Human Molecular Genetics, 2000, Vol. 9, No. 10 1473-1480
© 2000 Oxford University Press
Increased levels of apoptosis in the prefusion neural folds underlie the craniofacial disorder, Treacher Collins syndrome
School of Biological Sciences and Department of Dental Medicine and Surgery, 3.239 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK and 1Department of Experimental Pathology, Lund University Hospital, S-22185 Lund, Sweden
Received 22 February 2000; Revised and Accepted 10 April 2000.
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ABSTRACT |
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Treacher Collins syndrome (TCS) is an autosomal dominant disorder of human craniofacial development that results from loss-of-function mutations in the gene TCOF1. Although this gene has been demonstrated to encode the nucleolar phosphoprotein treacle, the developmental mechanism underlying TCS remains elusive, particularly as expression studies have shown that the murine orthologue, Tcof1, is widely expressed. To investigate the molecular pathogenesis of TCS, we replaced exon 1 of Tcof1 with a neomycin-resistance cassette via homologous recombination in embryonic stem cells. Tcof1 heterozygous mice die perinatally as a result of severe craniofacial anomalies that include agenesis of the nasal passages, abnormal development of the maxilla, exencephaly and anophthalmia. These defects arise due to a massive increase in the levels of apoptosis in the prefusion neural folds, which are the site of the highest levels of Tcof1 expression. Our results demonstrate that TCS arises from haploinsufficiency of a protein that plays a crucial role in craniofacial development and indicate that correct dosage of treacle is essential for survival of cephalic neural crest cells.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Facial development involves a complex series of events that are frequently disturbed, resulting in a wide variety of craniofacial anomalies. Such disorders are extremely distressing and are among the most common of the congenital malformations affecting mankind (1). Numerous craniofacial anomalies exist, each providing a unique opportunity to study a particular aspect of development. Treacher Collins syndrome (TCS) is one example of a well-characterized autosomal dominant disorder of craniofacial morphogenesis. The clinical features of TCS include hypoplasia of the facial bones, particularly the mandible and zygomatic complex; abnormalities of the external/middle ear, which result in conductive hearing loss; lateral downward sloping of the palpebral fissures; and cleft palate (2,3). There is, however, marked inter- and intra-familial variation in the phenotype (4).
The gene mutated in TCS, TCOF1, has been isolated using a positional cloning strategy (5) and shown to encode the nucleolar phosphoprotein, treacle (6–9). Analysis of TCOF1 has indicated that TCS arises as the result of loss-of-function mutations, suggesting that the underlying mechanism is haploinsufficiency (5,7,10,11). However, the specific role of treacle in craniofacial development and the molecular pathogenesis of TCS have remained elusive, particularly as expression studies have shown that murine Tcof1 is widely expressed (12,13). A number of mechanisms, which include abnormal neural crest cell migration (14), abnormal cell death (15,16) and inappropriate cellular differentiation (17), have been proposed; however, there is little experimental evidence to support any of them. Nevertheless, whole-mount in situ hybridization studies have indicated that the highest levels of Tcof1 expression are observed in the crests of the neural folds immediately prior to fusion, and in the first pharyngeal arch (12). These observations are consistent with the hypothesis that TCS results from haploinsufficiency and with a role for the gene in craniofacial morphogenesis. To elucidate the molecular pathogenesis of TCS and to analyse the function of treacle in vivo, we have generated Tcof1 heterozygous mice by targeted mutagenesis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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As a prelude to constructing the gene-targeting vector, we determined that Tcof1 was encompassed by 25 exons (data not shown). Subsequently, a more detailed restriction map of an 11 kb region encompassing exons 1, 2 and 3 was produced (Fig. 1A). To ensure complete functional inactivation of Tcof1 we replaced exon 1, which contains the translation initiation codon, with a neomycin-resistance cassette (Fig. 1A). Using two independent correctly targeted embryonic stem (ES) cell clones (Fig. 1B and C), chimeric males were produced and inter-crossed with C57BL6 females. Although the ES cell-derived coat colour gene was transmitted to the offspring, no Tcof1 heterozygous mice were detected by genotyping of 30 progeny. To investigate the possibility that loss of a single Tcof1 allele resulted in heterozygous lethality, a litter of embryonic day 13 (E13) mice was examined. Major craniofacial abnormalities were immediately apparent in three of the eight embryos, genotyping of which confirmed that they were heterozygotes. These observations prompted us to perform a detailed analysis of Tcof1+/– mice during embryogenesis (Fig. 2). Abnormalities in Tcof1+/– mice were first detected at E8, after which they exhibited a generalized developmental delay of ~0.5–1 day that continued throughout development (Fig. 2). In addition, Tcof1 embryos exhibited severe structural craniofacial anomalies that were not attributable solely to this delay.
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Analysis of E8.5 mice by scanning electron microscopy indicated that, although wild-type embryos displayed well-developed head folds and the first signs of optic development, Tcof1+/– embryos exhibited smaller, rounded neural folds and an absence of the optic evagination (Fig. 3A and B). While all wild-type embryos had completed the axial turning sequence and closed their rostral neuropore by E9, their Tcof1+/– littermates remained unturned with a patent rostral neuropore (Fig. 2A). By E9.5, structural anomalies in Tcof1+/– mice were clearly distinct, the frontonasal process being markedly hypoplastic with no evidence of division of the forebrain into telencephalic or optic vesicles. By E10.5 the formation of the olfactory pit in wild-type mice divided the frontonasal mass into medial and lateral nasal processes (Figs 2B and 3C). In contrast, Tcof1+/– embryos failed to develop either medial or lateral nasal processes (Figs 2B and 3D) and they exhibited exencephaly with neuroepithelium protruding through the open rostral neuropore. The brain was also hypoplastic with severely compromised fore- and midbrain development; however, this did not appear to be solely a secondary effect of the exencephaly as it was observed, albeit to a lesser extent, in the minority of mutant embryos that succeeded in closing their rostral neuropore. In addition, the mandibular component of the first arch was smaller and shorter than normal and the maxillary component was hypoplastic, less distinct and displaced rostrally (Fig. 3C and D).
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Histological analysis of E12.5 mice revealed that only a rudimentary upper lip had formed in Tcof1+/– embryos and that, unlike their wild-type littermates, these animals failed to develop nasal pits (Fig. 4A and B). The cranial ganglia and the otocysts of the mutant mice were also smaller than normal (Fig. 4A and B). By E14.5, the lack of nasal passages and anophthalmia in Tcof1+/– mice was clearly apparent (Figs 2C, 4C and D). Mutant mice also displayed mandibular hypoplasia, severe retrognathia of the middle third of the face, and low-set cup-shaped ears (Fig. 2C). While the secondary palate of wild-type mice had elevated and fused, the palatal shelves of Tcof1+/– mice were markedly disorganized with little evidence of normal development. Nevertheless, despite the fact that they were displaced, the incisor and molar tooth germs had developed to a stage similar to their wild-type counterparts (Fig. 4C and D).
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Whole-mount skeletal analysis was subsequently used to determine the extent of the abnormalities of the craniofacial skeleton. An absence of the frontal, parietal and inter-parietal bones of the vault of the skull was observed in Tcof1+/– mice (Fig. 4E and F). Strikingly, the nasal capsule and the maxilla were grossly malformed, and were replaced with an amorphous mass of bone and cartilage, the nasal capsule being represented by a cartilaginous spear (Fig. 4F and G). The zygomatic arch, the tympanic ring and the middle ear ossicles were also hypoplastic and misshapen. Immediately before birth (Fig. 2D), Tcof1+/– mice were alive and exhibited beating hearts and embryonic movements; however, post-natally they were unable to establish an airway, due to the lack of nasal passages, and, together with exencephaly, this resulted in death shortly after birth.
To investigate the developmental mechanism underlying the craniofacial malformations observed in the mutant mice, a whole-mount TUNEL assay, which labels individual apoptotic nuclei, was used to investigate the distribution of apoptotic cells in Tcof1+/– embryos. The pattern observed in wild-type embryos was consistent with previous reports (18). Small numbers of labelled nuclei were observed along the ridges of the closing neural tube at the level of the hindbrain, and in the cranial and pharyngeal arch mesenchyme (Fig. 5A and B). In contrast, the number of apoptotic cells was massively elevated in Tcof1+/– embryos. This was particularly apparent at E8.5 when the entire neuroepithelium of the cranial neural folds, and the neural tube, were decorated with a profusion of apoptotic cells (Fig. 5C and D). Abnormally high levels of apoptosis were observed in the cranial region and the neural tube of Tcof1+/– mice throughout E9 (Fig. 5E and F). Thereafter, the level of apoptosis decreased in the cranial region remaining noticeably high only in the post-fusion neural tube.
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These observations strongly suggest that in Tcof1+/– mice a subset of neural crest cells enter an apoptotic pathway. In support of these findings, immunostaining of E10.5 mice with the anti-neurofilament antibody, 2H3, indicated that the neural crest cell-derived cranial ganglia were severely hypoplastic, the ophthalmic branch of the trigeminal nerve and the glossopharyngeal ganglia/nerves being absent at this stage of development (Fig. 6A and B). In addition, the dorsal root ganglia, which are also derived from neural crest cells, were underdeveloped and markedly disorganized (Fig. 6C and D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Over recent years, the genetic mutations underlying several craniofacial-malformation syndromes have been identified (19). For example, branchio-oto-renal syndrome is caused by mutations in EYA1, the phenotype arising as the result of defective inductive tissue interactions and apoptotic regression of affected organ primordia (20–22). Similarly, a subset of cases of holoprosencephaly are due to mutations in Sonic Hedgehog (23), which is essential for medial patterning of the entire central nervous system (24). However, in the vast majority of these conditions, the underlying developmental mechanism has not been elucidated. To investigate the molecular pathogenesis of TCS, we have generated a mouse model of this condition by targeted mutagenesis. Tcof1 heterozygous mice died shortly after birth as a result of severe craniofacial anomalies that included agenesis of the nasal passages, abnormal development of the maxilla, exencephaly and anophthalmia. Although there is obvious overlap between the phenotype of Tcof1+/– mice and TCS patients (malformation of the maxilla, nasal complex and external/middle ear and hypoplasia of the mandible), that observed in the former is clearly more severe. While perinatal lethality resulting from a compromised airway has been documented in a small subset of TCS patients (25), exencephaly and anophthalmia have never been reported. This may be due to species-specific differences in craniofacial development between man and mouse. This is exemplified by a recent study which demonstrated that, despite being highly conserved, Wnt7a shows a different spatio-temporal expression pattern in the developing fore- and midbrain when compared with its human homologue WNT7A (26). However, due to our targeting strategy, Tcof1+/– embryos are hemizygous, whereas precisely comparable mutations are not observed in man (5,7,10,11). Nevertheless, analysis of this mouse model has allowed us to demonstrate that TCS results from haploinsufficiency of treacle, which causes a subset of neural crest cells to enter an apoptotic pathway. Interestingly, the most severe defects observed in Tcof1+/– mice occur in the frontonasal region and the maxilla, while the pharyngeal arch derivatives in these embryos, although hypoplastic, are morphologically similar to their wild-type counterparts. In this context, fate-mapping studies have shown that there is extensive mixing of neural crest-derived ectomesenchyme and paraxial mesoderm in the periocular and facial mesenchyme, but that the two populations are segregated in the pharyngeal arches (27,28).
Given its subcellular localization and similarities to rat nucleolar phosphoprotein 140 (29), it has been proposed that treacle may function in ribosome biogenesis (6–9). It is therefore possible that appropriate dosage of treacle is a requirement for sustaining a high rate of protein synthesis in rapidly proliferating cells, such as neural crest cells, whereas this is less so in cells that exhibit a slower rate of growth. In support of this hypothesis, it is significant that the sites at which elevated levels of apoptosis in Tcof1+/– mice are observed coincide with those at which the highest level of Tcof1 expression occurs, i.e. the pre-fusion neural folds (12). Moreover, Tcof1+/– embryos are smaller and developmentally delayed when compared with their wild-type littermates. In this context, mutation of the Drosophila minifly gene, which also encodes a ubiquitously expressed nucleolar phosphoprotein, results in small body size, developmental delay and reduced female fertility resulting from apoptotic cell death in the ovaries (30). As the protein encoded by the minifly gene plays a central role in ribosomal RNA processing, it will be interesting to investigate whether this process is inefficient in TCS patients/Tcof1+/– mice.
It is currently unclear precisely how the raised levels of apoptosis observed in the prefusion neural folds of Tcof1+/– embryos produce the observed phenotype. Clearly, as evidenced by our anti-neurofilament staining, there is a marked reduction of neural crest cells migrating into the developing craniofacial complex. The high levels of apoptosis may also lead to reduction or ablation of the expression domains of other key regulators of craniofacial development. Given the phenotypic overlap observed between the mice generated in the current study and those harbouring targeted mutations in a number of other genes, this seems to be a distinct possibility. For example, the distal-less-related genes, which act in concert to control forebrain development, are expressed in the craniofacial ectoderm and cranial neural crest where they co-operate to control craniofacial skeletogenesis. Mice with targeted mutations of the Dlx genes have craniofacial phenotypes (31,32). Dlx5 is of particular interest as it is expressed earlier than the other Dlx genes (33–35), being detected at all axial levels of the neural ridge in E8.25 mice. High levels of Dlx5 are detected in the anterior neural ridge that has been proposed to act as an organizing centre for forebrain and facial development (36). High levels of Dlx5 continue to be detected in the neural ridge and in the otic vesicle throughout E9. The expression of Dlx5 therefore broadly corresponds with the sites of apoptosis in Tcof1+/– mice. There is also considerable overlap between the phenotype of Tcof1+/– and Dlx5–/– mice in that both mutants exhibit exencephaly and defects of the nasal complex (34,35). Similarly, the anophthalmia and absence of nasal passages observed in Tcof1+/– embryos are reminiscent of the Small eye phenotype observed in both mouse and rat, both of which are due to disruption of the paired box transcription factor, Pax6 (37,38). Interestingly, the Small eye phenotype is also associated with impaired migration of midbrain neural crest cells, at least in the rat (38). In humans mutations of a single PAX6 allele are responsible for a range of congenital eye malformations, chiefly aniridia (39,40), while homozygosity for PAX6 loss-of-function results in loss of eyes and nasal cavities (41). Moreover, Pax6 is expressed in the lateral aspects of the prosencephalic neural plate during E8 (36). Finally, the cartilage homeobox gene Cart1 is expressed in the forebrain mesenchyme and pharyngeal arches, transcripts being particularly abundant in the mesenchyme of the frontonasal mass and that surrounding the optic vesicles at E9.5. Cart1 homozygous mutant mice, like Tcof1+/– mice, are acranic and exhibit abnormalities of the nasal complex (42). Obviously, these molecules represent only a small subset of the regulators of craniofacial development and a thorough molecular analysis of the Tcof1+/– mice generated in the current study will be important in further delineation of the molecular events underlying the observed phenotype.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Targeting strategy
A cDNA clone encompassing the entire Tcof1 coding sequence was used to screen a mouse 129 bacteriophage library. The genomic organization of Tcof1 was determined according to previously published methods (6). To generate the targeting vector, a 4 kb BamHI fragment was inserted into the XhoI site of pWH9 to provide the 5' arm of homology, and a 4 kb XhoI fragment was inserted into the SalI site of pWH9 to provide the 3' arm of homology. Upon homologous recombination, exon 1 of Tcof1, including the entire 5' untranslated region, is replaced by a neomycin-resistance cassette (Fig. 1A). Culture conditions for R1 ES cells, electroporation, isolation and blastocyst injection of ES cell clones, as well as breeding of chimeric mice, was performed as described previously (43). ES cell clones and embryos were screened by Southern hybridization of HindIII-digested genomic DNA using an external 1.1 kb BamHI–HindIII probe (Fig. 1B). To confirm the 3' integration event, and for subsequent genotyping, PCR analysis of genomic DNA was undertaken using the following primers in a duplex reaction: wild-type forward primer, 5'-TTC AGA CTC CTC TGC CCG TCT C-3'; targeted forward primer, 5'-TGA AGA ACG AGA TCA GCA GCC TC-3'; common reverse primer 5'-GAC TAC CCA TCA GCC ATT CCT GT-3' (Fig. 1C). In addition, screening with a probe derived from the neomycin cassette confirmed that additional random integration events had not occurred (data not shown). All protocols were performed in accordance with the Animals (Scientific Procedures) Act, UK, 1986.
Histology
Embryos were fixed in Bouin’s solution, dehydrated through a graded series of ethanol, cleared in chloroform and embedded in Paraplast wax. The embryos were serially sectioned at 5–7 µm in coronal, tranverse and/or sagittal planes, and stained using either haematoxylin and eosin (with or without alcian blue) or Mallory trichrome.
Scanning electron microscopy
E8.5–E10.5 mice were fixed in 2.5% glutaraldehyde/0.1 M sodium cacodylate, post-fixed in osmium tetroxide, washed in 0.1 M sodium cacodylate buffer, dehydrated, critical point dried, sputter-coated with gold and viewed in a Cambridge Stereoscan 360.
Whole-mount skeletal analysis
For skeletal analysis, E18 mice were skinned, eviscerated and fixed in 100% ethanol for 24 h prior to processing as detailed by Wallin et al. (44).
Whole-mount antibody TUNEL assay
E8.5–E10.5 embryos were fixed in 4% paraformaldehyde for 2 h at 4°C. The embryos were subsequently processed for the TUNEL assay as described by Conlon et al. (18). Apoptotic nuclei were labelled with 20 µM digoxygenin-dUTP (Roche Molecular Biochemicals, Lewes, UK), 20 µM dTTP and 0.3 U µl TdT in 1 x TdT buffer (30 mM Tris, 140 mM cacodylate pH 7.2 and 1 mM cobalt chloride) and detected with an anti-digoxygenin antibody coupled to alkaline phosphatase. The primary antibody was then detected using 0.3 mg ml nitroblue tetrazolium salt and 0.17 mg/ml 5-bromo-4-chloro-3-indolyl phosphate. To obtain sections of the embryos after this assay, the embryos were infiltrated with and embedded in 25% gelatin. Five micron cryosections were cut and mounted in DABCO mounting medium [nine parts glycerol containing 2% 1,4-diazobicyclo-(2,2,2)-octane and one part 0.2 M Tris–HCl, pH 7.5].
Whole-mount immunohistochemistry
E10.5 embryos were fixed as above and processed for immunohistochemical analyses as described by Conlon et al. 18). Neurofilaments were detected using the primary antibody 2H3 and a secondary antibody raised against mouse immunoglobulins, coupled to horseradish peroxidase (DAKO, Copenhagen, Denmark). The reaction was detected using 3, 3'-diaminobenzidine (Sigma, Poole, UK).
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ACKNOWLEDGEMENTS |
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We thank members of the Dixon and Fässler laboratories for their help with the preparation of this manuscript. The monoclonal antibody 2H3, developed by T. M. Jessell and J. Dodd, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences. The financial support of the Wellcome Trust (grant reference numbers 050591 and 058423) and the Swedish Medical Research Council is gratefully acknowledged.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 161 275 5620; Fax: +44 161 275 5620; Email: mike.dixon@man.ac.uk
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REFERENCES |
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