Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool.

The genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1 and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during development and tissue regeneration in adults following damage. In this report we demonstrate the involvement of a third transcription factor, the POU domain factor Brn-2. We show that Schwann cells express Brn-2 in a developmental profile similar to that of Oct-6 and that Brn-2 gene activation does not depend on Oct-6. Overexpression of Brn-2 in Oct-6-deficient Schwann cells, under control of the Oct-6 Schwann cell enhancer (SCE), results in partial rescue of the developmental delay phenotype, whereas compound disruption of both Brn-2 and Oct-6 results in a much more severe phenotype. Together these data strongly indicate that Brn-2 function largely overlaps with that of Oct-6 in driving the transition from promyelinating to myelinating Schwann cells. The high conduction velocity of nerve fibers is a hallmark of the nervous system of higher vertebrates and depends on structural and molecular specializations that are elaborated during development. These specializations occur through intimate and continued interactions between the neuron and its associated glial cells and result in the elaboration by glial cells of myelin, the important membranous structure that ensheaths and insulates axons (Arroyo and Scherer 2000; Fields and StevensGraham 2002;Mirsky et al. 2002). Two glial cell types produce myelin: the oligodendrocyte in the central nervous system (CNS) and the Schwann cell in the peripheral nervous system (PNS). Although very similarly organized, the molecular composition of CNS and PNS myelin differs significantly, and oligodendrocytes and Schwann cells have adopted different, but overlapping, sets of transcriptional regulators to coordinate myelogenesis (Hudson 2001; Topilko and Meijer 2001). These differences reflect their distinct embryonic origins. Whereas oligodendrocytes originate from the neuroepithelial precursors that line the lumen of the spinal cord and ventricles of the brain, Schwann cells derive mainly from the neural crest, a transient embryonic stem (ES) cell population that generates a wide variety of cell types including sensory and autonomic neurons and melanocytes (Le Douarin and Kalcheim 1999;Richardson 2001). Schwann cell precursors populate the early outgrowing nerve bundles, where they proliferate and segregate individual and groups of fibers until the number of Schwann cells and fibers is eventually matched. During the first few days of postnatal development, many Schwann cells establish a 1:1 relationship with axons, cease to proliferate, and initiate myelin formation such that by the end of the first postnatal week of development, all myelin-competent axons are actively being myelinated. Schwann cells that remain associated with groups of lower-caliber fibers will segregate these fibers in cytoplasmic cuffs without mye...

In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12−/− knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.

Progress in elucidating the molecular and cellular pathophysiology of neuropsychiatric disorders has been hindered by the limited availability of living human brain tissue. The emergence of induced pluripotent stem cells (iPSCs) has offered a unique alternative strategy using patient-derived functional neuronal networks. However, methods for reliably generating iPSC-derived neurons with mature electrophysiological characteristics have been difficult to develop. Here, we report a simplified differentiation protocol that yields electrophysiologically mature iPSC-derived cortical lineage neuronal networks without the need for astrocyte co-culture or specialized media. This protocol generates a consistent 60:40 ratio of neurons and astrocytes that arise from a common forebrain neural progenitor. Whole-cell patch-clamp recordings of 114 neurons derived from three independent iPSC lines confirmed their electrophysiological maturity, including resting membrane potential (−58.2±1.0 mV), capacitance (49.1±2.9 pF), action potential (AP) threshold (−50.9±0.5 mV) and AP amplitude (66.5±1.3 mV). Nearly 100% of neurons were capable of firing APs, of which 79% had sustained trains of mature APs with minimal accommodation (peak AP frequency: 11.9±0.5 Hz) and 74% exhibited spontaneous synaptic activity (amplitude, 16.03±0.82 pA; frequency, 1.09±0.17 Hz). We expect this protocol to be of broad applicability for implementing iPSC-based neuronal network models of neuropsychiatric disorders.