A Novel Mutant Allele of Pw1/Peg3 Does Not Affect Maternal Behavior or Nursing Behavior

Abstract

Parental imprinting is a mammalian-specific form of epigenetic regulation in which one allele of a gene is silenced depending on its parental origin. Parentally imprinted genes have been shown to play a role in growth, metabolism, cancer, and behavior. Although the molecular mechanisms underlying parental imprinting have been largely elucidated, the selective advantage of silencing one allele remains unclear. The mutant phenotype of the imprinted gene, Pw1/Peg3, provides a key example to illustrate the hypothesis on a coadaptation between mother and offspring, in which Pw1/Peg3 is required for a set of essential maternal behaviors, such as nursing, nest building, and postnatal care. We have generated a novel Pw1/Peg3 mutant allele that targets the last exon for the PW1 protein that contains >90% of the coding sequence resulting in a loss of Pw1/Peg3 expression. In contrast to previous reports that have targeted upstream exons, we observe that maternal behavior and lactation are not disrupted upon loss of Pw1/Peg3. Both paternal and homozygous Pw1/Peg3 mutant females nurse and feed their pups properly and no differences are detected in either oxytocin neuron number or oxytocin plasma levels. In addition, suckling capacities are normal in mutant pups. Consistent with previous reports, we observe a reduction of postnatal growth. These results support a general role for Pw1/Peg3 in the regulation of body growth but not maternal care and lactation.

Fig 1. Pw1 knockout strategy and characterization.

A. Pw1 knockout construct. Pink, blue, and green arrows-arrowheads correspond to location of Pw1 primers. B. Expression levels of Pw1 wildtype and Pw1 truncated knockout alleles from semi-quantitative RT-PCR analysis in postnatal day 0 (P0) and 2 months old (2 mo) Pw1^+/+ (+/+), Pw1^m+/p- (+/-), Pw1^m-/p+ (-/+), and Pw1^-/- (-/-) brains (n = 3). C. Expression level of Pw1 wild-type allele from real time PCR normalized to Hprt1 gene (n = 3). D. PW1 immunofluorescence (green) on 3–4 months old postpartum female hypothalamus (retrochiasmatic area) (n≥4). Nuclei were counterstained by DAPI. Scale bar: 50μm. E. Western blot analysis showing levels of PW1 at P0 in Pw1^+/+ (+/+), Pw1^m+/p- (+/-), Pw1^m-/p+ (-/+), and Pw1^-/- (-/-) brains (n = 3). F. Left panel: Postnatal growth of Pw1^+/+ (n = 26), Pw1^m+/p- (n = 12), Pw1^m-/p+ (n = 5), and Pw1^-/- (n = 5) female mice. Right panel: Postnatal growth of Pw1^+/+ (n = 18), Pw1^m+/p- (n = 10), Pw1^m-/p+ (n = 9), and Pw1^-/- (n = 4) male mice. Paternal loss of Pw1 leads to a reduced postnatal growth. G. Data shown in F presented additionally as percentage of Pw1^+/+ 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.

Fig 2. Maternal care is not impaired in Pw1 mutant mice.

A. 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. 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.

Fig 3. Pw1 deletion does not result in significant decrease in oxytocin production and release.

A. 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-C. Immunohistochemistry for oxytocin-expressing neurons in the PVN (B) and SON (C) of postpartum female brains (Pw1^+/+, n = 7; Pw1^m+/p-, n = 7; Pw1^m-/p+, n = 5; Pw1-/-, n = 6). Scale bar: 50μm. D. Total number of oxytocin (OT) positive neurons per nuclei as stained as in Fig 3B and 3C (Pw1^+/+, n = 7; Pw1^m+/p-, n = 7; Pw1^m-/p+, n = 5; Pw1-/-, n = 6). Bottom panel: total number of oxytocin (OT) positive neurons per medial preoptic area (MPOA) (Pw1^+/+, n = 6; Pw1-/-, n = 6). No significant differences were found between all four genotypes. E. Number of oxytocin-positive neurons per section as stained as in Fig 3B and 3C for Pw1^+/+ and Pw1^-/- postpartum female brains. F. Oxytocin plasma level in virgin (V) and postpartum (PP) females (V: n = 11, 9, 7, and 8; PP: n = 8, 8, 6, and 8; for Pw1^+/+, Pw1^m+/p-, Pw1^m-/p+, Pw1^-/- females, respectively). Pw1^-/- 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.

Fig 4. Lactation is not compromised in Pw1 mutant mice.

A. Birth weight of Pw1^+/+ pups born to Pw1^+/+, Pw1^m+/p-, or Pw1^m-/p+ mothers is unchanged (n = 7, 13, and 8 pups, respectively). B. Early postnatal growth of wild-type progeny of Pw1^m+/p- mothers is comparable to wild-type progeny of Pw1^+/+ 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 Pw1^+/+ x Pw1^+/+, and 7 breedings Pw1^m+/p- x Pw1^+/+, respectively). No significant differences were found. C. Early postnatal growth of Pw1^m-/p+ progeny to Pw1^m+/p- and Pw1^-/- mothers crossed with a Pw1^+/+ 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 Pw1^m+/p- x Pw1^+/+ and Pw1^-/- x Pw1^+/+, respectively). No significant differences were found. D. Milk intake was assessed by measuring the gain of pup weight after a 2 hour starvation period at postnatal day 7. Milk intake of Pw1^m+/p- pups was similar to Pw1^+/+ (Pw1^+/+: n = 20 pups; Pw1^m+/p-: n = 19 pups obtained from 5 independent breedings). The two-sided arrow indicates the 2 hour time-window when the pups were starved. E. Milk spot in day 0 pups (arrow). F and G. Percentage of postnatal day 2 pups showing a significant milk spot size from the following breedings: a female Pw1^m+/p- crossed with a male Pw1^+/+ (F) and a female Pw1^m+/p- crossed with a male Pw1^m+/p- (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.