User:Etchevers/Notebook/Conference notes/2010/05/26

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I can't get this to format. Attended the the Pasteur Conference – 2nd joint meeting of the Société Française de Biologie du Développement and Japanese Society for Developmental Biology from May 26th to 28th. [http://thenode.biologists.com/meeting-report-from-the-2nd-joint-meeting-of-the-sfbd-and-jsdb-2010-from-cells-to-organs/ A meeting report, polished, can be found here].
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|style="background-color: #EEE"|[[Image:C14.jpg|128px]]<span style="font-size:22px;"> Conference and seminar notes</span>
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Notes from the Pasteur Conference – 2nd joint meeting of the Société Française de Biologie du Développement and Japanese Society for Developmental Biology==
 
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Introduction of organizers by Margaret Buckingham. Thanks to sponsors.
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--[[User:Etchevers|Heather]] 17:45, 27 June 2010 (EDT)
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11:00-13:00 Session 1: Organogenesis and Tissue Homeostasis
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Chair : Josiane Fontaine-Pérus (Université de Nantes UMR CNRS6204, Nantes)
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11:00 Kenji Matsuno (Tokyo University of Science, Tokyo) Planar cell chirality contributes to the left-right asymmetric morphogenesis of epithelium in Drosophila
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Asymmetry in bilaterians. Genetic determination, small variations.
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Timelapse GFP in hindgut Drosophila cells during formation. Twisting then counterclockwise rotation, bringing hindgut to right. V. pretty movie.
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Screen 80% genome with 8K mutant lines by hitting chr 2 and 3 (non-acrosomal out of 4).
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MyoII, Wnt, JNK, septate junction complex, Dr no and 10 others for anterior; posterior gut genes cf Nature 2006 paper on Myosin31DF, DE-cadherin and others.
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Extramacrochaetae blocks ? which must block DE-cadherin, itself downstream of a + influence by Myosin31DF. M31DF mutants have mirror image posterior hindgut only – perfectly heterotaxic.
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Myosin31DF encodes unconventional MyosinId myosin, actin-based motor protein.
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DE-cad mutant randomizes laterality. Same tissues, same stage requirements.
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Centrosome position. Polarity in moving cells, they migrate with extension of protrusion on side where the centrosome is, away from the nucleus which gets dragged along.
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Express GFP-tagged centrosomin to look at centrosomes re: to cell membranes in red. The position of the centrosome (like the primary cilium) also has LR polarity in the cells of the epithelium. Plotted position of centrosome in quadrants, slight bias to the R side, looked at 327 cells from 10 embryos. About 40%-60% L-R bias. Then examine if shape of cells bias the chiral shape of the apical region of the hindgut. Compare to Myo31DF and DE-cad mutants. The measure is the angle of the top/bottom edges of cells relative to the anterior-posterior axis?
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The Myo31DF somewhat biased to the lower angles, the normal biased to the higher >90 angles, and the DE-cad mutants no more bias. Subtle effect to my neophyte eyes. "Planar Cell Chirality" Mimic with a mathematical model of tension on pentagonal (not hexagonal) virtual cells at a particular angle, the twist will induce release of tension and resumption of a more stable shape on the cell membrane.
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Questions:
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What sort of mutations, and can they be combined? LOF type (but some rescue from maternal)?
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Dorsal cells only can be examined, restricted set in the gut. Representative shapes? What about looking in fixed tissues? Answer – ventral side imaging is better, and live.
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Stochastic probabilities yet the final outcome is very reproducible – deterministic, even. How ensure the outcome?
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11:25 Reiko Kuroda (University of Tokyo) Chiral blastomere arrangement dictates the zygotic left-right asymmetry pathway in snails
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Chemist originally – molecular origin of snail chirality.
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Snail handedness determined by early cleavages – cf Crampton in 1894 and single gene locus ID'd in 1920's already.
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3rd cleavage is determinant at metaphase-anaphase before cytokinesis, sinistral vs dextral embryos now determined. Chirality visible in dextral snails. Cf Shibazaki et al Curr Biol 2004. Non-mirror image relationship within a species.
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Sinistral-dominant species vs dextral-dominant species, the alternatives are mirror image, but not L-R within the species – the cellular geometry different.
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Congenic snails – L. stagnalis. Can cross out to other animals. Backcrossing to get up to F10 generation only 0.1% dextral, rest are sinistral. Still the few dextral offspring, spiral deformation and spindle inclination. For the ADS backcrossed strain, 0.8% dextral, rest sinistral, same strong linkage between handedness-determination and third cleavage morphology.
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Reversal by applying forces on sinistral embryos to dextralize them, and vice-versa in other species. Can reverse these 8-cell embryos to snails with heterotaxia in shells and visceral situs inversus.
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Nodal and Pitx expression is asymmetric. Sinistral embryo, nodal on L, dextral nodal on R. Pitx roughly the same. When they physically reverse the forces that lead to chiral arrangement with glass electrodes, the gene expression is reversed by that mechanical manipulation. Leading to mirror-image organism.
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Dextralized adult snail by mechanical manipulation still gives rise to sinistral offspring (whew!)
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11:40 Jean-René Huynh (Institut Curie, Paris) How does the egg come first in Drosophila
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Oocyte fate specification – cells are all linked between them. Males all the cells become sperm, but in the females different animal species either keep all (Xen), 15% (mouse) or one to be oocyte and all the rest, nurse cells. Why that one? Symmetry-breaking step. Four preceding divisions, always symmetric and stereotyped.
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Position in germinarium over 2-3 days is important.
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Pre-pattern in original stem cell division asymmetry, propagated? Or choice between two pro-oocytes is stochastic, competition?
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2nd model supported by mutants. Eg. spindle-C-B has 2 oocytes both entering meiosis. egalitarian mutant they all do.
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Possible propagation by location of original machinery of the "fusome" – the connections between the cells, made of vesicles dynamically exchanging with the cytoplasm.
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But can't just dissect out and watch development in culture, also Gal4 labelling of one cell will diffuse to all of them because of fusome. Label the DNA with photoactivatable GFP-linked to histone. using 2 photon microscope. Label the germarium one cell's DNA, then implant the germarium into ovary-less Drosophila. cf A. Spradling in 1993 but still a cool technique. Then dissect them out a day or two later to get development and eggs produced.
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Made an improved photoconvertible transgene inserted into a histone locus instead. Conversion after 2-photon activation changes green to red. Wow. Much better signal/BG ratio. Another tool to label centrosomes, similar conversion method of red from green. No preferential inheritance in males of maternal centriole like in other stem cells. Prelim results presented to support idea that maternal centrosome could be inherited preferentially.
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Test fate determination by late photoablation of the prospective oocyte. The 2nd pro-oocyte can not replace the first. Par1-GFP distinguishes the pro-oocyte at the 2-cell stage, no seeming replacement at the earlier stage. Par-1 is cytoplasmic but more vesicle in the oocyte so correlates.
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Centrosome association with fusome but mechanistic hypotheses need to be developed!
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12:05 Willy Joly (Institut de Génétique Humaine, Montpellier) Translational control in the maintenance of Germ-line Stem Cells in Drosophila
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Nanos and pumilio are transcriptional repressors in stem cells, and Bam promotes diff''n in cystoblasts by perhaps repressing them. "Twin" gene encodes CCR4 deadenylase – wonder if mechanism of Pum and Nos in GSC need function of CCR4.
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mutant twin ovaries lose their GSCs. anti-Vasa to see germ cells progressively lost, all gone by 3 weeks. Cells not maintained because they differentiate, not because they die (no caspase-3 staining). This loss is enhanced by the removal of 1 of the 2 copies of either nos or pum.
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Co-IP with HA-tagged CCR4 in GSC complexes with pum, nanos and resistant to RNase. Not susceptible to differentiating factor mRNAs. In the cystoblasts, bam mutant needs to counteract to let them differentiate. It is in fact epistatic, because over time the double-mutants the GSCs do differentiate but then they are lost.
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CCR4 therefore deadenylates certain mRNAs, which is how Pum and Nos maintain GSCs. Looking for targets. Martine Simonelig lab.
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12:20 Atsuo Kawahara (National Cardiovascular Center Research Institute, Osaka) Involvement of sphingosine-1-phosphate (S1P) signaling in cardiac morphogenesis?
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Have lines with heart-specific promoter driving RFP expn and looking at heart beating in mutant screen. Found a cardia bifida line "ko157".
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Zebrafish one atrium and ventricle. Bilateral progenitors migrate medially to give rise to cone. atrial and ventrical myosin heavy chains are specific to those future segments. Another cardia bifida mutant is S1P2 morpholino treatment or in S1P receptor 2 mutants. ko157 mutant in this pathway, either in the production of S1P upstream, or in the reception of the signal downstream. Secretion not clear how it is regulated.
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ko157 may be a S1P transporter (reminds me of Stra6 for vitamin A). Gene = Spns2 with a R153S substitution. cf Osborne Curr Biol 2008 or Kawahara Science 2009.
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AS MO phenocopies, and mRNA of spns in ko157 will rescue. SPNS – spinster family genes, mutations in this membrane protein interferes with apoptosis.
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Spns2 independently ID'd by D Stainer's group.
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12 TM transporter – develop export system. 3H sphingosine via SphK1 converted in cell into 3H-S1P. See if introduction of Spns2 WT-EGFP or Spns2-mut-EGFP will regulate the S1P secretion from the cells. S1P in supernatant of zebrafish or human transfected cells with the WT isoform, not the mutant isoform. Use mass spec to examine C17-S1P to examine zSpns2 WT-EGFP (ok peak, secreted) or R153S
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Expression during early development in yolk layer marginal cells and during somitogenesis in somites/intermediate cell mass, as well as just under the migrating cardiac progenitors.
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KD in the YSL with MO's impairs the migration, causes bifida. Rescue with WT spns2 but not mutant form, in the ko157/ko157 cells. Exporter activity critical.
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SphK1 and K2 are kinases needed to convert Sphingosine to S1P. Look at Hait C et al Science 2009. Examining expn in zebrafish. Maternally supplied, ubiquitous during embryogenesis, including YSL.
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KD with MO against SphK1 at 1 cell stage – get elongation of embryo, no effect of SphK2 MO. Involvement in axis formation? Inject into YSL only at the dome stage, can get cardia bifida, even more severe (flat/wide). No effect with SphK2 MO similar injection.
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Expn pattern of S1P receptors now. 1a, 1b and 2. S1P2 being the most relevant one for the precursor migration.
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Questions: Soaking embryo in S1P or injection into body? Yes in rescue Spns2, discrete injection into YSL rescues, but overexposure leads to severe axis defects. Regulation of S1P is important in zebrafish. What about phosphatidic acid, ceramide, etc.? Have looked at another lipid without effect.
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Mouse mutants leading to cardia bifida – eg GATA4 and other TF. How would the TFs act – upstream of S1P or Spns? Answer: have not looked at the expn in the mutants. Spns2 only cardiac progenitors but the other vascular progenitors converging on the midline are not inhibited, so not clear where GATA4 would be in that pathway.
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Spns2-EGFP b/c no antibody exist. In cultured cells this is on the cell membrane, also in the embryo.
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12:45-15:15 Lunch and Poster session I (odd numbers)
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15:15-16:05 Keynote Lecture sponsored by Development, Company of Biologists
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Chair : François Schweisguth, Institut Pasteur, Paris
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Crossing boundaries: multilineage priming or reprogramming ? Yann Barrandon (Ecole Polytechnique Fédérale de Lausanne, Lausanne)
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Reprogramming is an increase in potency. Potency – range of commitment options available to a cell.
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Lineage priming is promiscuous stem cell expn of differentiation program genes.
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Relationship between different epithelial stem cells?
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Potency is interesting because of metaplasia – eg. cornea into epidermis, oesophagus into intestinal (simple epithelium), trachea into epidermis, bladder (transitional) into epidermal (stratified)…
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Conditional Notch-1 mutant in corneal epithelium. Get opaque plate in central cornea. Cf Nicolas et al Nat Genet 2003, Vauclair et al., Dev Cell 2007. Disrupts FGF and RA signaling. Can get a dog or human eye with hair shafts growing out of the cornea. See paper from Ferraris et al (Dhouailly lab) Development 2000 – corneal epithelium in co-explants can make hair.
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Ophthalmic surgeons would like to treat limbal stem cell deficiency with cells from other eye or from other eyes with autologous oral mucosal epithelia – cf Nishida et al., NEJM (Tano lab). Equates this effort to a metaplasia. However some improvement to patients but not a change of identity of the buccal cells – they seem to have a memory of their origin. Or not yet respecified by the microenvironment.
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Said microenvironment can be normal or diseased, or absent even.
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Stratified squamous epithelia – epidermis and hair follicle (also stem cells), ocular surface, oral cavity, vagina, oesophagus. Protection from envt'l aggressions, lots of turnover, most likely all contain stem cells. Either keratinized or not.
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Yang 1999 and Mills 1999 papers knocking out p63 TF in basal cells of stratified epithelia – no limb, epidermis absent or impaired, cf. review by van Bokhoven and McKeon 2002. p63 htz human has ectodactyly, facial clefting and mammany hypoplasia and absent teeth, as well as underdeveloped thymic epithelium.
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In vitro, epidermal cells can express differentiation markers of different epithelia or even subcompartments. Can clonogenic keratinocytes respond to other hair/skin morphogenetic signals? Yes, hair follicle multipotent stem cells can be serially cultivated and transplanted in the rat. Cf Claudinot et al., PNAS 2005. They make short term epidermis, then only sebaceous glands and hairs. If wound the follicles, will also make epidermis again. Cells from foot pads, vaginal epithelium, oesophagus, buccal cavity – in the right microenvironment they will make exactly the same hair/gland derivatives. However, upon the serial propagation, they will re-express keratin-4 as per their origin, when in culture.
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Thymus high keratin 7, Pax9, re: hair follicle SC's.
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Microarray analyses between the five cell types. The most distant cell type from the hair follicle SC's are those from the foot pad.
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Differential cell expression. Is the competence acquired in cell culture? Used Rosa26 adult mice with stratified or non-stratified epithelia, lots of blue cells. You can get cycling hair follicle stem cells being formed from central cornea, as per original Dhouailly lab papers. Even tooth epithelium (?) but trachea, bladder and gut epithelium can not as non-stratified epithelia, undergo this transdifferentiation when implanted into the developing skin.
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p63+ stem cell in the appropriate microenvironment give rise to all the stratified epithelia. Can get them all to become hairy skin stem cells, now would like to go the other direction.
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Endodermal thymus – Foxn1 gives rise to athymic phenotype. Bipotent progenitors which can give rise to cortical or medullary cells. Involuting with age, though possible to regenerate. Derived from 3rd PA. Cortical or medullary thymic epithelial cells are organized in a specific spongy 3D network, unlike all the other epithelia. Thymocytes circulate between. Cortical cells have Keratin 8 and 18 like simple epithelia; the medullary have K5 like in stratified epithelia, as well as the Hassalt's corpuscle which express involucrin.
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Just like in the skin.
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GFP+ rat thymus at E16-17 or postnatal stages. Cells in same skin human stem cell cultures. 0.1-0.5% cells form colonies. Bonfanti et al., Nature in press. The clonogenic cells appear from both cortex and medulla. K5/K14+ (medullary) and K8/K18+ - can be serially cultivated and grow well. However, they maintain a thymic ID in vitro because keep Hoxa3, barely Aire1 though. Thymic aggregates with freshly isolated cells will make a thymus-like structure when implanted under the kidney capsule assay, then they re-express Aire1 in vivo. Clonogenic hair SC won't do that, and they do not organize either into the right 3D network.
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In the other direction, the TEC's will contribute to long-term skin renewal (follow by an integrated GFP marker) – and hair follicles etc.
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Hairy skin potency increases upon serial passages through the skin microenvironment. Now do they keep thymic potency?
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Recovered cells from skin no longer express Pax1, Pax9 only later. Re-implant into thymic microenvironment, re-express MHC-II and Aire1. But it's not universal. Some cells will make thymic reconstitution and hair follicle reconstitution, as well as epidermal-like constitution in the thymus. More microarray analyses to compare more-TEC-like and more-hairy-skin-like – and they cluster with the phenotypes.
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The clonogenic TEC are of endodermal origin (in theory?) but have properties of ectodermal skin stem cells.
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Usually hair follicle stel cells don't make epidermis, but bladder can. Thymus only cell type so far that can make both kinds of derivatives from non-stratified and stratified epithelial cells.
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Drawing a "functional genealogy" – so hair follicles not anymore at the top but thinks now won't be able to get cornea from these, perhaps from buccal cavity or foot pad instead, thymus even higher up in potency. What other potency do the thymic ECs display? other than skin such as endodermal derivatives?
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Honor to Alessandro Amici, Paola Bonfanti, Stephanie Claudinot and others.
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yann.barraondon@epfl.ch
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Jean Deutsch – analogy between Drosophila and these questions of cell memory and crossing boundaries of differentiation. Effect on chromatin?. YB: so much to look at, not sure where to start. In thymus, cells remember and express their original lineage. Sort the stem cells from the hair follicle to answer this sort of questions. Check back in a few years.
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Margaret Buckingham – is there a ground state or is there some sort of transverse acquisition of ID? YB: Inclined to think thymus is already primed to be skin, needing p63. Over-ride thymic expression program in some but not all cases.
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16:05-19:00 Session 2: Early Embryonic Patterning
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Chair : Yumiko Saga (National Institute of Genetics, Mishima)
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16:05 Hiroshi Sasaki (Center for Developmental Biology, RIKEN, Kobe) Mechanisms of trophectoderm fate specification in preimplantation mouse embryos
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Preimplantation – TE needed for implantation and ICM for the embryo. Polarized outside cells but inside cells of ICM lose their polarity. The outer ones that become trophectoderm express Cdx2.
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Hippo signal in cell fate specification – it is a tumor suppressor in Drosophila – genetic screen id'd components of this pathway. Cadherin-like cell adhesion molecule, Fat, upstream. Mammalian counterparts of signaling components converge on TF Tead and co-activator Yap. (Hippo = Mst). Cell-cell contact regulates the signaling. Contact inhibition of proliferation is mediated by this signaling pathway. Low density, Yap is localized to nucleus in absence of signal by Hippo in cell cytoplasm, but when the signal is activated, Tead TF is inactive and cells stop dividing.
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Tead4 mutants (there are 14 or 40 Tead genes?) do not form cavitated blastocysts with a coelom. Proliferation is normal though. Mutants have no Cdx2+ TE but all cells express Oct3/4. Nishioka et al Mech Dev 2008.
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In fact Tead4 expressed in all the cells, but why Cdx2 only in outer cells? Yap RNA throughout, but the protein is only located in the nucleus of the outside cells. Precedex onset of Cdx2from 8 cell stage on, excluded from nuclei of the inner cells. Timing slightly earlier than Cdx2 expression. Tead4/Yap complex only in the outer cells to induce Cdx2; Tead4 activity modification affects Cdx2 expression.
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Activation of Hippo signal leads to Yap phosphorylation via Lats. Inner cells have more phosphor-Yap, sign of active Hip signaling – how is it activated? Remember that cell contact can do that in vitro, so decompacted embryos by treating with Ab inhibiting E-cadherin. Reduces phospho-Yap dramatically, leading to inner cells getting nuclear localization of Yap. (do they then start expressing less Oct3/4 and more Cdx2?)
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At 8 cell stage, all cells are "outside" cells. Reaggregate 3 embryos into a chimera even so, nuclear Yap only on outside cells and Cdx2 as well. Cell position suggestive to regulate cell fate specification. Cf Nishioka et al., Dev Cell 2009. – weak Hip signaling leads to nuclear Yap localization.
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How does polarity impinge on the Hippo pathway, keeping it low in the outer cells? Nuclear YAP localization correlates time-wise with polarity acquisition. Par-aPKC system. Knockdown Pard6b with shRNA injection. Disruption of polarity markers. This leads to much abolition of nuclear YAP and phospho-Yap is evenly distributed in both inner and outer cells.
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Not simple since Pard6b KD embryos actually have somewhat less Cdx2 expression, though still present in the non-nuclear YAP cells. Defects in nuclear localization not visible at up to 16 cell stage, though weaker than in controls; gone by 32 cell (blastocyst) stage (Excluded from nucleus.)
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Polarity-independent regulation up to 16 cells to promote nuclear YAP localization, then polarity-dependent regulation thereafter.
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Michel Labouesse – chicken/egg question. Polarity or cortical tension most important? Inhibit myosin II would lead to same result? Also, what happens to hippo signaling receptor Fat? Answer: Other Hip signaling components may distribute differently. Fat4 is the mouse counterpart, but the KD phenotype is a polarity defect, not a growth defect.
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Francois – if disrupt scribble, or other basolateral components, what would happen? Other ways of distrupting apical
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Another person – C elegans has mutant where Par6 can be basolateral – is it Par6 absence/presence itself the issue or the relocalization to the apical membrane that is important? Answer: not sure, but related overexpression of dnPar6 gives same results.
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16:30 Jérôme Collignon (Institut Jacques Monod, Paris) Wnt and TGFβ signalling regulate Nodal expression during epiblast development in the peri-implantation mouse embryo
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Nodal in ICM at E3.5 and maintained in epiblast and later primitive endoderm. Sometimes embryos w/o the expression in PE but transient, then by E5.5 they all do.
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Other components are present – smad2, 3, cripto, foxh1, lefty which is a target and a nodal antagonist. When is Nodal really required, since the mutant phenotype not visible <E5.5?
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transgenic PEE-GFP is heterogeneously expressed in ICM and epiblast and the single/few cells cluster rapidly. PEE is a promoter element containing LEF sites x2 upstream of Nodal. LOF and GOF in Wnt pathway to see expression of transgene. More cells express transgene in the APC(min/min) mutants.
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Ben Haim et al 2006 showed same sites and PEE activity depends on Wnt3 at E6.5 for gastrulation. Earlier use in the nascent epiblast.
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Symmetry-breaking signal at E4.5?
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TCF3 is a "pluripotency" factor in mESCs and its KD elicits Nodal upregulation. Best candidate. Conservation of canonical Wnt/B-cat signaling upstream of nodal means that blastocyst is really analogous to blastula of other vertebrates, even if these are determined by other TGFb family members (?) such as Vg1.
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Inhibitor of ALK4/5/7 is SB431542 (activin receptors) can reduce activity of ASE-YFP transgene in blastocysts.
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Vg1-related mouse factors Gdf1/3 in mouse don't really activate Smad2 and -3 and their expression are not affected by Nodal mutants even at E5.5. Other activins, present in oviduct and uterine epithelia, also in ICM and E4.5 TE, may be better candidates.
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ASE-YFP embryos for YFP and Gata4 (PrE-specific) show heterogeneity. Nanog is not specific to epiblast and also in PE. Bit in a few embryos can see complementarity in localization of ASE-YFP – maybe Nanog represses Nodal/activin signaling. Compatible with ChIP analysis of effect on "molecular machinery of pluripotency".
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Nanog complexes with phosphor-Smad2/3 in hESC and mEpiSCs, and dampens transcriptional activity. But by ChIP could not find that Nanog binds the ASE.
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Smad2/3 has to recruit a chromatin-remodeling complex before they can act as TF, so ASE-YFP may be marker of cells that have irreversibly begun their epiblastic identity.
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However, another highly bound element upstream (cf Chen et al., 2008 for hotspots of Nodal minding) conserved in eutherian mammals, separate from PSE and ASE and node-specific regulatory elements.
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HBE represses a strong promoter (pCAG) in luciferase assay. Reproduces effect of a known Nanog cis-regulatory element. Sub-units within the HBE have some enhancer effect but together they repress. The HBE may be repressed itself by Oct4/Nanog etc.
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No time for questions.
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16:55 Yuuta Moriyama (University of Tokyo, Tokyo) The zic genes as master regulators in dorsoventral patterning of fish trunk structures
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DV patterning.
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Double anal fin – two ventral halves as far as skin, end of notochord, iridophores dorsal when normally only ventral. However, NT and digestive tract are not affected.
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Enhancer mutant? First described in Ohtsuka et al 2004. Region contains Zic4 and in opposite orientation, Zic1. No CDS mutations. Both of their expressions though are lost in the dorsal somites at 18-19 somite stage. Continues in brain and spinal cord. Fin and pigment patterning are specifically changed, and this was effected by the somites.
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Cross back transgene overexpn of zic1 and zic4 to rescue the Da phenotype. Try to implant WT somites into DA phenotype from B-actin::dsRed at 13-16ss over 3-4 somites. Get rescue for melanophores and for fin fold morphology. Presumably for body shape too.
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BMP DV information is upstream of the instruction of the somite, which itself is upstream of the DV info that is imparted to the other tissues.
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Dr. Hayashi question: border also maintained in the dorsal myotome, followed up by Yoshiko Takahashi – the lateral line and the dorsal myotome
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17:10-17:40 Coffee break
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17:40 Hiroki Nishida (Osaka University, Osaka) How do embryos count the cell division rounds in each tissue lineage?: An ascidian case
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11 cell divisions on average, 3000 cells. But cf tissue type differences – muscle = 42 large postmitotic cells, 9 divisions. Notochord, 40 large postmitotic cells, 9 divisions. Mesenchyme = 600 cells, 13 times, small size. CNS 300 small postmitotic cells, 11 divisions.
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Inject into 64 cells stage blastomeres for fate-mapping. Even when cells isolated, acquired their fate.
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Developmental clock models – how know when should stop dividing? Digital clock – counting cell cycle rounds after fertilization? No evidence. Analog clock – measurement of elaped time ; Digital timer = counting cell cycle rounds after cell fate determination, or analog timer = measurement of elapsed time. Clock not start at fertilization but when fate has been determined.
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What if it's not timing? Cell volume – amount of cytoplasm, cultured cells can perceive own cell size. Or nucleo-cytoplasmic ratio, increases over embryogenesis eg in medioblastular transition in Xenopus, titration of some factor by nuclear DNA.
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Bisected fertilized eggs along different axes. With basal or animal ½ or sideways. Sometimes reduced cell numbers. Perhaps volume accounts for decrease. Sometimes removed paternal nucleus leaving volume intact, get actually more cells (1/2 NC ratio, and 1 volume). Nuclear/cytoplasmic ratio and total volume.
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Similarly tested these operations – endoderm, muscle and notochord cell numbers not affected by these. Maybe a developmental clock for these. However, mesenchyme and trunk epidermis are compatible with the cell volume regulation, not the nuclear/cell volume ratio.
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FGF helps FoxA Zic+ anterior cells to go toward notochord (3 divisons before hatching); emanates from vegetal pole. Otherwise default to CNS (many divisions). For posterior region, macho1+ cells with FGF are mesenchymal, w/o = muscle/
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In partial embryos, isolated cells are induced to Notochord by FGF. Overall, when cell fate already fixed, number of future cell divisions is determined and cell-autonomous. Compatible with the timer models, but cell number is independent of cell size in this case?
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If KD brachyury in determined notochord fate cells, what happens to cell divisions? MO meant cell division did not stop, though normally they do. Cycle is not accelerated but zygotic expression of Bra leads to division control normally. Start developmental timer.  GOF bra at the 1 cell stage control at 1 cell stage (egg) Usually starts at 64 cell stage. Stage of initiation does not determine the timing of cell division determination. 3 divisions after 64 cell stage, no matter when inject.
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cdc25 examination – important in muscle and notochord.
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18:05 Akatsuki (Aki) Kimura (National Institute of Genetics, Mishima) Quantitative measurement and modeling reveal cell size dependent mechanisms to elongate mitotic spindles in the C. elegans embryo
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Cell volume/size. "architectonics' – functional role of spatial organization. 3D + time. Iteration between modeling and simulation via model-based hypotheses to biological experiments
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Movie of C elegans fertilized 1-2 cell embryo. Forces regulating different aspects of cell cycle – streaming, centrosome centering (male pronucleus), spindle displacement and rotation, spindle elongation and then cytokinesis.
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Yuki Hara studying how the cells know their size – by quantifying cell size dependency in spindle elongation.
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http://learn.genetics.utah.edu/content/begin/cells/scale -  I was looking for that link to scales.
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Elongation of spindle pulled by the cortex via microtubules. This happens in cell size-dependent manner in a directly proportional manner but the nuclear size and length before elongation also correlate in the small cells. So looked only in larger cells. Elongation speed also correlates.
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GPR1/2 is a regulator of G-alpha, regulates cortical pulling forces. Use RNAi to KD expn and get some elongation, still cell-size dependent. There is a sensitive part, that is still dependent on cell size but the insensitive part is not dependent on size.
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Contact points of microtubule tips and cortex are fixed. This constant-pulling model can explain the Galpha-insensitive component. In the gpr1/2 RNAi experiments, they abolish the correlation between size and elongation length or speed. (I missed the critical part of this experiment, sorry)
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Idea that in large cells, force generation from inside, the number of the force generators at the cortex is limiting. This can account for the Ga activity sensitive mechanism.
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I missed a wonderful last slide about scaling and comparison of the models.
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Answer to a question – intraspindle pushing forces can be neglected because ablation of these microtubules do not affect length of elongation. Pulling from outside is dominant for the larger cells, but pushing from within for the smaller cells.
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Francois – sensing of geometry? The contact points are fixed during elongation so there is a kind of inward pulling force at certain points laterally and outward at each end.
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18:30 Pascal Thérond (Institut de Biologie du Développement et du Cancer, Nice) Regulation of Hedgehog long range activity in Drosophila
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How can hh signal spread dorsally at a 300 m distance in vertebrate NT as opposed to 50 m in the fly imaginal disc?
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Polarized epithelia may integrate at each pole a different sensation of morphogens from each side.
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Two targets as readouts in imaginal discs – En for 2-3 cells in anterior compartment; and long-range target is broader, dpp. Hh makes it further in the apical plane than in the basolateral one, as far as en and dpp.
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Deplete apical hh by sequestration of free luminal hh, and decrease dpp width, no effect on en. Block internalization in posterior cells to increase hh concentration, using dynamin mutants for hh-secreting cells. Get much more dpp expression but at expense of en expression.
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Glypican proteoglycan called Dally with HS chains, and a GPI anchor. Mutant clones in the imaginal posterior component where hh produced, get less dpp in anterior compartment. Possibly by cleavage and release into ECM. A GFP-Dally does seem to be released into the lumen and fill it up. Express therefore in the hh-expressing cells a constitutive secreted form of Dally and this increases hh spreading into the anterior compartment. Thus more dpp expression in anterior compartment.
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Notum is an extracellular PLC-like hydrolast that cleaves such GPI anchor proteins. KD in those cells with an RNAi gets less anterior dpp expression. Notum like Dally can regulate this long-range signaling.
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Dally does require Notum to augment that signaling, if express GFP-Dally again, the anchored version, but in absence of notum (mutant disc) get normal dpp expression once more (why not less?)
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Short-range basolateral gradient perhaps responsible for the short-range targets like en.
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18:45 Yoshihiro Morishita (Kyushu University, Fukuoaka) Optimal design of positional information encoding by morphogens : from an engineering viewpoint
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See notes in the abstract book, I have no more battery.
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19:00-20:30 Cocktail and Poster session I (odd numbers)
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__NOTOC__
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Current revision

I can't get this to format. Attended the the Pasteur Conference – 2nd joint meeting of the Société Française de Biologie du Développement and Japanese Society for Developmental Biology from May 26th to 28th. A meeting report, polished, can be found here.


--Heather 17:45, 27 June 2010 (EDT)

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