Research ArticleGeneticsNeuroscience Open Access | 10.1172/jci.insight.185159

Pierre-Yves Barelle,1,2 Alicia Sicardi,1,2 Fabienne Schaller,3 Julie Buron,3 Denis Becquet,4 Felix Omnes,3 Françoise Watrin,3 Marie-Sophie Alifrangis,3 Catarina Santos,5 Clément Menuet,3 Anne-Marie François-Bellan,4 Emilie Caron,1,2 Jessica Klucznik,1,2 Vincent Prevot,1,2 Sebastien G. Bouret,1,2 and Françoise Muscatelli3

1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

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1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

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1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

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1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

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1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

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1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

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1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

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1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

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1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Find articles by Santos, C. in: JCI | PubMed | Google Scholar

1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Find articles by Menuet, C. in: JCI | PubMed | Google Scholar

1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Find articles by François-Bellan, A. in: JCI | PubMed | Google Scholar

1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Find articles by Caron, E. in: JCI | PubMed | Google Scholar |

1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Find articles by Klucznik, J. in: JCI | PubMed | Google Scholar

1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Find articles by Prevot, V. in: JCI | PubMed | Google Scholar |

1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Find articles by Bouret, S. in: JCI | PubMed | Google Scholar

1University Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience and Cognition, UMR-S 1172, Lille F-59000, France.

2FHU 1000 Days for Health, School of Medicine, Lille F-59000, France.

3Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix Marseille Université, Marseille, France.

4University of Aix-Marseille, Inst Neurophysiopathol, Marseille, France.

5Phenotype Expertise, Marseille, France.

Address correspondence to: Sebastien G. Bouret, INSERM UMR-S 1172, 1 place de Verdun 59000 Lille, France. Phone: 33.0.3.5950.7551; Email: sebastien.bouret@inserm.fr. Or to: Françoise Muscatelli, INSERM UMR1249, Parc Scientifique de Luminy-BP 13 13273, Marseille, Cedex 09 France. Phone : 33.0.4.9182.8133; Email: francoise.muscatelli@inserm.fr.

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Find articles by Muscatelli, F. in: JCI | PubMed | Google Scholar |

Authorship note: PYB, AS, and FS contributed equally to this work. FM and SGB are co–senior authors.

Published March 6, 2025 - More info

Published in Volume 10, Issue 8 on April 22, 2025
JCI Insight. 2025;10(8):e185159. https://doi.org/10.1172/jci.insight.185159.
© 2025 Barelle et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. View PDF Abstract

Prader-Willi syndrome (PWS) is a multigenic disorder caused by the loss of 7 contiguous paternally expressed genes. Mouse models with inactivation of all PWS genes are lethal. KO mouse models for each candidate gene have been generated, but they lack the functional interactions between PWS genes. Here, we revealed an interplay between Necdin and Magel2 PWS genes and generated a mouse model (named Del Ndn-Magel2 mice) with a deletion including both genes. A subset of Del Ndn-Magel2 mice showed neonatal lethality. Behaviorally, surviving mutant mice exhibited sensory delays during infancy and alterations in social exploration at adulthood. Del Ndn-Magel2 mice had a lower body weight before weaning, persisting after weaning in males only, with reduced fat mass and improved glucose tolerance as well as altered puberty. Adult mutant mice displayed increased ventilation and a persistent increase in apneas following a hypercapnic challenge. Transcriptomics analyses revealed a dysregulation of key circadian genes and alterations of genes associated with axonal function similar to patients with PWS. At neuroanatomical levels, Del Ndn-Magel2 mice had an impaired maturation of oxytocin neurons and a disrupted development of melanocortin circuits. Together, these data indicate that the Del Ndn-Magel2 mouse is a pertinent and genetically relevant model of PWS.

Graphical Abstract Introduction

Prader-Willi syndrome (PWS) arises from the loss of expression of 7 contiguous paternally inherited genes located in the 15q11-q13 region, including the MAGEL2 and NECDIN genes. All the PWS genes are regulated by genomic imprinting, which is an epigenetic process where only 1 allele is expressed in a parent-of-origin–dependent manner. PWS is a complex neurodevelopmental disorder characterized by a lifelong spectrum of phenotypic features starting with severe feeding difficulties and respiratory distress at birth, early sensory deficits, and hypotonia, followed by growth deficiency, hypogonadism and delayed puberty, short stature, excessive weight gain with severe hyperphagia, and cognitive and behavioral problems throughout life (1, 2). Despite extensive clinical trials, there are no effective treatments for PWS, and comprehensive pathophysiological mechanisms have not yet been clearly identified, although converging evidence suggests that the PWS phenotype might result from hypothalamic dysfunctions (2, 3). The contribution of candidate genes in the pathogenesis of PWS and how they interact between each other is still under debate. Nevertheless, 7 rare patients with PWS have a chromosomal deletion including SNORD116 but not SNORD115 (noncoding small nucleolar RNA C/D clusters [SNORDs]) (4), suggesting an important role of SNORD116. However, 2 others patients have a normal expression of SNORD116 yet display a PWS-like phenotype (5, 6). In addition, point mutations in the paternal allele of MAGEL2 only are responsible for the Schaaf-Yang syndrome (SYS) that has a phenotypic overlap with PWS but with a more severe autistic phenotype in adolescence and adulthood (7, 8). These results suggest that the loss of function of MAGEL2 is responsible for overlapping symptoms in PWS and SYS.

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.

Results

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 and B). 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 and B; 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 (Ndntm1-Mus) (32) or Magel2 (Magel2tm1-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 overexpressed in Necdin-KO brain (Figure 1E) and Necdin immunoreactivity was increased in the brains of Magel2-KO mice (Figure 1F). Together, these data show that Magel2 and Necdin are spatiotemporally coregulated and share a putative enhancer with functions that partially overlap.

Figure 1

Coexpression and coregulation of Necdin and Magel2 genes in the mouse brain. (A and B) Images showing Necdin and Magel2 mRNA-expressing cells in the forebrain and brainstem of E12.5 mice (A) and in the hypothalamus of adult mice (B). (C) Map of the mouse genomic region including Necdin and Magel2 genes, ENCODE cis regulatory elements, and associations between enhancers and promoters of genes, extracted from UCSC Genome Browser. A coregulation of Magel2 and Necdin via shared enhancer is proposed (physical link). (D) qPCR analysis showing relative levels of Necdin and Magel2 mRNAs in the hypothalamus of WT, Magel2-KO, or Necdin-KO male and female mice at P0 (n = 3–5 animals per group). (E) Images showing Magel2 mRNA expression on horizontal brain sections (at the level of the presumptive hypothalamus) of WT and Necdin-KO embryos at E12.5. (F) Images showing Necdin immunoreactivity in coronal sections at the level of the hypothalamus of WT and Magel2-KO mice at P0. Data are presented as mean ± SEM. Statistical significance between groups was determined by a 2-way ANOVA with Šidák’s multiple-comparison test (D). *P < 0.05. Scale bars: 50 μm (A and E), 20 μm (B), 500 μm (F). cp, cortical plate; hp, hypothalamus; mge, median ganglion eminence; mo, medulla oblongata; ps, pons; tg, tongue; PVH, paraventricular nucleus; SCN, suprachiasmatic nucleus; V3, third ventricle.

Based on the findings described above, it appears that the combined loss of Magel2 and Necdin, by reflecting more accurately the genetics of PWS, will be a more appropriate model to study a PWS-like phenotype. We, therefore, generated mice with a large deletion including both Necdin and Magel2 genes (hereafter called Del Ndn-Magel2 mice) using both Ndntm1-Mus and Magel2 tm1-Mus–single KO mice and an in vivo chromosomal rearrangement based on the Cre-loxP system (Figure 2A). We created a potentially novel allele with a 32 kb deletion that includes Necdin, the fragment between Necdin and Magel2, the Magel2 promoter, and half of the Magel2 coding part (Figure 2A). We screened and validated the recombined Del Ndn-Magel2 allele by PCR using Necdin and Magel2 primers, and we sequenced the recombined allele confirming a recombination at the loxP sites and the expected deletion (Figure 2, B and C). Because Magel2 and Necdin are regulated by genomic imprinting resulting in transcriptional silencing of the maternal allele and expression of the paternal allele only, all studies described below were performed on heterozygous mice with a mutated paternal allele and a WT yet silent maternal allele (+m/–p), considered as KO. We measured Magel2 and Necdin mRNA levels in the hypothalamus of Del Ndn-Magel2 heterozygous male and female mice (+m/–p) using qPCR and confirmed the loss of expression of both transcripts (Figure 2, D and E). We also confirmed the loss of Necdin protein expression in Del Ndn-Magel2 brains using IHC (Figure 2F). The loss of Magel2 protein expression could not be checked due the lack of specific MAGEL2 antibodies. Because all PWS genes share a common spatiotemporal and imprinted regulation, it was possible that the genomic deletion of Necdin and Magel2 could also have an effect on the transcriptional regulation of other PWS genes. However, we did not find significant alterations in the expression of Snord115, Snord 116, Mkrn3, or Snurf-snrpn in the hypothalamus of Del Ndn-Magel2 mice (Figure 2, D and E).

Figure 2

Construction and validation of the Del Ndn-Magel2 mouse model. (A) Strategy to obtain a 32 kb deletion including both the Necdin and Magel2 genes using a transallelic recombination approach. First, a female mouse containing 1 maternal allele with the Magel2 deletion, 1 paternal allele with the Necdin deletion, and containing the Hprt-Cre gene was created using both Necdintm1-Mus– and Magel2tm1-Mus–KO models and a transgenic mouse line expressing the Cre recombinase under Hprt promoter. We then crossed these female mice with WT male mice and screened the litter for the recombinant Del Ndn-Magel2 allele using PCR with Necdin sense and Magel2 antisense primers. The Del Ndn-Magel2 allele being created in the ovocytes. (B) PCR product obtained from the recombinant Del Ndn-Magel2 allele (354 bp) in the F1 generation. (C) Sequence of the recombined Del Ndn-Magel2 allele with the Necdin upstream sequence (blue) and Ndn sense primer (bold blue), the LoxP sequence (bold black), and Magel2 sequence (green), and Ml2 anti-sense primer (bold green). This sequence validates the recombination at the loxP sites. (D and E) qPCR analysis showing relative levels of Snord115, Snord116, Mkrn3, Snurf-snron, Magel2, and Necdin mRNAs in the hypothalamus of WT and Del Ndn-Magel2 male (D) and female (E) mice at P60. (F) Images showing Necdin immunoreactivity on sagittal sections of WT and Magel2-KO mouse brains at P7. (G) Breeding strategy to generate Del Ndn-Magel2 and WT litters with an expected ratio of 1 Del Ndn-Magel2/1 WT. (H) Ratio of Del Ndn-Magel2 and WT mice at P21 from 6 cohorts produced in the different laboratories performing experiments for this study. (I) Photos showing cyanosis and lack of milk in the stomach associated to the death of Del Ndn-Magel2 pups at P1. Data are presented as mean ± SEM. Statistical significance between groups was determined by a Mann-Whitney U test (D and E). ***P ≤ 0.002. Scale bar: 400 μm. cer, cerebellum; cx, cortex; hp, hypothalamus; hip, hippocampus.

We generated several cohorts of Del Ndn-Magel2 heterozygous and WT mice in different laboratories. Although the expected Mendelian ratio of our cross was 1:1, we often observed a reduced number of Del Ndn-Magel2 mice compared with WT animals (Figure 2G). In 5 of 6 cohorts generated (representing 92 litters), we found a reduction of 40% (ranging from 20% to 60%) of Del Ndn-Magel2 mice compared with WT animals (Figure 2H). The variability in the postnatal lethality observed in Del Ndn-Magel2 pups appeared to be correlated with the level of sanitary status of the animal facility in which animals were housed; when animals were housed in a pathogen-free animal facility and fed sterile food, the phenotype was less severe (with 0% lethality) than when animals were housed in a conventional animal facility (with 60% lethality in mutant mice versus control littermates). The lethality occurred during the first day of life. Monitoring neonates shortly after birth revealed cyanosis in dead Del Ndn-Magel2 mice (Figure 2I), suggesting a lack of proper oxygenation due to respiratory dysfunction as observed in Ndntm1-Mus–KO mice (38, 43). Dead Del Ndn-Magel2 neonates also lacked milk in their stomachs (Figure 2I) as previously observed in the Magel2tm1-Mus–KO mouse model (20). Together, these data indicate that the possible causes of death in Del Ndn-Magel2–KO newborns include respiratory defects and/or feeding deficits at birth.

Del Ndn-Magel2 mice develop sensory alterations in infancy and social exploration deficits during adulthood.

Because patients with PWS present symptoms at birth evolving with age, we began to characterize the phenotype of Del Ndn-Magel2 during neonatal life. We first used 11 reliable tests established by Roubertoux et al. (44) to assess the sensory and motor abilities during the first 2 weeks of postnatal life (Supplemental Figure 2A). Each test was performed at specific postnatal ages defined by the critical period during which the response is established in 100% of control mice in the mouse strain that we used (i.e., C57BL6/J) (Supplemental Figure 2B). We compared Del Ndn-Magel2 with WT pups from the same litters. Since we did not observe differences between males and females, we pooled both sexes.

We found differences between mutant and WT mice in 5 of the 11 tests. Del Ndn-Magel2 pups were less efficient in righting response, rooting response, and paw position on the floor (Figure 3, A–C). The righting response reveals motor and sensory (proprioception) abilities, and its development was delayed in Del Ndn-Magel2 pups at P4 and P8 (Figure 3A). The rooting reflex involves cranial sensory nerves; this reflex disappeared in WT mice across postnatal development, but it was more frequent in Del Ndn-Magel2 pups at P7 and at P12 (tendency) compared with WT mice (Figure 3B). The paw position test is mainly a sensory test (proprioception, touch) and revealed a delay in sensory development in Del Ndn-Magel2 pups (Figure 3C). In contrast, mutant mice performed better in the bar-holding test at P11 (Figure 3D). This is a motor test, requiring muscle strength. Interestingly, Del Ndn-Magel2 pups also performed better in the pulling up on bar test at P12 (Figure 3E) — i.e., they were more efficient in bringing back the bar to them and using their hind legs. However, they were less efficient in standing up on the bar at P15, requiring more sensory abilities (Figure 3E). However, mutant and WT pups performed similarly in the vertical climbing test as well as the 2 other tests involving vestibular and motor activity (i.e., climbing the slope and cliff avoidance) (Supplemental Figure 3, C–E). Similarly, the age of eye opening and auditory canal opening, which normally occurs between P13 and P14, was similar between Del Ndn-Magel2 and WT mice (Supplemental Figure 3 A an