Genes and Immunity (2010) 11, 11–20 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 $32.00 www.nature.com/gene ORIGINAL ARTICLE Interleukin-7 (IL-7) and IL-7 splice variants affect differentiation of human neural progenitor cells M Moors1,4, NK Vudattu2,, J Abel1, U Kra¨mer1, L Rane2, N Ulfig3, S Ceccatelli4, V Seyfert-Margolies5, E Fritsche1 and MJ Maeurer2 1Group of Toxicology, Group of Epidemiology, Institut fu¨r Umweltmedizinische Forschung, Du¨sseldorf, Germany; 2Microbiology, Tumor and Cell Biology and Smittskyddsinstitutet, Stockholm, Sweden; 3Department of Anatomy, University of Rostock, Rostock, Germany; 4Division of Neurotoxicology, Department of Neuroscience, Karolinska Institutet, Sweden and 5Department of Medicine, University of California, San Francisco, CA, USA Alternative splicing of pre-mRNA increases proteomic diversity, a crucial mechanism in defining tissue identity. We demonstrate differentially spliced interleukin (IL)-7 in distinct anatomic areas in the adult, in developing human brains and in normal human neuronal progenitor (NHNP) cells. IL-7c (c, the canonical form spanning all six exons) or its variants IL-7d5, d4 or d4/5 were cloned and expressed as recombinant proteins. IL-7 and splice variants were able to shift the differentiation of NHNP cells as compared with the diluent control (Po0.01) defined by anti-b (III)-tubulin and glial fibrillary acidic protein expression, with different degrees (IL-7c4d4/54IL-7d5); IL-7d4 exhibited a significantly weaker potency. Differentiation was confirmed by transcriptome analysis of IL-7c-stimulated neural NHNP cells, resulting in 58 differentially expressed genes; some of these are involved in neural differentiation, for example, the developmentally regulated transcription factor kru¨ppel-like factor 12, musashi 2, a translational regulator of cell fate or the sonic hedgehog receptor patch 1. This suggests that IL-7 influences neural development at a molecular level by participating in human brain architecture through glia cell formation: a paradigm that alternative splicing in cytokines, for example, for IL-7, has a physiological role in human organ development and progenitor cell differentiation. Genes and Immunity (2010) 11, 11–20; doi:10.1038/gene.2009.77; published online 22 October 2009 Keywords: interleukin-7; neuronal progenitor cells; stem cells; differentiation; differential splicing Introduction This creates a soluble IL-7R, which is associated with an increased risk of developing multiple sclerosis.5–7 Cytokines have pleiotropic functions, which include The nominal IL-7R ligand, IL-7,8 is encoded by six targets in the central nervous system (CNS). They are exons (1–611 first, 612–718 second, 749–829 third, crucial factors in shaping neural plasticity, neuronal cell 830–961 fourth, 962–1015 fifth and 1016–2078 sixth) and differentiation and memory formation. For instance, forms a 177-amino-acid protein with a 25-amino-acid- impaired interleukin (IL)-1b expression in the hippo- long signal peptide. IL-7 has also been described to be campus is associated with spatial memory loss in mice,1 alternatively spliced.8–11 This is not driven by allelic interleukin (IL)-6 promotes sleep through trans-signaling variants of the human IL-7 gene, located on chromosome to neurons2 and IL-10, which exhibits predominantly 8q12–13. IL-7 splice variants have been identified in anti-inflammatory properties, provides direct trophic different conditions: malignant transformation,9 expo- support to neurons and may be useful for neuroprotec- sure to proinflammatory cytokines and tissue/organ- tive strategies.3 Other cytokines, including IL-1a, may specific expression.11 affect neurogenesis through transdifferentiation of adult Alternative splicing has not been studied for IL-7 in mesenchymal stem cells.4 Previous studies showed that the CNS, although alternative splicing of pre-mRNA is the IL-7 receptor (IL-7R) and IL-7 are involved in increasingly appreciated to increase proteomic diversity. neuronal development and pathophysiology. Three Up to 75% of the human genome is currently estimated independent studies recently showed that allelic differ- to be alternatively spliced.12 Alternative splicing ences of the IL-7R, located on chromosome 5p13.2, contributes to the evolution of protein diversity;13–15 lead to an alternative splicing of exon 6 of the IL-7R. both microRNAs and alternative pre-mRNA splicing are implicated in the development of the neuronal system. Neuron-specific microRNA (miR)-124 contri- Correspondence: Professor MJ Maeurer, Microbiology, Tumor and butes to progenitor cell differentiation and matures Cell Biology, Smittskyddsinstitutet, Nobels Va¨g 18, Stockholm neurons by regulating a complex network of alternative S-17182, Sweden. 16 E-mail: [email protected] splicing associated with neuronal development. Received 26 May 2009; revised 1 September 2009; accepted 3 Alternative pre-mRNA splicing has a pivotal role in the September 2009; published online 22 October 2009 establishment of neuronal identity,17 yet the regulatory IL-7 splice variants in neuronal differentiation M Moors et al 12 Figure 1 Interleukin (IL)-7 isoforms are expressed in adult brain tissue. A cDNA library from human brain, standardized for the expression for human b-actin and tested for mRNA integrity, was analyzed for IL-7 isoform expression. The canonical IL-7 isoform (IL-7c) spans six exons. Expression of different IL-7 mRNA isoforms is indicated with different colors (see figure legend, top right corner: skipped exons are in black, colors of the IL-7 isoforms correspond to IL-7 expression analysis from brain tissue). IL-7d2(À56 bp) represents a shifted reading frame resulting in the addition of seven new aa, the omitted 56 bp corresponds to nucleotides 476–531 of the IL-7c transcript. IL-7c mRNA is exclusively expressed in the cerebellum and pons; IL-7d4 is present in the caudate nucleus; the spinal cord shows IL-7c and IL-7d4/5 expression. The licence to use the brain image for publication was purchased from Fotosearch, LifeART, Waukesha, WI, USA, reference 20410757. The numbers in the boxes for tissues from the caudate nucleus and spinal cord indicate the ratio of differentially spliced IL-7 isoforms. network that governs neuronal-specific splicing is poorly IL-7 affects neuronal cell differentiation understood. To investigate the impact of IL-7c and splice variants on We report here the analysis of IL-7 splice variants neural development, we used neurospheres prepared identified in the CNS. The six-exon-spanning IL-7 is from normal human neural progenitor (NHNP) cells as crucial for T-cell homeostasis,18,19 yet the biological an in vitro model system. NHNP neurospheres represent functions of IL-7 splice variants are poorly understood. heterogeneous, self-regulating cell populations of human IL-7, lacking exon 5 (IL-7d5), showed superior activity on neural precursor and neural stem cells defined by the CD4 þ and CD8 þ T cells compared with the ‘canonical’ expression of nestin and other early neural markers.22 IL-7.11 As IL-7 has been identified in developing brains RNA expression analysis showed that NHNP neuro- from rats20 and data from animal studies suggested spheres express IL-7c and the splice variants IL-7d4 and that IL-7 represents a neural growth and differentia- d3/4 (Figure 3a); they express the full-length IL-7R and tion factor in vitro,21 we examined adult and developing the IL-7R splice variants IL-7d6 and IL-7Rd5/6 human brains for IL-7 mRNA expression patterns, (Figure 3a). IL-7 protein was detected by staining with followed by cloning and expression of IL-7 splice an anti-IL-7-specific mAb (Figure 3b), yet this does not variants as recombinant proteins, which were tested allow the discrimination of IL-7 splice variants, as for their capacity to drive neural progenitor cell monoclonal and polyclonal reagents recognize several differentiation. IL-7 isoforms.11 Human neurospheres were differentiated for 4 days in the presence or absence of IL-7 or its splice variants (for experimental set up see Figure 4). Immuno- Results cytochemical staining of NHNP cells harvested at day 4 revealed a distribution of approximately 15–30% b-(III)- Mapping of IL-7 isoforms in adult and developing brains tubulin-positive neurons and 70–85% glial fibrillary A cDNA library from human brains showed an exclusive acidic protein (GFAP)-positive glia cells (Figure 5a). expression of IL-7c (c, the canonical form spanning all six Treatment with IL-7c or IL-7 splice variants resulted in a exons) in the cerebellum and pons; the IL-7 exon 4 splice significant increase in the number of developed GFAP- variant (IL-7d4) is exclusively expressed in the caudate positive cells in relation to the number of formed nucleus. Tissue from spinal cord showed expression of neurons (Figures 5a and b) compared with the diluent IL-7c and the IL-7 splice variant lacking exon 4/5 (d4/5) control. The potency of the different IL-7 isoforms in (Figure 1). Human embryonic brains express IL-7c and shifting differentiation toward the glia cell lineage varied splice variants that lack either exon 5 (d5), IL-7d4, IL-7d4/ (Figure 5b): IL-7c4IL-7d4/54IL-7d5 were the most 5 or IL-7 lacking exon 3/4 (d3/4) (Figure 2). Each of the potent isoforms, whereas IL-7d4 exhibited a significantly individual IL-7 splice variants was confirmed by weaker potency. A shift in differentiation was not sequence analysis, cloned and expressed as recombinant detected at earlier time points (that is, days 1–3, data proteins. not shown) defined by immunocytochemical staining. Genes and Immunity IL-7 splice variants in neuronal differentiation M Moors et al 13 Figure 2 Interleukin (IL)-7 isoforms are expressed in embryonic human brain tissue. Brain tissues of different embryos (from natural miscarriage) were analyzed for a housekeeping gene (GAPDH), as well as for IL-7 Ra expression (cell membrane, M and soluble form, S) and IL-7 mRNA expression.