Comprehensive comparative homeobox gene annotation in human and mouse.

Homeobox genes are a group of genes coding for transcription factors with a DNA-binding helix-turn-helix structure called a homeodomain and which play a crucial role in pattern formation during embryogenesis. Many homeobox genes are located in clusters and some of these, most notably the HOX genes, are known to have antisense or opposite strand long non-coding RNA (lncRNA) genes that play a regulatory role. Because automated annotation of both gene clusters and non-coding genes is fraught with difficulty (over-prediction, under-prediction, inaccurate transcript structures), we set out to manually annotate all homeobox genes in the mouse and human genomes. This includes all supported splice variants, pseudogenes and both antisense and flanking lncRNAs. One of the areas where manual annotation has a significant advantage is the annotation of duplicated gene clusters. After comprehensive annotation of all homeobox genes and their antisense genes in human and in mouse, we found some discrepancies with the current gene set in RefSeq regarding exact gene structures and coding versus pseudogene locus biotype. We also identified previously un-annotated pseudogenes in the DUX, Rhox and Obox gene clusters, which helped us re-evaluate and update the gene nomenclature in these regions. We found that human homeobox genes are enriched in antisense lncRNA loci, some of which are known to play a role in gene or gene cluster regulation, compared to their mouse orthologues. Of the annotated set of 241 human protein-coding homeobox genes, 98 have an antisense locus (41%) while of the 277 orthologous mouse genes, only 62 protein coding gene have an antisense locus (22%), based on publicly available transcriptional evidence.

GENCODE: the reference human genome annotation for The ENCODE Project.

The GENCODE Consortium aims to identify all gene features in the human genome using a combination of computational analysis, manual annotation, and experimental validation. Since the first public release of this annotation data set, few new protein-coding loci have been added, yet the number of alternative splicing transcripts annotated has steadily increased. The GENCODE 7 release contains 20,687 protein-coding and 9640 long noncoding RNA loci and has 33,977 coding transcripts not represented in UCSC genes and RefSeq. It also has the most comprehensive annotation of long noncoding RNA (lncRNA) loci publicly available with the predominant transcript form consisting of two exons. We have examined the completeness of the transcript annotation and found that 35% of transcriptional start sites are supported by CAGE clusters and 62% of protein-coding genes have annotated polyA sites. Over one-third of GENCODE protein-coding genes are supported by peptide hits derived from mass spectrometry spectra submitted to Peptide Atlas. New models derived from the Illumina Body Map 2.0 RNA-seq data identify 3689 new loci not currently in GENCODE, of which 3127 consist of two exon models indicating that they are possibly unannotated long noncoding loci. GENCODE 7 is publicly available from gencodegenes.org and via the Ensembl and UCSC Genome Browsers.

Cell movement during chick primitive streak formation.

Gastrulation in amniotes begins with extensive re-arrangements of cells in the epiblast resulting in the formation of the primitive streak. We have developed a transfection method that enables us to transfect randomly distributed epiblast cells in the Stage XI-XIII chick blastoderms with GFP fusion proteins. This allows us to use time-lapse microscopy for detailed analysis of the movements and proliferation of epiblast cells during streak formation. Cells in the posterior two thirds of the embryo move in two striking counter-rotating flows that meet at the site of streak formation at the posterior end of the embryo. Cells divide during this rotational movement with a cell cycle time of 6-7 h. Daughter cells remain together, forming small clusters and as result of the flow patterns line up in the streak. Expression of the cyclin-dependent kinase inhibitor, P21/Waf inhibits cell division and severely limits embryo growth, but does not inhibit streak formation or associated flows. To investigate the role off cell-cell intercalation in streak formation we have inhibited the Wnt planar-polarity signalling pathway by expression of a dominant negative Wnt11 and a Dishevelled mutant Xdd1. Both treatments do not result in an inhibition of streak formation, but both severely affect extension of the embryo in later development. Likewise inhibition of myosin II which as been shown to drive cell-cell intercalation during Drosophila germ band extension, has no effect on streak formation, but also effectively blocks elongation after regression has started. These experiments make it unlikely that streak formation involves known cell-cell intercalation mechanisms. Expression of a dominant negative FGFR1c receptor construct as well as the soluble extracellular domain of the FGFR1c receptor both effectively block the cell movements associated with streak formation and mesoderm differentiation, showing the importance of FGF signalling in these processes.