Mouse RGMs : a three protein family with diverse function and localization

Identification and Functional Characterization of the Mouse RGM Family In the developing chick visual system, axons project from the retina to the optic tectum in a stereotypical manner to produce a topographic map. This topography conserves spatial information registered by the retina by preserving nearest neighbour relationships among the termination zones of projecting retinal ganglion cells (RGCs). Thus, two RGCs which lie next to each other in the retina will have axonal projections terminating very near each other within the optic tectum, while RGCs which are at opposite sides of the retina will have diametrically opposed termination zones. The establishment of the retinotectal topographic map relies on tight spatial and temporal control of molecules which control axon guidance, branching and termination. One such molecule, proposed to inhibit axonal growth into the tectum, Repulsive Guidance Molecule (RGM), has been implicated in control of RGC axon termination along the anterior-posterior axis of the chick optic tectum. We discovered three mouse genes homologous to chick RGM, the protein products of which share similarities in structure, proteolytic cleavage and putative GPI-anchoring, but which differ in spatio-temporal expression, cell surface targeting and most importantly function. Two members of this gene family (mRGMa and mRGMb) are expressed in the nervous system. In the visual system, mRGMa is prominently expressed in the superior colliculus, the mouse equivalent of the chick optic tectum, and mRGMb in the retinal ganglion cell layer at the time of anterior-posterior targeting of RGC axons. The third member of the family, mRGMc (independently identified as hemojuvelin (hjv)), is expressed most strongly in skeletal muscles, but also in liver and heart. Surprisingly, neither mRGMa nor mRGMb are expressed in a gradient in the superior colliculus. Moreover, disruption of either mRGMa or mRGMb does not affect the anterior-posterior targeting of the topographic map. Instead, half of mRGMa mutant mice show a severe defect in cephalic neural tube closure, known as exencephaly, while the remaining animals appear phenotypically normal. All mRGMb mutant mice die at approximately three weeks of age for unknown reasons, indicating an essential requirement for RGMb, however its specific function remains a mystery. Mice deficient in mRGMc suffer from severe iron overload. This condition is similar to juvenile hemochromatosis, a human disease resulting from mutations in the gene HFE2, the human homologue of mRGMc. At a molecular level, the severity of the disease state in Hjv mutant mice can be explained by dramatic decrease in hepcidin, a negative regulator of iron absorption produced by the liver in response to ingested iron. Interestingly, these mice retain the ability to produce hepcidin in response to inflammatory stimuli. Furthermore, induction of inflammatory response causes a rapid downregulation of Hjv in wildtype mice. Our findings define a key role for Hjv in dietary iron-sensing and reveal how Hjv acts a switch during inflammation, to prevent conflict between the pathway controlling dietary iron homeostasis and that controlling inflammatory iron sequestration as a defense mechanism against infection

The effect of growth factors on bulbospinal neurite outgrowth in an in vitro embryonic chick model

Injury to the spinal cord of higher vertebrates damages motor axons that connect the brain and brainstem with their targets in the spinal cord. Axotomized central nervous system (CNS) neurons often experience degeneration of the distal axon, retraction of the proximal end, and atrophy of the cell body. Injured neurons in the CNS do not experience significant functional regeneration, so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on the interplay between intrinsic and external cues. Regenerative failure in the mature axon is thought to be the result of: a) failure to survive primary and secondary damage, b) glial scarring, c) presence of inhibitory growth substrates that prevent neuronal extension, and d) decreased availability of neurotrophic factors and permissive substrates supporting neuronal process extension. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play the pivotal role in motor dysfunction, we still know relatively little about the trophic sensitivity of these populations. The experiments presented in this study will help to elucidate the role trophic molecules play in process extension in brainstem-spinal neuron populations. Using an assay specifically developed to examine the effect of trophic molecules on neuronal process extension, this study explores the response of bulbospinal populations to various trophic factors. Already investigated in our laboratory using these techniques are members of the fibroblast growth factor family, (FGF-1,-2, -5 and -9). Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) and epidermal growth factor (EGF) did not effect process outgrowth in the bulbospinal neurons of the vestibular complex (vestibulospinal neurons). Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and projection neurons from the raphe nuclei (raphespinal neurons). Neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) require further study to determine their contribution to process elongation. In order to examine the mechanism of trophic factor effects, immunohistochemistry to the cognate receptors for BDNF and IGF-1 was performed. At the developmental stages used in the study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology. It is hoped that this study will contribute to the growing pool of knowledge on spinal cord injury, and may one day play a role in development of a multi-faceted therapy for spinal cord injury.Medicine, Faculty ofGraduat

Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload

Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway

A rapid, nonradioactive in situ hybridization technique for use on cryosectioned adult mouse bone.

In situ hybridization (ISH) of adult bone is a difficult task that requires at least 3-5 weeks for decalcification, paraffin embedding, and sectioning. For that reason, bone ISH is often done only on embryonic or newborn animal tissue, leaving unanswered the question of gene expression in adults. Here, we report the development of an ISH system that requires only 7 days for acid-free decalcification, embedding, and sectioning, conditions that are conducive to preservation of tissue mRNA. The tissue cryosections, derived from adult mice 3-12 weeks old, were cut using the CryoJane Tape Transfer system. Paraffin-sectioned and cryosectioned tissue have comparable morphology. Examples are given of cryosections that were hybridized and stained enzymatically with digoxigenin-labeled riboprobes for mRNA found in either bone-forming osteoblasts (type I collagen, osteocalcin, Runx2) or the hypertrophic or proliferating chondrocytes (type X collagen, Runx2)

The BMP Coreceptor RGMb Promotes While the Endogenous BMP Antagonist Noggin Reduces Neurite Outgrowth and Peripheral Nerve Regeneration by Modulating BMP Signaling

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb−/− mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb−/− mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin