Neuronal lineage formation in the developing olfactory epithelium has been extensively studied at the cellular level, but little is known about the genes that control proliferation and differentiation of neuronal progenitor cells. Here, we report that the Wilms' tumour zinc-finger protein, Wt1, is required for normal formation of the olfactory epithelium. Wt1 was detected by immunohistochemistry in the developing olfactory epithelium of wild-type embryos between gestational days E9.5 and E18.5. Embryos with complete lack of Wt1 and embryos with selective ablation of the alternatively spliced Wt1(+KTS) isoform both had thinner olfactory epithelia and fewer neuronal progenitor cells than do normal animals. Mash1 and neurogenin 1, two basic helix-loop-helix transcription factors with critical functions during olfactory neuron development, were reduced in the Wt1(+KTS)^-/- mutants compared with the wild-type mice. Stable expression of the Wt1(+KTS) isoform, but not of the Wt1(-KTS) variant, upregulated Mash1 mRNA and protein in vitro. The olfactory epithelia of mouse embryos, which lacked the Wt1(-KTS) protein, appeared normal. However, formation of the neural retina was severely impaired in the Wt1(-KTS)^-/- mutants. These findings demonstrate that the Wt1(+KTS) protein, which has been proposed to play a role in mRNA processing, acts upstream of Mash1 to promote the development of the olfactory epithelium. Furthermore, neuron formation depends on distinct functions of alternatively spliced Wt1 products in the embryonic retina and the olfactory epithelium.

Molecular mechanisms for the developmental stage and tissue-specific regulation of the erythropoietin (EPO) gene are poorly understood. Recent findings indicate a role of the Wilms tumor suppressor, Wt1, in the formation of the hematopoietic system. Herein, we tested the hypothesis that Wt1 is a transcriptional regulator of the EPO gene. Binding of the transcriptionally competent Wt1(–KTS) isoform to the minimal EPO promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. Under normoxia, EPO expression was significantly increased in HEK 293 and HepG2 cells with forced expression of Wt1(–KTS). A reporter construct harboring the 117-bp minimal human EPO promoter was activated up to 20-fold by transient cotransfection of Wt1(–KTS) in different cell lines. Mutation of the Wt1 binding site in the EPO promoter abrogated this stimulatory effect of the Wt1(–KTS) protein. Hepatic Epo mRNA expression was significantly reduced in embryonic mice with homozygous Wt1 deletion. Furthermore, Wt1 and EPO were colocalized in hepatocytes of the liver and in neuronal cells of the dorsal root ganglia in developing mice. Both proteins were also detected in Sertoli cells of the adult murine testis. In conclusion, we identified Wt1(–KTS) as a novel transcriptional activator for the tissue-specific expression of the EPO gene.

Nestin is an intermediate filament protein originally described in neural stem cells and a variety of progenitor cells. More recently, nestin was detected in rat kidney podocytes. We show here that nestin is expressed in a developmentally regulated pattern in the kidney. Nestin was detected by immunohistochemistry in the condensing mesenchyme surrounding the ureter, in developing glomeruli, in podocytes of the adult kidney, and in a podocyte cell line. Nestin shared a striking overlap in expression with the Wilms' tumor suppressor Wt1. Nestin was significantly upregulated in a cell line with inducible Wt1 expression upon induction of Wt1. Cotransfection experiments in human embryonic kidney cells (HEK293) revealed stimulation of a nestin intron 2 enhancer element up to six-fold by the Wt1(-KTS) splice variant. Nestin expression was significantly reduced in an inducible mouse model of glomerular disease. This model is based on podocyte-specific overexpression of Pax2 and associated with a loss of Wt1 expression ( 33 ). Furthermore, also in the developing heart, nestin was found in an overlapping pattern with Wt1 in the epicardium and the forming coronary vessels. Strikingly, in the hearts of Wt1 knockout mice, nestin was barely detectable compared with the hearts of wild-type embryos. Our results show that nestin is expressed at different stages of kidney and cardiac development and suggest that its expression in these organs might be regulated by the Wilms' tumor suppressor Wt1.

Neurotrophins and their cognate receptors play a pivotal role in the development and function of the nervous system. High expression levels of the neurotrophin receptor TrkB and its ligands in neuroblastomas are associated with an unfavorable outcome. We report here that NTRK2, which encodes the TrkB receptor tyrosine kinase, is an oxygen-regulated gene, whose expression is stimulated by the hypoxia-inducible factor-1 (HIF-1). TrkB mRNA and protein levels were elevated nearly 30-fold in neuroblastoma-derived Kelly cells in hypoxia (1% O(2)) versus normoxia (21% O(2)). A luciferase reporter construct containing approximately 2.1 kilobases of the human TrkB promoter was activated about 6-fold both in hypoxia and after stimulation with the hypoxia mimetic 2,2'-dipyridyl (100 microm) at 21% O(2). Luciferase activity in the presence of 2,2'-dipyridyl was reduced significantly upon small interfering RNA knockdown of HIF-1alpha but not of HIF-2alpha. Accordingly, hypoxia failed to stimulate the TrkB promoter in mouse embryonic fibroblasts that lacked HIF-1alpha. The hypoxia-responsive promoter region could be mapped to three HIF-1 binding elements that were located between -923 and -879 bp relative to the transcription start site. The migration of cultured neuroblastoma cells was increased approximately 2-fold upon incubation at 1 versus 21% O(2). This effect of hypoxia was abrogated with the tyrosine kinase inhibitor K252a (200 nm). Our findings indicate that transcription of the NTRK2 gene is stimulated at low oxygen tension through a HIF-1-dependent mechanism. In conclusion, enhanced expression of TrkB could represent a critical switch for the previously reported dedifferentiation of neuroblastoma cells under hypoxic conditions.

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5′-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)₁₀ sequence located in the 5′-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation.

Wilms’ tumor (nephroblastoma) represents a unique example of an aberrant kidney formation that can result from mutations in a tumor suppressor gene, Wilms’ tumor 1 (WT1). Targeted gene inactivation in mice testifies that WT1 is a master switch for the development of the genitourinary system and other organs.