<i>Pw1</i> deletion does not result in significant decrease in oxytocin production and release.

<p><b>A.</b> Left panel: schematic sagittal section of the adult mouse brain showing sectioning direction (arrow) on interaural coordinates. Right panel: schematic coronal section of the adult mouse brain showing the paraventricular nuclei (PVN) and the supraoptic nuclei (SON) in pink. <b>B-C.</b> Immunohistochemistry for oxytocin-expressing neurons in the PVN (B) and SON (C) of postpartum female brains (<i>Pw1</i>^<i>+/+</i>, n = 7; <i>Pw1</i>^<i>m+/p-</i>, n = 7; <i>Pw1</i>^<i>m-/p+</i>, n = 5; <i>Pw1-/-</i>, n = 6). Scale bar: 50μm. <b>D.</b> Total number of oxytocin (OT) positive neurons per nuclei as stained as in Fig 3B and 3C (<i>Pw1</i>^<i>+/+</i>, n = 7; <i>Pw1</i>^<i>m+/p-</i>, n = 7; <i>Pw1</i>^<i>m-/p+</i>, n = 5; <i>Pw1-/-</i>, n = 6). Bottom panel: total number of oxytocin (OT) positive neurons per medial preoptic area (MPOA) (<i>Pw1</i>^<i>+/+</i>, n = 6; <i>Pw1-/-</i>, n = 6). No significant differences were found between all four genotypes. <b>E.</b> Number of oxytocin-positive neurons per section as stained as in Fig 3B and 3C for <i>Pw1</i>^<i>+/+</i> and <i>Pw1</i>^<i>-/-</i> postpartum female brains. <b>F.</b> Oxytocin plasma level in virgin (V) and postpartum (PP) females (V: <i>n</i> = 11, 9, 7, and 8; PP: n = 8, 8, 6, and 8; for <i>Pw1</i>^<i>+/+</i>, <i>Pw1</i>^<i>m+/p-</i>, <i>Pw1</i>^<i>m-/p+</i>, <i>Pw1</i>^<i>-/-</i> females, respectively). <i>Pw1</i>^<i>-/-</i> postpartum females tend to have a lower oxytocin plasma level but this observation is not statistically significant. In all graphs, values represent mean ± s.e.m. Statistical analysis was performed using nonparametric one-way ANOVA (Kruskal-Wallis test) (Fig 3D), multiple t-tests (Fig 3E) or two-way ANOVA test (Fig 3F). *P<0.05, **P<0.01, and ***P<0.001. NS: non-significant.</p

Lactation is not compromised in <i>Pw1</i> mutant mice.

<p><b>A.</b> Birth weight of <i>Pw1</i>^<i>+/+</i> pups born to <i>Pw1</i>^<i>+/+</i>, <i>Pw1</i>^<i>m+/p-</i>, or <i>Pw1</i>^<i>m-/p+</i> mothers is unchanged (n = 7, 13, and 8 pups, respectively). <b>B.</b> Early postnatal growth of wild-type progeny of <i>Pw1</i>^<i>m+/p-</i> mothers is comparable to wild-type progeny of <i>Pw1</i>^<i>+/+</i> mothers. Weights were measured at postnatal days 2, 7, 10, 14, and 21, prior to weaning (n = 15, and n = 14 pups from at least 7 breedings <i>Pw1</i>^<i>+/+</i> x <i>Pw1</i>^<i>+/+</i>, and 7 breedings <i>Pw1</i>^<i>m+/p-</i> x <i>Pw1</i>^<i>+/+</i>, respectively). No significant differences were found. <b>C.</b> Early postnatal growth of <i>Pw1</i>^<i>m-/p+</i> progeny to <i>Pw1</i>^<i>m+/p-</i> and <i>Pw1</i>^<i>-/-</i> mothers crossed with a <i>Pw1</i>^<i>+/+</i> male are comparable. Weights have been measured at postnatal days 2, 7, 10, 14, and 21, prior to weaning (n = 9, n = 11, for breedings <i>Pw1</i>^<i>m+/p-</i> x <i>Pw1</i>^<i>+/+</i> and <i>Pw1</i>^<i>-/-</i> x <i>Pw1</i>^<i>+/+</i>, respectively). No significant differences were found. <b>D.</b> Milk intake was assessed by measuring the gain of pup weight after a 2 hour starvation period at postnatal day 7. Milk intake of <i>Pw1</i>^<i>m+/p-</i> pups was similar to <i>Pw1</i>^<i>+/+</i> (<i>Pw1</i>^<i>+/+</i>: n = 20 pups; <i>Pw1</i>^<i>m+/p-</i>: n = 19 pups obtained from 5 independent breedings). The two-sided arrow indicates the 2 hour time-window when the pups were starved. <b>E.</b> Milk spot in day 0 pups (arrow). <b>F and G.</b> Percentage of postnatal day 2 pups showing a significant milk spot size from the following breedings: a female <i>Pw1</i>^<i>m+/p-</i> crossed with a male <i>Pw1</i>^<i>+/+</i> (F) and a female <i>Pw1</i>^<i>m+/p-</i> crossed with a male <i>Pw1</i>^<i>m+/p-</i> (G). The number of pups used is indicated on bars, with the number of independent breedings indicated in brackets. In all graphs, values represent mean ± s.e.m. Statistical analysis was performed using two-way ANOVA test.</p

Maternal care is not impaired in <i>Pw1</i> mutant mice.

<p><b>A.</b> Assessment of maternal behavior in 2 months old nulliparous (virgin) females (n≥12) using 3 foster pups of 1 to 3 days old chosen randomly. <b>B.</b> Assessment of maternal behavior in 3 to 4 months old primiparous females on the day of delivery (n≥12) using the female own litter. Nest quality is scored as followed: 0 = no nest building activity/no nest built; 1 = quick nest building activity, few nest materials/twigs have been retrieved; 2 = consequent nest building activity with some twigs remaining outside the nest. 3 = perfect nest without any twig left outside the nest. In all graphs, values represent mean ± s.e.m. Statistical analysis was performed using nonparametric one-way ANOVA (Kruskal-Wallis test). No significant differences were found between any of the four genotypes.</p

Reproduction parameters of <i>Pw1</i> mutant mice are comparable to <i>Pw1</i>^<i>+/+</i> mice.

<p>Reproduction parameters of <i>Pw1</i> mutant mice are comparable to <i>Pw1</i>^<i>+/+</i> mice.</p

<i>Pw1</i> knockout strategy and characterization.

<p><b>A.</b><i>Pw1</i> knockout construct. Pink, blue, and green arrows-arrowheads correspond to location of <i>Pw1</i> primers. <b>B.</b> Expression levels of <i>Pw1</i> wildtype and <i>Pw1</i> truncated knockout alleles from semi-quantitative RT-PCR analysis in postnatal day 0 (P0) and 2 months old (2 mo) <i>Pw1</i>^<i>+/+</i> (+/+), <i>Pw1</i>^<i>m+/p-</i> (+/-), <i>Pw1</i>^<i>m-/p+</i> (-/+), and <i>Pw1</i>^<i>-/-</i> (-/-) brains (n = 3). <b>C.</b> Expression level of <i>Pw1</i> wild-type allele from real time PCR normalized to <i>Hprt1</i> gene (n = 3). <b>D.</b> PW1 immunofluorescence (green) on 3–4 months old postpartum female hypothalamus (retrochiasmatic area) (n≥4). Nuclei were counterstained by DAPI. Scale bar: 50μm. <b>E.</b> Western blot analysis showing levels of PW1 at P0 in <i>Pw1</i>^<i>+/+</i> (+/+), <i>Pw1</i>^<i>m+/p-</i> (+/-), <i>Pw1</i>^<i>m-/p+</i> (-/+), and <i>Pw1</i>^<i>-/-</i> (-/-) brains (n = 3). <b>F. Left panel:</b> Postnatal growth of <i>Pw1</i>^<i>+/+</i> (n = 26), <i>Pw1</i>^<i>m+/p-</i> (n = 12), <i>Pw1</i>^<i>m-/p+</i> (n = 5), and <i>Pw1</i>^<i>-/-</i> (n = 5) female mice. <b>Right panel:</b> Postnatal growth of <i>Pw1</i>^<i>+/+</i> (n = 18), <i>Pw1</i>^<i>m+/p-</i> (n = 10), <i>Pw1</i>^<i>m-/p+</i> (n = 9), and <i>Pw1</i>^<i>-/-</i> (n = 4) male mice. Paternal loss of <i>Pw1</i> leads to a reduced postnatal growth. <b>G.</b> Data shown in F presented additionally as percentage of <i>Pw1</i>^<i>+/+</i> littermates weight. In all graphs except panel G, values represent mean ± s.e.m. Statistical analysis was performed using two-way ANOVA test. *P<0.05, **P<0.01, and ***P<0.001. NS: non-significant.</p

Table_1_Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle.docx

<p>Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70–80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.</p

Image_4_Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle.TIF

<p>Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70–80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.</p

Image_1_Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle.TIF

<p>Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70–80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.</p

Characterisation of the F2 CU hepatic and placental transcriptome.

<p>(A) Schematic of the F2 generation, the transcriptome of the CU and CC F2 crosses was assessed at E16.5. (B) Distribution of the ranked difference in gene expression in CU E16.5 liver according to FDR q-value. A list of genes comprising a network involved in lipid metabolism, identified as enriched in CU liver using IPA software was used as a positive control. Imprinted genes most closely resemble randomly selected genes. (C) Distribution of genes differentially expressed in CU E16.5 placenta according to FDR q-value. A list of genes comprising a network involved in lipid metabolism, identified as enriched in CU placenta using IPA software was used as a positive control. Imprinted genes most closely resemble randomly selected genes.</p

Analysis of F2 placental candidate imprinted gene expression by two-way ANOVA.

<p>Analysis of F2 placental candidate imprinted gene expression by two-way ANOVA.</p