Characterization of asrij expression, function and regulation in embryonic stem cells and the developing cardiovascular system

Abstract

This thesis analyses the expression, function and regulation of a novel gene, asrij. The early expression of Asrij allows its use as an embryonic stem- cell marker that is down-regulated in nonvascular tissues. Asrij belongs to a novel class of OCIA domain-containing conserved proteins, which localizes to endocytic vesicles. Chimeric- embryo analysis indicates that asrij is required for cardiovascular development and may be a haplo-insufficient gene. The asrij promoter is regulated by Sp transcription factors and shows highest activity in embryonic stem cells ;, vitro and the developing mouse heart and small blood vessels. Hence, the asrij promoter can be used to drive expression of genes in ES cells and the developing cardiovascular system. This is the first analysis of asrij, a gene that may play an important role in stem cells and cardiovascular development. Survival of the vertebrate embryo depends on its ability to correctly pattern and organize the cardiovascular system. We are interested in understanding mechanisms of cardiovascular development. Towards this aim, several genes expressed in the blood and vasculature during embryonic stem cell (ESC) differentiation, were identified on the basis of reporter expression, in a genome-wide screen. One such ESC clone (B2D2) shows reporter expression in undifferentiated ESCs that later gets restricted to vascular structures. My work involved identification and analysis of the endogenous gene in B2D2. Starting with the genomic sequence 5' to the insertion site in B2D2 we identified the eDNA and the endogenous gene. The insertion is associated with a 21.09 kb novel conserved gene, which we named asrij (Sanskrit asRij= blood), located on mouse chromosome 5C3.2. asrij encodes a predicted transmembrane protein of 247 amino acids. Using RNA probes for in situ hybridization and antibodies generated against Asrij, we analyzed the expression pattern in vitro and in vivo during mouse embryogenesis. During ES ce11-derived blood vessel formation in vitro, Asrij expression partially overlaps with the vascular markers Flk-1 and PECAM. Flk-1 is the earliest known marker for developing endothelial cells. Asrij expression in blood islands precedes that of Flk-1. During mouse embryogenesis, Asrij is expressed predominantly in yolk sac and embryonic blood islands and blood vessels. Asrij expression is also seen in branchial arches and the nervous system. The transcript is alternately spliced, and its expression is regulated in a tissue-specific manner. A shorter Asrij isoform of 196 amino acids is enriched in the adult mouse brain (Mukhopadhyay et al., 2003 Dev Dyn 227: 578-586). Asrij expressiOn m blood islands begins early-preceding that of Flk-1 (VEGFR-II)-suggesting a requirement at this stage. We analyzed the phenotypes of chimeric mice carrying cells heterozygous for an asrij gene- trap insert identifiable by reporter P-galactosidase expression. Chimeric adults showed very lO\\' levels of chimerism (less than 5%). Hence, we analyzed chimeric embryos to assess the level of chimerism and developmental abnormalities. Embryos with large patches of genetrap cells show extensive developmental defects. While several asrij-lacZ cells are seen at early developmental stages, heterozygous ESCs containing one copy of insertionally mutated asrij do not contribute significantly to live-born chimeric mice. Further, the insertionally mutated asrij allele is not transmitted to the germline. These results suggest that asrij may be a haplo- insufficient gene (Mukhopadhyay et al., submitted). Our analysis of asrij chimeric- mice and embryos is a valuable first step towards analysis of the function of this gene that has dominant organismal phenotypes while being cell- viable. Future studies will be aimed at creating a null allele of asrij by gene targeting and using additional gene trap clones now available for asrij. Asrij has 85% identity with the human ortholog OCIA (Ovarian Carcinoma Immunoreactive Antigen) and is conserved between vertebrates and invertebrates. Vertebrate asrij orthologs have a conserved N-terminal region ( aa 1-128), while shorter sequences containing the two hydrophobic stretches are conserved amongst all species. The conserved 128 aa sequence has been termed the OCIA domain (http://www.ncbi.nlm.nih.gov/Structure/cddlcddsrv.cgi) and assigned the family number pfam07051 (http://www.sanger.ac.uk/Software/Pf!!ml). This family consists of several orthologs of OCIA and a related eukaryotic sequence. The function of this family is unknown. Asrij protein sequence does not give any clue to its subcellular localization or function. To investigate the cellular function of Asrij, we first determined its intracellular localization by double immunostaining with several organelle markers. Asrij shows partial colocalization with vesicular markers such as LAMPl (Lysosomal associated membrane protein1), Rab4 and Rab5 but not with Go1gi marker GM130, endoplasmic reticulum marker EEA-1 or plasma membrane marker ICAM-2. These data show that asrij is localized to endocytic vesicles.