Transcription profiling of mouse branchial arches from Dlx2-/-, Dlx1/2 -/- or Dlx5/6 -/-
ABSTRACT: The Dlx homeobox genes have central roles in controlling patterning and differentiation of the brain and craniofacial primordia. In the brain, loss of Dlx function results in defects in the production, migration and differentiation of GABAergic neurons, that can lead to epilepsy. In the branchial arches, loss of Dlx function leads to craniofacial malformations that include trigeminal axon pathfinding defects. To determine how these genes function, we wish to identify the transcriptional circuitry that lies downstream of these transcription factors by comparing gene expression in wild type with Dlx mutant CNS and craniofacial tissues. 1) Compare gene expression in the maxillay branch of the first branchial arch (BA) of E10.5 wild type and Dlx2 -/- mutants. 2) Compare gene expression in the maxillary branch of the first BA of E10.5 wild type and Dlx1/2 -/- mutants. 3) Compare gene expression in wild type maxillary and mandibular branchial arches. 4) Compare gene expressionin mandibular branch of Dlx5/6 -/- mutants with wild type mandibular branch. The Dlx transcription factors are essential for controlling patterning of the brain and craniofacial primordia. In the brain, they control differentiation of GABAergic neurons of the basal ganglia. In the branchial arches, they control regional patterning. I hypothesize that there will be some conserved and some divergent mechanisms that the Dlx genes use in controlling brain and craniofacial development. We have already performed array analyses on Dlx function in the developing basal ganglia (with TGEN) by comparing expressed genes in wild type and Dlx1/2 mutants. Here we will compare gene expression in the brachial arches of wild type and Dlx mutant mice. 1) Generate E10.5 mouse embryos that are either wild type, Dlx2-/-, Dlx1/2 -/- or Dlx5/6 -/-. 2) Determine genotype by PCR. 3) Dissect branchial arches from the different genotypes. 4) Separate maxillary and mandibular branch of each branchial arch. 5) Prepare total RNA from the specimens. Obtain sufficient tissue to obtain 10 ug of total RNA - based on previous experience we anticipate that this will require ~ 10 branchial arches. We will pool the tissue from different embryos of the same genotype. 6) Send total RNA to TGEN for probe preparation, hybridization and array result analysis.
Project description:We present a gene expression atlas of early mouse craniofacial development. 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 We examined the facial mesenchyme and adjacent neuroepithelium at E8.5, at E9.5 we obtain cells from the facial mesenchyme, olfactory placode/epidermal ectoderm, underlying neuroepithileium, and emerging mandibular and maxillary arches. AT E10.5 we sampled the medial and lateral prominences, olfactory pit, multiple regions of the underlying neuroepithelium the mandibular and maxillary arches, and Rathke's pouch. Mouse emrbyos were harvested at developmental stage E8.5 , E9.5, and E10.5 and cells were captured from microregions responsible for the construction of the mammalian face. RNA was extracted, labelled, and quantified using the Mouse ST-l microarray.
Project description:Fusion of branchial arch derivatives is an essential event in the development of craniofacial architecture. A unique feature of the mandibular arch development is medial/lateral compartmentalization for the molecular networks. Those networks give rise to multiple region-specific organs, namely teeth, a tongue, salivary glands, and the supporting matrices such as bones and cartilages. We aimed to investigate molecular networks that govern the fusion process during mouse mandibular development. To this end, cDNA microarray technology was employed for screening of spatio-temporal gene expression in developing mandibular arch from E9.7 through E11.5. We conducted to divide a mandibular arch medially and laterally to compare both gene expression. From an embryo at E10.5, a medial (M) sample of the mandibular arch was dissected out -at just the distal end of opposed lateral lingual swellings-, and the bulk of remnant lateral region was collected as (L) sample under a stereomicroscope. Forty embryos for each time-point were used to obtain a pool of total RNA.
Project description:We report the RNA profiles of both control and talpid2 frontonasal, maxillary, and mandibular prominences of the chick face at Hamburger and Hamilton (HH) stage 25. For more details please see: "The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant, talpid2." Facial prominences (frontonasal, maxillary, and mandibular) from 8 control and 8 talpid2 HH 25 embryos were harvested, pooled, and RNA-seq was preformed on samples.
Project description:Neo/null loss of Tfap2a in E10.5 mouse facial prominences triplicate run comparing tissue dissected from the nasal, maxillary and mandibular comparing AP-2 mutant and control embryos
Project description:Growth and patterning of the face relies on several small buds of tissue, the facial prominences, which surround the primitive mouth. Beginning around E10 of mouse development the prominences undergo rapid growth and morphogenesis. By E11.5 the medial nasal prominences are in close apposition in the midline, as are the maxillary and medial nasal prominences on either side of the developing face. Subsequently, by E12.5 the nasal and maxillary prominences fuse to form a continuous shelf at the front of the face - the primary palate. Individual prominences are associated with specific developmental processes, and this is reflected by patterns of differential gene expression that give the prominences their unique identities. Thus, only the mandibular and maxillary prominences give rise to dentition while the frontonasal prominence has a unique role in olfaction, and the mandibular prominence in taste. We used microarrays to detail the differential gene expression program in each of the mandibular, maxillary, and frontonasal prominences during the key developmental timepoints of E10.0 through E12.5. Experiment Overall Design: Analysis of gene expression during growth and fusion of the facial prominences in the C57BL/6J mouse strain between embryonic (E) day 10.0 and 12.5. At the earliest timepoint, E10, only the mandibular prominence is a distinct entity that can be readily identified and dissected. The frontonasal prominence and the maxillary prominence are very small and not discrete from other components of the head such as the forebrain until E10.5. Analysis of these tissues at earlier timepoints would require laser capture and preamplification steps - techniques that were not used for the later timepoints. Thus samples were isolated from the mandibular prominence at E10.0 and from the mandibular, maxillary and frontonasal prominences of mouse embryos from E10.5 to E12.5, at 0.5 day intervals. In order to obtain sufficient sample for hybridization, each sample represents a pool of between 3 and 48 embryos depending on the timepoint. Specifically, the number of embryos were 40-48 for E10.0 (mandibular prominence only), 24-8 for E10.5, 8-9 for E11.0 and E11.5 and 3-4 for E12.0 and E12.5. Seven replicate samples were taken for each of the later five timepoints in each of the three prominences, with an additional seven samples for the mandibular E10.0 timepoint, for a total of 112 samples.
Project description:We performed chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (seq) from mouse E11.5 maxillary arches using anti-LHX6 antibody to identify LHX target cis-regulatory elements. Overall design: Genome binding/occupancy profiling by high throughput sequencing using only wild-type E11.5 maxillary arch tissue.
Project description:Gata6 ChIP-seq on mouse posterior branchial arches connected to outflow tract of the heart (PBA/OFT) at embryonic day (E) 11.5. H3K27Ac ChIP-seq on mouse second branchial arch (BA2) and posterior branchial arches connected to outflow tract of the heart (PBA/OFT) at embryonic day (E) 11.5.
Project description:We used laser capture microdissection to isolate maxillary arch mesenchyme from E10.5 embryos. This tissue was collected from both control (3x) and Lhx6-/-;Lhx8-/- mutant (3x) samples. Transcriptional profiling was performed using Affymetrix GeneChip Mouse Genome 430 2.0 arrays. The mutant mice are of mixed genetic background of C57BL/6J, 129 and CD-1 strains. The head of E10.5 embryos was collected and embedded in Optimal Cutting Temperature resin (Tissue-Tek) by flash freezing on dry ice. The frozen sections were collected on polyethylene naphthalate membrane slides (Leica). Leica LMD6000 Laser Micro-Dissection System was used to cut out the normal expression domain of Lhx6 and Lhx8 in the maxillary arch mesenchyme. The tissue was collected from the entire antero-posterior extent of the maxillary arches. Total RNA was extracted using RNeasy Micro Kit (Qiagen). Subsequent steps of transcriptional profiling were performed by the New York University Genome Technology Center, beginning with the amplification of RNA by Ovation Nano Amplification system (NuGen). RNA samples from three wild-type and three Lhx6−/−;Lhx8−/− mutant embryos, all somite count- and sex-matched (females), were analyzed with Affymetrix GeneChip Mouse Genome 430 2.0 arrays. A list of Lhx-regulated genes were generated from the microarray result based on the following criteria: fold change in the average expression between wild types and Lhx6−/−;Lhx8−/− mutants is >1.5, the difference is statistically significant (P < 0.05) and the average intensity of the probe signal is >100 for wild-type and/or mutant samples. The resulting list of 212 genes was used for a gene ontology analysis with DAVID (27,28).
Project description:Transcription profiling was performed of second branchial arches of E11.5 embryos from Hoxa2+/- intercrosses. After genotyping the embryos, wild type and Hoxa2-/- were profiled by microarray.
Project description:This investigation provides a robust multi-dimensional compendium of gene expression data relevant to mouse facial development. It profiles the transcriptome ofectoderm and mesenchyme from the three facial prominences in a time series encompassing their growth and fusion. Analysis of the dataset identified more than 8000 differentially expressed genes comprising dramatically different ectoderm and mesenchyme programs. The mesenchyme programs included many genes identified in earlier analyses as well hundreds of genes not previously implicated in craniofacial development. The ectoderm programs included over a thousand genes that highlight epithelial structure, cell-cell interactions and signaling. The dataset includes 45 .cel files, DABG probability and RMA log2 expression values for each probeset, and statistics for 9457 probesets representing 8575 genes. 45 total samples, with 15 conditions sampling three ages (E10.5, E11.5, E12.5), three facial prominences (mandibular, maxillary and fronto-nasal) and two tissue layers (ectoderm or mesenchyme), with 3 biological replicates per condition. Differential expression was determined after median filter for variance with three-way ANOVA, Benjamini-Hochberg multiple testing correction