User:Etchevers/Notebook/Conference notes/2009/09/12: Difference between revisions
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=== Hope Northrup | ===• Hope Northrup (I think) for AC Morrison on AD/HD and folate and glucose metabolism === | ||
Here I am eating a sugary maple candy and thinking I didn't want to take notes on this because I didn't want to pay attention. | Here I am eating a sugary maple candy and thinking I didn't want to take notes on this because I didn't want to pay attention. | ||
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=== Irene Zohn - developmental biologist discussing iron for forebrain patterning as well as NT closure. === | ===• Irene Zohn - developmental biologist discussing iron for forebrain patterning as well as NT closure. === | ||
Attended this conference 6 years ago. Mutagenesis screen from K Anderson a while ago. | Attended this conference 6 years ago. Mutagenesis screen from K Anderson a while ago. | ||
Revision as of 14:16, 12 September 2009
 Conference and seminar notes
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First day of 6th Intl Neural Tube Defects conferenceI arrived super late the first morning thanks to an epic 34-hour journey during which I slept 6 in a bed. First talk where I took notes: 6th Intl NTD Conference: first day = September 12, 2009 • Claudia Kappen – diabetic embryopathyRecent publication – effect on Wnt pathway genes in the induced diabetes model (injection of mice with streptozotocin, down insulin, blood sugar up, mate the dams at day 15. Get 10-15% NTDs at E10.5 and beyond. Genes mostly downreg in the embryo proper, also looked at and mostly reported the results in the placenta. Prevention of diabetic NTDs with folic acid (somewhat controversial). Breeder diet before mating dams >= 6 weeks. Timing of gene expression changes in induced diabetes model is different depending on Chow or Breeder diets (differ by 10% protein/fat, less folic acid in latter, same carbs). On Breeder-fed FPB mice, get more NTDs in diabetes-exposed (22% vs 6.6% on Chow diet) embryos. But revealed at the later stages in development, not seen by looking only at E10.5, also look at E12.5, E15.5, E18.5 (increasing incidence). Macrosomia in diabetic humans – different in mouse. How much related to the maternal obesity to start? Rarely observed in rodent models, but the diabetes is very severe. Questions: Genetic reasons for dietary effects of Chow vs Breeder in other strains of mice 4 misregulated imprinted genes (3 down, 1 up in diabetes) and 3 regulators of imprinting are misregulated. Since 10.5 is > neural tube closure (can ascertain if NTD then) then how relevant? Maternal diabetes changes in the affected embryos? Variance is as great as in the non-affected exposed embryos. • Mary Loeken - ChIP on Pax3 promoterDiabetes much reduces Pax3 – at E10.5 the exencephaly looks like the Splotch phenotype. Less expn at E8.5. Not all the embryos develop malformations. Stochastic NTD occurrence re: exposure to hyperglycemia (glucose necessary and sufficient to inhibit Pax3 by inducing oxidative stress) during a critical window. Drug induces oxidative stress: antimycin A; antioxidants reduce hyperglycemia. Using ES cells to plate and study molecular regulation. (Experiments described in D3 cells derived from 129 Sv strain.) Transfer ES cells to Petri dish and RA stimulation to form embryoid bodies. Select for nestin + precursors on ITSF + medium, get neuronal precursor-looking cells on the newly adherent plastic. Pax3 induced on differentiation, but abrogated with antomycin A, some other gene expression is maintained including nestin. Rather than high glucose: the D3 cells are cultured in a high glucose DMEM anyhow and have lost glucose response. Differentiating cells with or without ox stress. Perform ChIP. PCR Pax3 5’ elements. “Pan-H3” antibody, H3K27me3 (decreased expn), H3K4me3, H3K9ac (increased expn). 1.6kb upstream – strategy = primers amplifying overlapping 200bp amplicons, overlap by 100bp. 1.6kb directs Pax3 expn in NT aside from a part of the head and NCC. Doesn’t control expn in somites. -180 and -400 seems to be concentration of sites. What other chromatin modifications provide access? Get changes at best 1.5-2x, but error bars are very reproducible (relative IPm though to what?). Change at -1000 to -1200 on differentiation. H3K27me3 – no real changes but on a log scale and overall much noisier. H3K4me4, get changes over 3 conditions (before diffn, after selection/readhesion, and after but with antimycin A). Eg -200 to -400 v.low to start (0.01, then 1 for the after, and back down for antimycin treatment). Histone me inhibitor on lysine = chaetocin, HDAC inhibitor TSA. No effects in undiff condition, but addition instead of the antimycin treatment will inhibit Pax3 expression (relative expn, using qPCR).
Folic acid deficiency by up homocysteine, ionizing radiation, other causes of oxidative stress. Specific kinds of free radicals are responsible - increased superoxide production eg. What happens in Pax3 htz? Could not induce C57Bl/6 background maternal diabetes effect NTD even in the Pax3 – and this is a dominant effect. But Splotch on another background, maybe – difference due to glucose transporters? Would like to do ChIP-seq perhaps. Role for inositol supplementation cf diabetes? Alleviation reported in the rat in vitro… Fatty acids work as antioxidants. Or substrate for something playing a signaling role? (I wondered about available plasma vitamin A or retinoid levels in diabetic women - cf that study.)
• Paul (Kit Sing) AuGlucose metabolism genes associated with spina bifida risk or AD/HD in SB patients. 22% white US women obese, 24% men; Canada 25%; black and Hispanic populations are around 30%. Leptin and glucose transporter (glut1/2/3) genes SNPs associated with increased BMI. Cf. Davidson 2008 – SNPs and SB. How well do lymphocytes from patients take up glucose? Isolate from patients and mother, test under 3 conditions – normal, hi glucose, sucrose conditions, do they take up, according to GLUT1 haplotype? (or HK1, or LEPR) genotypes: the significant results are from the LEPR genotypes. Subdivide populations into these three subpopulations. Some SNPs have sig differences, using TDT instead. 140 SNPs analyzed using RC-TDT: BRCA1, GLUT1,/3/4/8, HK1, LEPR, INSR, LEP, SOD1/2, TP53. Plot –log p and get significant results for many SNPs in GLUT1 in particular rs3339682 reproduce previous findings from other groups as associated with SB in patients. Linkage disequilibrium of 10 sign GLUT1 SNPs comparing Hispanic vs Caucasian populations, but though seem different he thinks they are not actually… did not quite catch this.
• Mary E. CogswellFolic acid consumption large study including diet (enriched cereal grain products w/o ready to eat cereals), ECGP+cereals, ECGP+cereals+supplements. Used PC-SIDE software for intake distributions estimation, also accounted for race, ethnicity, sex, BMI, day of week as weekend diet can differ from weekdays. Calculate nutrient concentrations through a whole series of stat methods I am not qualified to assess but sound good. Complex sampling design. Usual total folic acid daily intake in 8,258 US adults. Median = 288 ug per day (160-462) and 2.7% at 95CI consumed >1mg. ECGP only 42% of adults, median = 138; ECGP+RTE cereals 274 ug, none exceeded tolerable upper limit. ECGP+supplements, 479 ug, 5.5% exceeded UL, 635, 9.4% exceeded UL. Consistent across all race, ethnicity, etc. Adults aged 65 and over 12.8% exceeded the upper limit and had ECGP, supplements and breakfast cereals. 11% adults up to 200 ug/day from supplements. Less than 1% exceeded UL. >400 ug/day, 48% of these exceeded the UL. 200-400 ug/day, again less than 1% exceeded UL % US adults with high serum folate and low B12 concentrations inverse relationship where up to 30% of ECGP+RTE cereals+supplements had >20ng/mL but decline with other sources of folic acid (from vitamin B12?). I didn’t get it – went too fast. But it keeps homocysteine down. Supplements for folic acid also have B12 reducing risk for B12 deficiency. Despite contributions from diet, RTE cereals and supplements <400 ug/day, 94% US adults won’t go over the recommended tolerable upper limit of 1 mg/day.
• Sarah Tinker, CDC also.Women of childbearing age but non pregnant in the cohort previously described. USDA and public health service recommendations – 400 ug/day if trying to get pregnant. Some recommendations up to 800 ug/day (USPSTF where TF is task force). Women with obesity or diabetes don’t particularly increase their folic acid intake and don’t make particular population-visible efforts to take supplements – conclusion, they should, because no women of childbearing age sub-populations are really able to get to the recommendations on diet alone. Questions: Tough with non-supplemented diet to get folate levels. Before supplements between 100 and 10000 years ago, was there a selective impact on the species? Was there another dietary source or were all those particular babies lost? Answer (of course): I don’t know! RCT’s were only done on synthetic folic acid. Other members of the audience chipped in: But although we think we are sophisticated in our eating habits, earlier populations may have actually eaten more appropriate food to prevent certain vitamin deficiencies, when the food itself was available. “Paleolithic diet” currently in fashion – yet would have it been up to 400 ug/day?
• Jenny Murdoch (Oxford) – exencephaly and SBHitchhiker mutant – modulator of the Shh pathway. Neural tube expansion. Sections are completely open book but sometimes large tissue oedema and luminal expansion, also not a proper roofplate. Dorsal view sometimes open SB but also splayed vertebral splay and thin skin covering that (SB occulta)? This looks like patterning DV defects. Vertebral defects: they are splayed. Mutant is tulp3, a splice site mutation. In WM of E10.5 Shh expression normal in head then much more in the caudal region of the embryo. In section, expands Shh expression all the way up to the alar plate. Get dramatic ventralization - Nkx2.2 and Pax6 looks pretty normal though says dorsalized, however, Msx really does look more restricted dorsally. Patched1 is overexpressed in caudal end of E9.5 mutants - increased activation of pathway by tulp3 at a point where Shh expression itself is still pretty normal. Loss of Gli3 repressor, activation of Gli1/2. Position tulp3 in there by genetic interactions - cross hhkr mice to Shh and Smo mutants. Souble hmz rescues the gross phenotypes especially exencephaly instead of holoprosencephaly, but no rescue of heart defect in Smo cross (hhkr acts downstream of Smo as well as of Shh). Rescue however does not compensate all head problems - synophthalmic rather than cyclopic. tulp3/gli2 double mutant looks more like gli2, so genetically upstream. Gli3 processing not altered. Gli3 mutant only subtle DV patterning problems. Somehow normal role of tulp3 to prevent activation of the Gli activators. Crossed to mutant lacking cilia - kif3a - need cilia to get the tulp3 ko phenotype. Ultrastructure from limb buds E11.5 embryos WT or mutants - same length, normal shape in hhkr mutants. Immunostain with polaris and still apparently normal cilia. Tulp3 expressed in the tip of the cilia! that is lost from the null allele - but it is also seen in the cell nucleus - wonder if it translocates. Want to try real time imaging for subcellular localization. Putative TF - nuclear localization, has a transcriptional activation domain in hybrid assay, and a DNA binding tubby domain. However, no evidence for role as a TF on components of the Shh pathway. Microarray analyses - caudal end of E9.5 - nothing but some DV markers changed. Protein ChIP with antibody but found no specific binding sites (admittedly not the best assay, but supports other data). Intracellular trafficking? PI linkage attaches to membrane. Doing a yeast two-hybrid assay (cf talk tomorrow from student) on a mouse embryonic brain library. (Vicki Patterson). Questions: What upregulates Shh in the mutant, then? But Shh pathway is activated and leads to increased expn in neural tube, not sure if perhaps Gli2 not properly repressed, and b/c activator more active than normal may lead to more ventralization. Expression pattern of Tulp3? Ubiquitous expression - no AP difference. Have not looked at protein level, though tried Westerns - why not immunohistochemistry? Vertebral phenotype. Somite patterning normal or not? (will look). If embryo develops further, double hmz eye malformations. Any notochord duplications? and any other anomalies? Only let them develop to E12.5. Narrow head and close-set eyes. Also limb defects. Double hmz forelimbs have two digits, hindlimbs have four. Incomplete activation of Shh by Tulp3. Also don't rescue floorplate marker expression. Think to mention to her Yuji and Anne-Helene's work in which Shh can inhibit Msx expression and thereby the formation of the spinous process. What is interesting is why the regulation is segment-specific along the length of the neural tube - why only caudal?
• Adriane Griffin- National Council on Folic AcidSpina bifida association - coalition building for change. 62 members of coalition - nonprofits, national organizations, etc. How get messages to women? Current shift from web-based portals to hand-held devices, as an example. Hooks for messages - need to present it eight times for it to register with the targeted audience. This is more about communication efforts and how to go about it, and not the right audience. But it's nice that someone who knows wheat they are doing in communication is behind the cause of making sure research results reach the women who could benefit from it.
• Andy Copp and bending of the neural plateHinge points. Transition from midline bending at E8.5 to dorsolateral hinges at alar plate. Loses midline bend at E9.5-E10 at same point when SB can occur. Is site where bend corresponding only to where somitic mesenchyme and neural crest are lateral to the neural tube? Examination of Zic2 mice. Mention of human embryo bank - Sandra Castro show DLHP also in humans at C12 in the open neural plate. From abstract: "Basal nuclear localization and prolongation of the cell cycle characterise midling neural plate bending" not DHLP. Dorsolateral neuroepithelium proliferates rapidly and also seems to have some convergent extension by integrating additional cells moving dorsally within neuroepithelium, speculate that cells change basal contact from paraxial mesoderm to surface ectoderm. Shh inhibits dorsolateral bending. Cf Ybot-Gonzalez 2002 and 2007 in Development. Bmp2 on dorsal side also inhibits, but noggin under regulation of Shh blocks Bmp2 and enables the DLHP to form. Lors of discussion in Edinburgh on Shh and cilia. Rab23, luzp gli3, gli2, patcc1 all give exencephaly. All of these give Shh overactivation - lead to NTDs. But complicated, and proteins eg from K Anderson have combined lack and overactivation of parts of the pathway cf dnchc2, ift172 and kif3a - all involved in ciliary transport, all give exencephaly (Huangfu 2005 PNAS and Nature papers). More work needed on Bmp side of the signaling. Mechanisms - pursestring model - apical constriction or also nuclear localization eg basal during S phase - if cells enriched for basal nuclei they are wider at that end and become wedge-shaped. Isn't that a bit hypothetical? is is possible to force nuclei into a particular part of an epithelial sheet? Could test by looking at other bent epithelia. I wonder about lines in the skin on the hand, by the way. Also, would this be a cause or an effect? Thinking of a counterexample - basal nuclei in the proliferating neuroepitheilium eg the cortex, the bending is the other way, but that is because the cells lose their basal attachment and wedge their whole bodies, including the nuclei, among the radial glia for example. Seductive concept. Anyhow, gets back to the tight attachment with surface ectoderm on dorsal side and to paraxial mesoderm for the neural tube ventral to that point. This is good. Susanne McShane for PhD did quantitative morphometric analyses. Number of NP cells in contact with surface ectoderm increases as you move from rostral to caudal (Andy said from younger to older, but that is not what the sections are). And cell width decreases, particular in region where the DLHP is. More cells per volume above the DLHP. Attraction to the matrix substrate? Cells "migrate" within neuroepithelium - couldn't they test this with iontophoretic dye injections just ventral to the hinge point at some moment? Some Laminin eg a5 or b1 and g1 are strongly in the dorsal ectoderm. Spent a couple months with Patrick Tam and indeed tested the hypothesis by marking cells very dorsal in DiO and ventral in DiI. Culture embryos and see if colors merge or not. Whole and must remain within the yolk sac. Why do this in the mouse and not in the chick embryo? The colors don't merge and in fact move away from one another if mark before the DLHP form. But at E9.5 do get merging where red seems to move dorsally and join the green - need to confirm on section. The dorsally directed relocation of cells can not lead to cell mixing much within the neuroepithelium. This reminds me of the "regeneration" experiments after neural fold extirpation which purportedly led to neural crest regeneration. Remember if you invert the neural tube (which doesn't really have a lot of DLHP in the chicken) you get repatterning. Questions: Why do Shh-activating mutants have exencephaly and not SB? Experiments applying beads showing Shh inhibitor = in spinal region. Expect to see same result cranially. Why not mutants SB? No real speculation. Are these genes expressed caudally? Some of them are not. Is ectoderm more adhesive or attractive? Answer: yes. Midbrain has DLHP. NCC migrate before closure in brain, some evidence that it would be necessary there for NT closure in brain, but NCC can migrate independently of neural tube closure in the spinal region. My question on neural fold ablation experiments in the chick. Tissue deletion - wound healing might bring tissues sufficiently close together for a tube to form. But Andy evoked experiments where remove surface ectoderm and the NT can not close. And in some of my experiments back in mid 90's where ablated rather far down, did not get fusion even though ectoderm came up to neural folds. Perhaps not in time. How does tube close in early stages without bending over? Not much of a lumen, and the tip cells at the folds meet and adhere.
• Hope Northrup (I think) for AC Morrison on AD/HD and folate and glucose metabolismHere I am eating a sugary maple candy and thinking I didn't want to take notes on this because I didn't want to pay attention. Certain MTHFR SNP allele associated with AD/HD. Reproduce and find two other SNPs, as well as 2 SNPs in GLUT1 that are significantly associated with ADHD. Questions: Connections between ADHD and obesity - wonder if SB kids would improve better with exercise (as possible) more than other immobilized children populations eg wheelchair bound (muscular dystrophy, accidents)? Interesting to me - given that 1/4 or almost 1/3 of moms are obese in the U.S. and maybe the kids develop to be that way or are
• Irene Zohn - developmental biologist discussing iron for forebrain patterning as well as NT closure.Attended this conference 6 years ago. Mutagenesis screen from K Anderson a while ago. Flatiron mutant for ferroportin 1. (Fpn1). Exports iron out of the cell from placenta and visceral endoderm to embryo. Required in extraembryonic lineage. Penetrance of NTDs is background dependent - NTDs on C3H/HeJ but not 129/SvJ background when hypomorphic mutations, whereas the htz hypomorph/KO always NTD. Cf. droopy-eye = p38IP and some others. So anemia leads to NTD? ffe also microphthalmic! (Common ground Patrick et Francois/Helene et Marie-Paule)? Iron might accumulate in VE disrupting organizing function and becoming toxic? VE particularly needed for forebrain patterning whereas mutants are forebrain-truncated. Indeed, AVE is induced and cerebrus (cer1) starts expression and then is diffuse in mutants. A day later during anterior neural induction the normally focal Six3 expression becomes diffuse and expanded. Are NTD due to severe iron deficiency? Culture embryos E7.5 to 8.5 in presence of iron chelators. Normal embryos treated like that can phenocopy the Six3 phenotype of mutants. What is iron required for? My own thoughts thereafter: BMP6 and BMP7 use same receptor. This review: "phosphorylation of SMAD1/5/8 and the formation of the SMAD1/5/8–SMAD4 complex, which, translocated to the nucleus, activates the promoter of the target HAMP gene (Fig. 1). Consistent with a role for BMP–SMAD signaling, inappropriately low hepcidin expression and iron overload are present in mice with a conditional deletion of Smad4 in the liver10, similar to Hfe2-deficient mice." This article says that BMP6 linked to IGF1 expression, at least in osteocytes. And controls miR-21 expression, for what that is worth (in breast cancer). Also, early embryonic BMP6 expression in lateral pharyngeal endoderm (BMP3 all around). Also: "Expression of BMP6 is detected in the branchial pouches (Fig. 1F) and, like BMP7, in the endodermal component of the visceral yolk sac (data not shown)." And "By 9.5 dpc, expression of BMP6 is detected in the roofplate of the neural tube (Fig. 1I)" - implication in NT closure, at least the rostral kind not involving dorsolateral hinge points? But not in the early developing eye - other BMPs are, though. BMP6 also strong in stromal mesenchyme around tubules in developing kidney.
Questions: Accumulation of heme is a sink for folate - keeping folate from being transported across the placenta? Heme may be not just an iron transporter. Other interactions possible with folate? Eg background-specific effects - NTD due to poor diet and other things may be missing from a folate-poor diet. Old data on ferritin in humans and NTD. Does anyone remember? Iron is a prooxidant - but when not bound to ferritin. Co-factor needed for metabolism....? Can not rescue ferroportin KO mutant embryos with additional iron (possibly) but what about the hypomorphs? Excited to find modifiers b.c may give insight into what contributes really to the NTD. These mutants die during mid-gestation b/c of the iron deficiency so other causes for human NTDs.
Coffee, juice etc. in this room from 8AM on so can start at 8:15 tomorrow instead of 8:40. Special edition of Birth Defects Research edited by Nick Greene and Susan Carmichael. Would be interested in publishing manuscipts from this meeting. Quick break then Clarke Fraser. Clarke Fraser from Montreal - genes, environment and multifactorial thresholds.Can everyone hear me? Reminds him of a meeting at which someone asked if everyone could hear him and someone at the back shouted, "No!" Whereupon a person seated at the front got up and walked to the back of the room. Mouse strains vary in frequency of missing the third molar. Strains with highest frequency had the smallest average molar size. Continuous distribution of tooth size, minimal threshold size of tooth anlage needed to induce the tooth? Hans Grueneberg. Cortisone-induced cleft palate related to stage at which shelves rise - this liability to CP has to do with which strain (A strain) later than C57 shelves elevate - closer to threshold. This is Michel's paper!! Another example of multifactorial threshold model - pyloric stenosis excess of males, but recurrence rate higher in female sibs. If proband is female, risk for brother = 18%; if proband male, risk for brother = 5%. Suggestion from London doctor because difference Why SB more responsive to folic acid than anencephaly? Work by shifting the distribution so takes more to get past threshold. Doesn't this just beg the question as to why the distribution is shifted? or what determines the arbitrary threshold?
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