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...

Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Krüppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in ␤-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.

Peripheral nerve development results from multiple cellular interactions between axons, Schwann cells and the surrounding mesenchymal tissue. The delayed axonal sorting and hypomyelination throughout the peripheral nervous system of claw paw (clp) mutant mice suggest that the clp gene product is critical for these interactions. Here we identify the clp mutation as a 225-bp insertion in the Lgi4 gene. Lgi4 encodes a secreted and glycosylated leucine-rich repeat protein and is expressed in Schwann cells. The clp mutation affects Lgi4 mRNA splicing, resulting in a mutant protein that is retained in the cell. Additionally, siRNA-mediated downregulation of Lgi4 in wild-type neuron-Schwann cell cocultures inhibits myelination, whereas exogenous Lgi4 restores myelination in clp/clp cultures. Thus, the abnormalities observed in clp mice are attributable to the loss of Lgi4 function, and they identify Lgi4 as a new component of Schwann cell signaling pathway(s) that controls axon segregation and myelin formation.

The remarkable high affinity (Kd approximately 10(-15) M) of avidin/streptavidin for biotin has been extensively exploited in purification methodologies. Recently a small peptide sequence (Avi-tag) has been defined that can be specifically and efficiently biotinylated by the bacterial BirA biotin ligase. Fusion of this small peptide sequence to a protein of interest and co-expression with the BirA gene in mammalian cells allowed purification of the biotinylated protein together with its associated proteins and other molecules. Ideally, one would like to apply these technologies to purify tagged proteins directly from mouse tissues. To make this approach feasible for a large variety of proteins we developed a mouse strain that expresses the BirA gene ubiquitously by inserting it in the ROSA26 locus. We demonstrate that the BirA protein is indeed expressed in all tissues tested. In order to demonstrate functionality we show that it biotinylates the transgene-encoded Avi-tagged Gata1 and Oct6 transcription factors in erythroid cells of the foetal liver and Schwann cells of the peripheral nerve respectively. Therefore, this mouse can be crossed to any transgenic mouse to obtain efficient biotinylation of an Avi-tagged protein for the purpose of protein (complex) purification.

The segregation and myelination of axons in the developing PNS, results from a complex series of cellular and molecular interactions between Schwann cells and axons. Previously we identified the Lgi4 gene (leucine-rich glioma-inactivated4) as an important regulator of myelination in the PNS, and its dysfunction results in arthrogryposis as observed in claw paw mice. Lgi4 is a secreted protein and a member of a small family of proteins that are predominantly expressed in the nervous system. Their mechanism of action is unknown but may involve binding to members of the Adam (A disintegrin and metalloprotease) family of transmembrane proteins, in particular Adam22. We found that Lgi4 and Adam22 are both expressed in Schwann cells as well as in sensory neurons and that Lgi4 binds directly to Adam22 without a requirement for additional membrane associated factors. To determine whether Lgi4-Adam22 function involves a paracrine and/or an autocrine mechanism of action we performed heterotypic Schwann cell sensory neuron cultures and cell typespecific ablation of Lgi4 and Adam22 in mice. We show that Schwann cells are the principal cellular source of Lgi4 in the developing nerve and that Adam22 is required on axons. Our results thus reveal a novel paracrine signaling axis in peripheral nerve myelination in which Schwann cell secreted Lgi4 functions through binding of axonal Adam22 to drive the differentiation of Schwann cells.

The POU domain transcription factor Pou3f1 (Oct6/Scip/Tst1) initiates the transition from ensheathing, promyelinating Schwann cells to myelinating cells. Axonal and other extra-cellular signals regulate Oct6 expression through the Oct6 Schwann cell enhancer (SCE), which is both required and sufficient to drive all aspects of Oct6 expression in Schwann cells. Thus, the Oct6 SCE is pivotal in the gene regulatory network that governs the onset of myelin formation in Schwann cells and provides a link between myelin promoting signalling and activation of a myelin related transcriptional network. In this study we define the relevant cis-acting elements within the SCE and identify the transcription factors that mediate Oct6 regulation. On the basis of phylogenetic comparisons and functional in vivo assays we identify a number of highly conserved core elements within the mouse SCE. We show that core element 1 is absolutely required for full enhancer function and that it contains closely-spaced inverted binding sites for Sox proteins. For the first time in vivo, the dimeric Sox10 binding to this element is shown to be essential for enhancer activity whereas monomeric Sox10 binding is non-functional. As Oct6 and Sox10 synergize to activate the expression of the major myelin-related transcription factor Krox20, we propose that Sox10 dependent activation of Oct6 defines a feed-forward regulatory module that serves to time and amplify the onset of myelination in the peripheral nervous system.

While an important role for the POU domain transcription factor Oct‐6 in the developing peripheral nerve has been well established, studies into its exact role in nerve development and regeneration have been hampered by the high mortality rate of newborn Oct‐6 mutant animals. In this study we have generated a Schwann cell‐specific Oct‐6 allele through deletion of the Schwann cell‐specific enhancer element (SCE) in the Oct‐6 locus. Analysis of mice homozygous for this allele (ΔSCE allele) reveals that rate‐limiting levels of Oct‐6 in Schwann cells are dependent on the SCE and that this element does not contribute to Oct‐6 regulation in other cell types. We demonstrate a Schwann cell autonomous function for Oct‐6 during nerve development as well as in regenerating nerve. Additionally, we show that Krox‐20, an important regulatory target of Oct‐6 in Schwann cells, is activated, with delayed kinetics, through an Oct‐6‐independent mechanism in these mice.

Various factors play an essential role in patterning the digestive tract. During development, Sox2 and Cdx2 are exclusively expressed in the anterior and the posterior parts of the primitive gut, respectively. However, it is unclear whether these transcription factors influence each other in determining specification of the naïve gut endoderm. We therefore investigated whether Sox2 redirects the fate of the prospective intestinal part of the primitive gut. Ectopic expression of Sox2 in the posterior region of the primitive gut caused anteriorization of the gut toward a gastric-like phenotype. Sox2 activated the foregut transcriptional program, in spite of sustained co-expression of endogenous Cdx2. However, binding of Cdx2 to its genomic targets and thus its transcriptional activity was strongly reduced. Recent findings indicate that endodermal Cdx2 is required to initiate the intestinal program and to suppress anterior cell fate. Our findings suggest that reduced Cdx2 expression by itself is not sufficient to cause anteriorization, but that Sox2 expression is also required. Moreover, it indicates that the balance between Sox2 and Cdx2 function is essential for proper specification of the primitive gut and that Sox2 may overrule the initial patterning of the primitive gut, emphasizing the plasticity of the primitive gut.