Catherine Roberts

Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein.

Fraser syndrome (OMIM 219000) is a multisystem malformation usually comprising cryptophthalmos, syndactyly and renal defects. Here we report autozygosity mapping and show that the locus FS1 at chromosome 4q21 is associated with Fraser syndrome, although the condition is genetically heterogeneous. Mutation analysis identified five frameshift mutations in FRAS1, which encodes one member of a family of novel proteins related to an extracellular matrix (ECM) blastocoelar protein found in sea urchin. The FRAS1 protein contains a series of N-terminal cysteine-rich repeat motifs previously implicated in BMP metabolism, suggesting that it has a role in both structure and signal propagation in the ECM. It has been speculated that Fraser syndrome is a human equivalent of the blebbed phenotype in the mouse, which has been associated with mutations in at least five loci including bl. As mapping data were consistent with homology of FRAS1 and bl, we screened DNA from bl/bl mice and identified a premature termination of mouse Fras1. Thus, the bl mouse is a model for Fraser syndrome in humans, a disorder caused by disrupted epithelial integrity in utero.

HIRA, a mammalian homologue of Saccharomyces cerevisiae transcriptional co-repressors, interacts with Pax3.

HIRA maps to the DiGeorge/velocardiofacial syndrome critical region (DGCR) at 22q11 (Refs 1,2) and encodes a WD40 repeat protein similar to yeast Hir1p and Hir2p. These transcriptional co-repressors regulate cell cycle-dependent histone gene transcription, possibly by remodelling local chromatin structure. We report an interaction between HIRA and the transcription factor Pax3. Pax3 haploinsufficiency results in the mouse splotch and human Waardenburg syndrome (WSI and WSIII) phenotypes. Mice homozygous for Pax3 mutations die in utero with a phenocopy of DGS, or neonatally with neural tube defects. HIRA was also found to interact with core histones. Thus, altered stoichiometry of complexes containing HIRA may be important for the development of structures affected in WS and DGS.

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.

Great vessel development requires biallelic expression of Chd7 and Tbx1 in pharyngeal ectoderm in mice

Aortic arch artery patterning defects account for approximately 20% of congenital cardiovascular malformations and are observed frequently in velocardiofacial syndrome (VCFS). In the current study, we screened for chromosome rearrangements in patients suspected of VCFS, but who lacked a 22q11 deletion or TBX1 mutation. One individual displayed hemizygous CHD7, which encodes a chromodomain protein. CHD7 haploinsufficiency is the major cause of coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies/deafness (CHARGE) syndrome, but this patient lacked the major diagnostic features of coloboma and choanal atresia. Because a subset of CHARGE cases also display 22q11 deletions, we explored the embryological relationship between CHARGE and VCSF using mouse models. The hallmark of Tbx1 haploinsufficiency is hypo/aplasia of the fourth pharyngeal arch artery (PAA) at E10.5. Identical malformations were observed in Chd7 heterozygotes, with resulting aortic arch interruption at later stages. Other than Tbx1, Chd7 is the only gene reported to affect fourth PAA development by haploinsufficiency. Moreover, Tbx1+/-;Chd7+/- double heterozygotes demonstrated a synergistic interaction during fourth PAA, thymus, and ear morphogenesis. We could not rescue PAA morphogenesis by restoring neural crest Chd7 expression. Rather, biallelic expression of Chd7 and Tbx1 in the pharyngeal ectoderm was required for normal PAA development.

Retinoic acid down-regulates Tbx1 expression in vivo and in vitro

Both Tbx1 and retinoic acid (RA) are key players in embryonic pharyngeal development; loss of Tbx1 produces DiGeorge syndrome‐like phenotypes in mouse models as does disruption of retinoic acid homeostasis. We have demonstrated that perturbation of retinoic acid levels in the avian embryo produces altered Tbx1 expression. In vitamin A‐deficient quails, which lack endogenous retinoic acid, Tbx1 expression patterns were disrupted early in development and expression was subsequently lost in all tissues. “Gain‐of‐function” experiments where RA‐soaked beads were grafted into the pharyngeal region produced localized down‐regulation of Tbx1 expression. In these embryos, analysis of Shh and Foxa2, upstream control factors for Tbx1, suggested that the effect of RA was independent of this regulatory pathway. Real‐time polymerase chain reaction analysis of retinoic acid‐treated P19 cells showed a dose‐dependent repression of Tbx1 by retinoic acid. Repression of Tbx1 transcript levels was first evident after 8–12 hr in culture in the presence of retinoic acid, and to achieve the highest levels of repression, de novo protein synthesis was required. Developmental Dynamics 232:928–938, 2005.

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome.

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of β-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1lacZ (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1−/− mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures.

Isolation of a new marker and conserved sequences close to the DiGeorge syndrome marker HP500 (D22S134).

End fragment cloning from a YAC at the D22S134 locus allowed the isolation of a new probe HD7k. This marker detects hemizygosity in two patients previously shown to be dizygous for D22S134. This positions the distal deletion breakpoint in these patients to the sequences within the YAC, and confirms that HD7k is proximal to D22S134. In a search for coding sequences within the region commonly deleted in DGS we have identified a conserved sequence at D22S134. Although no cDNAs have yet been isolated, genomic sequencing shows a short open reading frame with weak similarity to collagen proteins.

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

Endogenous Retinoic Acid Activity in Principal Cells and Intercalated Cells of Mouse Collecting Duct System

Background Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys. Methodology/Principal Findings RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henles loop and distal tubules. Conclusions/Significance Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the collecting duct system.

Sequence homology between HLA-bound cytomegalovirus and human peptides: A potential trigger for alloreactivity

Human cytomegalovirus (hCMV) reactivation may often coincide with the development of graft-versus-host-disease (GVHD) in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n = 50; matched related donor (MRD), n = 27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (≥6) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50<500nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, an average of 29,658 matched the MRD DRP alloreactive peptides and 52,910 matched MUD DRP peptides (p<0.001). In silico analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner. hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset. Analysis of patients with GVHD identified potential cross reactive peptide expression within affected organs. We propose that hCMV peptide sequence homology with human alloreactive peptides may contribute to the pathophysiology of GVHD.