Intracellular mediators of transforming growth factor β superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo

<p>Abstract</p> <p>Background</p> <p>Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs.</p> <p>Results</p> <p>Proteins that are downstream of the transforming growth factor-β superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFβ superfamily for their normal development. Phosphorylated Smad1 (pSmad1), pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA) and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. <it>Bmpr1a </it>conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells.</p> <p>Conclusion</p> <p>Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-β superfamily to endosomes is important for the regulation of growth factor signaling.</p

Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain.

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6⁺/⁻ lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6⁻/⁻ lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues

The mechanism of lens placode formation: A case of matrix-mediated morphogenesis

AbstractAlthough placodes are ubiquitous precursors of tissue invagination, the mechanism of placode formation has not been established and the requirement of placode formation for subsequent invagination has not been tested. Earlier measurements in chicken embryos supported the view that lens placode formation occurs because the extracellular matrix (ECM) between the optic vesicle and the surface ectoderm prevents the prospective lens cells from spreading. Continued cell proliferation within this restricted area was proposed to cause cell crowding, leading to cell elongation (placode formation). This view suggested that continued cell proliferation and adhesion to the ECM between the optic vesicle and the surface ectoderm was sufficient to explain lens placode formation. To test the predictions of this “restricted expansion hypothesis,” we first confirmed that the cellular events that accompany lens placode formation in chicken embryos also occur in mouse embryos. We then showed that the failure of lens placode formation when the transcription factor, Pax6 was conditionally deleted in the surface ectoderm was associated with greatly diminished accumulation of ECM between the optic vesicle and ectoderm and reduced levels of transcripts encoding components of the ECM. In accord with the “restricted expansion hypothesis,” the Pax6-deleted ectoderm expanded, rather than being constrained to a constant area. As a further test, we disrupted the ECM by deleting Fn1, which is required for matrix assembly and cell–matrix adhesion. As in Pax6CKO embryos, the Fn1CKO lens ectoderm expanded, rather than being constrained to a fixed area and the lens placode did not form. Ectoderm cells in Fn1CKO embryos expressed markers of lens induction and reorganized their cytoskeleton as in wild type ectoderm, but did not invaginate, suggesting that placode formation establishes the minimal mechanical requirements for invagination

Climatology of surface fronts

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The function of FGF signaling in the lens placode

AbstractPrevious studies suggested that FGF signaling is important for lens formation. However, the times at which FGFs act to promote lens formation, the FGFs that are involved, the cells that secrete them and the mechanisms by which FGF signaling may promote lens formation are not known. We found that transcripts encoding several FGF ligands and the four classical FGF receptors are detectable in the lens-forming ectoderm at the time of lens induction. Conditional deletion of Fgfr1 and Fgfr2 from this tissue resulted in the formation of small lens rudiments that soon degenerated. Lens placodes lacking Fgfr1 and 2 were thinner than in wild-type embryos. Deletion of Fgfr2 increased cell death from the initiation of placode formation and concurrent deletion of Fgfr1 enhanced this phenotype. Fgfr1/2 conditional knockout placode cells expressed lower levels of proteins known to be regulated by FGF receptor signaling, but proteins known to be important for lens formation were present at normal levels in the remaining placode cells, including the transcription factors Pax6, Sox2 and FoxE3 and the lens-preferred protein αA-crystallin. Previous studies identified a genetic interaction between BMP and FGF signaling in lens formation and conditional deletion of Bmpr1a caused increased cell death in the lens placode, resulting in the formation of smaller lenses. In the present study, conditional deletion of both Bmpr1a and Fgfr2 increased cell death beyond that seen in Fgfr2CKO placodes and prevented lens formation. These results suggest that the primary role of autocrine or paracrine FGF signaling is to provide essential survival signals to lens placode cells. Because apoptosis was already increased at the onset of placode formation in Fgfr1/2 conditional knockout placode cells, FGF signaling was functionally absent during the period of lens induction by the optic vesicle. Since the expression of proteins required for lens formation was not altered in the knockout placode cells, we can conclude that FGF signaling from the optic vesicle is not required for lens induction

The physical activity experiences of men with serious mental illness: Three short stories

Objectives: Although a considerable amount of research has explored the effects of physical activity on mental health, the voices of people with mental illness have been largely excluded from published reports. Through this study we aim to foreground service users' voices in order to shed light on the personal and subjective nature of the relationship between physical activity and serious mental illness (SMI). Methods: An interpretive case study approach was used to explore in depth the physical activity experiences of three men with SMI. Creative analytic practice was used to write three creative non-fictions which, as first-person narratives, foreground the participants' voices. Results: We present three short stories in an effort to communicate participants' personal and subjective experiences of physical activity in an accessible, engaging, and evocative manner. We hope to: (i) provide potentially motivating physical activity success stories for others who live with SMI; (ii) increase awareness among mental health professionals of the possibilities of physical activity; and (iii) provide an empathetic understanding of possibilities and problems of living with SMI which may help challenge the stigma surrounding mental illness. Conclusions: For us, the stories communicate the diversity and difference inherent in the ways men with SMI experience physical activity. We reflect on how the short story form allows these differences to be preserved and respected. We resist making further interpretations of the stories preferring instead to encourage the reader to form her or his own conclusions. © 2007 Elsevier Ltd. All rights reserved