Wendy L. Kimber

Transcriptome analysis of mouse stem cells and early embryos.

Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

Targeted Mutagenesis of the Hira Gene Results in Gastrulation Defects and Patterning Abnormalities of Mesoendodermal Derivatives Prior to Early Embryonic Lethality

ABSTRACT The Hira gene encodes a nuclear WD40 domain protein homologous to the yeast transcriptional corepressors Hir1p and Hir2p. Using targeted mutagenesis we demonstrate that Hira is essential for murine embryogenesis. Analysis of inbred 129Sv embryos carrying the null mutation revealed an initial requirement during gastrulation, with many mutant embryos having a distorted primitive streak. Mutant embryos recovered at later stages have a range of malformations with axial and paraxial mesendoderm being particularly affected, a finding consistent with the disruption of gastrulation seen earlier in development. This phenotype could be partially rescued by a CD1 genetic background, although the homozygous mutation was always lethal by embryonic day 11, with death probably resulting from abnormal placentation and failure of cardiac morphogenesis.

Cloning and mapping of murine Dgcr2 and its homology to the Sez-12 seizure-related protein.

Hemizygosity for a region of human Chromosome (Chr) 22q11 has been associated with a wide range of congenital malformation syndromes. The major abnormalities encountered are cardiac defects, dysmorphic facies, T cell dysfunction, clefting, hypocalcemia, and learning or behavioral problems (Wilson et al. 1993). Individually, patients may be diagnosed as DiGeorge syndrome (DGS), velo-cardio-facial syndrome (VCFS), conotruncal anomaly face (CTAF), Cayler syndrome, or Opitz GBBB syndrome. The deletions detected in these conditions are large, encompassing 2 Mb or more of 22q11. Comparisons of terminal and submicroscopic interstitial deletions have been made in order to establish a shortest region of deletion overlap for these disorders. A minimal DiGeorge critical region has recently been proposed (MDGCR, Budarf et al. 1995). A family segregating a 2;22 balanced translocation has been described (Augusseau et al. 1986). The proband, ADU, has DiGeorge syndrome, and his mother, although very mildly affected, has VCFS. Two other family members have the translocation, but there is very little clinical information available, other than they are not severely affected by either condition. The translocation breakpoint (ADUBP) maps within the MDGCR, implying that the translocation disrupts a gene haploinsufficient in DiGeorge syndrome. DGCR2 was isolated during attempts to isolate genes at or adjacent to the ADU breakpoint. The DGCR2 gene encodes a transmembrane protein (Demczuk et al. 1995). The alternative name of IDD was an acronym for Integral membrane protein, Deleted in DiGeorge syndrome (Wadey et al. 1995). The putative extracellular region contains domains with similarity to both the LDL-receptor binding domain and C-type lectins, suggesting a ligand-binding function for this part of the molecule. It has been suggested that this might involve mediation of the interaction of cephalic and cardiac neural crest cells with the substratum or with other cells during their migration. A defective neural crest cell contribution can mimic the DGS in some experimental systems (Kirby et al. 1983). However, no point mutations of DGCR2 have been detected despite extensive searches (Wadey et al. 1995, and unpublished data). Recent data have suggested that the ADUBP exerts a position effect on the gene or genes haploinsufficient in DGS, rather than directly disrupting a protein-encoding locus (Levy et al. 1995; Sutherland et al. 1996). Thus, as the closest structural gene to the ADU breakpoint, DGCR2 remains of interest despite the lack of mutations. To investigate the gene further, we have cloned and mapped the murine homolog of DGCR2. A 10.5 dpc (days post coitum) mouse embryo cDNA library was screened with the human DGCR2 clone, and three positives were obtained. The clone with the largest insert, KT4, was subcloned into M13 and sequenced. An open reading frame of 548 amino acids was detected, and the GCG program bestfit revealed that this ORF was 92% identical to the human DGCR2 sequence, with three gaps (Fig. 1a). The accession number for Dgcr2 is X95480. Database searches were repeated with BLAST and Maspar in order that similarity to recent submissions might be detected. A highly significant match was obtained with GB:D78641 over the entire length of the gene (Fig. 1a), including identity within the 38 UTR (not shown). This gene, Sez-12, is described as encoding a membrane glycoprotein and was isolated from a murine neuronal precursor cDNA library (Kajiwara et al. 1996). Sez-12 is over 99% identical to Dgcr2 at the amino acid level, although there are two gaps in the alignment (Fig. 1a). Differences between the Sez-12 and Dgcr2 sequences were checked and confirmed. The main difference is a gap of three amino acids at Dgcr2 residue 109, in the LDL binding domain of Sez-12. The human and murine Dgcr2 sequences are identical at this point, as are sequences from additional mouse clones, indicating that the sequence presented here is most likely to be correct. No other informative database matches were obtained. Partial sequence of a chick Dgcr2 cDNA was also obtained providing information concerning the aminoterminal 92 amino acids of Cdgcr2 (accession number X95885). This region contains the signal peptide and the LDL-receptor binding domain. This chick sequence was 75% identical and 83% similar to the murine Dgcr2 in this region, with two gaps. A schematic of the Dgcr2 gene showing the position of the conserved motifs is given in Fig. 1b. Northern analysis of mRNA from 10.5 to 15.5 dpc embryos detected a single transcript of 4.4kb (not shown), as demonstrated for the human gene and the Sez-12 transcripts. Comparison with the level of expression of b-actin suggests no great change in the expression of Dgcr2 takes place during this period. Expression of Dgcr2 was studied further by wholemount hybridization of 9.5 dpc embryos, in an initial attempt to identify whether Dgcr2 might be expressed in regions containing neural crest cells and their derivatives. Dgcr2 expression was detected throughout the embryo, but expression was particularly strong in the first and second branchial arches, and in the limb buds (Fig. 2). At 9.5–10 dpc, sections through wholemount embryos detected expression of Dgcr2 in the dorsal half of the mid and hindbrain, and the optic lobes (Fig. 2iii). Dorsal mesenchyme surrounding the neuroepithelium was also positive, particularly around the hindbrain. High levels of expression were found throughout the branchial arches (Fig. 2iv). There Correspondence to: P.J. Scambler

Modeling Genetic Diseases in the Mouse

The use of powerful linkage strategies for the mapping of genetic disease genes has led to the positional cloning of a number of genes associated with human genetic diseases. At the same time, techniques for manipulating the mammalian genome have been refined, so that the modification of the mouse genome via transgenic technology is now routine throughout the world. These two technologies have advanced and proliferated simultaneously to the point that among the first experiments planned upon the cloning of a human disease gene is the creation of an appropriate transgenic or knock-out mouse, with the hope of modeling that disease. A mammalian model is extremely valuable in the understanding of the function of a disease gene in normal animals, as well as its role in the pathophysiology of the disease. A good disease model can be used to test therapeutic options, especially gene therapy vectors. Finally, the genetic and biochemical pathways that a disease gene is part of can be dissected and investigated in animal models.