Necdin, a Prader-Willi syndrome candidate gene, regulates gonadotropin-releasing hormone neurons during development - PubMed (original) (raw)

. 2009 Jan 15;18(2):248-60.

doi: 10.1093/hmg/ddn344. Epub 2008 Oct 17.

Affiliations

Necdin, a Prader-Willi syndrome candidate gene, regulates gonadotropin-releasing hormone neurons during development

Nichol L G Miller et al. Hum Mol Genet. 2009.

Abstract

Prader-Willi syndrome (PWS) is a complex genetic disorder characterized by hyperphagia, obesity and hypogonadotrophic hypogonadism, all highly suggestive of hypothalamic dysfunction. The NDN gene, encoding the MAGE family protein, necdin, maps to the PWS chromosome region and is highly expressed in mature hypothalamic neurons. Adult mice lacking necdin have reduced numbers of gonadotropin-releasing hormone (GnRH) neurons, but the mechanism for this reduction is unknown. Herein, we show that, although necdin is not expressed in an immature, migratory GnRH neuronal cell line (GN11), high levels are present in a mature GnRH neuronal cell line (GT1-7). Furthermore, overexpression of necdin activates GnRH transcription through cis elements bound by the homeodomain repressor Msx that are located in the enhancer and promoter of the GnRH gene, and knock-down of necdin expression reduces GnRH gene expression. In fact, overexpression of Necdin relieves Msx repression of GnRH transcription through these elements and necdin co-immunoprecipitates with Msx from GnRH neuronal cells, indicating that necdin may activate GnRH gene expression by preventing repression of GnRH gene expression by Msx. Finally, necdin is necessary for generation of the full complement of GnRH neurons during mouse development and extension of GnRH axons to the median eminence. Together, these results indicate that lack of necdin during development likely contributes to the hypogonadotrophic hypogonadal phenotype in individuals with PWS.

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Figures

Figure 1.

Figure 1.

Necdin is expressed in GT1-7 but not detected in GN11 cells. (A) RNA from GT1-7 versus GN11 cells was compared using Affymetrix microarray MOE430A. Fold difference between GT1-7 and Gn11 are shown for MAGE family members Necdin, Maged1 (encoding Dlxin-1) and Magel2, and for Nhlh1. Data were analyzed using GeneSpring software and the mean of two arrays is presented. (B) RT–PCR analysis to confirm expression of MAGE family members, necdin, Dlxin-1 and Magel2 in GN11 and GT1-7 cell lines. Adult mouse brain cDNA was used as a positive control for Mage mRNAs. Cyclophilin B (CyB) was used as a control for equal loading. Ethidium-stained 1% gel pictured. (C) Immunoblots of Necdin and cyclophilin B proteins from GT1-7, GN11, NIH3T3, and LβT2 cells.

Figure 2.

Figure 2.

Necdin activates GnRH gene expression. (A) Two luciferase reporters were studied: −5 kb GnRH and GnRH enhancer reporter, which is a condensed combination of the three well-characterized rat GnRH regulatory elements: upstream enhancer (UE), enhancer (E) and promoter (P) (36). (B) Each reporter was co-transfected into GN11 cells with a necdin expression vector (necdin) or empty vector (control). (C) GT1-7 cells were co-transfected with pGL3-luciferase or GnRH enhancer reporter and either necdin expression vector or empty vector control. (D) GN11 cells were co-transfected with GnRH enhancer reporter and either necdin, Magel2, Dlxin-1, or empty vector control. Experiments were performed in quadruplicate and repeated at least three times. Results shown are mean ± SEM. The means were compared by one-way ANOVA and Tukey–Kramer HSD. *indicates significant difference (P < 0.05) relative to empty vector control.

Figure 3.

Figure 3.

Necdin specifically disrupts Msx repression of GnRH gene expression. (A) The GnRH enhancer reporter was co-transfected into GN11 cells with either empty vector control or Msx1 expression vector ± necdin expression vector. *Significant difference (P < 0.05) from empty vector control, while #Significant difference (P < 0.05) relative to Msx1 alone, as indicated by brackets. (B) The GnRH enhancer reporter was co-transfected with either empty vector control, or with Dlx1, Dlx2 and Dlx5 expression vectors (Dlx) ± necdin expression vector. Experiments were performed in quadruplicate and repeated at least three times. Results shown are mean ± SEM. One-way ANOVA and Tukey–Kramer HSD compared the means.

Figure 4.

Figure 4.

Necdin activation requires the Msx-binding sites in the GnRH enhancer and promoter. (A) The rat GnRH enhancer reporter contains four Msx/Dlx-binding sites (bold) in the GnRH promoter and enhancer at −1637, −1624, −58, and −42 bp relative to the mRNA start site. Mutations are shown as deletions (-) or base changes (underline) in the below sequence. (B) Necdin expression vector was co-transfected into GN11 cells with either GnRH enhancer reporter or mutant GnRH enhancer reporter. Experiments were performed in quadruplicate and repeated at least three times. Results shown are mean ± SEM. Means were compared by Student’s _t_-test and *Significant values (P < 0.05) relative to empty vector control.

Figure 5.

Figure 5.

Knockdown of necdin in GT1-7 cells decreases GnRH mRNA and gene expression. (A) GT1-7 cells were either mock-transfected or transfected with non-targeting (control), cyclophilin B (CyB) or necdin (Ndn) siRNA duplexes. Specific knockdown of necdin and cyclophilin B proteins was detected by immunoblot. (B) GT1-7 cells were transfected as in (A) followed by RNA isolation. GnRH was measured by quantitative RT–PCR and data are shown as pg GnRH mRNA when normalized to CyB mRNA. Results are the means of three experiments performed in triplicate and *****indicates significantly different from control by Student’s _t_-test (P < 0.05). (C) GT1-7 cells were co-transfected with control or Ndn siRNA duplexes and either GnRH enhancer reporter or mutant GnRH enhancer reporter. Experiments were performed in quadruplicate and repeated at least three times. Results shown are mean ± SEM. One-way ANOVA and Tukey–Kramer HSD compared the means. *P < 0.05.

Figure 6.

Figure 6.

Necdin interacts with Msx in GT1-7 cells. (A) Proteins from GT1-7 cells were immunoprecipitated (IP) with either anti-necdin antibody or rabbit IgG control. Msx1 and necdin were detected by immunoblotting the immunoprecipitated proteins. Ten percent of the input was used for Msx1 and 2% for necdin. (B) GT1-7 cells were transiently transfected with Msx1-FLAG, Dlxin1-FLAG expression vector or empty vector control. Cell extract was immunoprecipitated with anti-FLAG affinity gel. Immunoprecipitates and 4% of the input were immunoblotted for necdin.

Figure 7.

Figure 7.

Necdin expression does not change the cellular localization of Msx. (A) Msx1-FLAG overexpression in GN11 was detected with an anti-FLAG-Cy3 antibody conjugate (red). (B) GN11 cells were transiently transfected with both necdin and Msx1-FLAG expression vectors and then subjected to immunofluorescence using the anti-FLAG-Cy3 antibody (red) or anti-necdin (green). Blue staining is DAPI nuclear labeling. Image shown is at 40× magnification.

Figure 8.

Figure 8.

Embryonic day 13.5 Necdin-null mouse embryos have significantly fewer GnRH neurons and lower density of GnRH fibers in the median eminence. (A) Diagram of e13 mouse head anatomy in sagittal showing location of migrating GnRH neurons and the orientation of all sections. Immunohistochemistry for GnRH in e13.5 wild-type embryos (B) or necdin-null embryos (C). Images are shown at 4×. (D) Average numbers of total GnRH neurons at e13.5. (E) Average numbers of GnRH neurons counted in the nasal, cribriform plate or brain regions at e13.5 as depicted in (B). e17.5 Necdin-null mice have fewer GnRH neurons in the brain. (F) Diagram of e17 mouse head anatomy in sagittal showing location of migrating GnRH neurons. The red box represents the field shown in images in (G) and (H), while the green box indicates the field shown in images in (K) and (L). All images are in the orientation shown in the illustration. Immunohistochemistry for GnRH in e17.5 wild-type embryos (G) or necdin-null embryos (H). Images are shown at 10×. (I) Average numbers of total GnRH neurons at e17.5. (J) Average numbers of GnRH neurons counted in the nasal, cribriform plate or brain regions at e17.5 as depicted in (B). Means were compared with wild-type littermates by Student’s _t_-test and *P < 0.05. Error bars are standard deviation. Sagittal sections of littermate e17.5 wild-type (K) and necdin-null (L) embryos were stained for GnRH immunoreactivity in the median eminence (ME). Sections also reveal the structure of the developing pituitary (P). Images are representative sections from those used for GnRH neuron quantitation.

Figure 9.

Figure 9.

A model for necdin action on GnRH gene expression. Early in their development, the majority of GnRH neurons is located in the nasal septum and is migrating toward the brain. During this spatiotemporal period, Msx homeodomain repressors are expressed in vivo and may be bound to specific elements within the GnRH promoter and enhancer to repress GnRH, resulting in low GnRH expression (26). Late in their development, most GnRH neurons have crossed into the forebrain and hypothalamus (areas of Dlx activator expression) and are expressing high levels of GnRH. It is thought that Dlx proteins are then binding to the same homeodomain elements within the GnRH regulatory region, to increase GnRH expression (26). The ‘switch’ from Msx to Dlx factors on the GnRH promoter may be the result of the interaction of Msx repressors with the MAGE protein, necdin, which is being expressed along the GnRH migratory route at this time, resulting in an inactivation of Msx repression and allows Dlx activators to exert their function. Necdin may require co-factors such as MAGE family member Dlxin-1 for this functional complex.

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