In this study, we showed that adrenocorticotropic hormone (ACTH) promoted erythroblast differentiation and increased the enucleation ratio of erythroblasts. Because ACTH was contained in hematopoietic medium as contamination, the ratio decreased by the addition of anti-ACTH antibody (Ab). Addition of neutralizing Abs (nAbs) for melanocortin receptors (MCRs) caused erythroblast accumulation at specific stages, i.e., the addition of anti-MC2R nAb led to erythroblast accumulation at the basophilic stage (baso-E), the addition of anti-MC1R nAb caused accumulation at the polychromatic stage (poly-E), and the addition of anti-MC5R nAb caused accumulation at the orthochromatic stage (ortho-E). During erythroblast differentiation, ERK, STAT5, and AKT were consecutively phosphorylated by erythropoietin (EPO). ERK, STAT5, and AKT phosphorylation was inhibited by blocking MC2R, MC1R, and MC5R, respectively. Finally, the phosphorylation of myosin light chain 2, which is essential for the formation of contractile actomyosin rings, was inhibited by anti-MC5R nAb. Taken together, our study suggests that MC2R and MC1R signals are consecutively required for the regulation of EPO signal transduction in erythroblast differentiation, and that MC5R signal transduction is required to induce enucleation. Thus, melanocortin induces proliferation and differentiation at baso-E, and polarization and formation of an actomyosin contractile ring at ortho-E are required for enucleation.

Hypoxia-inducible factor 1 α (Hif1α) plays a crucial role in brain development. To study the function of Hif1α in early brain development, we generated neuroepithelial cell-specific Hif1α-knockout mice. Hif1α-knockout mice died soon after birth; these mice exhibited an abnormal head shape, indicating the presence of brain defects. Morphological analysis revealed that Hif1α ablation reduced the overall size of the brain, especially affecting the telencephalon. Neuronal apoptosis predominantly occurred in deep-layer neurons, consequently the alignment of cortical layers was severely disorganized in Hif1α knockout mice. Furthermore, we demonstrated that Vegf signaling contributes to the survival of deep-layer neurons as a downstream effector of Hif1α-dependent hypoxia signaling. Taken together, our findings demonstrate that Hif1α plays a critical role in the early stages of telencephalon development.

We previously showed that maternal leukemia inhibitory factor (LIF) induces placental production of adrenocorticotropic hormone (ACTH), which stimulates fetal nucleated red blood cells to further secrete LIF and promote neurogenesis in rodent brains. However, the underlying mechanism of LIF-dependent ACTH induction remains unclear. Recently, we found that LIF induces corticotropin-releasing hormone (CRH) in mouse trophoblast stem cells. This finding supports the results of a previous study that CRH, which is produced by the placenta, induces placental ACTH production. In this study, we examined whether the effects of LIF are mediated by the induction of Pomc via CRH upregulation in mouse trophoblast. In vivo, protein levels of LIF and CRH peak in mouse placenta at 13.5 days post coitum. In mouse placenta, Crh mRNA and protein levels significantly increased 3 h after intraperitoneal injection of LIF (5 μg/kg body weight) into dams at 13.5 days post coitum. We also examined the effect of LIF-induced CRH on the expression of Pomc induced by LIF in mouse trophoblast stem cells in vitro. After LIF supplementation for 3 days, we found that the increased expression of Crh-induced by new supplementation of LIF was earlier than that of Pomc. Furthermore, LIF-induced upregulation of Pomc in mouse trophoblast stem cells was attenuated by inhibition of the CRH/CRHR1 pathway, whereas LIF-induced secretion of ACTH was attenuated by inhibition of the JAK/STAT3 pathway. Therefore, LIF indirectly increases placental Pomc expression through the CRH/CRHR1 pathway, and placental ACTH secretion is induced directly by LIF via the JAK/STAT3 pathway.

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Hypoxia-inducible factor 1 a (Hif1α) plays a crucial role in brain development. To study the function of Hif1α in early brain development, we generated neuroepithelial cell-specific Hif1α-knockout mice. Hif1α-knockout mice died soon after birth; these mice exhibited an abnormal head shape, indicating the presence of brain defects. Morphological analysis revealed that Hif1α ablation reduced the overall size of the brain, especially affecting the telencephalon. Neuronal apoptosis predominantly occurred in deep-layer neurons, consequently the alignment of cortical layers was severely disorganized in Hif1α knockout mice. Furthermore, we demonstrated that Vegf signaling contributes to the survival of deep-layer neurons as a downstream effector of Hif1α-dependent hypoxia signaling. Taken together, our findings demonstrate that Hif1α plays a critical role in the early stages of telencephalon development.