i>Ndn</i> mutant allele (−m/+p) or a paternal <i>Ndn</i> mutant allele (+m/−p), then there is no difference in the copy number of <i>Ndn</i> maternal transcripts between the <i>Ndn+m/−p</i> individuals of the offspring of both types of crosses (n = 25 and n = 46, respectively) (D,E,F). However, we observed an effect of the maternal genotype, with a significant difference in the level of <i>Ndn</i> maternal transcripts between the <i>Ndn+m/−p</i> individuals (n = 22) issued from a (−m/+p female X −/− male) compared with the <i>Ndn+m/−p</i> individuals (n = 29) issued from a (+m/−p female X −/− male) (D,E,F); the +/+ or <i>Ndn+m/−p</i> maternal genotype is correlated with a significant three times higher level of <i>Ndn</i> maternal expression in the <i>Ndn+m/−p</i> offspring. Values are represented as Median (Q1, Q3). WMW test, two-tailed. * P value<0.05 and ** <0.01.</p

<i>Ndn</i> expression in the 5HT raphe nuclei of <i>Ndn+m/−p</i> individuals.

<p>(A) % of Necdin immunoreactive cells among 5HT positive cells located in the B1 to B9 raphe nuclei. Wild-type (n = 3), <i>Ndn</i>−/− (n = 1) and <i>Ndn+m/−p</i> (n = 10) newborn mice were analyzed. (B) Number of 5HT-expressing neurons in the B1/B2 raphe nuclei of +/+ (n = 9), <i>Ndn +m/−p</i> (n = 18) and <i>Ndn−/−</i> (n = 8) individuals. <i>Ndn+m/−p</i> (n = 18) individuals are divided in two populations: a population in which Necdin immunolabeling is detected in a mean of 46% of 5HT neurons of <i>Ndn+m/−</i> (Ndn+, n = 9) individuals and a population in which no Necdin/5HT colabelling is detected in the B1/B2 raphe nuclei of <i>Ndn+m/−p</i> (Ndn−, n = 9) individuals. Scale bar: 10 µm.</p