Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse Dll3 and Notch1 homozygous mutant embryos to investigate craniofacial malformations of human birth defects

ABSTRACT: Mutations in the Notch1 receptor and delta-like 3 (Dll3) ligand cause global disruptions in axial segmental patterning. Genetic interactions between members of the notch pathway have previously been shown to cause patterning defects not observed in single gene disruptions. We examined Dll3-Notch1 compound mouse mutants to screen for potential gene interactions. While mice heterozygous at either locus appeared normal, 30% of Dll3-Notch1 double heterozygous animals exhibited localized, stochastic segmental anomalies similar to human congenital vertebral defects. Unexpectedly, double heterozygous mice also displayed statistically significant decreases in mandibular height and elongated maxillary hard palate. Examination of somite-stage embryos and perinatal anatomy and histology did not reveal any organ defects, so we used microarray-based analysis of Dll3 and Notch1 mutant embryos to identify gene targets that may be involved in notch-regulated segmental or craniofacial development. Therefore, Dll3-Notch1 double heterozygous mice model human congenital scoliosis and craniofacial disorders. Experiment Overall Design: Given the reduced penetrance and stochastic nature of the segmental and craniofacial defects in Dll3-Notch1 double heterozygous animals, we next sought to identify candidate genes that may be down or up-regulated in Dll3 and Notch1 homozygous mutant embryos during development of these structures. We carried out microarray analysis using Affymetrix MOE430 microarrays, comparing 9.5 dpc homozygous embryos in duplicate to littermates with wild-type alleles at both loci, and comparing with heterozygous littermate embryos. Affymetrix analysis software (Microarray Suite 5.0) was used to determine whether gene probes were present, marginal or absent. Probes with present flags in replicates of Dll3, Notch1, or embryos with wild-type alleles at both loci were considered present for further analysis. Altogether, out of 22,690 probe sets on the MOE430A array, we identified 12,820 probes that were present in replicates of at least one genotypic group. To identify genes that were increased or decreased in expression in mutant embryos, we carried out robust multichip average (RMA) normalization of microarray data sets using the RMA module of Genespring GX 7.3 (Agilent). After RMA normalization, housekeeping genes such as Gapdh showed steady expression (probe AFFX-GapdhMur/M32599_M_at, normalized expression 1.00 Â± 0.02). As a general indicator of variability between samples, we calculated Pearsonâs correlation coefficients. We found that the correlations between duplicates were moderately high: wild-type embryos (0.623), Dll3 embryos (0.584), Notch1 embryos (0.531).

ORGANISM(S): Mus musculus

SUBMITTER: Kenro Kusumi

PROVIDER: E-GEOD-8027 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Dll3 and Notch1 genetic interactions model axial segmental and craniofacial malformations of human birth defects.

Loomes Kathleen M KM Stevens Stacey A SA O'Brien Megan L ML Gonzalez Dorian M DM Ryan Matthew J MJ Segalov Michelle M Dormans Nicholas J NJ Mimoto Mizuho S MS Gibson Joshua D JD Sewell William W Schaffer Alyssa A AA Nah Hyun-Duck HD Rappaport Eric F EF Pratt Stephen C SC Dunwoodie Sally L SL Kusumi Kenro K

Developmental dynamics : an official publication of the American Association of Anatomists 20071001 10

Mutations in the Notch1 receptor and delta-like 3 (Dll3) ligand cause global disruptions in axial segmental patterning. Genetic interactions between members of the notch pathway have previously been shown to cause patterning defects not observed in single gene disruptions. We examined Dll3-Notch1 compound mouse mutants to screen for potential gene interactions. While mice heterozygous at either locus appeared normal, 30% of Dll3-Notch1 double heterozygous animals exhibited localized, segmental a ...[more]

PMID: 17849441

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Project description:Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rphn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function. Experiment Overall Design: To enrich for genes in the presomitic mesoderm that are specifically disrupted by Dll3 mutation, we compared microdissected tissues from wild-type and Dll3 mutant embryos. We generated biological replicate pools from Dll3+/+ (wild-type) or Dll3neo/neo embryos for a total of six pools. Microarray analysis using Affymetrix MOE430A arrays was carried out on the biological pool triplicates for both wild-type and mutant genotypes.

Project description:Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rphn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a; regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function. Experiment Overall Design: To enrich for genes in the somite level tissues that are specifically disrupted by Dll3 mutation, we compared microdissected tissues from wild-type and Dll3 mutant embryos. We generated biological replicate pools from Dll3+/+ (wild-type) or Dll3neo/neo embryos for a total of six pools. Microarray analysis using Affymetrix MOE430A arrays was carried out on the biological pool triplicates for both wild-type and mutant genotypes.

Project description:Purpose: Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for craniofacial and rib defects in patients with Cerebrocostomandibular Syndrome (CCMS). However, why the mutations in SNRPB causes tissue specific CCMS abnormalities such as craniofacial defects are unknown. We hypothesize that splicing of tissue specific transcripts required for craniofacial development is disrupted due to SNRPB deficiency, during embryonic development. The purpose of the study was to identify those developmentally important transcripts that are dysregulated due to Snrpb mutation. Methods: We mated Snrpbloxp mice that we have developed with commercially available Wnt-1-Cre2 transgenic mice to remove one allele of Snrpb from neural crest cells. The mutant (Snrpb \ncc+/-) embryos showed craniofacial abnormalities at E9.5 and onward. We collected morphologically normal embryos at E9.0 for both Snrpb wildtype and Snrpbncc+/- mutants. We pooled two embryo heads of similar somites for each genotype and extracted the RNA for RNAseq. Results: We found that Snrpbncc+/- mutants show aberrant splicing that includes both increased exon skipping and intron retention. A strong tendency towards increased exon skipping and intron inclusion in the mutant samples were observed; there were more SE (273 in Het versus 83 in WT) and RI (191 in Het versus 21 in WT). We identified 13 transcripts required for craniofacial development or stem cell development with significant increases in exon skipping including Smad2, Rere and Pou2f1. From pathway analysis of differentially expressed genes, we found they were associated with the spliceosome and the P53-pathway. We also found an increase in splicing of two P53 regulator, Mdm2 and Mdm4. Conclusions: We propose that abnormal splicing underlies the defects found in Snrpbncc+/-mutant embryos. We confirmed differential splicing of the P53 regulators, Mdm2 and Mdm4 and identified several transcripts important for craniofacial development which were abnormally spliced in Snrpbncc+/- embryos.

Dataset Information

Transcription profiling of mouse Dll3 and Notch1 homozygous mutant embryos to investigate craniofacial malformations of human birth defects

Publications

Dll3 and Notch1 genetic interactions model axial segmental and craniofacial malformations of human birth defects.

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