Analysis of premature loss of the extraembryonic Amnioserosa in Drosophila morphogenetic mutants

During Drosophila embryogenesis, an extra-embryonic tissue, known as the amnioserosa (AS), is required for the morphogenetic processes of germ band retraction (GBR) and dorsal closure (DC). Being extra-embryonic, the AS is not part of the embryo proper but is eliminated via programmed cell death (PCD) in the late stages of embryogenesis. Programmed elimination of the AS during normal development can be prevented by directly inhibiting apoptosis, either through the deletion of the pro-apoptotic genes hid, grim and reaper, or through the expression of the pan-caspase inhibitor P-35. PCD in the AS can also be prevented by indirect inhibition of apoptosis via inactivation of autophagy, either through activation of the InR/PI3K pathway, or through activation of the Ras signalling pathway. The timing of AS elimination is critical to development as mutants associated with premature AS loss fail in GBR. To better characterize this premature AS death, a detailed phenotypic analysis of the AS behaviour in the GBR mutant hindsight (hnt) was performed. Direct inactivation of apoptosis failed to rescue the GBR defects in hnt mutant, though the premature AS death was completely rescued. Inactivation of autophagy, however, rescued AS cell behaviour and contacts during GBR, with partial rescue of the GBR defects in the hnt mutant. The nature of premature AS loss is characterized as a possible model for anoikis, a form of cell death that is triggered through reduced cell-cell or cell- matrix contact

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope

Kaiso-induced intestinal inflammation is preceded by diminished E-cadherin expression and intestinal integrity.

Chronic intestinal inflammation contributes to pathologies such as inflammatory bowel disease (IBD) and colon cancer. While the precise etiology remains controversial, IBD is believed to manifest as a result of various factors. We previously reported that intestinal-specific overexpression of the transcription factor Kaiso results in an intestinal inflammatory response; however, the cause of this inflammation is unknown. To elucidate the underlying mechanism(s) of the Kaiso-mediated intestinal inflammatory phenotype, we evaluated two independent transgenic mouse lines that express varying levels of Kaiso (KaisoTg). Histological analyses of KaisoTg mice revealed intestinal damage including thickening of the mucosa, intestinal "lesions" and crypt abscesses, which are reminiscent of IBD pathology. Additionally, higher Kaiso levels induced intestinal neutrophilia as early as 12 weeks, which worsened as the mice aged. Notably, the Kaiso-induced intestinal inflammation correlated with a leaky intestinal barrier and mis-regulation of E-cadherin expression and localization. Interestingly, Kaiso overexpression resulted in reduced proliferation but enhanced migration of intestinal epithelial cells prior to the onset of inflammation. Collectively, these data suggest that Kaiso plays a role in regulating intestinal epithelial cell integrity and function, dysregulation of which contributes to a chronic inflammatory phenotype as mice age

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Not AvailableResearch Leadership Building System (RLBS) is envisaged as a single window platform for applying in various Leadership programmes /schemes. Schemes covered under RLBS are as follows: ICAR-Emeritus Scientist (ES) : The ICAR Emeritus Scientist Programme started during 4th five-year plan (1973-74) with an objective of tapping the brain and skill bank of superannuated professionals of NARES by allowing them to complete the work in hand for its conclusion, utilize their talent in teaching specialized courses and use their experience in addressing nationally important policy issues. ICAR-Emeritus Professor (EP): The ICAR Emeritus Professor Programme is a structural method of tapping Brain and skill bank of the outstanding superannuated professionals of National Agricultural Research and Education system (NARES) by utilizing their talent in teaching courses and uses their experience in addressing nationally important policy issues. ICAR- National Fellow (NF) & National Prof. (NP): The National Fellow (NF) Programme was initiated in 1978-79 with the objective to promote excellence at national level in agricultural research & education and recognize the meritorious contribution of individual agricultural scientists/teachers and facilitate their research and related activities in agriculture.Not Availabl

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Not AvailableIndian Council of Agricultural Research (ICAR) in its continued efforts towards enhancing and sustaining the standards, quality and relevance of higher agricultural education in the country has instituted International Fellowships with dual purpose of: (i) Human resource development in cutting edge technologies, and (ii) Demonstrating the strength of Indian agricultural system in the world. The objective is to develop competent human resource that are trained in the identified best laboratories in the world (for Indian candidates) and similarly expose overseas candidates to the best Indian Agricultural Universities (AUs) in the ICAR-AU system (comprising of State Agricultural Universities, Central Agricultural Universities (CAUs), ICAR-Deemed Universities, Allahabad Agricultural Institute-DU, Central Universities having agricultural faculty) for creating a pool of scientist-envoys for enhanced future co-operation.Not Availabl

The POZ-ZF Transcription Factor Kaiso (ZBTB33) Induces Inflammation and Progenitor Cell Differentiation in the Murine Intestine

<div><p>Since its discovery, several studies have implicated the POZ-ZF protein Kaiso in both developmental and tumorigenic processes. However, most of the information regarding Kaiso’s function to date has been gleaned from studies in <i>Xenopus laevis</i> embryos and mammalian cultured cells. To examine Kaiso’s role in a relevant, mammalian organ-specific context, we generated and characterized a Kaiso transgenic mouse expressing a murine Kaiso transgene under the control of the intestine-specific <i>villin</i> promoter. Kaiso transgenic mice were viable and fertile but pathological examination of the small intestine revealed distinct morphological changes. Kaiso transgenics (<i>Kaiso^Tg/+</i>) exhibited a crypt expansion phenotype that was accompanied by increased differentiation of epithelial progenitor cells into secretory cell lineages; this was evidenced by increased cell populations expressing Goblet, Paneth and enteroendocrine markers. Paradoxically however, enhanced differentiation in <i>Kaiso^Tg/+</i> was accompanied by reduced proliferation, a phenotype reminiscent of Notch inhibition. Indeed, expression of the Notch signalling target HES-1 was decreased in <i>Kaiso^Tg/+</i> animals. Finally, our Kaiso transgenics exhibited several hallmarks of inflammation, including increased neutrophil infiltration and activation, villi fusion and crypt hyperplasia. Interestingly, the Kaiso binding partner and emerging anti-inflammatory mediator p120^ctn is recruited to the nucleus in <i>Kaiso^Tg/+</i> mice intestinal cells suggesting that Kaiso may elicit inflammation by antagonizing p120^ctn function.</p></div

Generation of transgenic mouse lines ectopically expressing <i>villin</i>-Kaiso.

<p>(<b>A</b>) Myc-tagged murine <i>Kaiso</i> cDNA was cloned downstream of the 9 kb v<i>illin</i> promoter sequence. (<b>B</b>) The transgene copy number in each transgenic line was evaluated via PCR. Line A transgenic animals have the greatest copy number. (<b>C</b>) RT-PCR confirmed expression of the Kaiso transgene in <i>villin</i>-expressing tissues of transgenic mice, <i>i.e.</i> the small intestine, large intestine, and kidneys. (<b>D</b>) Immunoblot analysis shows increased Kaiso expression in both small and large intestines in Kaiso transgenic (<i>Kaiso^Tg</i>^/+) Line A mice compared to non-transgenic (Non-Tg) siblings.</p

<i>Kaiso^Tg</i>^/+ mice display decreased HES-1 expression in the small intestine.

<p>Both Non-Tg and <i>Kaiso^Tg</i>^/+ tissues displayed nuclear HES-1 expression in the crypts of the small intestine, however <i>Kaiso^Tg</i>^/+ tissue displays significantly decreased HES-1 expression in the villi. Quantitative RT-PCR showed a significant decrease in HES-1 expression in <i>Kaiso^Tg</i>^/+ mice. Values were first normalized to the GAPDH housekeeping gene, followed by normalizing to non-Tg HES-1 expression (** represents p<0.05).</p

Secretory cell lineages are expanded in the intestines of <i>Kaiso^Tg</i>^/+ mice.

<p>(<b>A</b>) PAS stain for Goblet cells (black arrowheads) revealed increased numbers of Goblet cells in both the villi and crypts of <i>Kaiso^Tg</i>^/+ intestines, p = 0.011 & 0.002. (<b>B</b>) Lysozyme staining revealed increased Paneth cell numbers in <i>Kaiso^Tg</i>^/+ mice, p = 0.017. (<b>C</b>) Synaptophysin positive enteroendocrine cells (arrowheads) are increased in <i>Kaiso^Tg</i>^/+ mice, p = 0.031. n = 3 mice/genotype; measurements performed by two independent blind observers; T-test used for p-value. ** represents significance.</p

Kaiso transgenic mice exhibit inflammation of the intestinal mucosa.

<p>(<b>A</b>) <b><u>H</u></b>ematoxylin and <b><u>e</u></b>osin (H&E) stained sections were used to measure villi length (red bracket; ∼80 villi/mouse) and crypt depth (black bracket; ∼800 open crypts/mouse). <i>Kaiso^Tg</i>^/+ display increased crypt depth compared to their Non-Tg siblings, p = 0.001. (<b>B</b>) <i>Kaiso^Tg</i>^/+ mice exhibit increased immune cell infiltration of the lamina propria (yellow demarcated area) accompanied by increased MPO activity compared to their Non-Tg siblings, p = 0.014. (<b>C</b>) Line B mice do not exhibit immune cell infiltration or enhanced MPO activity compared to Non-Tg siblings. ** represents significance.</p