Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin-and interleukin 6-stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.Signals mediated by BMP ligands serve diverse roles throughout the life of vertebrates. During embryogenesis, the dorsoventral axis is established by BMP signaling gradients formed by the coordinated expression of ligands, receptors, co-receptors and soluble antagonists 1-3 . Excess BMP signaling causes ventralization (an expansion of ventral structures at the expense of dorsal structures) whereas diminished BMP signaling causes dorsalization (an expansion of dorsal structures at the expense of ventral structures) 1,3,4 . BMPs are key regulators of gastrulation,

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Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the “candy store” effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.

Bone morphogenetic proteins (BMPs) 2 are members of the TGF-␤ family that play essential roles in early embryonic patterning, gastrulation, and organogenesis as well as in the remodeling of mature tissues (1). BMP signals are involved in the commitment of vascular precursor cells in vasculogenesis and regulate the growth, differentiation, and turnover of vascular cell populations (2-4). There is increasing evidence that BMP signals contribute to vascular calcific disease of the intima and tunica media (5). Further underscoring the importance of BMP signaling in vascular tissues, defects in the BMP signaling apparatus have been linked to two genetic vascular syndromes, hereditary hemorrhagic telangectasia syndrome and idiopathic pulmonary arterial hypertension (4). In idiopathic pulmonary arterial hypertension, diverse loss-of-function mutations in the canonical BMP type II receptor (BMPRII) are associated with the majority of familial and a subset of sporadic cases (6 -9). Advanced idiopathic pulmonary arterial hypertension is marked by an obstructive and proliferative vasculopathy involving endothelial and myofibroblast lineages (10). Although it has been suggested that defective BMP-mediated regulation of cell differentiation and turnover could contribute to abnormal vascular remodeling, the precise contribution of BMPRII to these functions is incompletely understood.Signaling by BMP ligands is accomplished by at least three type II receptors, BMPRII, ActRIIa, and ActRIIb, and three type I receptors, ALK2, ALK3, and ALK6 (11)(12)(13)(14)(15)(16)(17)(18)(19). At least 25 different BMP ligands can facilitate the assembly of type II and type I receptor heteromers to cause phosphorylation and activation of type I receptor kinases by constitutively active type II receptor kinases. Activated type I receptors phosphorylate BMP-responsive SMAD (BR-SMAD) intracellular effectors (SMAD1, -5, and -8). Downstream effects of BR-SMAD activation are mediated in part by the transcription of the dominant negative bHLH Inhibitor of differentiation (Id) proteins, as well as by

Despite their physiological roles, Müller glial cells are involved directly or indirectly in retinal disease pathogenesis and are an interesting target for therapeutic approaches for retinal diseases and regeneration such as CRB1 inherited retinal dystrophies. In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells. ShH10Y and AAV9 were the most powerful capsids to infect mouse Müller glial cells. Retinaldehyde-binding protein 1 (RLBP1) promoter was the most powerful promoter to transduce Müller glial cells. ShH10Y capsids and RLBP1 promoter targeted human Müller glial cells in vitro. We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors. Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller glial cells. In summary, ShH10Y and AAV9 capsids, and RLBP1 or minimal CMV promoters are of interest as specific tools to target and express in mouse or human Müller glial cells.

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Fibroblast growth factor 2 (FGF-2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF-2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF-2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF-2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF-2 (LV-FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF-2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV-FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV-FGF2 into the rat sciatic nerve resulted in increased expression of FGF-2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF-2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV-FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV-FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow-up.

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