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Loss-of-function Studies (loss-of-function + studies)
Selected AbstractsProprotein convertase genes in Xenopus developmentDEVELOPMENTAL DYNAMICS, Issue 3 2005Sylvia Nelsen Abstract Proprotein convertases (PCs) are a family of serine endoproteases that proteolytically activate many precursor proteins within various secretory pathway compartments. Loss-of-function studies have demonstrated a critical role for these proteases in embryonic patterning and adult homeostasis, yet little is known about how substrate selectivity is achieved. We have identified Xenopus orthologs of three PCs: furin, PC6, and PC4. In addition to previously described isoforms of PC6 and furin, four novel splice isoforms of PC6, which are predicted to encode constitutively secreted proteases, and a putative transmembrane isoform of PC4 were identified. Furin and PC6 are expressed in dynamic, tissue-specific patterns throughout embryogenesis, whereas PC4 transcripts are restricted primarily to germ cells and brain in adult frogs. Developmental Dynamics 233:1038,1044, 2005. © 2005 Wiley-Liss, Inc. [source] Xenopus aristaless-related homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain developmentDEVELOPMENTAL DYNAMICS, Issue 2 2005Daniel W. Seufert Abstract Mutations in the aristaless-related homeobox (ARX) gene have been found in patients with a variety of X-linked mental retardation syndromes with forebrain abnormalities, including lissencephaly. Arx is expressed in the developing mouse, Xenopus, and zebrafish forebrain. We have used whole-mount in situ hybridization, overexpression, and loss-of-function studies to investigate the involvement of xArx in Xenopus brain development. We verified that xArx is expressed in the prospective diencephalon, as the forebrain is patterned and specified during neural plate stages. Expression spreads into the ventral and medial telencephalon as development proceeds through neural tube and tadpole stages. Overexpression of xArx resulted in morphological abnormalities in forebrain development, including loss of rostral midline structures, syn- or anophthalmia, dorsal displacement of the nasal organ, and ventral neural tube hyperplasia. Additionally, there is a delay in expression of many molecular markers of brain and retinal development. However, expression of some markers, dlx5 and wnt8b, was enhanced in xArx -injected embryos. Loss-of-function experiments indicated that xArx was necessary for normal forebrain development. Expansion of wnt8b expression depended on xArx function as a transcriptional repressor, whereas ectopic expression of dlx5, accompanied by development of ectopic otic structures, depended on function of Arx as a transcriptional activator. These results suggest that Arx acts as a bifunctional transcriptional regulator in brain development. Developmental Dynamics 232:313,324, 2005. © 2004 Wiley-Liss, Inc. [source] The canonical Wnt signaling pathway plays an important role in lymphopoiesis and hematopoiesisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2008Frank Abstract The evolutionarily conserved canonical Wnt-,-catenin-T cell factor (TCF)/lymphocyte enhancer binding factor (LEF) signaling pathway regulates key checkpoints in the development of various tissues. Therefore, it is not surprising that a large body of gain-of-function and loss-of-function studies implicate Wnt-,-catenin signaling in lymphopoiesis and hematopoiesis. In contrast, recent papers have reported that Mx-Cre-mediated conditional deletion of ,-catenin and/or its homolog ,-catenin (plakoglobin) did not impair hematopoiesis or lymphopoiesis. However, these studies also report that TCF reporter activity remains active in ,-catenin- and ,-catenin-deficient hematopoietic stem cells and all cells derived from these precursors, indicating that the canonical Wnt signaling pathway was not abrogated. Therefore, these studies in fact show that the canonical Wnt signaling pathway is important in hematopoiesis and lymphopoiesis, even though the molecular basis for the induction of the reporter activity is currently unknown. In this perspective, we provide a broad background to the field with a discussion of the available data and create a framework within which the available and future studies may be evaluated. [source] Nestin-Cre transgenic mouse line Nes-Cre1 mediates highly efficient Cre/loxP mediated recombination in the nervous system, kidney, and somite-derived tissuesGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 8 2006Nicole C. Dubois Abstract Here we describe the generation of the Nes-Cre1 transgenic mouse line in which Cre recombinase expression is controlled by the rat nestin promoter and intron 2 enhancer. This line has previously been used for conditional loss-of-function studies of various genes in the central nervous system and first branchial arch ectoderm. Here we report the detailed temporal and spatial recombination pattern of Nes-Cre1 using three different reporters of Cre-mediated recombination, ROSA26R (R26R), Z/AP, and Z/EG. Cre/loxP recombination was detected in embryos as early as the head-fold stage. By embryonic day (E)15.5 recombination occurred in virtually all cells of the nervous system and unexpectedly also in somite-derived tissues and kidneys. Tissues with little or no recombination included heart, liver, thymus, and lung. This study suggests that Nes-Cre1-mediated recombination occurs in progenitor cell types present in the neuroectoderm, the developing mesonephros, and the somites. genesis 44:355,360, 2006. © 2006 Wiley-Liss, Inc. [source] An ABA-responsive bZIP protein, OsBZ8, mediates sugar repression of , -amylase gene expressionPHYSIOLOGIA PLANTARUM, Issue 1 2003Yi-Ching Lee Expression of some , -amylase genes in cereals is suppressed by sugars and activated by sugar starvation. A 100-bp sugar response sequence (SRS) identified in the promoter of a rice , -amylase gene, ,Amy3, contains three essential motifs: the GC box, the G box, and the TATCCA element. To study the mechanism of sugar regulation of ,Amy3 transcription, an ABA-responsive bZIP protein, OsBZ8, which binds specifically to the G box in ,Amy3 SRS was characterized and function analysed. In sucrose-starved rice suspension cells and embryos, decline in OsBZ8 mRNA levels coincided with the induction of ,Amy3 mRNA accumulation. In vivo gain- and loss-of-function studies by transient expression assays in rice embryos revealed that OsBZ8 suppresses SRS activity through the G box and overrides the activity of an activator, OsMYBS1, which binds to the TATCCA element. Gel mobility shift assays revealed that OsBZ8 binds specifically to the G box in vitro. These studies suggest that OsBZ8 is a suppressor responsible for sugar repression of ,Amy3 expression, and OsMYBS1 is responsible for sugar starvation induced expression of ,Amy3. [source] T-cadherin loss induces an invasive phenotype in human keratinocytes and squamous cell carcinoma (SCC) cells in vitro and is associated with malignant transformation of cutaneous SCC in vivoBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2010D. Pfaff Summary Background, Cadherins play important roles in controlling keratinocyte growth, differentiation and survival. Atypical glycosylphosphatidylinositol-anchored T-cadherin (T-cad) is highly expressed in the basal keratinocyte layer of skin. The role of T-cad in keratinocyte biology and pathology is unclear. Objectives, To define the role of T-cad in the pathogenesis of cutaneous squamous cell carcinoma (SCC) through gain-of-function and loss-of-function studies in vitro and through examination of T-cad expression patterns in human cutaneous SCC specimens in relation to histological classification of degree of tumour differentiation. Methods,In vitro studies employed lentiviral-mediated overexpression/silencing of T-cad in normal human keratinocyte (HaCaT) and SCC (A431) cell lines, monolayer and multicellular spheroid culture models, cell morphology analyses and assays of random motility and invasion. Immunohistochemistry was performed on skin specimens from patients with actinic keratosis, Bowen disease or SCC. Results,In vitro, silencing of T-cad induced a morphologically elongated and disorganized cell phenotype, increased random motility and markedly enhanced invasive potential. Overexpression of T-cad induced a morphologically spread and compact cell phenotype and blunted invasive potential. In vivo, regional loss of T-cad expression was more frequent and prominent in SCC classified as moderately-to-poorly differentiated than in SCC classified as well differentiated. However, in both categories aberrant and/or absence of T-cad expression was associated with histological features of a potentially more malignant and invasive phenotype of cutaneous SCC. Conclusions, T-cad is a controlling determinant of SCC phenotype and invasive behaviour and its loss is associated with the process of malignant transformation from noninvasive to invasive SCC. [source] | |||||||||||||