Project description:This study analyzed mRNA profiles in rhombomere 4 of E10.5 mouse knock-in embryos expressing either normal endogenous Hox-B1 protein or the paralogous Hox-A1 protein from the Hoxb1 locus. The Hox-A1 protein was found to be detectably less efficacious than Hox-B1 in promoting neurogenesis in the basal plate of rhombomere 4 and its transcriptional profile shared several similarities with the Hoxb1 mutant. Keywords: gene swap, knock-in, hindbrain development, rhombomere 4
Project description:This study analyzed mRNA profiles in rhombomere 4 of E10.5 mouse knock-in embryos expressing either normal endogenous Hox-B1 protein or the paralogous Hox-A1 protein from the Hoxb1 locus. The Hox-A1 protein was found to be detectably less efficacious than Hox-B1 in promoting neurogenesis in the basal plate of rhombomere 4 and its transcriptional profile shared several similarities with the Hoxb1 mutant. Experiment Overall Design: GFP-positive cells were FACS-sorted from dissected hindbrains of entire litters of E10.5 mouse embryos expressing either normal endogenous Hox-B1 protein or the paralogous Hox-A1 protein from the Hoxb1 locus, either one tagged with IRES-tauGFP. Three independent biological replicates of each genotype were analyzed. Total RNA was isolated, amplified and hybridized to Affymetrix Mouse Genome 430 2.0 Arrays.
Project description:Hoxb1 is required for proper specification of rhombomere 4 and the facial motor neurons. This study analyzed gene expression in the corresponding hindbrain segment of E10.5 mutant embryos. Several genetic pathways were found altered, including transcription factors such as Phox2b, Gata3, Nkx2-2 and Nkx6-1. Keywords: hindbrain development, rhombomere 4, Hoxb1
Project description:Hoxb1 is required for proper specification of rhombomere 4 and the facial motor neurons. This study analyzed gene expression in the corresponding hindbrain segment of E10.5 mutant embryos. Several genetic pathways were found altered, including transcription factors such as Phox2b, Gata3, Nkx2-2 and Nkx6-1. Experiment Overall Design: Each of the three mutant and control samples was an independent biological replicate. Pools of r4 segments from multiple embryos were snap frozen on dry ice and stored at -80ºC. Total RNA was isolated, processed with standard Affymetrix protocols and hybridized to GeneChip Mouse Expression Sets 430A.
Project description:Despite 30 years of Hox gene study we have a remarkably limited knowledge of the downstream target genes that Hox transcription factors regulate to confer regional identity. Here, we have used a microarray approach to identify genes that function downstream of a single vertebrate Hox gene, zebrafish hoxb1a. This gene plays a critical and conserved role in vertebrate hindbrain development, conferring identity to hindbrain rhombomere 4. For example, zebrafish Hoxb1a, similar to mouse Hoxb1, is required for the migration of r4-derived facial branchiomotor neurons into the posterior hindbrain. We have screened microarrays carrying more than 16,000 expressed sequence tags (ESTs) for genes that are differentially regulated in normal versus Hoxb1a-deficient rhombomere 4 tissue. Using this approach, we have identified both positively and negatively regulated candidate Hoxb1a target genes. We have used in situ hybridization to validate twelve positively regulated Hoxb1a targets. These downstream targets are expressed in a variety of subdomains within r4, with one gene, a novel prickle homolog (pk1b), expressed specifically within the facial branchiomotor neurons. Using morpholino knock-down we show that the Hoxb1a target Pk1b is required for facial neuron migration, a single aspect of rhombomere 4 identity. Keywords: Comparison of normal and Hox-deficient tissue
Project description:Following neural tube closure at around E9.5, the rhombic lip within the rhombomere 1/isthmus region ("upper rhombic lip") produces a sequence of neuronal lineages that populate the brainstem and cerebellum. The transcription factor Atoh1 (Math1) is required for this specialized neurogenesis, although the genetic programs that delineate the temporal cell fate changes downstream of Atoh1 are not well characterized. We examined the gene expresion changes that take place within Atoh1 lineages We purified early (E10.5) and late (E13.5) born Atoh1 expressing cells from E14.5 embryos using a transgenic labeling strategy, and analyzed differences in gene expression across the two populations using the microarray data shown below.
Project description:Limb patterning relies in a large part on the function of the Hox family of developmental genes. While the differential expression of Hox genes shifts from the anterior-posterior (A-P) to the proximal-distal (P-D) axis around embryonic day 11 (E11), whether this shift coincides with a more global change of P-D versus A-P patterning program remains unclear. By performing and analyzing the transcriptome of the developing limb bud from E10.5 to E12.5, at single cell resolution, we have uncovered transcriptional trajectories which revealed a general switch from A-P to P-D genetic program between E10.5 and E11.5. Interestingly, the transcriptional trajectories at E10.5 all end with cells expressing either proximal or distal markers suggesting a progressive acquisition of P-D identity. Moreover, we observed that distally expressed Hox genes, namely Hoxa13 and Hoxd13, act as a key determinant for P-D patterning as their transcriptional control results in their distal restricted expression, which in turn restricts Hoxa11 in the proximal limb bud domain, in progenitor cells of the zeugopod. Finally, we identified three categories of genes expressed in the distal limb mesenchyme characterized by distinct temporal expression dynamics. As anticipated from previous results, HOX13 binding was observed within or in the neighborhood of several of these genes consistent with previous evidence suggesting that the transition from the early/proximal to the late/distal transcriptome of the limb mesenchyme largely relies on HOX13 function.
Project description:We found that, whereas Hoxb1 does not affect NMP specification per se, it promotes NMP survival through the upregulation of Fgf8 and other components of Fgf signaling as well as the repression of components of the apoptotic pathway. Additionally, it upregulates the expression of some, primarily anterior, Hox genes suggesting that it plays an active role in the early steps of AP specification. In hindbrain neural progenitors Hoxb1 synergizes with shh to repress the expression of dorsal markers, regulate the expression of ventral markers and direct the specification of facial branchiomotorneuron (FBM) - like progenitors. Additionally, Hoxb1 and shh synergize in regulating the expression of diverse signals and signaling molecules in neural progenitors, including the Ret tyrosine kinase receptor. Hoxb1 synergizes with GDNF to strengthen Ret expression and further promote the generation of facial branchiomotorneuron (FBM) – like progenitors
Project description:Skeletal muscle tissue demonstrates global hypermethylation with aging. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 hours, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; ‘pathways-in-cancer’ (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), ‘rap1-signaling’, ‘axon-guidance’ and ‘hippo-signalling’. Aged cells also demonstrated a hypermethylated profile in pathways; ‘axon-guidance’, ‘adherens-junction’ and ‘calcium-signaling’, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alteration in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in the ‘focal adhesion’ and ‘PI3K-AKT signalling’ pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells (on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P). Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3.