Olivier De Backer

Structure, Chromosomal Localization, and Expression of 12 Genes of the Mage Family

We reported previously that human geneMAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with aMAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12MAGE genes are located in the q terminal region of chromosome X. LikeMAGE-1, the 11 additionalMAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that ofMAGE-1. The coding sequences of theMAGE genes predict the same main structural features for allMAGE proteins. In contrast, the promoters and first exons of the12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of eachMAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of theMAGE family includingMAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

Protocadherin Celsr3 is crucial in axonal tract development

In the embryonic CNS, the development of axonal tracts is required for the formation of connections and is regulated by multiple genetic and microenvironmental factors. Here we show that mice with inactivation of Celsr3, an ortholog of Drosophila melanogaster flamingo (fmi; also known as starry night, stan) that encodes a seven-pass protocadherin, have marked, selective anomalies of several major axonal fascicles, implicating protocadherins in axonal development in the mammalian CNS for the first time. In flies, fmi controls planar cell polarity (PCP) in a frizzled-dependent but wingless-independent manner. The neural phenotype in Celsr3 mutant mice is similar to that caused by inactivation of Fzd3, a member of the frizzled family. Celsr3 and Fzd3 are expressed together during brain development and may act in synergy. Thus, a genetic pathway analogous to the one that controls PCP is key in the development of the axonal blueprint.

Early forebrain wiring: genetic dissection using conditional Celsr3 mutant mice.

Development of axonal tracts requires interactions between growth cones and the environment. Tracts such as the anterior commissure and internal capsule are defective in mice with null mutation of Celsr3. We generated a conditional Celsr3 allele, allowing regional inactivation. Inactivation in telencephalon, ventral forebrain, or cortex demonstrated essential roles for Celsr3 in neurons that project axons to the anterior commissure and subcerebral targets, as well as in cells that guide axons through the internal capsule. When Celsr3 was inactivated in cortex, subcerebral projections failed to grow, yet corticothalamic axons developed normally, indicating that besides guidepost cells, additional Celsr3-independent cues can assist their progression. These observations provide in vivo evidence that Celsr3-mediated interactions between axons and guidepost cells govern axonal tract formation in mammals.

MAGE‐A4, a germ cell specific marker, is expressed differentially in testicular tumors

Testicular germ cell tumors are the most common malignancy in young males, and the frequency of these tumors has risen dramatically over the last century. Because it is known that the MAGE genes are expressed in a wide variety of tumors but are expressed only in the mitotic spermatogonia (germ cells) and in the primary spermatocytes in the normal testis, the authors screened the expression of MAGE‐A4 in a panel of testicular germ cell tumors.

ProNGF induces TNFα-dependent death of retinal ganglion cells through a p75NTR non-cell-autonomous signaling pathway

Neurotrophin binding to the p75 neurotrophin receptor (p75NTR) activates neuronal apoptosis following adult central nervous system injury, but the underlying cellular mechanisms remain poorly defined. In this study, we show that the proform of nerve growth factor (proNGF) induces death of retinal ganglion cells in adult rodents via a p75NTR-dependent signaling mechanism. Expression of p75NTR in the adult retina is confined to Müller glial cells; therefore we tested the hypothesis that proNGF activates a non-cell-autonomous signaling pathway to induce retinal ganglion cell (RGC) death. Consistent with this, we show that proNGF induced robust expression of tumor necrosis factor alpha (TNFα) in Müller cells and that genetic or biochemical ablation of TNFα blocked proNGF-induced death of retinal neurons. Mice rendered null for p75NTR, its coreceptor sortilin, or the adaptor protein NRAGE were defective in proNGF-induced glial TNFα production and did not undergo proNGF-induced retinal ganglion cell death. We conclude that proNGF activates a non-cell-autonomous signaling pathway that causes TNFα-dependent death of retinal neurons in vivo.

Loss of Maged1 results in obesity, deficits of social interactions, impaired sexual behavior and severe alteration of mature oxytocin production in the hypothalamus

MAGED1, NECDIN and MAGEL2 are members of the MAGE gene family. The latter two of these genes have been involved in Prader-Willi syndrome (PWS), which includes hyperphagia, repetitive and compulsive behaviors, and cognitive impairment. Here, we show that Maged1-deficient mice develop progressive obesity associated with hyperphagia and reduced motor activity. Loss of Maged1 also results in a complex behavioral syndrome that includes reduced social interactions and memory, deficient sexual behavior, as well as increased anxiety and self-grooming. Oxytocin (OT), which is produced in the hypothalamus, can act as a neurotransmitter that reduces anxiety, promotes social behaviors and regulates food intake. Growing evidences indicate that OT is involved in autism. We found that Maged1 mutants showed a severe reduction in the levels of mature OT, but not of its precursors, in the hypothalamus. Moreover, the administration of OT rescued the deficit in social memory of these mice. We conclude that Maged1 is required for OT processing or stability. A decrease in mature OT levels in Maged1 mutants affects social interactions and possibly other behavioral processes. Our observations suggest that, in human, MAGED1 could play a role in autism or cause a neurodevelopmental condition that is reminiscent of the PWS.

Polyhydramnios, Transient Antenatal Bartter’s Syndrome, and MAGED2 Mutations

BACKGROUND Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartters syndrome. METHODS To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein-protein interaction studies. RESULTS We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartters syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartters syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein. CONCLUSIONS We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartters syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).

Loss of Function but No Gain of Function Caused by Amino Acid Substitutions in the Hexapeptide of Hoxa1 In Vivo

ABSTRACT Homeodomain containing transcription factors of the Hox family play critical roles in patterning the anteroposterior embryonic body axis, as well as in controlling several steps of organogenesis. Several Hox proteins have been shown to cooperate with members of the Pbx family for the recognition and activation of identified target enhancers. Hox proteins contact Pbx via a conserved hexapeptide motif. Previous biochemical studies provided evidence that critical amino acid substitutions in the hexapeptide sequence of Hoxa1 abolish its interaction with Pbx. As a result, these substitutions also abolish Hoxa1 activity on known target enhancers in cellular models, suggesting that Hoxa1 activity relies on its capacity to interact with Pbx. Here, we show that mice with mutations in the Hoxa1 hexapeptide display hindbrain, cranial nerve, and skeletal defects highly reminiscent of those reported for the Hoxa1 loss of function. Since similar hexapeptide mutations in the mouse Hoxb8 and the Drosophila AbdA proteins result in activity modulation and gain of function, our data demonstrate that the functional importance of the hexapeptide in vivo differs according to the Hox proteins.

Systems genetic analysis of osteoblast-lineage cells.

The osteoblast-lineage consists of cells at various stages of maturation that are essential for skeletal development, growth, and maintenance. Over the past decade, many of the signaling cascades that regulate this lineage have been elucidated; however, little is known of the networks that coordinate, modulate, and transmit these signals. Here, we identify a gene network specific to the osteoblast-lineage through the reconstruction of a bone co-expression network using microarray profiles collected on 96 Hybrid Mouse Diversity Panel (HMDP) inbred strains. Of the 21 modules that comprised the bone network, module 9 (M9) contained genes that were highly correlated with prototypical osteoblast maker genes and were more highly expressed in osteoblasts relative to other bone cells. In addition, the M9 contained many of the key genes that define the osteoblast-lineage, which together suggested that it was specific to this lineage. To use the M9 to identify novel osteoblast genes and highlight its biological relevance, we knocked-down the expression of its two most connected “hub” genes, Maged1 and Pard6g. Their perturbation altered both osteoblast proliferation and differentiation. Furthermore, we demonstrated the mice deficient in Maged1 had decreased bone mineral density (BMD). It was also discovered that a local expression quantitative trait locus (eQTL) regulating the Wnt signaling antagonist Sfrp1 was a key driver of the M9. We also show that the M9 is associated with BMD in the HMDP and is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In conclusion, we have identified a physiologically relevant gene network and used it to discover novel genes and regulatory mechanisms involved in the function of osteoblast-lineage cells. Our results highlight the power of harnessing natural genetic variation to generate co-expression networks that can be used to gain insight into the function of specific cell-types.

Comparative Expression Analysis of the MAGED Genes During Embryogenesis and Brain Development

The MAGED gene subfamily contains three genes in mouse and four in human. The MAGED1, D2, and D3 proteins are highly conserved between mouse and human, whereas paralogues are less conserved between each other. This finding suggests that each MAGED protein exerts a distinct function. To get a better insight into their physiological roles, we have analyzed their expression patterns during embryogenesis and brain development. In the mouse, Maged3 expression is restricted to the central nervous system where it was mostly detected in postmitotic neurons. Maged2 is mainly expressed in tissues of mesodermal origin. The expression pattern of Maged1 roughly summarizes that of Maged2 and Maged3; however, contrary to that of Maged3, it includes the proliferative zones of the nervous system. We observed a discrepancy between Maged1 expression levels of RNA and protein, suggesting that its expression is regulated at a posttranscriptional level during the mouse development. Developmental Dynamics 230:325–334, 2004.