Mouse ENU mutagenesis programmes have yielded a series of independent mutations on proximal chromosome 4 leading to dominant head-bobbing and circling behaviour due to truncations of the lateral semicircular canal of the inner ear. Here, we report the identification of mutations in the Chd7 gene in nine of these mutant alleles including six nonsense and three splice site mutations. The human CHD7 gene is known to be involved in CHARGE syndrome, which also shows inner ear malformations and a variety of other features with varying penetrance and appears to be due to frequent de novo mutation. We found widespread expression of Chd7 in early development of the mouse in organs affected in CHARGE syndrome including eye, olfactory epithelium, inner ear and vascular system. Closer inspection of heterozygous mutant mice revealed a range of defects with reduced penetrance, such as cleft palate, choanal atresia, septal defects of the heart, haemorrhages, prenatal death, vulva and clitoral defects and keratoconjunctivitis sicca. Many of these defects mimic the features of CHARGE syndrome. There were no obvious features of the gene that might make it more mutable than other genes. We conclude that the large number of mouse mutants and human de novo mutations may be due to the combination of the Chd7 gene being a large target and the fact that many heterozygous carriers of the mutations are viable individuals with a readily detectable phenotype.

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.

CHARGE syndrome is a multiple congenital anomaly syndrome characterised by Coloboma, Heart defects, Atresia of choanae, Retardation of growth and/or development, Genital hypoplasia, and Ear anomalies often associated with deafness. It is caused by heterozygous mutations in the CHD7 gene and shows a highly variable phenotype. Anosmia and hypogonadotropic hypogonadism occur in the majority of the CHARGE patients, but the underlying pathogenesis is unknown. Therefore, we studied the ability to smell and aspects of the reproductive system (reproductive performance, gonadotropin-releasing hormone (GnRH) neurons and anatomy of testes and uteri) in a mouse model for CHARGE syndrome, the whirligig mouse (Chd7Whi/+). We showed that Chromodomain Helicase DNA-binding protein 7 (Chd7) is expressed in brain areas involved in olfaction and reproduction during embryonic development. We observed poorer performance in the smell test in adult Chd7Whi/+ mice, secondary either to olfactory dysfunction or to balance disturbances. Olfactory bulb and reproductive organ abnormalities were observed in a proportion of Chd7Whi/+ mice. Hypothalamic GnRH neurons were slightly reduced in Chd7Whi/+ females and reproductive performance was slightly less in Chd7Whi/+ mice. This study shows that the penetrance of anosmia and hypogonadotropic hypogonadism is lower in Chd7Whi/+ mice than in CHARGE patients. Interestingly, many phenotypic features of the Chd7 mutation showed incomplete penetrance in our model mice, despite the use of inbred, genetically identical mice. This supports the theory that the extreme variability of the CHARGE phenotype in both humans and mice might be attributed to variations in the fetal microenvironment or to purely stochastic events.

DEVELOPMENT 3399 RESEARCH ARTICLE INTRODUCTIONIn mouse embryos, extra-embryonic mesoderm cells of the prospective visceral yolk sac, chorion, allantois and amnion are among the first mesoderm cells to emerge early at gastrulation. Cell labelling studies have shown that a population of posterior and posterolateral epiblast cells that enters the primitive streak proximally will give rise to this extra-embryonic mesoderm and also to primordial germ cells (PGCs) (Lawson and Hage, 1994;Parameswaran and Tam, 1995;Kinder et al., 1999). Genetic evidence and epiblast culture experiments have shown that germ cell competence and formation of the allantois are induced in the proximal epiblast before gastrulation, in response to bone morphogenetic proteins (Bmps) produced in the extra-embryonic ectoderm, but depend also on the presence of visceral endoderm (reviewed by Zhao, 2003;de Sousa Lopes et al., 2004).Bmps are ligands of the transforming growth factor ␤ (Tgf␤) family that use various receptor complexes to directly activate intracellular effector proteins (Smad1/5/8). Smads transmit the signal to the nucleus and participate in the regulation of target gene expression (Shi and Massagué, 2003). Analysis of conventional knockout mice showed that the repertoire of Bmp signalling proteins involved in PGC induction comprise Bmps (Bmp2, -4 and -8b), a type I Bmp receptor (Alk2; Acvr1 -Mouse Genome Informatics) and two Bmp-Smads (Smad1/5) (Lawson et al., 1999;Ying et al., 2000;Chang and Matzuk, 2001;Tremblay et al., 2001;Hayashi et al., 2002;de Sousa Lopes et al., 2004;Okamura et al., 2005). Recently, it was shown that Blimp1 (Prdm1 -Mouse Genome Informatics), a transcriptional regulator thought to be induced by Bmp4, is a key regulator of germ cell specification (Vincent et al., 2005;Ohinata et al., 2005). Subsequent PGC localization and survival, as well as allantois differentiation, also depend on the presence of Bmp4 in the extra-embryonic mesoderm (Fujiwara et al., 2001).Several Bmps have been implicated in the specification and growth of extra-embryonic mesoderm. The induction of the allantois crucially depends on Bmp4 (Winnier et al., 1995;Fujiwara et al., 2001), and on Alk2-mediated signalling in visceral endoderm (Mishina et al., 1999;Gu et al., 1999). Bmp5;Bmp7 double mutant mouse embryos display impaired allantois maturation (Solloway and Robertson, 1999). Bmp2 plays a unique role in amnion development, as the pro-amniotic canal does not close in Bmp2 mutant mice, which has been ascribed to reduced and thus insufficient production of extra-embryonic mesoderm (Zhang and Bradley, 1996).Smad1 and Smad5 null embryos die at mid-gestation and have defects in PGC specification and allocation, and in the development of extra-embryonic tissues (Chang et al., 1999;Chang et al., 2000;Yang et al., 1999;Lechleider et al., 2001;Tremblay et al., 2001;Hayashi et al., 2002;Umans et al., 2003). The presence on the amnion of aggregates of cells that contain ectopic primitive red blood cells, and endothelial and PGC-like cells, is a un...