Syracuse

Animal models for PWS are instrumental in understanding mechanisms and identifying novel pathways involved in the pathophysiology of PWS. The mouse chromosome 7C presents a conserved synteny to the human PWS region. However, mouse models with inactivation of all PWS genes display a 100% lethality rate within the first week after birth and have, therefore, not been very useful for understanding postnatal symptoms (9, 10). Mouse KO models for single candidate genes have also been generated (11). However, these single KO models have limitations, since it is likely that the PWS phenotype is the result of the lack of expression of several genes that are coexpressed in the same brain regions (12) and that may interact with each other, creating a more complex and integrative phenotype.

Among the genes inactivated in PWS, SNORDs NECDIN and MAGEL2 are of particular interest. Snord116-KO mice display some lethality before weaning, and they display growth delay but consume proportionally more food, considered as hyperphagia (13-15), although they do not become obese (13, 14). They also exhibit cognitive deficits (16) and sleep behavior alterations (17). However, Snord115-KO mice appear normal with no obvious behavioral or metabolic alterations (18, 19). Necdin and Magel2-single KO mouse models are also of particular interest since they display several distinct phenotypes mimicking part of the PWS clinical features, although there is variability among the different models depending on the genomic construction. Magel2-KO mice exhibit suckling deficits at birth (20), growth retardation (20), altered metabolism (21, 22), circadian activity disturbances ( 23), and deficits in cognitive, social, and parental behaviors (24-27). Our teams and others also reported impaired hypothalamic regulation in Magel2-KO mice with abnormal oxytocin (OT) maturation (20, 24, 28, 29) and disrupted development and function of proopiomelanocortin (POMC) neurons (29, 30). Necdin-KO mice display variable lethality after birth (31, 32) due to respiratory distress (33), growth retardation, motor deficit in infancy (34), sensory deficits (35), high scraping, cognitive alterations (32), and alterations of social and circadian behaviors (36, 37). At the neuroanatomical level, Necdin-KO mice display a reduction in the number of OT and gonadotropin-releasing hormone-producing (GnRH-producing) neurons, alterations in perinatal serotonergic metabolism and development (32, 38), and alterations in clock gene expression (36).

MAGEL2 and NECDIN belong to the melanoma antigen gene expression (MAGE) gene family (39). They are physically close in the genome (30 kb), are without introns, and have probably evolved through sequential retrotransposition events (12). In addition, they are coexpressed in many brain structures, including in the developing hypothalamus (12, 40, 41). At the molecular level, both proteins act through a ubiquitin-dependent mechanism to turn over and recycle proteins (42). Overall, their molecular function and expression pattern suggest that their roles in cellular processes may partially overlap (42). Therefore, an animal model with the combined loss of Magel2 and Necdin versus a single invalidation of each gene should reveal the complex interaction of these 2 genes and avoid the potential functional redundancy or compensatory mechanism between them. This model would therefore be more relevant, compared with single KO mice, to study the complexity of PWS.

In the present study, we investigated the interplay between Necdin and Magel2 genes, generated a mouse model with a deletion including both Magel2 and Necdin genes, and provided a comprehensive characterization of the behavioral, physiological, neurodevelopmental, and transcriptomic alterations of this model.

Coexpression and coregulation of Magel2 and Necdin genes and generation of a mouse model with a deletion including both genes. We previously found that Necdin and Magel2 mRNAs were highly expressed in the developing brain (12). Here, we showed a striking overlapping expression pattern of both genes in the embryonic and adult brain (Figure 1, A andB). We used a single-cell RNA-Seq-based (scRNA-Seq-based) interactive atlas (mousebrain.org) to examine which cell types expressed Necdin and Magel2 and found that these genes were mainly expressed in neurons in various brain regions and neuronal systems and that nearly all Magel2 cells also express Necdin (Supplemental Figure 1, A andB; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.185159DS1). Our previous studies (12) and genomic analysis (https://genome.ucsc.edu) predicted that both genes share a common enhancer (Figure 1C). We have previously generated 2 mouse models in which the promoter and 5′ of the coding region of Necdin (Ndn tm1-Mus ) (32) or Magel2 (Magel2 tm1-Mus ) (20) were deleted, preventing the expression of Necdin and Magel2 transcripts, respectively. In the present study, we used quantitative PCR (qPCR) and found that Necdin mRNA was overexpressed in the hypothalamus of Magel2-KO mice, and Magel2 mRNA was overexpressed in Necdin-KO hypothalami at P0 (Figure 1D). We also showed by in situ hybridization that Magel2 transcript was