User:Etchevers/Notebook/Conference notes/2009/09/14: Difference between revisions
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NTDs (craniorachischisis), open eyelids during development, and inner ear defects. | NTDs (craniorachischisis), open eyelids during development, and inner ear defects. | ||
KO of Fz3/Fz6 has the same phenotype. | |||
Cecr2 chromatin modifier protein expressed many places including limb, neural tube, nose or whisker follicles? ear. Mouse has exencephaly. BBS4 mutant has exencephaly, open eyelids and inner ear pbs. Interaction with Vangl protein in zebrafish. Cecr2 other phenotypes? Gorgeous stain - + in crania and - in caudal NT, and in hindgut - ectopically? | |||
Measured inner ear defects as basal more mature than apical eclls of the cochlea, so misalignment more pronounced basally than apically (predicted) - imaged along axis to see inner ear cells. Size of cochlea is already macroscopically different between wt and mutants. | |||
(My thoughts: eyelid closure prevention leads to any eye problems? further development of retinal pigment epithelia? Jun and target EGFR reduced in skin keratincytes leads to [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WW3-48SHT2J-D&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1010997617&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1a24c1b56336dcae81350f8fa741fff7 open eyelids at birth] in mutants. | |||
Painstaking analysis of cells and their orientation to demonstrate misalignment in Cecr -/-, +/- and +/+ mice. Chi-squared with Bonferroni downward adjustment to compare the measurements. at 50% maturation, wt to mutant comparison of misaligned cells statistically different. The most affected as expected from spatiotemporal maturation. | |||
Similar to Cecr1, Ptk1, Fz7 phenotypes. (Not sure I heard gene names right.) | |||
Want to cross htz to Vangl htz to see if worsen phenotypes. | |||
''Question:'' | |||
BBS4 as example not considered PCP gene - link between ciliogenesis and PCP but will extend to other members involved in ciliogenesis? thinks it would be valuable. How will study chromatin remodeling? Answer where Cecr2 is remodeling - using ChIP-Seq? Expression of other PCP genes in mutants to see if affected? Not by in situs. | |||
Simon: Correction of orientation of cells over time? Yes, they do. | |||
Wnt5a and Vangl expressed in cochlear floor - Cecr2 not directly within cells. Prospective regulation of a signal eg as a Wnt but also not close spatially? | |||
=== Nicolas Fairbridge on cecr2 phenotype and metanephros (Cecr2 22q11.2) === | |||
Kidney defects - loss of glomeruli, duplex kidney disorder, tips of fingers in limbs, also meso/pronephros (segmented). | |||
'''Kidney defects only show up on the FVB/N strain''' - will look if Arhgap19 involved. | |||
Only expressed in outer edge of cortex where glomeruli are. Maturation from least (cortex) to most (medullar) differentiated glomeruli. Convergent-extension defects in collecting ducts. Not in the tubule, but in the outer mesenchyme aggregating into early glomeruli and in S-bodies as they differente into definitive glomeruli and connect to collecting duct. Then turned off. | |||
40% "duplex" kidney - two separate ureters or bifurcation, leads to fused kidney. Is this similar to horseshoe kidney? ALso kidneys are smaller, so developmental progression delayed. Size related to fewer glomeruli. | |||
Adult FVB/N Cecer2m/m get L/R size asymmetry, loss of one kidney (12/5%), or hydronephrosis unilaterally. Double ureter increases its risk. PCP: loss of Fat4 leads to cystic and smaller kidneys when double KO with Fjx. Worsening of cysts, which are not observed in Cecr2. | |||
Polycystic kidneys in PCP defects (eg Wnt9b) , rather than from the glomerular side. | |||
Same microarray assay as above. | |||
Cecr2 itself down in mutants, bc mutant is only hypomorph (splice around). Zfp9, Csn3, Alx1, Rgn, Cd36, Hey2, Hoxd1, Tshz3, '''Wnt5b''', Lix1, Frzb, Rab2, Procr, Perp are downregulated when comparing Balb/c and FV. Other 19 other PCP-related genes significant but more minor expression changes. Alx1/Cart1 strain-dependent exencephaly, TF needed for forebrain "mesenchyme" cf Gray et al., 2004. Cecr2 in the retina and ciliary ganglion, forebrain, nose - but Alx1 is not obviously connected to PCP, more so to beta-catenin (canonical pathway). | |||
Exon 1 deletion of Cecr2 get 96% penetrance of exencephaly. '''No limb defects ever observed'''. | |||
Questions: wonder if short list b/c whole embryo and why not dissect affected tissues? Underway. | |||
I asked if anyone looked at the histology of the liver for potential effect on biliary ducts - answer, no. | |||
Claudia - why expn based on bgal gene trap? ISH never worked well for Cecr2, currently redoing. | |||
Andy - Alx1 also a chromatin remodelling agent but not overlapping - how explain? Answer - might Wnt5b be an intermediary, upstream of Alx1? | |||
*'''[[User:Etchevers|Heather]] 09:34, 14 September 2009 (EDT)''': | |||
=== Philip Lupo on NBDPS study === | === Philip Lupo on NBDPS study === | ||
Revision as of 06:34, 14 September 2009
 Conference and seminar notes
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Third day of 6th Intl Neural Tube Defects conferenceMegan Kooistra on Arhgap2 and Cecr2 (McDermid group)Balb/c different susceptibility to NTD on cecr2 hypomorph allele. Whole genome linkage analysis for modifiers suggestive on X, present on Chr19. Dominant or semi- within FBV/N strain. Human susceptibility? Marcy Speer study 2005 with screen chr 7 (largest, from one family) and chr10 (q25.3) - syntenic to mouse chr 19D2. Gene expn analysis using microarray and embryos E8.5 cranial NT closure. Compared wt between two strains and mutants on same BGs. Other genes lipid metabolism also foxd4. Most differential = Arhgap19. One paper showing fetal expn by RT-PCR. Rho GTPase protein acts as a negative regulator of GTPases. Fits into PCP by potential interaction with RhoA to lead to actin cytoskeleton remodeling. qRT-PCR confirm change in two strains (Balb/c very reduced re: FVB/N, C57 and 129S2), then check for sequence changes, and exencephaly penetrance when move cecr2 genetrap onto 129S2 and C57Bl/6 strains. 35 SNPs and 2 insertions, as well as 1 deletion of 132bp in 3' UTR. 3 synonmymous mutations and one nonsense mutation. T insertion after a stretch, end of exon 6 leads to fs and stop codon. Mutation not present in other strains. NMD mediating degradation? If residual mRNA translated, predicted truncation removes everything after the RhoGAP domain, perhaps affecting folding. Bred mice onto different backgrounds to see if change penetrance of exencephalic Cecr2 phenotype. Encouraging preliminary data correlating - in the 129S2 penetrance 43% whereas on BALB/c background penetrance >70%. Screening other strains now, will carry out ISH. Developing new mouse line gene trap fusion Arhgap19-beta-gal. Questions: Muriel - any defects caused by Arhgap19 on cilia, subtle effect on the BALB/c strain? not looked yet. Any back-rescue to increase penetrance by adding back in? Need superovulation but the BALB/c strain doesn't superovulate well. Examining actin cytoskeleton to see if changes in genetrap mutant. Irene asks if have checked on Vangl mice to backcross? Rick comment thinks he has the Arhgap19 gene trap with B-gal reporter and offers it. Cool! This is what conferences should be all about!
Christine Dawe on Cecr2 phenotype (McDermid group)PCP pathway review. She did it all for me! And everyone has their own modification from Kibar 2007! NTDs (craniorachischisis), open eyelids during development, and inner ear defects. KO of Fz3/Fz6 has the same phenotype. Cecr2 chromatin modifier protein expressed many places including limb, neural tube, nose or whisker follicles? ear. Mouse has exencephaly. BBS4 mutant has exencephaly, open eyelids and inner ear pbs. Interaction with Vangl protein in zebrafish. Cecr2 other phenotypes? Gorgeous stain - + in crania and - in caudal NT, and in hindgut - ectopically? Measured inner ear defects as basal more mature than apical eclls of the cochlea, so misalignment more pronounced basally than apically (predicted) - imaged along axis to see inner ear cells. Size of cochlea is already macroscopically different between wt and mutants. (My thoughts: eyelid closure prevention leads to any eye problems? further development of retinal pigment epithelia? Jun and target EGFR reduced in skin keratincytes leads to open eyelids at birth in mutants. Painstaking analysis of cells and their orientation to demonstrate misalignment in Cecr -/-, +/- and +/+ mice. Chi-squared with Bonferroni downward adjustment to compare the measurements. at 50% maturation, wt to mutant comparison of misaligned cells statistically different. The most affected as expected from spatiotemporal maturation. Similar to Cecr1, Ptk1, Fz7 phenotypes. (Not sure I heard gene names right.) Want to cross htz to Vangl htz to see if worsen phenotypes. Question: BBS4 as example not considered PCP gene - link between ciliogenesis and PCP but will extend to other members involved in ciliogenesis? thinks it would be valuable. How will study chromatin remodeling? Answer where Cecr2 is remodeling - using ChIP-Seq? Expression of other PCP genes in mutants to see if affected? Not by in situs. Simon: Correction of orientation of cells over time? Yes, they do. Wnt5a and Vangl expressed in cochlear floor - Cecr2 not directly within cells. Prospective regulation of a signal eg as a Wnt but also not close spatially?
Nicolas Fairbridge on cecr2 phenotype and metanephros (Cecr2 22q11.2)Kidney defects - loss of glomeruli, duplex kidney disorder, tips of fingers in limbs, also meso/pronephros (segmented). Kidney defects only show up on the FVB/N strain - will look if Arhgap19 involved. Only expressed in outer edge of cortex where glomeruli are. Maturation from least (cortex) to most (medullar) differentiated glomeruli. Convergent-extension defects in collecting ducts. Not in the tubule, but in the outer mesenchyme aggregating into early glomeruli and in S-bodies as they differente into definitive glomeruli and connect to collecting duct. Then turned off. 40% "duplex" kidney - two separate ureters or bifurcation, leads to fused kidney. Is this similar to horseshoe kidney? ALso kidneys are smaller, so developmental progression delayed. Size related to fewer glomeruli. Adult FVB/N Cecer2m/m get L/R size asymmetry, loss of one kidney (12/5%), or hydronephrosis unilaterally. Double ureter increases its risk. PCP: loss of Fat4 leads to cystic and smaller kidneys when double KO with Fjx. Worsening of cysts, which are not observed in Cecr2. Polycystic kidneys in PCP defects (eg Wnt9b) , rather than from the glomerular side. Same microarray assay as above. Cecr2 itself down in mutants, bc mutant is only hypomorph (splice around). Zfp9, Csn3, Alx1, Rgn, Cd36, Hey2, Hoxd1, Tshz3, Wnt5b, Lix1, Frzb, Rab2, Procr, Perp are downregulated when comparing Balb/c and FV. Other 19 other PCP-related genes significant but more minor expression changes. Alx1/Cart1 strain-dependent exencephaly, TF needed for forebrain "mesenchyme" cf Gray et al., 2004. Cecr2 in the retina and ciliary ganglion, forebrain, nose - but Alx1 is not obviously connected to PCP, more so to beta-catenin (canonical pathway). Exon 1 deletion of Cecr2 get 96% penetrance of exencephaly. No limb defects ever observed. Questions: wonder if short list b/c whole embryo and why not dissect affected tissues? Underway. I asked if anyone looked at the histology of the liver for potential effect on biliary ducts - answer, no. Claudia - why expn based on bgal gene trap? ISH never worked well for Cecr2, currently redoing. Andy - Alx1 also a chromatin remodelling agent but not overlapping - how explain? Answer - might Wnt5b be an intermediary, upstream of Alx1?
Philip Lupo on NBDPS studyPedro Rocha on Med12Michelle O'Byrne on an association study of MTHFR and other folate genesRick Finnell on maternal immune factors and NTDsBermans Iskandar on folates and repair of the CNSSteve Whitehead on folates and MCP-1My talk which is available upon requestRobert Cabrera on autoantibodies to folate receptor alpha in Norwegian cohortLeah Burke on genetic counseling for NTDs, including Melissa Baker and Karen Lawliss, parents, and Denise Boyun, genetic counselor | |
