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Variation of external craniofacial morphology of Otx2 heterozygous mutant embryos at 18.5 dpc. (A) Wild-type mouse backcrossed Otx2 knockout chimeras with wild-type B6 females. Otx2 heterozygous mutant mice (N2) backcrossed Otx2 mutant chimeras with wild type CBA females (B) and with wild type B6 females (C-I). No noticeable malformations are evident in the mutant mouse on the CBA strain genetic background (B). The severity of the phenotype varies from normal to acephaly (C-I). No apparent external abnormalities are observed in the mutant mouse on the B6 strain genetic background (C). The mutant mouse displays reduction of the lower jaw (D). The mutant mouse lacks an entire lower jaw (E). The mutant mouse displays excencephaly (F). The distal region of the face is shortened in the mutant mouse (short nose) (G). The face is cleft in the mutant mouse (cleft face) (H). The entire head is lacking (acephaly) in the mutant mouse (I).
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Mice heterozygous for the Otx2 mutation display a craniofacial malformation, known as otocephaly or agnathia-holoprosencephaly complex. The severity of the phenotype is dependent on the genetic background of a C57BL/6 (B6) strain; most of the offspring of Otx2 knock-out chimeras, which are equivalent to the F(1) of CBA and B6 strains, backcrossed w...
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... an effort to map loci responsible for modification of the severity of craniofacial defects in Otx2 heterozygous mutant mice, two strains of mice, displaying disparate phenotypes of Otx2 heterozygosity, were used (Figs 1, 2). We have generated Otx2 mutant chimeras employing a TT2 ES cell line derived from F1 embryos of B6 females and CBA males ( Yagi et al., 1993;Matsuo et al., 1995). ...
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... have generated Otx2 mutant chimeras employing a TT2 ES cell line derived from F1 embryos of B6 females and CBA males ( Yagi et al., 1993;Matsuo et al., 1995). Upon backcross of chimeric males with wild-type B6 females to generate heterozygous mutant mice, severe craniofacial malformations occurred in the majority of the Otx2 heterozygous mutants at 18.5 dpc (Figs 1, 2). External abnormalities were mainly characterized as the reduction or loss of the lower jaw and eyes ( Fig. 1D,E; Fig. 2). ...
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... al., 1993;Matsuo et al., 1995). Upon backcross of chimeric males with wild-type B6 females to generate heterozygous mutant mice, severe craniofacial malformations occurred in the majority of the Otx2 heterozygous mutants at 18.5 dpc (Figs 1, 2). External abnormalities were mainly characterized as the reduction or loss of the lower jaw and eyes ( Fig. 1D,E; Fig. 2). Additionally, the severity of the phenotype varied greatly from a normal condition to the appearance of acephaly ( Fig. 1C-I). By contrast, when chimeric males were backcrossed with wildtype CBA females, craniofacial malformations were not observed (Fig. ...
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... craniofacial malformations occurred in the majority of the Otx2 heterozygous mutants at 18.5 dpc (Figs 1, 2). External abnormalities were mainly characterized as the reduction or loss of the lower jaw and eyes ( Fig. 1D,E; Fig. 2). Additionally, the severity of the phenotype varied greatly from a normal condition to the appearance of acephaly ( Fig. 1C-I). By contrast, when chimeric males were backcrossed with wildtype CBA females, craniofacial malformations were not observed (Fig. ...
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... were mainly characterized as the reduction or loss of the lower jaw and eyes ( Fig. 1D,E; Fig. 2). Additionally, the severity of the phenotype varied greatly from a normal condition to the appearance of acephaly ( Fig. 1C-I). By contrast, when chimeric males were backcrossed with wildtype CBA females, craniofacial malformations were not observed (Fig. ...
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... in order to investigate the variation of severity of craniofacial malformations, the chimeras were backcrossed with wild-type B6 females, resulting in N2 heterozygous offspring. Subsequently, the external abnormalities of these offspring were examined at 18.5 dpc (Fig. 1, Fig. 2A). Descriptions of eye and holoprosencephaly malformations were excluded in this investigation owing to the difficulty associated with judging defects from an external perspective; moreover, further histological analysis is required for the precise description of these abnormalities ( Matsuo et al., 1995). Thirty-seven percent of ...
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... investigation owing to the difficulty associated with judging defects from an external perspective; moreover, further histological analysis is required for the precise description of these abnormalities ( Matsuo et al., 1995). Thirty-seven percent of heterozygous pups (N2) did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. ...
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... histological analysis is required for the precise description of these abnormalities ( Matsuo et al., 1995). Thirty-seven percent of heterozygous pups (N2) did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly ...
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... ( Matsuo et al., 1995). Thirty-seven percent of heterozygous pups (N2) did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these ...
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... percent of heterozygous pups (N2) did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these craniofacial abnormalities is characteristic ...
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... did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these craniofacial abnormalities is characteristic of a monogenic trait that is caused ...
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... (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these craniofacial abnormalities is characteristic of a monogenic trait that is caused by modifier loci (Figs 1, 2) (Lander and Schork, ...
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... remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these craniofacial abnormalities is characteristic of a monogenic trait that is caused by modifier loci (Figs 1, 2) (Lander and Schork, 1994). ...
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... order to define the genetics underlying this dramatic variation in mandible phenotype, a whole genome search for modifier loci involved in the modulation of mandible abnormalities was conducted. Thus, all mutant individuals exhibiting no apparent abnormalities, reduction of lower jaw and loss of lower jaw (Fig. 2), were genotyped; however, mutant embryos displaying other external phenotypes, such as excencephaly, short nose, cleft face, acephaly, etc., were not investigated with respect to further genotyping experiments (Figs 1, 2). ...
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... order to map the regions of the genome containing modifying loci, 199 mutant pups displaying no apparent abnormalities, reduction of lower jaw and loss of lower jaw (Figs 1, 2) were initially selected from the first generation of B6 backcrossed animals (N2). These were subjected to further skull staining and the lengths of each mandible were measured. ...
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... mutant individuals displaying normal mandible (the mandible length is longer than 5.0 mm) and no mandible, or those displaying normal mandible and small mandible, respectively (Fig. 6). Consequently, one significant linkage on chromosome 18, which was defined as Otx2 modifier (Otmf) 18, was obtained exhibiting a peak LOD score of 3.33 at 11.1 cM (Fig. 6C, Table 1). One suggestive linkage was found on chromosome 10, with a peak LOD score of 2.56 at 38.1 cM (Fig. 6B, Table 1). These two loci exert effects on both the no mandible and small mandible phenotypes (Fig. 6B,C, Table 1). Unexpectedly, Otmf18 was derived from the CBA strain (Table 1), suggesting epistatic interactions between modifiers. ...
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... displaying normal mandible and small mandible, respectively (Fig. 6). Consequently, one significant linkage on chromosome 18, which was defined as Otx2 modifier (Otmf) 18, was obtained exhibiting a peak LOD score of 3.33 at 11.1 cM (Fig. 6C, Table 1). One suggestive linkage was found on chromosome 10, with a peak LOD score of 2.56 at 38.1 cM (Fig. 6B, Table 1). These two loci exert effects on both the no mandible and small mandible phenotypes (Fig. 6B,C, Table 1). Unexpectedly, Otmf18 was derived from the CBA strain (Table 1), suggesting epistatic interactions between modifiers. Additionally, two weak linkages were also detected on chromosome 2; these linkages exhibited peak LOD scores of ...
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... Otx2 heterozygous mutant N3 embryos (n=200) at 18.5 dpc obtained by backcrossing the N2 male with wild-type B6 females are phenotypically classified into eight groups according to their external morphology. Genetic loci modifying craniofacial malformations Linkage analysis using N3 offspring As described previously, small numbers of N2 backcross heterozygous mutant mice survived to weaning, followed by fertility that afforded further progeny (Figs 1, 2) ( Matsuo et al., 1995). In order to refine modifier location, a single N2 male was selected; subsequently, allele distribution between B6 and CBA was genotyped employing the 92 polymorphic markers from the N2 initial genome scan (Fig. 5). ...
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... this N2 male was backcrossed with wild type B6 females, resulting in heterozygous N3 animals. External phenotypes were classified as described above (Fig. 1). The frequency of external phenotype in these N3 mutant embryos is summarized in Fig. 2B. Twenty-nine percent of heterozygous pups did not display prominent abnormalities in jaw, nose or head (Fig. 2B). Mutant progeny exhibited reduction (28.5%) of and loss (31.5%) of the lower jaw (Fig. 2B). All mutant animals exhibiting no apparent ...
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... the otocephalic phenotype. Furthermore, the findings indicate that these loci are genetically associated with Otx2 locus. In addition, these modifier may interact with other unidentified modifier loci epistatically. One locus, Otmf18, was mapped on the CBA allele (Table 1). As the otocephalic phenotype is not evident on the CBA genetic background (Fig. 1), the Otmf18 locus on the CBA strain alone appears to be insufficient to induce mandible abnormalities. Thus, a second undetermined modifier, probably located on the B6 strain, may be required for expression of mandible ...
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... an effort to map loci responsible for modification of the severity of craniofacial defects in Otx2 heterozygous mutant mice, two strains of mice, displaying disparate phenotypes of Otx2 heterozygosity, were used (Figs 1, 2). We have generated Otx2 mutant chimeras employing a TT2 ES cell line derived from F1 embryos of B6 females and CBA males ( Yagi et al., 1993;Matsuo et al., 1995). ...
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... have generated Otx2 mutant chimeras employing a TT2 ES cell line derived from F1 embryos of B6 females and CBA males ( Yagi et al., 1993;Matsuo et al., 1995). Upon backcross of chimeric males with wild-type B6 females to generate heterozygous mutant mice, severe craniofacial malformations occurred in the majority of the Otx2 heterozygous mutants at 18.5 dpc (Figs 1, 2). External abnormalities were mainly characterized as the reduction or loss of the lower jaw and eyes ( Fig. 1D,E; Fig. 2). ...
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... al., 1993;Matsuo et al., 1995). Upon backcross of chimeric males with wild-type B6 females to generate heterozygous mutant mice, severe craniofacial malformations occurred in the majority of the Otx2 heterozygous mutants at 18.5 dpc (Figs 1, 2). External abnormalities were mainly characterized as the reduction or loss of the lower jaw and eyes ( Fig. 1D,E; Fig. 2). Additionally, the severity of the phenotype varied greatly from a normal condition to the appearance of acephaly ( Fig. 1C-I). By contrast, when chimeric males were backcrossed with wildtype CBA females, craniofacial malformations were not observed (Fig. ...
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... craniofacial malformations occurred in the majority of the Otx2 heterozygous mutants at 18.5 dpc (Figs 1, 2). External abnormalities were mainly characterized as the reduction or loss of the lower jaw and eyes ( Fig. 1D,E; Fig. 2). Additionally, the severity of the phenotype varied greatly from a normal condition to the appearance of acephaly ( Fig. 1C-I). By contrast, when chimeric males were backcrossed with wildtype CBA females, craniofacial malformations were not observed (Fig. ...
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... were mainly characterized as the reduction or loss of the lower jaw and eyes ( Fig. 1D,E; Fig. 2). Additionally, the severity of the phenotype varied greatly from a normal condition to the appearance of acephaly ( Fig. 1C-I). By contrast, when chimeric males were backcrossed with wildtype CBA females, craniofacial malformations were not observed (Fig. ...
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... in order to investigate the variation of severity of craniofacial malformations, the chimeras were backcrossed with wild-type B6 females, resulting in N2 heterozygous offspring. Subsequently, the external abnormalities of these offspring were examined at 18.5 dpc (Fig. 1, Fig. 2A). Descriptions of eye and holoprosencephaly malformations were excluded in this investigation owing to the difficulty associated with judging defects from an external perspective; moreover, further histological analysis is required for the precise description of these abnormalities ( Matsuo et al., 1995). Thirty-seven percent of ...
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... investigation owing to the difficulty associated with judging defects from an external perspective; moreover, further histological analysis is required for the precise description of these abnormalities ( Matsuo et al., 1995). Thirty-seven percent of heterozygous pups (N2) did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. ...
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... histological analysis is required for the precise description of these abnormalities ( Matsuo et al., 1995). Thirty-seven percent of heterozygous pups (N2) did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly ...
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... ( Matsuo et al., 1995). Thirty-seven percent of heterozygous pups (N2) did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these ...
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... percent of heterozygous pups (N2) did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these craniofacial abnormalities is characteristic ...
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... did not exhibit prominent abnormalities in jaw, nose or head externally (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these craniofacial abnormalities is characteristic of a monogenic trait that is caused ...
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... (Fig. 1C, Fig. 2A). Nineteen and 21.5% of these offspring displayed reduction and absence of the lower jaw, respectively (Fig. 1D,E, Fig. 2A). A small percentage of mutants exhibited excencephaly (7.0%; Fig. 1F, Fig. 2A), short nose (3.0%; Fig. 1G, Fig. 2A), cleft face (2.0%; Fig. 1H, Fig. 2A) and acephaly, showing loss of the entire head (5.5%; Fig. 1I, Fig. 2A). The remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these craniofacial abnormalities is characteristic of a monogenic trait that is caused by modifier loci (Figs 1, 2) (Lander and Schork, ...
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... remaining small percentage of mutants revealed additional phenotypes, including ethmocephaly (5.0%; Fig. 2A; and data not shown). Consequently, the distribution of these craniofacial abnormalities is characteristic of a monogenic trait that is caused by modifier loci (Figs 1, 2) (Lander and Schork, 1994). ...
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... order to define the genetics underlying this dramatic variation in mandible phenotype, a whole genome search for modifier loci involved in the modulation of mandible abnormalities was conducted. Thus, all mutant individuals exhibiting no apparent abnormalities, reduction of lower jaw and loss of lower jaw (Fig. 2), were genotyped; however, mutant embryos displaying other external phenotypes, such as excencephaly, short nose, cleft face, acephaly, etc., were not investigated with respect to further genotyping experiments (Figs 1, 2). ...
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... order to map the regions of the genome containing modifying loci, 199 mutant pups displaying no apparent abnormalities, reduction of lower jaw and loss of lower jaw (Figs 1, 2) were initially selected from the first generation of B6 backcrossed animals (N2). These were subjected to further skull staining and the lengths of each mandible were measured. ...
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... mutant individuals displaying normal mandible (the mandible length is longer than 5.0 mm) and no mandible, or those displaying normal mandible and small mandible, respectively (Fig. 6). Consequently, one significant linkage on chromosome 18, which was defined as Otx2 modifier (Otmf) 18, was obtained exhibiting a peak LOD score of 3.33 at 11.1 cM (Fig. 6C, Table 1). One suggestive linkage was found on chromosome 10, with a peak LOD score of 2.56 at 38.1 cM (Fig. 6B, Table 1). These two loci exert effects on both the no mandible and small mandible phenotypes (Fig. 6B,C, Table 1). Unexpectedly, Otmf18 was derived from the CBA strain (Table 1), suggesting epistatic interactions between modifiers. ...
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... displaying normal mandible and small mandible, respectively (Fig. 6). Consequently, one significant linkage on chromosome 18, which was defined as Otx2 modifier (Otmf) 18, was obtained exhibiting a peak LOD score of 3.33 at 11.1 cM (Fig. 6C, Table 1). One suggestive linkage was found on chromosome 10, with a peak LOD score of 2.56 at 38.1 cM (Fig. 6B, Table 1). These two loci exert effects on both the no mandible and small mandible phenotypes (Fig. 6B,C, Table 1). Unexpectedly, Otmf18 was derived from the CBA strain (Table 1), suggesting epistatic interactions between modifiers. Additionally, two weak linkages were also detected on chromosome 2; these linkages exhibited peak LOD scores of ...
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... Otx2 heterozygous mutant N3 embryos (n=200) at 18.5 dpc obtained by backcrossing the N2 male with wild-type B6 females are phenotypically classified into eight groups according to their external morphology. Genetic loci modifying craniofacial malformations Linkage analysis using N3 offspring As described previously, small numbers of N2 backcross heterozygous mutant mice survived to weaning, followed by fertility that afforded further progeny (Figs 1, 2) ( Matsuo et al., 1995). In order to refine modifier location, a single N2 male was selected; subsequently, allele distribution between B6 and CBA was genotyped employing the 92 polymorphic markers from the N2 initial genome scan (Fig. 5). ...
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... this N2 male was backcrossed with wild type B6 females, resulting in heterozygous N3 animals. External phenotypes were classified as described above (Fig. 1). The frequency of external phenotype in these N3 mutant embryos is summarized in Fig. 2B. Twenty-nine percent of heterozygous pups did not display prominent abnormalities in jaw, nose or head (Fig. 2B). Mutant progeny exhibited reduction (28.5%) of and loss (31.5%) of the lower jaw (Fig. 2B). All mutant animals exhibiting no apparent ...
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... the otocephalic phenotype. Furthermore, the findings indicate that these loci are genetically associated with Otx2 locus. In addition, these modifier may interact with other unidentified modifier loci epistatically. One locus, Otmf18, was mapped on the CBA allele (Table 1). As the otocephalic phenotype is not evident on the CBA genetic background (Fig. 1), the Otmf18 locus on the CBA strain alone appears to be insufficient to induce mandible abnormalities. Thus, a second undetermined modifier, probably located on the B6 strain, may be required for expression of mandible ...
Citations
... There are many examples of digenic interactions that lead to a disease phenotype, including HPE, retinitis pigmentosa, hirschsprung disease, glaucoma and insulin resistance (37,(86)(87)(88)(89)(90)(91). There is also mounting evidence of digenic disease in patients with hypopituitarism and clear evidence of genetic background effects on the severity of pituitary disorders in mice (4,44,(92)(93)(94)(95). Our observations extend this paradigm to include Six3 and Pou1f1. ...
Congenital hypopituitarism is a genetically heterogeneous condition that is part of a spectrum disorder that can include holoprosencephaly. Heterozygous mutations in SIX3 cause variable holoprosencephaly in humans and mice. We identified two children with neonatal hypopituitarism and thin pituitary stalk who were doubly heterozygous for rare, likely deleterious variants in the transcription factors SIX3 and POU1F1. We used genetically engineered mice to understand the disease pathophysiology. Pou1f1 loss of function heterozygotes are unaffected; Six3 heterozygotes have pituitary gland dysmorphology and incompletely ossified palate; and the Six3+/−; Pou1f1+/dw double; heterozygote mice have a pronounced phenotype, including pituitary growth through the palate. The interaction of Pou1f1 and Six3 in mice supports the possibility of digenic pituitary disease in children. Disruption of Six3 expression in the oral ectoderm completely ablated anterior pituitary development, and deletion of Six3 in the neural ectoderm blocked development of the pituitary stalk and both anterior and posterior pituitary lobes. Six3 is required in both oral and neural ectodermal tissues for activation of signaling pathways and transcription factors necessary for pituitary cell fate. These studies clarify the mechanism of SIX3 action in pituitary development and provide support for a digenic basis for hypopituitarism.
... Experiments with mice, heterozygous for the Otx2 mutation, provide new insights into the genetic pathogenesis of human otocephaly. 13 It is discovered that genetic modifications of Otx2, Otmf18, and Otmf2, located on chromosomes 18 and 2, have been found to correlate with the phenotype of otocephaly and are thought to be related to the development of agnathy with holoprosencephaly in humans. 1 Prenatal diagnosis of otocephaly is extremely difficult and that is why it is rarely reported. 1 Since its first description by Kerckring in 1717, until this moment, only a little bit more than 100 cases were reported worldwide. ...
... It should be noted, however, that two internal controls of our database harboring chromosome 1p34.1-p33 duplications encompassing DMBX1 had normally shaped head and set ears (seeTable 4for details), suggesting incomplete penetrance for DMBX1-related phenotypes, in line with what observed in knock-out Otx2 heterozygous mice.56 Another interesting CNV was found in a patient with azygos con-tinuation of the inferior vena cava. ...
Oculo‐auriculo‐vertebral spectrum (OAVS) is a developmental disorder of craniofacial morphogenesis. Its etiology is unclear, but assumed to be complex and heterogeneous, with contribution of both genetic and environmental factors. We assessed the occurrence of copy number variants (CNVs) in a cohort of 19 unrelated OAVS individuals with congenital heart defect. Chromosomal microarray analysis identified pathogenic CNVs in 2/19 (10.5%) individuals, and CNVs classified as variants of uncertain significance in 7/19 (36.9%) individuals. Remarkably, two subjects had small intragenic CNVs involving DACH1 and DACH2, two paralogs coding for key components of the PAX‐SIX‐EYA‐DACH network, a transcriptional regulatory pathway controlling developmental processes relevant to OAVS and causally associated with syndromes characterized by craniofacial involvement. Moreover, a third patient showed a large duplication encompassing DMBX1/OTX3, encoding a transcriptional repressor of OTX2, another transcription factor functionally connected to the DACH‐EYA‐PAX network. Among the other relevant CNVs, a deletion encompassing HSD17B6, a gene connected with the retinoic acid signaling pathway, whose dysregulation has been implicated in craniofacial malformations, was also identified. Our findings suggest that CNVs affecting gene dosage likely contribute to the genetic heterogeneity of OAVS, and implicate the PAX‐SIX‐EYA‐DACH network as novel pathway involved in the etiology of this developmental trait.
... and Twsg1 have been shown to cause AGOTC phenotype in mice, while Alx4 was previously proposed to be a genetic modifier of the otocephalic phenotype observed in an Otx2 heterozygous mutant mice [37,40,[46][47][48][49][50]. However, to date, no mutation was found in patients after screening of these different candidate genes [20,44]. ...
Objectives
Agnathia-otocephaly complex is a rare condition characterized by mandibular hypoplasia or agnathia, ear anomalies (melotia/synotia) and microstomia with aglossia. This severe anomaly of the first branchial arch is most often lethal. The estimated incidence is less than 1 in 70.000 births, with etiologies linked to both genetic and teratogenic factors. Most of the cases are sporadic. To date, two genes have been described in humans to be involved in this condition: OTX2 and PRRX1. Nevertheless, the overall proportion of mutated cases is unknown and a significant number of patients remain without molecular diagnosis. Thus, the involvement of other genes than OTX2 and PRRX1 in the agnathia-otocephaly complex is not unlikely. Heterozygous mutations in Cnbp in mice are responsible for mandibular and eye defects mimicking the agnathia-otocephaly complex in humans and appear as a good candidate. Therefore, in this study, we aimed (i) to collect patients presenting with agnathia-otocephaly complex for screening CNBP, in parallel with OTX2 and PRRX1, to check its possible implication in the human phenotype and (ii) to compare our results with the literature data to estimate the proportion of mutated cases after genetic testing.Materials and methodsIn this work, we describe 10 patients suffering from the agnathia-otocephaly complex. All of them benefited from array-CGH and Sanger sequencing of OTX2, PRRX1 and CNBP. A complete review of the literature was made using the Pubmed database to collect all the patients described with a phenotype of agnathia-otocephaly complex during the 20 last years (1998–2019) in order (i) to study etiology (genetic causes, iatrogenic causes…) and (ii), when genetic testing was performed, to study which genes were tested and by which type of technologies.ResultsIn our 10 patients’ cohort, no point mutation in the three tested genes was detected by Sanger sequencing, while array-CGH has allowed identifying a 107-kb deletion encompassing OTX2 responsible for the agnathia-otocephaly complex phenotype in 1 of them. In 4 of the 70 cases described in the literature, a toxic cause was identified and 22 out the 66 remaining cases benefited from genetic testing. Among those 22 patients, 6 were carrying mutation or deletion in the OTX2 gene and 4 in the PRRX1 gene. Thus, when compiling results from our cohort and the literature, a total of 32 patients benefited from genetic testing, with only 34% (11/32) of patients having a mutation in one of the two known genes, OTX2 or PRRX1.Conclusions
From our work and the literature review, only mutations in OTX2 and PRRX1 have been found to date in patients, explaining around one third of the etiologies after genetic testing. Thus, agnathia-otocephaly complex remains unexplained in the majority of the patients, which indicates that other factors might be involved. Although involved in first branchial arch defects, no mutation in the CNBP gene was found in this study. This suggests that mutations in CNBP might not be involved in such phenotype in humans or that, unlike in mice, a compensatory effect might exist in humans. Nevertheless, given that agnathia-otocephaly complex is a rare phenotype, more patients have to be screened for CNBP mutations before we definitively conclude about its potential implication. Therefore, this work presents the current state of knowledge on agnathia-otocephaly complex and underlines the need to expand further the understanding of the genetic bases of this disorder, which remains largely unknown.Clinical relevanceWe made here an update and focus on the clinical and genetic aspects of agnathia-otocephaly complex as well as a more general review of craniofacial development.
... For example, Otx2 +/− mice display microphthalmia and otocephaly, alongside reduced fertility in males, reflective of the abnormal development of the hypothalamic-pituitarygonadal axis seen in humans with OTX2 mutations causing syndromic microphthalmia 5 (MCOPS5 -OMIM #610125). 53,[168][169][170][171][172] These features coincide as, in addition to controlling oculogenesis, Otx2 regulates the expression of genes involved in pituitary development, such as Hesx1.168,173,174 Investigation of extraocular phenotypes in mice can provide information on the effect of different variants and genetic/environmental factors on systemic involvement, thus unlocking genotypephenotype relationships.Identification of novel variants through mouse studies. ...
Microphthalmia is a rare developmental eye disorder affecting 1 in 7000 births. It is defined as a small (axial length ⩾2 standard deviations below the age-adjusted mean) underdeveloped eye, caused by disruption of ocular development through genetic or environmental factors in the first trimester of pregnancy. Clinical phenotypic heterogeneity exists amongst patients with varying levels of severity, and associated ocular and systemic features. Up to 11% of blind children are reported to have microphthalmia, yet currently no treatments are available. By identifying the aetiology of microphthalmia and understanding how the mechanisms of eye development are disrupted, we can gain a better understanding of the pathogenesis. Animal models, mainly mouse, zebrafish and Xenopus, have provided extensive information on the genetic regulation of oculogenesis, and how perturbation of these pathways leads to microphthalmia. However, differences exist between species, hence cellular models, such as patient-derived induced pluripotent stem cell (iPSC) optic vesicles, are now being used to provide greater insights into the human disease process. Progress in 3D cellular modelling techniques has enhanced the ability of researchers to study interactions of different cell types during eye development. Through improved molecular knowledge of microphthalmia, preventative or postnatal therapies may be developed, together with establishing genotype–phenotype correlations in order to provide patients with the appropriate prognosis, multidisciplinary care and informed genetic counselling. This review summarises some key discoveries from animal and cellular models of microphthalmia and discusses how innovative new models can be used to further our understanding in the future.
Plain language summary
Animal and Cellular Models of the Eye Disorder, Microphthalmia (Small Eye) Microphthalmia, meaning a small, underdeveloped eye, is a rare disorder that children are born with. Genetic changes or variations in the environment during the first 3 months of pregnancy can disrupt early development of the eye, resulting in microphthalmia. Up to 11% of blind children have microphthalmia, yet currently no treatments are available. By understanding the genes necessary for eye development, we can determine how disruption by genetic changes or environmental factors can cause this condition. This helps us understand why microphthalmia occurs, and ensure patients are provided with the appropriate clinical care and genetic counselling advice. Additionally, by understanding the causes of microphthalmia, researchers can develop treatments to prevent or reduce the severity of this condition. Animal models, particularly mice, zebrafish and frogs, which can also develop small eyes due to the same genetic/environmental changes, have helped us understand the genes which are important for eye development and can cause birth eye defects when disrupted. Studying a patient’s own cells grown in the laboratory can further help researchers understand how changes in genes affect their function. Both animal and cellular models can be used to develop and test new drugs, which could provide treatment options for patients living with microphthalmia. This review summarises the key discoveries from animal and cellular models of microphthalmia and discusses how innovative new models can be used to further our understanding in the future.
... To increase the frequency of craniofacial phenotypes and facilitate future molecular analyses, the Ttc21b aln allele was serially backcrossed onto the C57BL/6J (B6) strain. The B6 genetic background has previously been shown to be more susceptible to craniofacial phenotypes in a number of studies [23][24][25][26]. This initial, exploratory backcross was done informally and was not genotyped. ...
The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.
... OTX2 patients encompass extremely variable phenotypes, including additional eye malformations such as anterior segment dysgenesis, retinal dystrophy and hypoplasia or aplasia of the optic nerve and optic chiasm, as well as syndromic features including pituitary abnormalities, hypopituitarism, brain anomalies, seizures and developmental delay (Table 1) [8,91,93,215]. Homozygous mouse knockouts of homeobox gene Otx2 lack eyes, while heterozygous knockout mouse models also show a highly variable phenotype, from acephaly, micrognathia, anophthalmia, microphthalmia to normal development, depending on the genetic background [8,25,63,80,[215][216][217][218][219]. A relationship between genotype and clinical phenotype has been observed, as mutations in the second half of OTX2 seem to occur more frequently with abnormal pituitary function, although this correlation is not always seen [8,91,215]. ...
Human eye development is coordinated through an extensive network of genetic signalling pathways. Disruption of key regulatory genes in the early stages of eye development can result in aborted eye formation, resulting in an absent eye (anophthalmia) or a small underdeveloped eye (microphthalmia) phenotype. Anophthalmia and microphthalmia (AM) are part of the same clinical spectrum and have high genetic heterogeneity, with >90 identified associated genes. By understanding the roles of these genes in development, including their temporal expression, the phenotypic variation associated with AM can be better understood, improving diagnosis and management. This review describes the genetic and structural basis of eye development, focusing on the function of key genes known to be associated with AM. In addition, we highlight some promising avenues of research involving multiomic approaches and disease modelling with induced pluripotent stem cell (iPSC) technology, which will aid in developing novel therapies.
... Proper regulation and function of this gene is crucial, as the mutation of Otx2 leads to defects in the rostral head in several developmental processes (Acampora et al., 1995;Acampora et al., 1997;Matsuo et al., 1995;Ang et al., 1996;Suda et al., 1996;Suda et al., 1997;Suda et al., 2001;Tian et al., 2002;Hide et al., 2002). However, very little is known about how Otx2 gene expression is dynamically regulated. ...
... 1997). Consistently, Otx2 is expressed in the cephalic mesenchyme, and functions in the formation of premandibular and mandibular skulls ( Matsuo et al., 1995;Kimura et al., 1997;Hide et al., 2002). The F11 region governs expression in the dorsal pretectum and mesencephalon, including in the posterior commissure, the mesencephalic trigeminal neurons, the oculomotor nerve, the first and second branches of the trigeminal nerve, and the trigeminal ganglions (Figs 8, 10). ...
The Otx2 gene, containing a highly conserved paired-type homeobox, plays a pivotal role in the development of the rostral head throughout vertebrates. Precise regulation of the temporal and spatial expression of Otx2 is likely to be crucial for proper head specification. However, regulatory mechanisms of Otx2 expression remain largely unknown. In this study, the Otx2 genome of the puffer fish Fugu rubripes, which has been proposed as a model vertebrate owing to its highly compact genome, was cloned. Consistently, Fugu Otx2 possesses introns threefold smaller in size than those of the mouse Otx2 gene. Otx2 mRNA was transcribed after MBT, and expressed in the rostral head region throughout the segmentation and pharyngula periods of wild-type Fugu embryos. To elucidate regulatory mechanisms of Otx2 expression, the expression of Otx2-lacZ reporter genes nearly covering the Fugu Otx2 locus, from -30.5 to +38.5 kb, was analyzed, by generating transgenic mice. Subsequently, seven independent cis-regulators were identified over an expanse of 60 kb; these regulators are involved in the mediation of spatiotemporally distinct subdomains of Otx2 expression. Additionally, these expression domains appear to coincide with local signaling centers and developing sense organs. Interestingly, most domains do not overlap with one another, which implies that cis-regulators for redundant expression may be abolished exclusively in the pufferfish so as to reduce its genome size. Moreover, these cis-regions were also able to direct expression in zebrafish embryos equivalent to that observed in transgenic mice. Further comparative sequence analysis of mouse and pufferfish intergenic regions revealed eight highly conserved elements within these cis-regulators. Therefore, we propose that, in vertebrate evolution, the Otx2 promoter acquires multiple, spatiotemporally specific cis-regulators in order to precisely control highly coordinated processes in head development.
... The subsequent breeding and mapping of the causative mutation involved an outcross to the FVB/NJ (FVB) strain (Herron et al. 2002;Tran et al. 2008). Ttc21b aln/aln homozygous mutants on this mixed susceptible to craniofacial phenotypes in a number of studies (Hide et al. 2002;Dixon and Dixon 2004;Mukhopadhyay et al. 2012;Percival et al. 2017). As the Ttc21b aln was serially backcrossed to the B6 background, we noticed the relative size of the forebrain tissues in B6.Cg-Ttc21b aln/aln mutants increased substantially, although they remained smaller than control brains and still lacked olfactory bulbs (Fig.1 E,F). ...
The primary cilium is a critical signaling center for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background. Ttc21baln/aln mutants on an FVB/NJ background develop a forebrain significantly smaller than mutants on a C57BL/6J background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified a brain specific orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP between the FVB and B6 strains in Gpr63, which creates a missense mutation predicted to be deleterious in the FVB protein. We first demonstrated that Gpr63 can localize to the cilium and then used CRISPR-Cas9 genome editing to create FVB congenic mice with the B6 sequence of Gpr63 and a deletion allele leading to a truncation of the GPR63 C-terminal tail. These alleles genetically interact with Ttc21baln/aln, validating Gpr63 as a forebrain modifier of Ttc21b and strongly supporting Gpr63 as the variant causal gene (i.e., the quantitative trait gene, QTG) for Moaq1.
... In vitro studies showed that while wildtype and mutant OTX2 protein bound equally well to two specific sites in the 5-prime flanking region of the HESX1 gene, mutant OTX2 revealed decreased transactivation, resulting in a dominant negative inhibitor of HESX1 gene expression. While homozyogous Otx-knockout mice die during midgestation, Otx heterozygous mutant mice present eye, pituitary and craniofacial defects (61,62). In zebrafish, morpholinos targeting otx2 result in mild microphthalmia and shortening of the pharyngeal skeleton at 5 days post fertilization (dpf) (63). ...
The GH/IGF axis plays an important role in the control of pre and postnatal growth. At least 48 monogenic defects have been described affecting the production, secretion, and action of GH and IGFs. Molecular defects of the GH/IGF axis resulting in short stature were arbitrarily classified into 4 groups: 1. Combined pituitary hormone deficiency (CPHD) (a. syndromic CPHD and b. non-syndromic CPHD), 2. Isolated GH deficiency (IGHD), 3. GH insensitivity, and 4. IGF-I insensitivity. Genetic diagnosis is obtained in about 30-40% of children with growth retardation, severe IGHD, CPHD, apparent GH or IGF-I insensitivity, and small for gestational age. Increased accessibility to next generation sequencing (NGS) techniques resulted in a significant number of likely pathogenic variants in genes previously associated with short stature as well as in completely novel genes. Functional in vitro assays and in vivo animal models are required to determine the real contribution of these findings.
