Present address: Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami Cho, Chuou-Ku, Kobe, Hyougo 650-0047, Japan
1 Department of Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan
2 Department of Neurosurgery, Kumamoto University School of Medicine, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan
3 Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami Cho, Chuou-Ku, Kobe, Hyougo 650-0047, Japan
4 Division of Transgenic Technology, Center for Animal Resources and Development (CARD), Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan
5 Department of Mammalian Development, National Institute of Genetics, Mishima, Japan
*Author for correspondence (e-mail: saizawa{at}cdb.riken.go.jp)
Accepted 11 June 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Otx2, Otocephaly, Agnathia-holoprosencephaly complex, Genetic modifier, Neural crest, Mandible, Mouse
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mouse Otx2 is a paired-like type homeobox gene functioning as a transcriptional activator (Simeone et al., 1992; Simeone et al., 1993
). It is sequentially expressed in the epiblast, anterior visceral endoderm, anterior definitive endoderm and anterior neuroectoderm prior to and during gastrulation; at the subsequent neurula stage, Otx2 is expressed in the entire rostral brain region (Simeone et al., 1992
; Ang et al., 1994
; Acampora et al., 1998
; Kimura et al., 2000
). Indeed, several knockout and compound mutations of the Otx2 gene suggest that it is involved in several steps for early AP patterning and rostral brain development in cooperation with other regulatory genes (Matsuo et al., 1995
; Acampora et al., 1995
; Acampora et al., 1997
; Acampora et al., 1998
; Ang et al., 1996
; Suda et al., 1996
; Suda et al., 1997
; Suda et al., 2001
; Kimura et al., 2000
; Kimura et al., 2001
; Tian et al., 2002
).
In addition, Otx2 is expressed in the cephalic mesenchyme, including the mesencephalic neural crest cells, which are distributed to the premandibular and distal regions of the mandibular regions (Kimura et al., 1997). Reflected by Otx2 expression in the cephalic mesenchyme, Otx2 also plays a crucial role in craniofacial development. Otx2 single heterozygous mutant mice displayed craniofacial malformations that were strictly dependent on the genetic background of the murine strains (Matsuo et al., 1995
). Previously, the Otx2 knockout chimera has been generated in TT2 ES cells, which are derived from F1 embryos obtained from crosses of inbred C57BL/6 (B6) and CBA strains (Yagi et al., 1993
; Matsuo et al., 1995
). Upon backcross of these chimeras with B6 females, the majority of heterozygous mutants are dead at birth, accompanied by severe craniofacial malformations, which are designated as otocephaly in many mammalian species and agnathia-holoprosencephaly complex in humans (Bixler et al., 1985
; Juriloff et al., 1985
; Winter, 1996
; Wallis and Muenke, 2000
). Notably, these mutants displayed reduction or absence of the lower jaw and/or eyes externally as well as holoprosencephaly by histological examination (Matsuo et al., 1995
). However, when the chimeras were crossed with CBA females, most of the Otx2 heterozygous mutant pups exhibited no noticeable phenotype. This evidence explicitly suggests the presence of several genetic modifier genes exerting strong effects on the expressivity of the Otx2 heterozygous mutant phenotype.
On the basis of facilitated recognition and quantitation of small changes in Otx2 activity through lower jaw development, in particular, the length of the mandible, this phenotype provides a simple and sensitive assay for allelic differences at secondary loci interacting with the Otx2 gene product. Changes in the gene products of secondary loci that lie upstream, downstream or interact directly with the Otx2 protein would all impact the expression of the Otx2 mutant mandible phenotype. With the advent of simple sequence length polymorphism (SSLP) markers, which are distributed throughout the entire genome (Dietrich et al., 1992), it is now possible to rapidly map the loci that contribute to such complex genetic traits. This situation provides an ideal opportunity for defining the genes that control the severity of otocephaly. Moreover, genetic analysis of well-defined experimental models of otocephaly offers the potential to markedly accelerate the genetic analysis of human agnathia-holoprosencephaly complex. In this study, two different mouse strains, B6 and CBA, were employed in order to identify and map modifier loci acting upon the expression of mandible abnormalities of Otx2 heterozygous mutant mice. The modifier loci thus identified regulate a genetic pathway of craniofacial development interacting with Otx2; furthermore, these loci may also be possible genetic causes of human agnathia-holoprosencephaly complex diseases.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The most frequently observed phenotype was lower jaw abnormality; consequently, we focused on the jaw anomalies. In order to investigate the phenotype of lower jaws more precisely, the morphology of the mandibular skull following bone and cartilage staining by Alcian Blue and Alizarin Red was further examined (Fig. 3). We found that even in reduced lower jaws, mandible formation was affected to varying extents, ranging from simple fusion of the anterior tips of the incisors to involution of the entire mandible in a small single median bone (Fig. 3F,H,J). Furthermore, to determine the severity of the anomalous mandibles, the length of each was measured (Figs 3, 4). Normally, lengths of wild-type mandibles of B6 and CBA strains were consistently longer than 5.0 mm at 18.5 dpc (Fig. 3B, Fig. 4A; data not shown). Similarly, lengths of Otx2 heterozygous mutant mandibles on CBA genetic background were also longer than 5.0 mm (data not shown). By contrast, mandibles of heterozygous mutants backcrossed to B6 females exhibited varying lengths (Fig. 3, Fig. 4B). Mutant mandibles demonstrated lengths in excess of 5.0 mm as well as in the range of 0.5 to 4.9 mm; additionally, the mandible was absent in several samples (Fig. 3C-K, Fig. 4B).
In 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).
Linkage analysis using N2 offspring
We hypothesized that the variable severity in the Otx2 heterozygous mutant mandible of the B6 strain was due to the variation in genetic background, particularly involving modifier loci, the alleles of which differed between B6 and CBA. With the discovery of the highly polymorphic and simple genotyping protocols of simple sequence length polymorphisms (SSLPs) (Love et al., 1990), these markers are very applicable to the mapping of the location of genetic loci involved in genetic background-dependent phenotypic differences. However, the usefulness of CBA for genetic mapping studies has been limited by the lack of information regarding DNA variants alleles (Dietrich et al., 1992
; Dietrich et al., 1994
) (http://www.informatics.jax.org/). In order to map locations of modifiers of Otx2 mutant mice, we first surveyed variant SSLP markers between CBA and B6 strains for the entire genome scan (Fig. 5). Of the 293 markers tested, 180 were variant based on agarose gel electrophoresis (data not shown). This rate of variant alleles is comparable with that observed in other inbred laboratory mouse strains (Dietrich et al., 1994
). Given the high frequency of variant alleles and large litter size, the CBA strain could be useful in mapping studies of genetic modifiers in transgenic or knockout mice that are widely generated in the CBA and B6 genetic background.
|
Thus, linkage analysis was conducted with the composite interval mapping of QTL-cartographer program (Fig. 6) (Basten et al., 2001); in addition, to investigate whether the genetic loci can modify the phenotype for small mandible (the mandible length corresponds to 0.5 to 4.9 mm) or no mandible (0 mm) qualitatively, genetic analysis was also performed with 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. Additionally, two weak linkages were also detected on chromosome 2; these linkages exhibited peak LOD scores of 1.59 at 17 cM and 1.8 at 66.9 cM, respectively (Fig. 6A). Thus, these findings acquired via the survey of N2 offspring indicate that at least one modifier locus Otmf18 is significantly involved in the severity of mandible phenotypes in Otx2 mutant embryos.
|
Next, 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 abnormalities, reduction of lower jaw and loss of lower jaw (Fig. 2) were subjected to skeletal staining and the lengths of each mandible were measured (Fig. 4C). Then, the severity of the mandibular phenotype was designated as normal mandible (the mandible length is longer than 5.0 mm), small mandible (the mandible length corresponds to 0.5 to 4.9 mm) and no mandible (the length is 0 mm). For the modifier mapping, these 202 mutant N3 pups were genotyped initially with 51 microsatellite markers that were not homozygous for B6 strain allele in this male (Fig. 5, marked in gray). Thus, genetic analysis was conducted with the composite interval mapping of QTL-cartographer program as described (Fig. 7) (Basten et al., 2001). For markers (e.g. chromosome 2) with potential linkage (P<0.05), extended genotyping was performed along with 16 additional SSLP markers (Fig. 7). Furthermore, to investigate whether the loci can modify the phenotype for small mandible or no mandible qualitatively, genetic analysis was also performed with mutant individuals displaying normal mandible and no mandible or those displaying normal mandible and small mandible, respectively (Fig. 7). Consequently, we found that one significant locus was mapped on chromosome 2. Otmf2, which was also linked weakly in the N2 linkage analysis (Fig. 6A), regulates the phenotype displaying no mandible with a peak LOD score of 3.93 at 77 cM (Fig. 7). Furthermore, one suggestive locus, which was characterized by a peak LOD score of 3.13 at 96 cM on chromosome 2, regulates the phenotype of the small mandible (Fig. 7). The above results, in conjunction with N2 linkage data, indicate that at least two distinct modifier loci, Otmf2 and Otmf18, regulate the severity of the otocephalic phenotypes in Otx2 heterozygous mutant mice.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Therefore, these findings, in conjunction, suggest that the genetic mechanism of the otocephalic phenotype is substantially more complex than originally expected. Nevertheless, the modifier loci account for the genetic effect between B6 and CBA strains and can, in part, explain the distribution of craniofacial malformations brought about by haploinsufficiency of the Otx2 gene. Indeed, identification and characterization of these genetic loci provide new insights into mechanistic pathways of mandible development derived from mesencephalic neural crest. Furthermore, the otocephalic mouse model may afford a powerful approach with respect to identification and characterization of candidate genes that may contribute to human agnathia-holoprosencephaly complex diseases.
Otx2 modifier loci may control several distinct steps for the formation of neural crest cells
The modifier loci identified in this study are considered to regulate the developmental processes of mandible, which originates from mesencephalic neural crest. Fate-mapping experiments in chicks have suggest that skull bones of the premandibular and the distal regions of the mandibular regions originate from cephalic neural crest mainly at the level of mesencephalon (Couly et al., 1993; Koentges and Lumsden, 1996
). Similarly in mouse, mesencephalic neural crest cells contribute to the mesenchyme of premandibular and mandibular regions (Osumi-Yamashita et al., 1994
; Imai et al., 1996
). Notably, endogenous Otx2 is expressed in neural plate, neural crest and neural crest cells at the level of mesencephalon; moreover, distal elements of mandibular arch skeletons are lacking or severely affected in Otx2 heterozygous mutants (Matsuo et al., 1995
; Kimura et al., 1997
) (this study). Thus, the Otx2 heterozygous mutant defects relate primarily to Otx2 function in the formation of mesencephalic neural crest (Kimura et al., 1997
).
As the genetic modifier loci were crucial for development of neural crest-derived structures, it is likely that they play an important role in the induction, guidance, migration or differentiation of mesencephalic neural crest in the identical genetic pathway of the Otx2 gene. Neural crest is induced at the dorsolateral edge of the neural plate; from that point, neural crest cells delaminate and migrate along specific routes to many destinations in the vertebrate embryo (Le Douarin, 1982). Grafting experiments in the chick have shown that interactions between embryonic non-neural ectoderm (presumptive epidermis) and neural plate induce the formation of neural crest cells at their interface, and that each of these tissues contributes to the neural crest (Selleck and Bronner-Fraser, 1995
; Liem et al., 1995
). After induction, neural crest delaminates from neural tube; that is, neural crest undergoes an epithelial to mesenchymal conversion and begins to migrate along specific pathways, differentiating into several structures. An important link exists between the guidance and differentiation of neural crest cells. In some cases, specified cells are targeted to the correct destinations, whereas in other instances, cells migrate to sites where they encounter inductive signals. These crest cells finally differentiate into a wide variety of cell types, including neurons and glial cells of the peripheral nervous system, melanocytes and smooth muscle cells, and cartilaginous and skeletal elements in the head (Le Douarin, 1982
).
We have found two genetic loci that significantly modify the severity of mandible phenotypes of Otx2 heterozygous mutants. Notably, the Otmf18 locus appeared to be linked to phenotypes of no mandible and small mandible (Fig. 6C; Table 1). This finding suggests that Otmf18 may direct the formation of mesencephalic neural crest cells fated to the entire mandible. The Otmf2 locus was linked solely with the phenotype of no mandible (Fig. 7; Table 1), indicating that this locus may regulate earlier processes in neural crest formation, i.e. induction or delamination of neural crest. By contrast, one suggestive locus at 96.0 cM on chromosomes 2, was linked with the small mandible phenotype but not with the no mandible phenotype (Fig. 7), suggesting that this locus may regulate later processes, such as the migration or differentiation of mesencephalic neural crest cells, which exclusively contribute to the most distal region of the mandible. Thus, Otx2 may regulate several distinct steps of neural crest formation at that stage, interacting with distinct modifier genes. Further precise mechanisms of mandible development by modifiers await the identification of modifier genes.
Candidate genes and mechanism of interaction with Otx2
We have identified two modifiers; however, mapping resolution is not sufficiently fine to determine the single gene that is responsible for modification of the mandible phenotype. Nevertheless, from this survey, many genes that are believed to interact with Otx2, such as the Emx1, Emx2, Otx1, Cripto and Lim1 genes (Matsuo et al., 1995; Suda et al., 1996
; Suda et al., 1997
; Suda et al., 2001
; Acampora et al., 1997
; Acampora et al., 1998
; Kimura et al., 2001
; Zoltewicz et al., 1999
), were excluded as a genetic modifier of Otx2 in craniofacial development. A potential Otmf2 candidate is Alx4. The modifier, Otmf2, identified on proximal chromosome 2, was located near the Alx4 gene, which is located at 65.0 cM of chromosome 2 (Table 1) (Qu et al., 1998
). Alx4 is a closely related member of the family of paired-related homeobox genes named as Prx family (Qu et al., 1998
). The Prx family consists of Prx1 (previously referred to as Mhox), Prx2, Cart1, Alx3 and Alx4. All of these genes are expressed in the cranial mesenchyme of the mandibular arch (Zhao et al., 1994
; Zhao et al., 1996
; Qu et al., 1997
; Berge et al., 1998a
; Berge et al., 1998b
; Lu et al., 1999
). Indeed, Alx4/ mutation in mouse and haploinsufficiency of human ALX4 cause ossification defects of the skull (Qu et al., 1997
; Wu et al., 2000
; Wuyts et al., 2000
; Mavrogiannis et al., 2001
). Furthermore, the Alx4 heterozygous mutant phenotype is subject to strain-specific genetic modifying loci in mouse (Forsthoefel, 1962
; Forsthoefel, 1968
; Qu et al., 1999
). Moreover, in Alx4/; Cart1/ double mutant mice, the distal region of the mandible was severely truncated (Qu et al., 1999
). Indeed, based on our N2 analysis, no mandible and small mandible phenotypes were suggestively associated in chromosome 10, on which Cart1 is located (Fig. 6B) (Zhao et al., 1994
). Furthermore, expression of Prx family and Otx2 genes was consistently co-localized in the mesenchyme of the mandibular arch (data not shown). These results support our hypothesis that Alx4 may genetically interact with Otx2 in skull development.
One possible interaction between Otx2 and Alx4 involves direct transactivation by these transcriptional factors of Otx2 expression in cephalic mesenchyme. Consistent with this hypothesis, we previously found that DNA sequences, termed motif B (TAATTA), were highly conserved in cis-regulatory elements between mouse and pufferfish Otx2; additionally, these sequences were essential for Otx2 expression in cephalic mesenchyme (Kimura et al., 1997). Motif B is a suitable candidate for the Prx family homeodomain binding sites (Cserjesi et al., 1992
; Kimura et al., 1997
; Cai, 1998
; Qu et al., 1999
). These Prx family proteins exhibit similar DNA-binding activity; moreover, these proteins also form heterodimers and activate transcription in a similar fashion (Qu et al., 1999
). Furthermore, they are dose-sensitive genes and function in a partially redundant manner in mandible development (see above). These lines of evidences supports our hypothesis that Prx family transcription factors directly transactivate the level of Otx2 expression in cephalic mesenchyme.
Differences in the amino acid sequences of these candidate genes between B6 and CBA could underlie subtle changes in the function of these proteins, affecting the mechanisms by which interaction occurs with downstream target genes or transcription factor complex. Alternatively, slight differences may exist between the B6 and CBA alleles in the temporal or spatial patterns and level of expression of these genes. Therefore, assessment of the aforementioned candidates as modifiers of Otx2 will require high-resolution mapping studies employing congenic strains to obtain a more precise localization of these loci. Moreover, sequence comparisons and analysis of relative timing and expression levels in the B6 and CBA alleles are necessary.
Human agnathia-holoprosencephaly complex
Otocephaly, also referred to as agnathia-holoprosencephaly, is a lethal developmental field complex that is characterized by extreme hypoplasia or absence of the mandible, microstomia, aglossia and synotia (Bixler et al., 1985). Significant advances in the study of this disease have revealed the genetic and gene-environment bases of numerous common and rare craniofacial disorders (Winter, 1996
; Wallis and Muenke, 2000
). In humans, this condition can occur alone or in association with various other anomalies, including cyclopia, holoprosencephaly, cerebellar hypoplasia and other visceral anomalies (Opitz, 1980
; Pauli et al., 1983
). Moreover, the otocephalic phenotype has been observed in many animal species, including mouse (Juriloff et al., 1985
), sheep (Willson, 1966
; Smith, 1968
), guinea pig (Wright and Wagner, 1934
) and rabbit (Faller and Rossier, 1969
). In mouse, the otocephaly (oto) mutation was identified in a screen for lethal mutations on chromosome 1 (Juriloff et al., 1985
). This locus has been mapped between D1Mit79 and D1Mit134 in a region of synteny with human 2q35-36 (Zoltewicz et al., 1999
). Strong linkage with the oto locus for mandible phenotypes of Otx2 heterozygous mutants was not detected in the current investigation; however, further consomic or congenic analysis is required in order to finally determine whether the oto locus is associated with the Otx2 mutant phenotype.
In addition to mandible abnormalities, most Otx2+/ mutant mice also displayed holoprosencephaly (Matsuo et al., 1995). In humans, holoprosencephaly is the most common developmental defect of the forebrain (Wallis and Muenke, 1999
). It exhibits an incidence as high as 1 in 250 during early embryogenesis (Matsunaga and Shiota, 1977
). The phenotype of holoprosencephaly is quite variable and proceeds in a continuous spectrum from severe manifestations with major brain and face anomalies to clinically normal individuals (Wallis and Muenke, 1999
). Several distinct human genes for holoprosencephaly have been identified recently, including SHH, ZIC2, SIX3, TGIF and HESX1 (Roessler et al., 1996
; Brown et al., 1998
; Wallis et al., 1999
; Gripp et al., 2000
; Dattani et al., 1998
). Intrafamilial variability of clinical findings exists in kindreds carrying specific mutations in either SHH or SIX3 (Nanni et al., 1999
; Brown et al., 1998
). Indeed, heterozygous carriers for mutations in either SHH or SIX3 can appear phenotypically normal; by contrast, other heterozygous mutation carriers within the same family may be severely affected. This observation suggests the possibility of the occurrence of an undetermined second mutation in the same gene. Alternatively, other gene products or environmental factors may act in these pathways and alterations in the identical or additional genes or factors could be required for severe holoprosencephaly manifestations (Nanni et al., 1999
; Brown et al., 1998
). It is not known as to whether OTX2 is involved in human holoprosencephaly. The modifier loci identified in this study might be suitable candidates for genetic causes of human craniofacial congenital diseases. Identification of human mutations of OTX2 modifier genes and evaluation of interaction between these genes and environmental causes awaits molecular identification of these modifier genes.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acampora, D., Mazan, S., Lallemand, Y., Avantaggiato, V., Maury, M., Simeone, A. and Brûlet, P. (1995). Forebrain and midbrain regions are deleted in Otx2/ mutants due to a defective anterior neuroectoderm specification during gastrulation. Development 121, 3279-3290.
Acampora, D., Avantaggiato, V., Tuorto, F. and Simeone, A. (1997). Genetic control of brain morphogenesis through Otx gene dosage requirement. Development 124, 3639-3650.
Acampora, D., Avantaggiato, V., Tuorto, F., Briata, P., Corte, G. and Simeone, A. (1998). Visceral endoderm-restricted translation of Otx1 mediates recovery of Otx2 requirements for specification of anterior neural plate and normal gastrulation. Development 125, 5091-5104.
Ang, S. L., Conlon, R. A., Jin, O. and Rossant, J. (1994). Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants. Development 120, 2979-2989.
Ang, S.-L., Jin, O., Rhinn, M., Daigle, N., Stevenson, L. and Rossant, J. (1996). A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development 122, 243-252.
Basten, C. J., Weir, B. S. and Zeng, Z.-B. (2001). QTL Cartographer: A reference manual and tutorial for QTL mapping. Program in Statistical Genetics, Department of Statistics, North Carolina State University.
Berge, D. t., Brouwer, A., Korving, J., Martin, J. F. and Meijlink, F. (1998a). Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs. Development 125, 3831-3842.
Berge, D. t., Brouwer, A., Bahi, S. E., Guenet, J.-L., Robert, B. and Meijlink, F. (1998b). Mouse Alx3: An aristaless-like homeobox gene expressed during embryogenesis in ectomesenchyme and lateral plate mesoderm. Dev. Biol. 199, 11-25.[Medline]
Bixler, D., Ward, R. and Gale, D. D. (1985). Agnathia-holoprosencephaly: a developmental field complex involving face and brain. Report of 3 cases. J. Craniofac. Genet. Dev. Biol. Suppl. 1, 241-249.
Brown, S. A., Warburton, D., Brown, L. Y., Yu, C. Y., Roeder, E. R., Stengel-Rutkowski, S., Hennekam, R. C. and Muenke, M. (1998). Holoprosencephaly due to mutations in ZIC2, a homologue of Drosophila odd-paired. Nat. Genet. 20, 180-183.[Medline]
Cai, R. L. (1998). Human CART1, a paired-class homeodomain protein, activates transcription through palindromic binding sites. Biochem. Bioph. Res. Comm. 250, 305-311.[Medline]
Couly, G. F., Coltey, P. M. and le Douarin, N. M. (1993). The triple origin of skull in higher vertebrates: a study in quail-chick chimeras. Development 117, 409-429.
Cserjesi, P., Lilly, B., Bryson, L., Wang, Y., Sassoon, D. A. and Olson, E. N. (1992). MHox: a mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer. Development 115, 1087-1101.
Darvasi, A. (1998). Experimental strategies for the genetic dissection of complex traits in animal models. Nat. Genet. 18, 19-24.[Medline]
Dattani, M. T., Martinez-Barbera, J. P., Thomas, P. Q., Brickman, J. M., Gupta, R., Martensson, I.-L., Toresson, H., Fox, M., Wales, J. K. H., Hindmarsh, P. C. et al. (1998). Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse. Nat. Genet. 19, 125-133.[Medline]
Dietrich, W., Katz, H., Lincoln, S. E., Shin, H.-S., Friedman, J., Dracopoli, N. C. and Lander, E. S. (1992). A genetic map of the mouse suitable for typing intraspecific crosses. Genetics 131, 423-447.
Dietrich, W. F., Miller., J. C., Steen, R. G., Merchant, M., Damron, D., NAhf, R., Gross, A., Joyce, D. C., Wessel, M., Dredge, R. D. et al. (1994). A genetic map of the mouse with 4,006 simple sequence length polymorphisms. Nat. Genet. 7, 220-245.[Medline]
Faller, A. and Rossier, B. (1969). Reconstruction of brain and ventricle system in an anchyote prosophthalmic otocephalic newborn cephalothoracopagus rabbit. Acta Anat. 73, 2-31.
Forsthoefel, P. F. (1962). Genetics and manifold effects of Strongs luxoid gene in the mouse, including its interactions with Greens luxoid and Carters luxate genes. J. Morphol. 110, 391-420.
Forsthoefel, P. F. (1968). Responses to selection for plus and minus modifiers of some effects of Strongs luxoid gene on the mouse skeleton. Teratology 1, 339-51.[Medline]
Gripp, K. W., Wotton, D., Edwards, M. C., Roessler, E., Ades, L., Meinecke, P., Richieri-Costa, A., Zackai, E. H., Massague, J., Muenke, M. and Elledge, S. J. (2000). Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination. Nat. Genet. 25, 205-208.[Medline]
Horan, G. S. B., Kovacs, E. N., Behringer, R. R. and Featherstone, M. S. (1995). Mutations in paralogous Hox genes result in overlapping homeotic transformations of the axial skeleton: evidence for unique and redundant function. Dev. Biol. 169, 359-372.[Medline]
Imai, H., Osumi-Yamashita, N., Ninomiya, Y. and Eto, K. (1996). Contribution of early-emigrating midbrain crest cells to the dental mesenchyme of mandibular molar teeth in rat embryos. Dev. Biol. 176, 151-165.[Medline]
Jiang, C. and Zeng, Z.-B. (1995) Multiple trait analysis of genetic mapping for quantitative trait loci. Genetics 140, 1111-1127.
Juriloff, D. M., Sulik, K. K., Roderick, T. H. and Hogan, B. K. (1985). Genetic and developmental studies of a new mouse mutation that produces otocephaly. J. Craniofac. Genet. Dev. Biol. 5, 121-145.[Medline]
Kelly, W. L. and Bryden, M. M. (1983). A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetuses and small vertebrates. Stain Technol. 58, 131-134.[Medline]
Kimura, C., Takeda, N., Suzuki, M., Oshimura, M., Aizawa, S. and Matsuo, I. (1997). Cis-acting elements conserved between mouse and pufferfish Otx2 genes govern the expression in mesencephalic neural crest cells. Development 124, 3929-3941.
Kimura, C., Yoshinaga, K., Tian, E., Suzuki, M., Aizawa, S. and Matsuo, I. (2000). Visceral endoderm mediates forebrain development by suppressing posteriorizing signals. Dev. Biol. 225, 304-321.[Medline]
Kimura, C., Shen, M. M., Takeda, N., Aizawa, S. and Matsuo, I. (2001). Complementary functions of Otx2 and Cripto in initial patterning of mouse epiblast. Dev. Biol. 235, 12-32.[Medline]
Koentges, G. and Lumsden, A. (1996). Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development 122, 3229-3242.
Lander, E. S. and Botstein, D. (1989). Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121, 185-199.
Lander, E. S. and Schork, N. J. (1994). Genetic dissection of complex traits. Science 265, 2037-2048.[Medline]
Lander, E. and Kruglyak, L. (1995). Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat. Genet. 11, 241-247.[Medline]
LeCouter, J. E., Kablar, B., Whyte, P. F. M., Ying, C. and Rudnicki, M. A. (1998). Strain-dependent embryonic lethality in mice lacking the retinoblastoma-related p130 gene. Development 125, 4669-4679.
Le Douarin, N. (1982). The neural crest. In Development and Cell Biology Series 12. Cambridge: Cambridge University Press.
Liem, K. F., Jr, Tremml, G., Roelink, H. and Jessell, T. M. (1995). Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82, 969-979.[Medline]
Love, J. M., Knight, A. M., McAleer, M. A. and Todd, J. A. (1990). Towards construction of a high resolution map of the mouse genome using PCR-analyzed microsatellites. Nucleic Acids Res. 18, 4123-4130.[Abstract]
Lu, M.-F., Cheng, H.-T., Kern, M. J., Potter, S. S., Tran, B., Diekwisch, T. G. H. and Martin, J. F. (1999). prx-1 functions cooperatively with another paired-related homeobox gene, prx-2, to maintain cell fates within the craniofacial mesenchyme. Development 126, 495-504.
Matsunaga, E. and Shiota, K. (1977). Holoprosencephaly in human embryos: Epidemiologic studies of 150 cases. Teratology 16, 261-272.[Medline]
Matsuo, I., Kuratani, S., Kimura, C., Takeda, N. and Aizawa, S. (1995). Mouse Otx2 functions in the formation and patterning of rostral head. Genes Dev. 9, 2646-2658.[Abstract]
Mavrogiannis, L. A., Antonopoulou, I., Baxova, A., Kutilek, S., Kim, C. A., Sugayama, S. M., Salamanca, A., Wall, S. A., Morris-Kay, G. M. and Wilkie, A. O. M. (2001). Haploinsufficiency of the human homeobox gene ALX4 causes skull ossification defects. Nat. Genet. 27, 17-18.[Medline]
Nanni, L., Ming, J. E., Bocian, M., Steinhaus, K., Bianchi, D. W., Die-Smulders, C., Giannotti, A., Imaizumi, K., Jones, K. L., Campo, M. D. et al. (1999). The mutational spectrum of the Sonic hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly. Hum. Mol. Genet. 8, 2479-2488.
Opitz, J. M. (1980). Letter to the editors. Clin. Genet. 17, 69-71.
Osumi-Yamashita, N., Ninomiya, Y., Doi, H. and Eto, K. (1994). The contribution of both forebrain and midbrain crest cells to the mesenchyme in the frontonasal mass of mouse embryos. Dev. Biol. 164, 409-419.[Medline]
Pauli, R. M., Pettersen, J. C., Arya, S. and Gilbert, E. F. (1983). Familial agnathia-holoprosencephaly. Am. J. Med. Genet. 14, 677-698.[Medline]
Proetzel, G., Pawlowski, S. A., Wiles, M. V., Yin, M., Boivin, G. P., Howles, P. N., Ding, J., Ferguson, M. W. J. and Doetschman, T. (1995). Transforming growth factor-ß3 is required for secondary palate fusion. Nat. Genet. 11, 409-414.[Medline]
Qu, S., Li, L. and Wisdom, R. (1997). Alx4:cDNA cloning and characterization of a novel paired-type homeodomain protein. Gene 203, 217-223.[Medline]
Qu, S., Tucker, S. C., Ehrlich, J. S., Levorse, J. M., Flaherty, L. A., Wisdom, R. and Vogt, T. F. (1998). Mutations in mouse Aristaless-like4 cause Strongs luxoid polydactyly. Development 125, 2711-2721.
Qu, S., Tucker, S. C., Zhao, Q., deCrombrugghe, B. and Wisdom, R. (1999). Physical and genetic interactions between Alx4 and Cart1. Development 126, 359-369.
Roessler, E., Belloni, E., Gaudenz, K., Jay, P., Berta, P., Scherer, S. W., Tsui, L. C. and Muenke, M. (1996). Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat. Genet. 14, 357-360.[Medline]
Rozmahel, R., Wilschanski, M., Matin, A., Plyte, S., Oliver, M., Auerbach, W., Moore, A., Forstner, J., Durie, P., Nadeau, J., Bear, C. and Tsui, L.-C. (1996). Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nat. Genet. 12, 280-287.[Medline]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Selleck, M. A. and Bronner-Fraser, M. (1995). Origins of the avian neural crest: the role of neural plate-epidermal interactions. Development 121, 525-538.
Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A. and Boncinelli, E. (1992). Nested expression domains of four homeobox genes in developing rostral brain. Nature 358, 687-690.[Medline]
Simeone, A., Acampora, D., Mallamaci, A., Stornaiuolo, A., DApice, M. R., Nigro, V. and Boncinelli, E. (1993). A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo. EMBO J. 12, 2735-2747.[Abstract]
Smith, I. D. (1968). Agnathia and micrognathia in the sheep. Aust. Vet. J. 44, 510-511.[Medline]
Suda, Y., Matsuo, I., Kuratani, S. and Aizawa, S. (1996). Otx1 function overlaps with Otx2 in development of mouse forebrain and midbrain. Genes Cells 1, 1031-1044.
Suda, Y., Matsuo, I. and Aizawa, S. (1997). Cooperation between Otx1 and Otx2 genes in developmental patterning of rostral brain. Mech. Dev. 69, 125-141.[Medline]
Suda, Y., Hossain, Z. M., Kobayashi, C., Hatano, O., Yoshida, M., Matsuo, I. and Aizawa, S. (2001). Emx2 directs the development of diencephalon in cooperation with Otx2. Development 128, 2433-2450.
Tian, E, Kimura, C., Takeda, N., Aizawa, S. and Matsuo, I. (2002). Otx2 is required to respond to signals from anterior neural ridge for forebrain specification. Dev. Biol. 242, 204-223.[Medline]
Wallis, D. E. and Muenke, M. (1999). Molecular mechanisms of holoprosencephaly. Mol. Genet. Metab. 68, 126-138.[Medline]
Wallis, D. E. and Muenke, M. (2000). Mutations in holoprosencephaly. Hum. Mutat. 16, 99-108.[Medline]
Wallis, D. E., Roessler, E., Hehr, U., Nanni, L., Wiltshire, T., Richieri-Costa, A., Gillessen-Kaesbach, G., Zackai, E. H., Rommens, J. and Muenke, M. (1999). Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly. Nat. Genet. 22, 196-198.[Medline]
Wawersik, S., Purcell, P., Rauchman, M., Dudley, A. T., Robertson, E. J. and Maas, R. (1999). BMP7 acts in murine lens placode development. Dev. Biol. 207, 176-188.[Medline]
Willson, J. E. (1966). Congenital otocephalus in a lamb. Vet. Med. Small Anim. Clin. 61, 58-59.[Medline]
Winter, R. M. (1996). Whats in a face? Nat. Genet. 12, 124-129.[Medline]
Wojnowski, L., Stancato, L. F., Zimmer, A. M., Hahn, H., Beck, T. W., Larner, A. C., Rapp, U. R. and Zimmer, A. (1998). Craf-1 protein kinase is essential for mouse development. Mech. Dev. 76, 141-149.[Medline]
Wright, S. and Wagner, K. (1934). Types of subnormal development of the head from inbred strains of guinea pigs and their bearing on the classification and interpretation of vertebrate monsters. Am. J. Anat. 54, 383-447.
Wu, Y. Q., Badano, J. L., McCaskill, C., Vogel, H., Potocki, L. and Shaffer, L. G. (2000). Haploinsufficiency of ALX4 as a potential cause of parietal foramina in the 11p11.2 contiguous gene-deletion syndrome. Am. J. Hum. Genet. 67, 1327-1332.[Medline]
Wuyts, W., Cleiren, E., Homfray, T., Rasore-Quartino, A., Vanhoenacker, F. and van Hul, W. (2000). The ALX4 homeobox gene is mutated in patients with ossification defects of the skull (foramina parietalia permagna, OMIM 168500). J. Med. Genet. 37, 916-920.
Yagi, T., Tokunaga, T., Furuta, Y., Nada, S., Yoshida, M., Tsukada, T., Saga, Y., Takeda, N., Ikawa, Y. and Aizawa, S. (1993). A novel ES cell line, TT2, with high germline-differentiating potency. Anal. Biochem. 214, 70-76.[Medline]
Zeng, Z.-B. (1994). Precision mapping of quantitative trait loci. Genetics 136, 1457-1468.
Zhao, G.-Q., Eberspaecher, H., Seldin, M. F. and de Crombrugghe, B. (1994). The gene for homeodomain-containing protein Cart-1 is expressed in cells that have a chondrogenic potential during embryonic development. Mech. Dev. 48, 245-254.[Medline]
Zhao, Q., Behringer, R. R. and de Crombrugghe, B. (1996). Prenatal folic acid treatment suppresses acrania and meroanencephaly in mice mutant for the Cart1 homeobox gene. Nat. Genet. 13, 275-283.[Medline]
Zoltewicz, J. S., Plummer, N. W., Lin, M. I. and Peterson, A. S. (1999). oto is a homeotic locus with a role in anteroposterior development that is partially redundant with Lim1. Development 126, 5085-5095.