Project description:To better understand the role of Geminin during germ layer specification, we bred floxed Geminin mice to Mox2-Cre mice. Mutants (fl/fl; cre/o) had spinal neural tube defects with complete penetrance. We obtained neural tube and paraxial tissue by laser capture microdissection and analyzed total RNA by microarray. Spinal neural tube and paraxial tissue were obtained separately by laser capture microdissection from wild-type and mutant E10.5 embryos. There were 2 replicates of each sample.
Project description:To better understand the role of Geminin during germ layer specification, we bred floxed Geminin mice to Mox2-Cre mice. Mutants (fl/fl; cre/o) had spinal neural tube defects with complete penetrance. We obtained neural tube and paraxial tissue by laser capture microdissection and analyzed total RNA by microarray.
Project description:Laser capture microdissection (LCM) was used to isolate cells from the principal critical micro-regions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the facial mesenchyme, composed of neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium We performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm, and present microarray data detailing expression patterns within these embryonic cell populations. Mouse emrbyos were harvested at developmental stage E8.5 and single cells were captured from the neuroepithilium, neural crest, and paraxial mesoderm. RNA was extracted, labelled, and quantified using the Mouse ST-l microarray.
Project description:The nucleoprotein Geminin (Gmnn) promotes neural cell fate acquisition of embryonic stem cells, while knockdown reduces the efficiency of neural gene activation. This occurs, at least in part, through Geminin’s ability to promote histone hyperacetylation at neural genes, to activate their expression. In mouse models in vivo, Geminin deficiency in the embryonic neural tube between embryonic days 8.5-10.5 also reduces the expression of genes controlling neural specification and/or differentiation, contributing to neural tube defects. To determine where Geminin binding and Gmnn-dependent acetylation occurs throughout the genome, we performed ChIP-chip analysis (these data) and ChIP-seq analysis (GSE77246) to define Geminin-bound chromatin locations in mouse embryonic stem cells (ESCs) and in ESC-derived neuroectoderm. We also performed ChIP-chip analysis of H3K9 acetylation in ESC-derived neuroectoderm, with or without Doxycycline-dependent Gmnn knockdown, to define the requirements for Geminin activity for histone acetylation of promoters during neural fate specification.
Project description:Maternal diabetes is a teratogen that can lead to neural tube closure defects in the offspring. In neurulation-stage embryos from diabetic dams, we detected abnormal tissue protruding from the open neural tube. To determine the origin of such protrusions, we compared gene expression profiles between open neural plate with normal morphology, and protrusion tissue. Neurulation-stage mouse embryos at 8.5 days of gestation were used to prepare open neural tube at the anterior aspect of neural tube closure site 1 by laser capture microdissection. For each sample, 10 sections were pooled, total RNA was extracted, and 7 ng of total RNA were used for expression profiling by Tag sequencing using an Applied Biosystems SolidSAGE kit for library construction, and an AB SOLiD 5500 XL instrument for sequencing. Protrusion tissue was prepared from whole embryos by microdissection, and 12ng of total RNA per sample was used for Tag sequencing. Sequence reads were mapped to RefSeq RNA, and count data per gene were obtained using a modified version of the Applied Biosystems SOLiD™ SAGE™ Analysis Software.
Project description:Laser capture microdissection (LCM) was used to isolate cells from the principal critical micro-regions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the facial mesenchyme, composed of neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium. For these 11 samples, we used a single cell isolation and cDNA generation protocol followed by a standard Affymetrix labeling protocol to better define the gene expression states of the early E8.5 pioneer neural crest and paraxial mesoderm cells. Using a combination of statistical and expression level filtering criteria, we have identified expression patterns present within these embryonic cell populations. The genes identified through this study are highly enriched for known critical genes as well as many genes not previously known to be cell type specific at this stage.
Project description:Maternal diabetes is a teratogen that can lead to neural tube closure defects in the offspring. In neurulation-stage embryos from diabetic dams, we detected abnormal tissue protruding from the open neural tube. To determine the origin of such protrusions, we compared gene expression profiles between open neural plate with normal morphology, and protrusion tissue. Neurulation-stage mouse embryos at 8.5 days of gestation were used to prepare open neural tube at the anterior aspect of neural tube closure site 1 by laser capture microdissection. For each sample, 10 sections were pooled, total RNA was extracted, and 7 ng of total RNA were used for expression profiling by Tag sequencing using an Applied Biosystems SolidSAGE kit for library construction, and an AB SOLiD 5500 XL instrument for sequencing. Protrusion tissue was prepared from whole embryos by microdissection, and 12ng of total RNA per sample was used for Tag sequencing. Sequence reads were mapped to RefSeq RNA, and count data per gene were obtained using a modified version of the Applied Biosystems SOLiD⢠SAGE⢠Analysis Software. Neural plate protrusion compared to open neural plate anterior of closure site 1 with normal morphology
Project description:Maternal diabetes is a teratogen that can lead to neural tube closure defects in the offspring. We therefore sought to compare gene expression profiles at the site of neural tube closure between stage-matched embryos from normal dams, and embryos from diabetic dams. Neurulation-stage mouse embryos at 8.5 days of gestation were used to prepare neural tissue at the anterior aspect of neural tube closure site 1. Tissue was procured from the open neural tube immediately anterior of the closure site, and from the closed neural tube immediately posterior to the closure site by laser microdissection. For each sample, 10 sections were pooled, total RNA was extracted, and 7 ng of total RNA were used for expression profiling by Tag sequencing using an Applied Biosystems SolidSAGE kit for library construction, and an AB SOLiD 5500 XL instrument for sequencing. Sequence reads were mapped to RefSeq RNA, and count data per gene were obtained using a modified version of the Applied Biosystems SOLiDâ?¢ SAGEâ?¢ Analysis Software. diabetic dam - closed neural tube // diabetic dam - open neural tube // normal dam - closed neural tube // normal dam - open neural tube
Project description:Cell intrinsic factors that control neuroectoderm specification of early embryonic cells include the nucleoprotein Geminin (Gmnn) and the Zic family of zinc finger transcription factors. Gmnn modulates chromatin state to activate neural gene expression during neural cell fate acquisition, while Gmnn deficiency in the forming neural plate disrupts transcriptional programs that control neural cell specification, neural plate patterning and neurogenesis, resulting in neural tube defects. Likewise, Zic1 over-expression promotes neural gene expression, while heterozygous deletion of Zic1/4 leads to Dandy-Walker malformation, the most common congenital cerebellar malformation. During embryonic development, Geminin and Zic1 expression is enriched in neuroectoderm from gastrula stages, with broad expression in the forming CNS during post-gastrula stages, when neural tube closure and neurogenesis are initiated. To gain a greater understanding of the molecular events that regulate neural cell specification, here we used ChIP-seq to define genome-wide chromatin binding profiles for Gmnn in embryonic stem cells (ESCs) and for Gmnn and Zic1 during specification of ESCs into neuroectoderm.