The leukocyte-specific adapter molecule SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kilodaltons) is rapidly phosphorylated on tyrosine residues after receptor ligation in several hematopoietically derived cell types. Mice made deficient for SLP-76 expression contained no peripheral T cells as a result of an early block in thymopoiesis. Macrophage and natural killer cell compartments were intact in SLP-76-deficient mice, despite SLP-76 expression in these lineages in wild-type mice. Thus, the SLP-76 adapter protein is required for normal thymocyte development and plays a crucial role in translating signals mediated by pre-T cell receptors into distal biochemical events.

Antigen receptor engagement on T lymphocytes activates transcription factors important for stimulating cytokine gene expression. This is critical for clonal expansion of antigen-specific T cells and propagation of immune responses. Additionally, under some conditions antigen receptor stimulation initiates apoptosis of T lymphocytes through the induced expression of CD95 ligand and its receptor. Here we demonstrate that the transcription factor, NFAT, which is critical for the inducible expression of many cytokine genes, also plays a critical role in the regulation of T cell receptor-mediated CD95 ligand expression. Two sites within the CD95 ligand promoter, identified through DNase I footprinting, bind NFAT proteins from nuclear extracts of activated T cells. Although both sites appear important for optimal expression of CD95 ligand in activated T cells, mutational analysis suggests that the distal NFAT site plays a more significant role. Furthermore, these sites do not appear to be required for constitutive CD95 ligand expression in Sertoli cells.Adaptive immune responses require the activation of T lymphocytes through antigen-specific T cell receptor (TCR) 1 stimulation. Signaling events initiated by TCR ligation lead to the activation of transcription factors that regulate expression of cytokine genes, such as IL-2. This is important for the clonal expansion of antigen-specific T cells and propagation of immune responses (1, 2). However, once the antigenic stimulus has been cleared, the expanded population of cells must be eliminated to prevent accumulation of excessive lymphocytes (2, 3). Recently, it has been proposed that one mechanism by which this occurs is through the induced expression of CD95 ligand (4 -7). Once expressed, CD95 ligand engages its receptor, CD95, also expressed on the population of activated lymphocytes (8). In the absence of costimulatory signals that can delay apoptosis (9 -12), CD95 ligation rapidly initiates the programmed cell death machinery thus efficiently eliminating excessive activated lymphocytes (8). Additionally, autoreactive T cells that are inappropriately activated in the periphery are believed to undergo apoptosis through a similar process (13).The significance of CD95 ligand expression in the process of activation-induced cell death has been highlighted by recent studies that demonstrate that blocking CD95/CD95 ligand interactions prevents apoptosis of TCR-stimulated lymphocytes (5, 7). Interestingly, in addition to its inducible expression on activated lymphocytes, CD95 ligand is constitutively expressed on epithelial cells within the eye and Sertoli cells within the testes (14 -16). Constitutive CD95 ligand expression participates in maintenance of the "immune privileged" status of these tissues by inducing apoptosis in infiltrating, CD95-bearing, activated lymphocytes (17). Despite improved understanding of the important physiological roles for CD95 ligand in immune privileged sites and in controlling T cell homeostasis, little is yet known about the regulation of ...

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The interaction between CD95 (Fas) and CD95L (Fas ligand) initiates apoptosis in a variety of cell types. Although the regulation of CD95L expression on activated T cells is an area of intense study, knowledge related to the induction of CD95L promoter activity in primary T cells is lacking. In this report we describe the generation of a novel transgenic mouse strain, CD95LP-Luc, in which murine CD95L promoter sequence controls the expression of a luciferase reporter gene. We use these mice to illustrate several important findings related to transcriptional regulation of CD95L in primary T cells. We demonstrate that maximal CD95L promoter activity occurs only after prolonged T cell stimulation and requires costimulation through CD28. We provide evidence that thymocytes express CD95L/luciferase after strong TCR ligation and that inducible CD95L promoter activation is present, but unequal, in both Th1 and Th2 effector cells. We also illustrate that while agonist peptide presentation by APCs generates robust proliferation during a primary T cell response, the same stimulus induces only modest CD95L promoter activity. These results suggest alternate explanations for the well-characterized delay in CD95-mediated activation-induced cell death following initial ligation of the TCR.

The developmental role of Lef-1 in ectodermal organs has been characterized using Lef-1 murine knockout models. We generated a Lef-1 conditional over-expression (COEL) mouse to determine the role of Lef-1 expression in epithelial structures at later stages of development after endogenous expression switches to the mesenchyme. Lef-1 over expression (OE) in the oral epithelium creates a new dental epithelial stem cell niche that significantly increases incisor growth. These data indicate that Lef-1 expression is switched off in the dental epithelial at early stages to maintain the stem cell niche and regulate incisor growth. Bioinformatics analyses indicated that miR-26b expression increased coinciding with decreased Lef-1 expression in the dental epithelium. We generated a murine model over-expressing miR-26b that targets endogenous Lef-1 expression and Lef-1-related developmental mechanisms. miR-26b OE mice have ectodermal organ defects including a lack of incisors, molars, and hair similar to the Lef-1 null mice. miR-26b OE rescues the Lef-1 OE phenotype demonstrating a critical genetic and developmental role for miR-26b in the temporal and spatial expression of Lef-1 in epithelial tissues. Lef-1 expression regulates Wnt signaling and Wnt target genes as well as cell proliferation mechanisms, while miR-26b OE reduced the levels of Wnt target gene expression. The extra stem cell compartment in the COEL mice expressed Lef-1 suggesting that Lef-1 is a stem cell factor, which was absent in the miR-26b OE/COEL rescue mice. This is the first demonstration of a microRNA OE mouse model that has ectodermal organ defects. These findings demonstrate that the levels of Lef-1 are critical for development and establish a role for miR-26b in the regulation of ectodermal organ development through the control of Lef-1 expression and an endogenous stem cell niche.

ObjectiveThe objective of the study was to investigate differential gene expression between murine right and left maxilla–mandibular (MxMn) complexes.Setting and Sample PopulationWild‐type (WT) C57BL/6 embryonic (E) day 14.5 (n = 3) and 18.5 (n = 3) murine embryos.MethodsThe E14.5 and 18.5 embryos were harvested and hemi‐sectioned the MxMn complexes into right and left halves in the mid‐sagittal plane. We isolated total RNA using Trizol reagent and further purified using the RNA‐easy kit (QIAGEN). We confirmed equal expression of house‐keeping genes in right and left halves using RT‐PCR and then performed paired‐end whole mRNA sequencing in LC Sciences (Houston, TX) followed by differential transcript analyses (>1 or <−1 log fold change; p < .05; q < .05; and FPKM >0.5 in 2/3 samples). The Mouse Genome Informatics and Online Mendelian Inheritance in Man databases as well as gnomAD constraint scores were used to prioritize differentially expressed transcripts.ResultsThere were 19 upregulated and 19 downregulated transcripts at E14.5 and 8 upregulated and 17 downregulated transcripts at E18.5 time‐points. These differentially expressed transcripts were statistically significant and shown to be associated with craniofacial phenotypes in mouse models. These transcripts also have significant gnomAD constraint scores and are enriched in biological processes critical for embryogenesis.ConclusionsWe identified significant differential expression of transcripts between E14.5 and 18.5 murine right and left MxMn complexes. These findings when extrapolated to humans, they may provide a biological basis for facial asymmetry. Further experiments are required to validate these findings in murine models with craniofacial asymmetry.

Objective Wolf‐Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate (CLP), hypodontia and taurodontism. However, the causative factors and underlying mechanisms of these oral anomalies are relatively unknown. Wolf‐Hirschhorn syndrome candidate 1 (WHSC1) is a H3K36‐specific methyltransferase that is frequently deleted in WHS. This gene has been associated with craniofacial defects including CLP and defects in occipital ossification. In our study, we aim to understand the role of WHSC1 in tooth development. Methods To characterize the role of Whsc1 in tooth development, we profiled the Whsc1 expression pattern during mouse tooth development by immunofluorescence staining (IF). To investigate the regulatory effects between Whsc1 and Pitx2, we overexpressed either Whsc1 or Pitx2 in oral epithelial cell line LS8 and detected their expression. Then, ChIP‐PCR and luciferase assays were performed to confirm the binding on the promoter regions. To determine the negative regulation of Whsc1 by miR‐23‐3p and miR‐24‐3p, miRs were inhibited by our Plasmid‐based microRNA inhibition system in PMIS‐miR‐23‐27‐24 embryos and LS8 cells followed by detecting the mRNA and protein level of Whsc1. Luciferase assays were also conducted to confirm the direct binding between miRs and Whsc1 3’UTR. Results Whsc1 expresses from early stage of tooth development and restricts to stem cell niches in homeostatic tooth germ. Whsc1 and Pitx2 reciprocally activate each other's expression through chromosomal and transcriptional regulation. miR‐23‐3p and miR‐24‐3p, two miRs that regulated by Pitx2, directly inhibit Whsc1. Conclusion Whsc1 expresses in the developing tooth germ and participates the Whsc1‐Pitx2‐miR23/24 regulatory network in oral epithelium cells. Our observations provide new insights into the potential role of Whsc1 in regulating tooth development and a possible causer of the dental defects in WHS.