Sonic hedgehog signalling is essential for the embryonic development of many tissues including the central nervous system, where it controls the pattern of cellular differentiation. A genome-wide screen of neural progenitor cells to evaluate the Shh signalling-regulated transcriptome identified the forkhead transcription factor Foxj1. In both chick and mouse Foxj1 is expressed in the ventral midline of the neural tube in cells that make up the floor plate. Consistent with the role of Foxj1 in the formation of long motile cilia, floor plate cells produce cilia that are longer than the primary cilia found elsewhere in the neural tube, and forced expression of Foxj1 in neuroepithelial cells is sufficient to increase cilia length. In addition, the expression of Foxj1 in the neural tube and in an Shh-responsive cell line attenuates intracellular signalling by decreasing the activity of Gli proteins, the transcriptional mediators of Shh signalling. We show that this function of Foxj1 depends on cilia. Nevertheless, floor plate identity and ciliogenesis are unaffected in mouse embryos lacking Foxj1 and we provide evidence that additional transcription factors expressed in the floor plate share overlapping functions with Foxj1. Together, these findings identify a novel mechanism that modifies the cellular response to Shh signalling and reveal morphological and functional features of the amniote floor plate that distinguish these cells from the rest of the neuroepithelium.

Regulatory elements that lie outside the basal promoter of a gene may be revealed by local changes in chromatin structure and histone modifications. The promoter of the CFTR (cystic fibrosis transmembrane conductance regulator) gene is not responsible for its complex pattern of expression. To identify important regulatory elements for CFTR we have previously mapped DHS (DNase I-hypersensitive sites) across 400 kb spanning the locus. Of particular interest were two DHS that flank the CFTR gene, upstream at -20.9 kb with respect to the translational start site, and downstream at +15.6 kb. In the present study we show that these two DHS possess enhancer-blocking activity and bind proteins that are characteristic of known insulator elements. The DHS core at -20.9 kb binds CTCF (CCCTC-binding factor) both in vitro and in vivo; however, the +15.6 kb core appears to bind other factors. Histone-modification analysis across the CFTR locus highlights structural differences between the -20.9 kb and +15.6 kb DHS, further suggesting that these two insulator elements may operate by distinct mechanisms. We propose that these two DHS mark the boundaries of the CFTR gene functional unit and establish a chromatin domain within which the complex profile of CFTR expression is maintained.

SummaryCD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, b-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)-antigen-CD1d complex show how docking between CD1d-antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3b loop determines iNKT TCR affinity for ligand-CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails. Ligands loaded into CD1d on the cell surface promote Th2 responses, whereas ligands with long hydrophobic tails are loaded endosomally and promote Th1 responses. This information is informing the design of synthetic iNKT-cell antigens. The iNKT cells may be activated by exogenous antigen, or by a combination of dendritic cell-derived interleukin-12 and iNKT TCR-self-antigen-CD1d engagement. The iNKT-cell activation is further modulated by recent foreign or self-antigen encounter. Activation of dendritic cells through pattern recognition receptors alters their antigen presentation and cytokine production, strongly influencing iNKT-cell activation. In a range of bacterial infections, dendritic cell-dependent innate activation of iNKT cells through interleukin-12 is the dominant influence on their activity.

The early growth response (Egr) transcription factor family regulates multiple steps during T-cell development. We examine here the role played by Egr2 in positive selection. In double-positive cells, Egr2 is upregulated immediately following TCR ligation, and its expression requires both the MAPK and calcineurin signaling pathways. Inducible transgenic and knockout mice were generated to cause gain-or loss-of-function of Egr2 in double-positive cells, and had reciprocal effects; more mature single-positive cells were made when Egr2 was overexpressed, and fewer when Egr2 was absent. These defects were associated with changes in the survival of positively selected cells rather than perturbation of positive selection or immediate post-selection signaling. The survival function of Egr2 at least partly depends upon its ability to activate the cytokine-mediated survival pathway, likely through negative regulation of both the IL-7R and suppressor of cytokine signaling 1 (Socs1), the molecular switch whose downregulation normally results in restored responsiveness to cytokine signaling following selection. While gain of Egr2 caused a decrease in Socs1 mRNA, loss of Egr2 resulted in downregulation of IL-7R, upregulation of Socs1, and inhibition of Stat5 phosphorylation and IL-7-mediated survival post-selection. Therefore, expression of Egr2 following positive selection links the initial TCR signaling event to subsequent survival of signaled cells. , and Egr3 is also required for the post-b-selection proliferative burst to occur [12]. These transcription factors are also induced rapidly following ligation of the abTCR, both during thymocyte selection [15] and in mature T cells responding to antigen-MHC, where Egr1 has a role in upregulation of IL2 transcription [16], and Egr2 and Egr3 are required for induction of anergy [17,18], and regulate expression of FasL [19,20]. Through its control of self-tolerance, Egr2 has also been implicated in the development of late-onset autoimmune disease [21] and is required for the development and maturation of NKT cells [22].Upregulation of Egr proteins during positive selection is dependent upon the Ras/MAPK pathway [13]. Egr proteins are direct transcriptional targets of ternary complex factor Sap-1, which is itself a substrate of Erk and essential for positive selection [23]. In addition, Egr2 and Egr3 are regulated by calcineurin signaling, likely via NFAT [13,20,22]. Both Egr1 and Egr3 have roles in positive selection. Egr1 overexpression enhances positive selection of cells with low affinity TCR [24]. Conversely, Egr1-deficient mice have impaired positive selection [25]; although the initial TCR signal is transduced, cells stall at the DP to SP transition, resulting in a numerical decrease in CD4 and CD8 SP. Animals doubly deficient for both Egr1 and Egr3 have a similar but more marked selection phenotype, and CD8 differentiation is significantly impaired [14]. For both Egr1 and Egr3, the principal reason for the alterations in SP cell number is a change in the cells' susceptibi...

The transcription factors mediating the development of CD1d-restricted invariant NKT (iNKT) cells remain incompletely described. Here, we show that loss of the AP-1 transcription factor Fra-2 causes a marked increase in the number of both thymic and peripheral iNKT cells, without affecting the development of other T-lineage cells. The defect is cell-autonomous and is evident in the earliest iNKT precursors. We find that iNKT cells expressing the lower affinity TCRVβ8 are proportionally overrepresented in the absence of Fra-2, indicating altered selection of iNKT cells. There is also widespread dysregulation of AP-1-directed gene expression. In the periphery, mature Fra-2-deficient iNKT cells are able to participate in an immune response, but they have an altered response to Ag, showing increased expansion and producing increased amounts of IL-2 and IL-4 compared with their wild-type counterparts. Unusually, naive Fra-2-deficient T cells also rapidly produce IL-2 and IL-4 upon activation. Taken together, these data define Fra-2 as necessary for regulation of normal iNKT cell development and function, and they demonstrate the central role played by the AP-1 family in this lineage.