Effects of 2,3-DPG knockout on SCD phenotype in Townes SCD model mice.

Sickle cell disease (SCD) is a severe, multisystemic hematological disorder that impacts nearly every major organ in adults. The current approved treatments for SCD directly target mutant hemoglobin or address downstream disease pathology. Several compounds targeting reduction of 2,3-DPG by activation of Pyruvate Kinase-R are currently being evaluated in SCD patients. In this study, we genetically engineered a mouse lacking 2,3-DPG on the Townes SCD mouse model background and evaluated the effects of 2,3-DPG loss on disease pathology. Animals lacking 2,3-DPG showed improvements in hematological markers and reductions in RBC sickling relative to native Townes mice, however, minimal difference in organ damage was observed in 2,3-DPG deficient mice compared to native Townes animals. When animals lacking 2,3-DPG were dosed with a compound designed to increase hemoglobin oxygen affinity, oxygen delivery related toxicity was observed.

Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia.

Erythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with ß-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of ß-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE's mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti-ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE-BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.

Erythroferrone inhibits the induction of hepcidin by BMP6.

Decreased hepcidin mobilizes iron, which facilitates erythropoiesis, but excess iron is pathogenic in ß-thalassemia. Erythropoietin (EPO) enhances erythroferrone (ERFE) synthesis by erythroblasts, and ERFE suppresses hepatic hepcidin production through an unknown mechanism. The BMP/SMAD pathway in the liver is critical for hepcidin control, and we show that EPO suppressed hepcidin and other BMP target genes in vivo in a partially ERFE-dependent manner. Furthermore, recombinant ERFE suppressed the hepatic BMP/SMAD pathway independently of changes in serum and liver iron. In vitro, ERFE decreased SMAD1, SMAD5, and SMAD8 phosphorylation and inhibited expression of BMP target genes. ERFE specifically abrogated the induction of hepcidin by BMP5, BMP6, and BMP7 but had little or no effect on hepcidin induction by BMP2, BMP4, BMP9, or activin B. A neutralizing anti-ERFE antibody prevented ERFE from inhibiting hepcidin induction by BMP5, BMP6, and BMP7. Cell-free homogeneous time-resolved fluorescence assays showed that BMP5, BMP6, and BMP7 competed with anti-ERFE for binding to ERFE. We conclude that ERFE suppresses hepcidin by inhibiting hepatic BMP/SMAD signaling via preferentially impairing an evolutionarily closely related BMP subgroup of BMP5, BMP6, and BMP7. ERFE can act as a natural ligand trap generated by stimulated erythropoiesis to regulate the availability of iron.

The mouse Wnt/PCP protein Vangl2 is necessary for migration of facial branchiomotor neurons, and functions independently of Dishevelled.

During development, facial branchiomotor (FBM) neurons, which innervate muscles in the vertebrate head, migrate caudally and radially within the brainstem to form a motor nucleus at the pial surface. Several components of the Wnt/planar cell polarity (PCP) pathway, including the transmembrane protein Vangl2, regulate caudal migration of FBM neurons in zebrafish, but their roles in neuronal migration in mouse have not been investigated in detail. Therefore, we analyzed FBM neuron migration in mouse looptail (Lp) mutants, in which Vangl2 is inactivated. In Vangl2(Lp/+) and Vangl2(Lp/Lp) embryos, FBM neurons failed to migrate caudally from rhombomere (r) 4 into r6. Although caudal migration was largely blocked, many FBM neurons underwent normal radial migration to the pial surface of the neural tube. In addition, hindbrain patterning and FBM progenitor specification were intact, and FBM neurons did not transfate into other non-migratory neuron types, indicating a specific effect on caudal migration. Since loss-of-function in some zebrafish Wnt/PCP genes does not affect caudal migration of FBM neurons, we tested whether this was also the case in mouse. Embryos null for Ptk7, a regulator of PCP signaling, had severe defects in caudal migration of FBM neurons. However, FBM neurons migrated normally in Dishevelled (Dvl) 1/2 double mutants, and in zebrafish embryos with disrupted Dvl signaling, suggesting that Dvl function is essentially dispensable for FBM neuron caudal migration. Consistent with this, loss of Dvl2 function in Vangl2(Lp/+) embryos did not exacerbate the Vangl2(Lp/+) neuronal migration phenotype. These data indicate that caudal migration of FBM neurons is regulated by multiple components of the Wnt/PCP pathway, but, importantly, may not require Dishevelled function. Interestingly, genetic-interaction experiments suggest that rostral FBM neuron migration, which is normally suppressed, depends upon Dvl function.

Atypical cadherins Celsr1-3 differentially regulate migration of facial branchiomotor neurons in mice.

During hindbrain development, facial branchiomotor neurons (FBM neurons) migrate from medial rhombomere (r) 4 to lateral r6. In zebrafish, mutations in planar cell polarity genes celsr2 and frizzled3a block caudal migration of FBM neurons. Here, we investigated the role of cadherins Celsr1-3, and Fzd3 in FBM neuron migration in mice. In Celsr1 mutants (knock-out and Crash alleles), caudal migration was compromised and neurons often migrated rostrally into r2 and r3, as well as laterally. These phenotypes were not caused by defects in hindbrain patterning or neuronal specification. Celsr1 is expressed in FBM neuron precursors and the floor plate, but not in FBM neurons. Consistent with this, conditional inactivation showed that the function of Celsr1 in FBM neuron migration was non-cell autonomous. In Celsr2 mutants, FBM neurons initiated caudal migration but moved prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3 in FBM neurons and mimicked by inactivation of Fzd3. Furthermore, Celsr2 was epistatic to Celsr1. These data indicate that Celsr1-3 differentially regulate FBM neuron migration. Celsr1 helps to specify the direction of FBM neuron migration, whereas Celsr2 and 3 control its ability to migrate.

The cell adhesion molecule Tag1, transmembrane protein Stbm/Vangl2, and Lamininalpha1 exhibit genetic interactions during migration of facial branchiomotor neurons in zebrafish.

Interactions between a neuron and its environment play a major role in neuronal migration. We show here that the cell adhesion molecule Transient Axonal Glycoprotein (Tag1) is necessary for the migration of the facial branchiomotor neurons (FBMNs) in the zebrafish hindbrain. In tag1 morphant embryos, FBMN migration is specifically blocked, with no effect on organization or patterning of other hindbrain neurons. Furthermore, using suboptimal morpholino doses and genetic mutants, we found that tag1, lamininalpha1 (lama1) and stbm, which encodes a transmembrane protein Vangl2, exhibit pairwise genetic interactions for FBMN migration. Using time-lapse analyses, we found that FBMNs are affected similarly in all three single morphant embryos, with an inability to extend protrusions in a specific direction, and resulting in the failure of caudal migration. These data suggest that tag1, lama1 and vangl2 participate in a common mechanism that integrates signaling between the FBMN and its environment to regulate migration.